JP6655245B2 - Cartridge for protein or pathogen detection and automatic detection device - Google Patents

Description

  The present invention relates to a cartridge for detecting a protein or a pathogen and an automatic detection device. More specifically, the present invention relates to a protein or pathogen detection cartridge and an automatic detection device provided with a conductive diamond electrode having a protein or pathogen recognition element immobilized on the surface.

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

  Have 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 an Au electrode via a gold thiol bond, thereby simultaneously detecting both hemagglutinin (HA) and neuraminidase (NA) oligonucleotide targets. It is possible to Kamikawa et al. Report IFV detection using magnetic nanoparticles covered with an electroactive polyaniline and modified with an H5N1 HA monoclonal antibody on the surface (Non-patent Document 2). In this method, nanoparticles interacting with the H5N1 virus are recovered from serum by strong magnetic force (MPC), and the amount of virus binding is quantified by electrochemical measurement.

  Sakurai et al. Report a rapid and highly sensitive typing of seasonal influenza using fluorescent immunochromatography (Non-Patent Document 3). Immunochromatography, a clinically used method for detecting IFV, has a detection sensitivity of 1000 pfu and is difficult to detect in the early stage of infection. In the same document, Sakurai et al. Used both a virus capture antibody and a sensitizer-labeled detection antibody to capture the virus in a sandwich form, and to use a densitometry analyzer or a fluorescent immunochromatography analyzer to detect the virus. It is reported that the sensitivity was improved 100 times as much as the conventional one. However, a complementary oligonucleotide for hybridization or an antibody immobilized on a substrate requires enormous labor and cost for production, and has a problem of storage stability.

Hassen et al. Report 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 a gold electrode surface.

  On the other hand, a boron-doped diamond electrode has excellent characteristics as compared with other conventional electrode materials such as glassy carbon and a platinum electrode, and has recently attracted attention. In addition to diamond's well-known properties of 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. The boron-doped diamond electrode is physically and chemically stable and has 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 reference, oxalic acid is electrochemically detected using a diamond electrode modified with 4-pentenoic acid.

  The conventional IFV detection device needs to perform wavelength measurement after adding a hybridization reagent or gold nanoparticles to a sample. There is a demand for a device that can detect viruses, various pathogenic bacteria, and pathogens with high sensitivity and at an early stage without using antibodies or expensive devices.

JP 2007-292717 A (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 an apparatus for quickly and highly sensitively detecting a protein or a pathogen which solves the conventional problems.

  The present invention provides a cartridge for detecting a protein or a pathogen and an automatic detection device provided with a conductive diamond electrode having an element for recognizing the protein or the pathogen immobilized on a surface thereof, in order to solve the above-described problem. By performing an electrochemical measurement using an apparatus equipped with this cartridge, it is possible to detect influenza virus quickly and with high sensitivity without adding a special reagent or gold nanoparticles to a specimen.

That is, the present invention includes the following.
[1] A cartridge for detecting a protein or a 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 having the same or different kind, a conductive diamond electrode having an element for recognizing a protein or a pathogen immobilized on the surface thereof,
The working electrode, the counter electrode, and the reference electrode are configured so that electrical conduction with the outside is possible,
The working electrode, the counter electrode, and the liquid junction of the reference electrode are arranged in the container so that the supporting electrolyte solution can be contacted when the supporting electrolyte solution is present in the container,
The container has an outside air inlet, an outside air outlet side vent and an outlet,
The inlet and the vent on the outside air outlet side are configured so that the outside air introduced from the inlet passes through the supporting electrolyte solution held in the container and is led to the vent on the outside air outlet side. The outside air that has passed through the outside air outlet side vent is configured to be further led out to the outlet of the cartridge,
The container and the working electrode are configured so that the outside air can contact one or more working electrodes when passing through the supporting electrolyte solution, and when the outside air is in contact with one or more working electrodes, The counter electrode and the reference electrode are configured such that the liquid junction of the counter electrode and the reference electrode are always in contact with the supporting electrolyte solution,
It is configured to be able to inject the supporting electrolyte solution and the internal electrolyte solution of the reference electrode into the container,
When the outside air is brought into contact with the working electrode and when performing electrochemical measurement with the working electrode, the surface on which the element of the working electrode is fixed is configured to face downward,
The outside air inlet and the outside air outlet and the vent on the outside air outlet side are arranged at a position higher than the highest level of the supporting electrolyte solution.
A cartridge for detecting the protein or pathogen.
[2] The cartridge according to 1, wherein the ventilation port on the side of the outside air introduction port is provided with an air bubble generation filter that converts the outside air into air bubbles.
[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 side, and the other working electrode on the outlet side is provided. 3. The cartridge according to 1 or 2, wherein the cartridge is arranged at a position higher 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 mechanism capable of raising and lowering the liquid level. Or a cartridge for detecting a pathogen,
The one or more working electrodes, each having the same or different kind, a conductive diamond electrode having an element for recognizing a protein or a pathogen immobilized on the surface thereof,
The working electrode, the counter electrode, and the reference electrode are configured so that electrical conduction with the outside is possible,
The container includes an outside air inlet, an outside air outlet side vent and an outlet, and further includes an air inlet 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 level of the supporting electrolyte solution 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 more working electrodes,
The vent on the outside air outlet side is arranged so that the outside air blown to one or a plurality of working electrodes can pass therethrough, and the outside air passing through the vent on the outside air outlet side is further led to the outlet of the cartridge. Is composed of
When adsorbing proteins or pathogens 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 liquid 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 level of the supporting electrolyte solution is raised, and the liquid junctions of one or more working, counter, and reference electrodes are in contact with the supporting electrolyte solution. Has been
It is configured to be able to inject the supporting electrolyte solution and the internal electrolyte solution of the reference electrode 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 element of the working electrode is fixed is configured to face downward,
The outside air inlet and the outside air outlet and the vent on the outside air outlet side are arranged at a position higher than the highest level of the supporting electrolyte solution.
A cartridge for detecting the protein or pathogen.
[5] When the cartridge has a plurality of working electrodes, the working electrode on the outside air inlet side is arranged at a position higher than other working electrodes on the outlet side, and the other working electrode on the outlet side is provided. 5. The cartridge according to 4, wherein the cartridge 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 level up / down mechanism is at a position higher than the highest liquid level position of the supporting electrolyte solution.
[7] The cartridge according to any one of 1 to 6, wherein the supporting electrolyte solution is injected from a counter electrode insertion port, and the container is sealed by attaching the counter electrode.
[8] Any one of 1 to 7, wherein the internal electrolyte solution for the reference electrode is injected from the metal electrode insertion opening of the reference electrode, and the container can be sealed by attaching the metal electrode. A cartridge according to claim 1.
[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 storage / reproducible element is recordable / reproducible from outside 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 a pathogen, comprising one or a plurality of 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 that is configured to be capable of being electrically connected to the outside are electrically connected to the detection device,
When the cartridge includes an element capable of storage and reproduction, the element capable of storage and reproduction is electrically connected to the detection device,
Equipped with an electrochemical measurement circuit capable of performing cyclic voltammetry measurement and / or electrochemical impedance measurement,
A display circuit that displays the measurement result, or an output terminal that outputs or transmits the measurement result,
Power supply circuit,
An external air intake port for sucking external air into the detection device is provided, and the external air intake port is connected to an external air inlet of the cartridge,
An outside air exhaust port for discharging outside air from the detection device is provided, and the outside air exhaust port is connected to an outside air outlet of the cartridge,
It has an outside air suction pump connected to the outside air intake port,
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 has a liquid level raising / lowering mechanism, and the detection device has a pump for the liquid level raising / lowering mechanism, and the pump is connected to a control circuit of the detection device. 13. The apparatus of claim 12, wherein
[14] The apparatus according to [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] The device according to any one of items 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 to / from the element when the cartridge includes an element capable of storing / reproducing.
[17] The apparatus according to any one of [12] to [16], further comprising an outside air filter between the outside air intake port of the apparatus and the outside air introduction port of the cartridge.
[18] Any one of 12 to 17, which is provided with an outside air tank and a circulation pump, and between the outside air exhaust port and the outside air tank, an outside air tank exhaust valve capable of closing an exhaust pipe connected to the outside air exhaust port. An apparatus according to claim 1.

[19] A method for detecting a protein or a pathogen using the cartridge according to any one of 1 to 11 or the device according to any one of 12 to 18.
[20] When the cartridge includes elements capable of storing and reproducing data, information of a protein or a pathogen to be measured by one or more working electrodes, initial characteristics of one or more working electrodes, and , For cartridges already used for measurement, the time used for measurement, the step of reading the information of the detected numerical value,
A step of operating the outside air suction pump and sending the sucked outside air into the cartridge;
An adsorption step in which the inhaled outside air is blown onto a surface on which the element of one or more working electrodes is fixed, or is brought into contact with one or more working electrodes as bubbles in a supporting electrolyte solution.
An exhaust process in which the outside air after the adsorption step is passed through a vent on the outside air outlet side of the cartridge and discharged from the outside air outlet and the outside air exhaust port of the device;
Stopping the outside air suction pump, while sequentially switching one or more working electrodes of one or more cartridges, an electrochemical measurement step of performing cyclic voltammetry measurement or electrochemical impedance measurement,
A step of recording the measurement result in the element of each cartridge when the cartridge has an element capable of storing and reproducing, and communicating the recording information to the outside if the apparatus has a wireless or wired communication circuit. Process,
20. The detection method according to 19, comprising:
[21] The following steps were performed using the detection device according to 18 provided with an outside air tank, a circulation pump, and an outside air tank exhaust valve.
If the cartridge is equipped with elements that can be stored and played back, from each element, information on the protein or pathogen to be measured by one or more working electrodes, the initial characteristics of one or more working electrodes, and already measured For the cartridge used for, the time used for the measurement, the step of reading the information of the detected numerical value,
Opening the outside air tank exhaust valve, activating the outside air suction pump, and introducing unmeasured outside air into the outside air tank;
Closing the outside air tank exhaust valve and stopping the outside air suction pump,
Activating the circulation pump to send the outside air in the outside air tank into the cartridge,
An adsorption step in which the inhaled outside air is blown onto a surface on which one or more working electrode elements are fixed, or is brought into contact with one or more working electrodes as bubbles in a supporting electrolyte.
A step of passing outside air after the adsorption step through a vent on the outside air outlet side of the cartridge and exhausting the outside air from the outside air outlet to an outside air tank,
Stopping the circulation pump, while sequentially switching one or more working electrodes of one or more cartridges, an electrochemical measurement step of performing cyclic voltammetry measurement or electrochemical impedance measurement,
A step of recording the measurement result in the element of each cartridge when the cartridge includes an element capable of storing and reproducing, and
A step of communicating the recorded information to the outside if the device has a wireless or wired communication circuit,
A method for detecting a protein or a pathogen, comprising:

  Proteins or pathogens can be rapidly, sensitively and automatically detected using the device provided with the cartridge of the present invention. Compared to other electrochemical detection methods, diamond electrodes have a more inert surface, so non-specific adsorption of proteins and the like can be suppressed, and noise is small and the potential window is wide, enabling high-sensitivity detection. . Further, the present invention not only can detect proteins or pathogens with high sensitivity without using expensive molecules such as antibodies, but also requires the addition of a hybridization reagent or gold nanoparticles to the sample. And rapid and highly sensitive detection is possible. In addition, there is no need to manually prepare a test sample, and secondary infection to test personnel can be prevented.

FIG. 3 is a perspective view of the cartridge of the present invention. FIG. 3 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 a left view (a) and a right view (b) of the cartridge of the present invention. FIG. 3 is a cross-sectional view of the cartridge 100 of the present invention as viewed from a cross section AA in FIG. 2. 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 viewed from a cross section BB in FIG. 2. FIG. 3 is a cross-sectional view of the cartridge 200 of the present invention as viewed from a cross section AA in FIG. 2. The vertical movement mechanism 201 is not expanded, and the liquid surface 230 is shown in a low state. FIG. 3 is a cross-sectional view of the cartridge 200 of the present invention as viewed from a cross section AA in FIG. 2. The state in which the liquid level 230 is raised by expanding the vertical movement mechanism 201 is shown. FIG. 3 is a perspective view of a detection unit 300 including a working electrode element 110 according to the present invention. (a) is a diagram of the detection unit 300 viewed from diagonally above, and (b) is a diagram of the detection unit 300 viewed from diagonally below. 4B, the surface 301 to which the element of the working electrode is fixed is shown below the detection unit 300. (a) is a plan view of the detection unit 300. FIG. 11B is a cross-sectional view of the detection unit 300 as viewed from a GG cross section in FIG. FIG. 3 is a cross-sectional view of the cartridge 100 of the present invention as viewed from a cross section taken along line CC in FIG. 2. FIG. 3 is a cross-sectional view of the cartridge 100 of the present invention as viewed from a cross section taken along line DD in FIG. 2. FIG. 4 is a cross-sectional view of the cartridge 100 of the present invention as viewed from a cross section EE in FIG. 3. FIG. 4 is a cross-sectional view of the cartridge 100 of the present invention as viewed from a cross section taken along line FF in FIG. 3. 5 shows an automatic detection device 500 provided with the cartridge 100 of the present invention. 9 shows an automatic detection device 600 including a cartridge 100, an outside air tank 610, and a circulation pump 620 according to the present invention. 6 shows an automatic detection device 700 including a cartridge 200, an outside air tank 610, a circulation pump 620, and a pump 701 for a vertical movement mechanism of the present invention. 1 shows a scheme for solid-phase synthesis of an azide group-introduced s2 (1-5) peptide dendrimer. The detection of HA protein by CV measurement using peptide unmodified (left) and peptide-modified (right) diamond electrodes is shown. The detection of influenza virus (IFV) by CV measurement using peptide unmodified (left) and peptide-modified (right) diamond electrodes is shown. FIG. 4 shows detection of HA protein by electrochemical impedance (EIS) measurement using a peptide-modified diamond electrode. 3 shows the results of a target HA protein and a control protein BSA in EIS measurement using a peptide-modified diamond electrode. Squares are HA (R ² = 0.989) and circles are BSA (R ² = 0.784). FIG. 4 shows detection of IFV by EIS measurement using a peptide-modified diamond electrode.

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

Cartridge for detecting protein or pathogen of the present invention The cartridge for detecting protein or pathogen of the present invention (hereinafter sometimes simply referred to as the cartridge of the present invention) is a conductive cartridge having an element for recognizing a protein or a pathogen immobilized on the surface. A conductive diamond electrode (hereinafter sometimes simply referred to as a conductive diamond electrode). Here, "recognizing" a protein or a pathogen by an element means that the element interacts, binds, or associates with the protein or the pathogen. When the device recognizes a protein or a pathogen, when an electric potential is applied, a current flows due to an electrochemical reaction. By measuring this current, the 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 capable of holding a supporting electrolyte solution 104. This cartridge is for detecting proteins or pathogens. The number of one or a plurality of working electrodes included in the cartridge of the present invention is not particularly limited, and may be one, two, three, four, six, eight, 10, 12, 14, 16, or the like. it can.

  The one or more working electrodes 101 each have a conductive diamond electrode 110 having the same or different types of proteins or pathogen recognition elements immobilized on the surface.

  The working electrode 101, the counter electrode 102, and the reference electrode 103 are configured so as to be able 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 that the supporting electrolyte solution 104 can be in contact with the supporting electrolyte solution when the solution is present in the container 105. I have.

  The container 105 includes an outside air inlet 111 and a vent 150 on the outside air outlet side. The outside air inlet 111 and the outside air outlet side vent 150 have the outside air introduced from the inlet 111 held by the container. It is configured to pass through the supporting electrolyte solution and be led to the vent 150 on the outside air outlet side, and the outside air passing through the vent 150 on the outside air outlet side is further led to the outlet 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 one or more working electrodes 101 when passing through the supporting electrolyte solution 104, and the outside air has one or more working electrodes. The entire counter electrode 102 and the reference electrode are configured such that the liquid junction 123 of the counter electrode 102 and the reference electrode is always in contact with the supporting electrolyte solution 104 when in contact with the electrode. The entire reference electrode includes a metal electrode (hereinafter simply referred to as a reference electrode) 103 of the reference electrode, a metal electrode insertion opening 107 of the reference electrode, a liquid junction 123 of the reference electrode, a member 125 for holding an internal electrolyte solution, and If present, it refers to the whole including the internal electrolyte solution 124.

  The cartridge is configured so 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 the electrochemical measurement is performed by the working electrode 101, the surface 301 on which the element of the working electrode is fixed is configured to face downward. That is, the detection unit 300 is attached to the cartridge such that the surface 301 on which the element of the working electrode is fixed faces downward.

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

  In one embodiment, the ventilation port 113 on the outside air introduction port side may include a bubble generation filter 120 that converts outside air into bubbles. The ventilation port 113 on the outside air introduction port side may have any shape such as a circle or a square. In some embodiments, the vent 113 on the side of the outside air inlet may be circular with a diameter of 1-10 mm, for example 3-4 mm. In one embodiment, the vent 113 on the side of the outside air inlet is provided with an inflow prevention mechanism so that the supporting electrolyte solution 104 does not flow into the side of the outside air inlet 111. The bubble generation filter 120 may also serve as an inflow prevention mechanism. The inflow prevention mechanism may be a valve that prevents the liquid from flowing.

  Although the dimensions of the cartridge of the present invention are not particularly limited, for example, when the vent 113 on the inlet side of the outside air has a circular shape with a diameter of 3 to 4 mm, the width through which bubbles in the container pass and the lateral width of the detecting unit 300 also 3 to 5 mm, for example, about 3 to 4 mm. In some embodiments, the cartridge of the present invention can have a vertical length of about 3-30 cm, a horizontal length of about 4-40 cm, and a depth of about 1.5-15 cm.

  In one embodiment, when the cartridge 100 includes a plurality of working electrodes 101, the working electrode 140 on the outside air inlet side is disposed at a lower position than the other working electrode 145 on the outside air outlet side, and The other working electrode 145 is arranged at a higher position than the working electrode 140 on the outside air inlet side. Here, the low position and the high position are as follows. When the outside air introduced from the outside air introduction port 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 and the upper position are the higher positions. 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 comes into contact with one or a plurality of working electrodes in order, Is disposed at a position lower than the one or more working electrodes and at a position lower than the level of the supporting electrolyte solution, and the vent 150 on the outside air outlet side is provided with the one or more working electrodes. Are arranged at a position higher than the working electrode and at a position higher than the liquid level of the supporting electrolyte solution.

  The cartridge of the present invention includes a cartridge provided with a liquid level vertical movement mechanism. In one embodiment, as a cartridge including the liquid level raising / lowering mechanism 201, a cartridge 200 illustrated in FIGS.

  The liquid level raising / lowering mechanism 201 can be, for example, a balloon made of a material that can be inflated and contracted. By filling the inside with air, the balloon can be inflated and the liquid level 230 can be raised. Further, the balloon can be contracted by sucking the air inside, and the liquid level 230 can be lowered.

  In this embodiment, the container 105 includes an outside air inlet 111, a vent 150 on the outside air outlet side, and an outlet 155, and further includes an air inlet 202 for a liquid level vertical movement mechanism. The vent 150 on the outside air outlet side is arranged so that the outside air blown to one or a plurality of working electrodes 101 can pass therethrough, and the outside air passing through the vent 150 on the outside air outlet side further flows to the outlet 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 disposed in the container so that the liquid level 230 of the supporting electrolyte solution 104 in the container 105 can come into contact with the supporting electrolyte solution when the liquid level 230 rises. Have been. FIG. 9 shows an example.

  When proteins or pathogens contained in the outside air are adsorbed to the element, the liquid surface 230 of the supporting electrolyte solution 104 is lowered so that one or a plurality of working electrodes 101 do not come into contact with the supporting electrolyte solution. FIG. 8 shows an example. When the liquid level 230 of the supporting electrolyte solution 104 is lowered, the liquid junction 123 of the counter electrode 102 and the reference electrode may be in contact with the supporting electrolyte solution 104. When detecting a protein or a pathogen adsorbed on the element, the liquid level 230 of the supporting electrolyte solution 104 is raised, and one or more working electrodes 101, the counter electrode 102, and the liquid junction 123 of the reference electrode are connected to the supporting electrolyte solution 104. It is configured to be in contact.

  In the case where 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 position higher than other working electrodes 245 on the outlet side, and Another 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 some embodiments, the working electrode 240 on the outside air inlet side may be arranged at the same height as the other working electrodes 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 connected to the outside air inlet side. It may be arranged at a position higher than the working electrode. Here, the low position is a position on the side where the liquid level 230 is lowered by the liquid level up / down movement mechanism 201, and the high position is a position on the side where the liquid level 230 is raised by the liquid level up / down movement mechanism 201. It is.

  In one embodiment, with respect to the cartridge 200, the air inlet 202 for the liquid level raising / lowering mechanism can be arranged at a position higher than the highest liquid level position of the supporting electrolyte solution 104. Conversely, the highest liquid level position of the supporting electrolyte solution 104 can be set to a position lower than the air inlet 202 for the liquid level up / down movement mechanism. In one embodiment, the maximum level 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). Conversely, the outside air inlet 111, the outside air outlet 155, and the vent 150 on the outside air outlet side (specifically, the lower ends thereof) are arranged at positions higher than the highest level of the supporting electrolyte solution 104. .

  In one embodiment, the cartridge of the present invention has a structure capable of injecting the supporting electrolyte solution 104 and the internal electrolyte solution 124 of the reference electrode. In one embodiment, the cartridge of the present invention may be configured such that the supporting electrolyte solution 104 is injected from the counter electrode insertion port 106 and the container 105 can be sealed by attaching the counter electrode 102. In one embodiment, the cartridge of the present invention is configured such that the internal electrolyte solution 124 for the reference electrode is injected from the metal electrode insertion opening 107 of the reference electrode, and the container can be sealed by attaching the metal electrode. May be.

  In one embodiment, the cartridge of the present invention further includes an element 401 capable of storing and reproducing. The storage / reproducible element 401 can be made recordable / reproducible from outside the cartridge via the substrate 402. The substrate 402 can include the wiring 403. The element 401 capable of storing and reproducing data may 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 storage / reproducible element 401 includes information on a protein or a pathogen to be measured by one or more working electrodes 101, initial characteristics of one or more working electrodes 101, and a cartridge 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, for example, 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, in the case of a USB memory, four wires (GND, D +, D-, and VBUS) can be used.

  In one 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 virus particles can pass therethrough.

  The cartridge of the present invention has a detection unit 300. This is illustrated in FIGS. The detection unit 300 includes the conductive diamond electrode 110 of the working electrode 101. The detector may have one or more conductive diamond electrodes 110. For convenience, the surface 301 to which the element of the working electrode is fixed is defined as the lower side of the detection unit 300. That is, a surface 301 to which the element of the working electrode is immobilized is located below the detection unit 300, and this is the side that comes into contact with the outside air or electrolyte containing proteins or pathogens (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 able to be electrically connected to the outside.

Automatic detection device of the present invention The present invention provides an automatic detection device for detecting a protein or a pathogen (hereinafter, may be simply referred to as the device of the present invention). The device of the invention comprises one or more cartridges of the invention. Examples of the cartridge include the cartridge 100 and the cartridge 200. As the automatic detection device of the present invention, for example, an automatic detection device 500 provided with the cartridge 100 of the present invention, an automatic detection device 600 provided with the cartridge 100 of the present invention and the outside air tank 610, and a cartridge having a liquid level up and down movement mechanism 201 200, an outside air tank 610, and an automatic detection device 700 including a pump 701 for a liquid level up / down 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, which are configured to be electrically connected to the outside, are electrically connected to the detection device.

  When the cartridge of the present invention includes the element 401 capable of storing and reproducing data, the element 401 capable of storing and reproducing data may be electrically connected to the device.

  Further, the apparatus of the present invention includes an electrochemical measurement circuit 501 capable of performing 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 accessory circuits.

  Further, the device of the present invention may include a data display circuit 520 for displaying the measurement result, or an output terminal for outputting or transmitting the measurement result. The data display circuit 520 may also serve as an operation circuit.

  Further, 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 intake port 505 for sucking outside air into the detection device, and the outside air intake port 505 is connected to the outside air introduction port 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 an outside air outlet 155 of the cartridge.

  Further, the device of the present invention includes an outside air suction pump 506 connected to the outside air intake port 505.

  The device of the present invention includes a control circuit 510. The control circuit 510 can control all operations such as measurement. Control circuit 510 can include a CPU. The control circuit 510 may have a function of recording the measurement result on the 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 can 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, when an abnormality is found in the measurement result, displaying the abnormality on the display circuit 520 and outputting an alarm to the outside. The control circuit 510 stops the measurement when "the set number of measurements has been completed", "the cartridge has exceeded the detection capacity", or the like, and can record and reproduce the measurement results in the respective cartridges. The function can be recorded in the element 401, communicated to the outside by a wireless or wired communication circuit 530, displayed on the display circuit 520, and stopped. Further, the device of the present invention may include a wireless or wired communication circuit 530.

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

  In the apparatus of the present invention, when the cartridge is provided with a liquid level up / down mechanism, for example, in the case of 200, the apparatus of the present invention may include a pump 701 for the liquid level up / down 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 raising / lowering mechanism may have an inlet 702 and an outlet 703. An example is shown in FIG. The inlet 702 and the outlet 703 may be inside the device of the present invention or may be connected to the outside.

  When the device 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 device in series or in parallel. In series, of the n cartridges included in 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 connected to the third air outlet. And then similarly connected to the outside air inlet of the nth cartridge. The term “parallel” means that, for a plurality of cartridges of the apparatus of the present invention, the outside air inlet and the outside air outlet of each cartridge are connected to the outside air intake port and outside air exhaust port of the apparatus without being connected to each other.

  When the cartridge of the present invention includes the element 401 capable of storing and reproducing data, the apparatus of the present invention may include a data recording / reproducing circuit 540 for recording and reproducing data to and from 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 intake port 505 of the apparatus and the outside air introduction port 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 capable of removing cotton dust, sand particles, dust, and the like is preferable.

  In one embodiment, the apparatus of the present invention further comprises an outside air tank 610 and a circulation pump 620, and between the outside air exhaust port 507 and the outside air tank 610, the exhaust pipe 509 connected to the outside air exhaust port 507. An external air tank exhaust valve 630 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 a pathogen using the cartridge of the present invention or the device of the present invention.

In one 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 data, information on a protein or a pathogen to be measured by one or more working electrodes 101, initial characteristics of one or more working electrodes 101, and , For cartridges already used for measurement, the time used for measurement, the step of reading the information of the detected numerical value,
Actuating the outside air suction pump 506 to feed the sucked outside air into the cartridge;
An adsorption step in which the inhaled outside air is blown onto the surface 301 on which the element of the one or more working electrodes is fixed, or is brought into contact with the 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 step 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 device;
An external air suction pump 506 is stopped, and one or more working electrodes of one or a plurality of cartridges are sequentially switched to perform a cyclic voltammetry measurement or an electrochemical impedance measurement. A step of recording the measurement result on the element 401 of each cartridge when the element 401 is provided, and a step of communicating the recorded information to the outside if the apparatus includes a wireless or wired communication circuit 530.

  In the above-described method, an adsorption step of blowing the sucked outside air onto the surface 301 on which the element of the one or more working electrodes is fixed, or contacting the 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 the outside air tank 610, the circulation pump 620, and the 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 elements 401 capable of storing and reproducing data, from each element 401, information on a protein or a pathogen to be measured by one or more working electrodes 101, initial characteristics of one or more working electrodes 101, And, for cartridges already used for measurement, the time used for measurement, the step of reading the information of the detected numerical value,
Opening the outside air tank exhaust valve 630, operating the outside air suction pump 506, and introducing 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 to feed the outside air in the outside air tank into the cartridge;
An adsorption step in which the inhaled outside air is blown onto the surface 301 on which the element of the one or more working electrodes is fixed, or is brought into contact with the one or more working electrodes 101 as bubbles 121 in the supporting electrolyte solution 104;
A step of passing outside air after the adsorption step through the vent 150 on the outside air outlet side of the cartridge and exhausting the outside air from the outside air outlet 155 to the outside air tank 610;
Stopping the circulation pump 620, and sequentially switching one or a plurality of working electrodes of one or a plurality of cartridges, an electrochemical measurement step of performing a cyclic voltammetry measurement or an electrochemical impedance measurement,
A step of recording the measurement result in the element of each cartridge when the cartridge includes the element 401 capable of storing and reproducing, and
Communicating the recorded information to the outside if the device has a wireless or wired communication circuit 530;

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

  The above is merely an example, and the present invention is not limited to these embodiments. For example, as a variation of the device 700 illustrated in FIG. 18, a device 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. It is preferable to dope this conductive diamond electrode with a small amount of impurities. By doping impurities, desired properties as an electrode can be obtained. Examples of the impurities include boron (B), sulfur (S), nitrogen (N), oxygen (O), silicon (Si), and the like. For example, in a source gas containing a carbon source, diborane, trimethoxyborane, boron oxide to obtain boron, sulfur oxide, hydrogen sulfide to obtain sulfur, oxygen or carbon dioxide 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 a wide potential window and advantageous properties such as a small background current as compared with other electrode materials. Therefore, a boron-doped diamond electrode will be described below by way of example. A conductive diamond electrode doped with another impurity may be used. In this specification, the term “potential” and “voltage” are used interchangeably and are interchangeable unless otherwise specified. In this specification, a conductive diamond electrode may be simply described as a diamond electrode, and a boron-doped diamond electrode may be described as a BDD electrode.

The electrode portion of the working electrode has a diamond layer on a substrate surface on which diamond mixed with 0.01 to 8% w / w boron material is deposited. The size of the substrate is not particularly limited, but preferably has an area capable of measuring a sample solution in mL units or μL units. The substrate may have a diameter of, for example, 1 to 10 cm and a thickness of 0.1 to 5 mm. The substrate may be a glass substrate such as Si substrate, SiO ₂ or quartz substrate, a ceramic substrate such as Al ₂ O ₃ , or a metal such as tungsten or molybdenum. All or part of the surface of the substrate can be a diamond layer.

The size of the electrode portion of the conductive diamond electrode can be appropriately designed depending on the measurement object. 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 part of the working electrode has a diamond layer in which the surface of the Si substrate is deposited with diamond containing a high boron raw material (0.01 to 8% w / w boron raw material as raw material preparation). A preferable boron raw material mixing ratio is 0.05 to 5% w / w, and particularly preferably about 0.3% w / w. After forming the diamond layer on the substrate, a modification layer can be provided thereon.

The deposition process of the diamond mixed with the boron raw material on the substrate may be performed at 700 to 900 ° C. for 2 to 12 hours. The conductive diamond thin film is formed by a usual microwave plasma chemical vapor deposition (MPCVD). That is, a substrate such as a silicon single crystal (100) is set in a film forming apparatus, and a film forming gas using high-purity hydrogen gas as a carrier gas is flowed. The film forming gas contains carbon and boron. When microwaves are applied to a film-forming apparatus in which a high-purity hydrogen gas containing carbon and boron is flowing to cause plasma discharge, carbon radicals are generated from a 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 thickness of the diamond thin film can be controlled by adjusting the deposition 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 boron-doped diamond deposition process on the substrate surface may be determined according to the substrate material. As an example, the plasma power can be between 500 and 7000 W, for example between 3 kW and 5 kW, and preferably 5 kW. When the plasma output is in this range, the synthesis proceeds efficiently, and a high-quality conductive diamond thin film with few by-products is formed.

  The above-mentioned electrodes are disclosed in JP-A-2006-98281, JP-A-2011-152324, JP-A-2015-172401, and the like, and can be manufactured according to the descriptions in these publications.

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

  The cartridge of the present invention has a triode. The resistance on the reference electrode side is set high, and no current flows between the working electrode and the reference electrode. The counter electrode is not particularly limited, but for example, a silver wire or a platinum wire may 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. In the present specification, unless otherwise specified, the measured voltage is assumed to be measured with reference 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 configuration 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 an internal electrolyte solution 124, and a liquid junction 123 of the reference electrode is disposed between the internal electrolyte solution 124 and the supporting electrolyte solution 104 in the container 105. Have been. In the liquid junction 123 of the reference electrode, the internal electrolyte solution 124 and the supporting electrolyte solution 104 are electrically connected to each other by a porous Vycor glass or ceramic or a salt bridge. The salt bridge may be a glass tube or filter paper, and may be gelled with agar. The salt bridge is usually filled with an inert electrolyte, such as sodium chloride.

  The shape of the working electrode, the counter electrode, and the reference electrode of the cartridge of the present invention is not particularly limited as long as the working electrode, the counter electrode, and the reference electrode are arranged so as to be able to come in contact with the supporting electrolyte solution during the 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. In one embodiment, immobilization means that the device is connected to the surface of the conductive diamond electrode by a covalent bond. The layer to which the element is fixed is sometimes called a modification layer. When the modified layer is brought into contact with the protein or pathogen of interest, the device recognizes the protein or pathogen. At this time, when a potential is applied, a current flows, and by measuring this, a protein or a pathogen in the sample can be detected.

  As the pathogen detected by the cartridge or the device of the present invention, any pathogen can be used as long as it is recognized by an element on the surface of the conductive diamond electrode and a current flows. Pathogens include viruses and pathogenic bacteria. Pathogenic bacteria include, but are not limited to, mycoplasma, botulinum, pertussis, tetanus, diphtheria, cholera, shigella, anthrax, pathogenic Escherichia coli, staphylococci, salmonella, welsh and cereus. Absent.

  The virus detected by the cartridge or the device of the present invention may be any virus as long as it is recognized by an element on the surface of the conductive diamond electrode and a current flows. Viruses detected are DNA virus, RNA virus, double-stranded DNA virus, single-stranded DNA virus, double-stranded RNA virus, single-stranded RNA (+) strand virus, single-stranded RNA (-) strand virus , 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 their various subtypes.

  The protein detected by the cartridge or device 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 some embodiments, the protein detected has tryptophan residues. In certain embodiments, the protein detected is a protein from a pathogen, pathogenic bacterium, or virus as 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 recognized by the element may be a hemagglutinin (HA) or neuraminidase (NA) protein derived from the influenza virus. The proteins to be detected further include M1 protein and M2 protein of influenza virus. When the detection target is a mycoplasma bacterium, the element recognized by the element may be a P1 protein, a membrane antigen protein or a ribosomal protein L7 / L12 derived from a mycoplasma bacterium. In certain embodiments, the protein 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, vero toxin, anthrax toxin (PA, EF, or LF), and , Escherichia coli, Staphylococci, Salmonella, Bacillus cereus, and the like, but not limited thereto.

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

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

Electrode- (L) -R
[Wherein Electrode is a conductive diamond electrode, L is a linker that may optionally be present, and R represents a molecule that recognizes a protein or a pathogen. ]

  In some embodiments, the protein or pathogen-recognizing element has a peptide that specifically interacts with the protein or pathogen of interest. Explained in the above formula, the molecule R that recognizes a protein or pathogen can have a peptide. As the peptide, those 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. As the peptide, for example, those 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 has a drastically increased HA binding property by dendrimerization, and the 4-branch type is about 750 times as large as 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 from the pathogen, pathogenic bacterium or virus described above. The amino acid sequence of such a peptide can be obtained from known databases or known databases such as GenBank. The peptide may be arbitrarily modified or dendrimerized as described above.

  In certain embodiments, the element that recognizes a protein or pathogen has a peptide that recognizes an influenza virus. As the peptide recognizing the influenza virus, any known peptides can be used. For example, WO 2007/105565, WO 2010/024108, JP 2010-209052 (these are incorporated herein by reference). (The contents of which are incorporated herein), but are not limited thereto. The following are examples of peptides that recognize influenza virus.

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

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

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

  If desired, a linker molecule can be used for immobilizing the device. Hereinafter, molecules for forming the linker portion are exemplified.

AL ₁ -B (-P)
[Wherein, A is a functional group capable of reacting with the conductive diamond electrode, L ₁ is a linker moiety, B is a functional group capable of reacting with a protein or a molecule recognizing a pathogen, and P is an optional functional group. Represents a good protecting group. ]

  In this case, the above molecules are first connected 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.

  The following are examples of molecules R that recognize proteins or pathogens.

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

  The linking reaction between the linker moiety and the molecule R recognizing a protein or a pathogen (the linking reaction between the functional group B and the functional group C) is a Heusgen cycloaddition reaction, a Glacer reaction, a coupling thereof, a Suzuki-Miyaura coupling reaction. And the like can be used, and those forming a covalent bond are preferable, but not limited thereto.

The functional group A capable of reacting 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, or the like. The linker moiety L ₁ is an aromatic ring, carbocyclic, heterocyclic, such as a phenyl group, a naphthyl group, a polyether group, a polyethylene glycol group may have a hydrocarbon group. These may be optionally further substituted by a suitable substituent such as an alkyl group, an aryl group, a halogen group, a hydroxyl group and the like. Further, the functional groups B and C may be an alkynyl group, a boranyl group, a boryl group, a halogenated aryl group, an azide group, or the like. When a Huisgen 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. Functional group B may optionally be protected by protecting group P. Examples of the protecting group include trisubstituted silyl groups such as triisopropylsilyl group, trimethylsilyl group, butyldiphenylsilyl group, and dimethylcumylsilyl group, benzyl group, lower alkoxycarbonyl group, halogeno lower alkoxycarbonyl group, benzyloxycarbonyl group, and acetyl group. Groups, acyl groups such as a benzoyl group, a triphenylmethyl group, a tetrahydropyranyl group, 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 and immobilizing it on the electrode surface and deprotecting it. In certain embodiments, the molecule R that recognizes a protein or pathogen may have a peptide group as R 1 and an azide group as the functional group C. When an azide group (functional group C) of R reacts with an alkynyl group (functional group B) presented on the electrode surface, a 1,2,3-triazole ring is formed, and a molecule R that recognizes a protein or a pathogen is formed. Is immobilized on the electrode surface.

About the electrochemical measurement method The target protein or pathogen can be detected using the cartridge or the device of the present invention. When the working electrode of the cartridge of the present invention is brought into contact with air (outside air) or an electrolyte solution as a sample, the element on the surface of the working electrode recognizes a protein or a pathogen. At this time, when a potential is applied to the working electrode, a 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 electrode potential is swept from the initial potential (E _i ) to the inversion potential (E _λ ) at the sweep speed (v), then reversed, and the current obtained when the electrode potential is returned to Ei is observed. The initial potential E _i not happen the electrode reaction potential, also by the electrode reaction the reversal potential E _lambda is set to a potential such that the diffusion rate-determining, it is possible to obtain a current-potential graph (cyclic voltammogram). The initial potential, the sweep speed, the inversion potential, and the like can be set as appropriate.

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

Electrochemical impedance measurement The electrochemical impedance measurement uses a potentiostat connected to an AC transmitter. A constant DC potential is applied to the electrodes 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 also input to the lock-in amplifier. As a result, the current flowing is a combination of the DC current and the AC current, and the lock-in amplifier compares the AC component of the current with the AC 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 a complex plane plot is performed based on the impedance and phase difference obtained at each frequency.

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

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

  In this case, a chronoamperometric measurement is performed on a protein or pathogen sample of known concentration or amount at a constant applied voltage. A current value at a certain time after voltage application is recorded, and a calibration curve is created by plotting a relationship between the concentration or amount of the protein or pathogen sample and the current value. Next, the concentration or amount of the protein or pathogen sample in the test sample solution is calculated by measuring the current value of the measurement sample at the fixed time after the application of the voltage and comparing the current value with the calibration curve.

  The test sample to be measured by the method of the present invention is not particularly limited, and for example, the atmosphere may include a virus or a pathogen. For example, the air in clinical facilities such as hospitals where schools may have influenza viruses, schools, train stations, airports, and all public facilities. The outside air may be compressed or may be directly used for measurement.

  In the present invention, an aqueous solvent is mainly used for the measurement. The solution in which the measurement is performed 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.

  As used herein, “detection” of a protein means that the protein of interest can be specifically measured in a sample other than the protein of interest or in a sample mixed with viruses or bacteria. In addition, “detection” of a pathogen means that the target pathogen can be specifically measured in a sample in which proteins other than the target pathogen or viruses or bacteria are mixed.

  In the present specification, the viral load is represented by the number of plaque forming units (pfu). In one embodiment, the pfu when describing the amount of influenza virus is based on the influenza virus A / PR / 8/34 (H1N1) strain. The method of measuring pfu can be estimated by measuring plaque 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. The pfu unit may be appropriately converted to the ng unit. In one embodiment, the device of the present invention can detect an influenza virus with high sensitivity, for example, an influenza virus of 20 pfu or more, or 40 pfu or more and 1000 pfu or less.

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

  In some embodiments, the protein or pathogen-recognizing element is a virus or pathogen-recognizing element. The types of viruses and pathogens that are recognized are as described above, and in particular, influenza viruses.

  In certain embodiments, the protein or pathogen-recognizing element has a peptide that recognizes a pathogen protein. Recognized pathogen proteins are as described above, in particular the HA protein of influenza virus. 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 portion, and the protein or pathogen-recognizing molecule is immobilized on the surface of the diamond electrode via the linker portion. Have been. Molecules that recognize proteins or pathogens, linker moieties, and immobilization methods are as described above.

Production Examples and Use Examples of the Conductive Diamond Electrode of the Present Invention The following procedures are intended for illustration only, and are not intended to limit the technical scope of the present invention in any way. Unless otherwise stated, reagents are commercially available or are obtained or prepared according to procedures conventional in the art, procedures in the known literature.

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: triiropropylsilane
_{TIPS-Eth-Ar-N 2} + BF 4 -: 4 - (( triisopropylsilyl) ethynyl) benzene diazonium 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 noted, compounds and chemicals were used without further purification as they were commercially available. Those skilled in the art can modify the described procedures without departing from the spirit of the invention.

1. Synthesis of Peptide and Linker A two-branched peptide dendrimer (ARLPR) 2-K-KN3 having an azidolysine Lys (N ₃ ) introduced into the terminal was synthesized using the Fmoc solid phase method. The peptide ARLPR is a hemagglutinin-binding peptide (JP-A-2006-68020). In peptide synthesis by the solid phase method, a deprotection reagent is added to a peptide resin (a solid support on which amino acids are immobilized) to remove the N-α protecting group. The activated amino acids are reacted there, and the chain length of the peptide is extended one after another. The carboxy terminus of the peptide was amidated to eliminate unnecessary charge. In addition, a branched structure was created by introducing Lys to the C-terminal side of the peptide. FIG. 19 shows a scheme of solid-phase synthesis of an azide group-introduced s2 (1-5) peptide dendrimer.

(A) Introduction of Lys (N ₃ ) into Resin by Manual Synthesis A reaction column (PD-10, Amersham) was charged with 169 mg (0.1 mmol) of Fmoc-NH-SAL resin (Watanabe Chemical, 0.59 mmol / g). 2 mL of DMF was added thereto, and the mixture was shaken gently, and then DMF was removed from the lower part 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 one minute, the solvent was removed with an aspirator. Similarly, 2 mL of 20% PIP / DMF was poured, and the mixture was shaken for 15 minutes to remove the solvent. The operation of pouring 2 mL of DMF and removing with an aspirator was repeated four times to wash the resin. This resulted in deprotection.

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, and added for 1 hour. Shake. Thus, a coupling reaction was performed.

  One hour later, the operation of removing the reaction solution with an aspirator, pouring 2 mL of DMF into a reaction column, shaking lightly, and removing with an aspirator was repeated four times (washing). Further, the operation of adding 2 mL of DCM and removing with an aspirator was repeated four times. The reaction column was placed in a desiccator and dried with a vacuum pump for 1 hour. When the sample was sufficiently dried, it was stored at 4 ° C.

(B) Quantification of amino acid introduction rate
20 mg of the 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. This supernatant was diluted 50-fold with DMF, and the absorbance at 301 nm was measured. The blank was measured using DMF, and the introduction ratio of the amino acid was calculated from the following formula based on 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) × 50/7800 × (2 × 10 -3) × (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 produced in the above (A), the introduction rate was 0.413 mmol / g.

(C) Elongation of peptide
Fmoc-Lys (N ₃ ) -NH-SAL resin 242 mg (0.05 mmol) was charged into PD-10 empty columns (17-0435-01, Amersham Biosciences), and the following (C-1) to (C-4) The elongation reaction of the peptide was performed by the operation described in (the amount of the resin in which the peptide became 0.1 mmol was used).

(C-1) Deprotection of Fmoc group The resin into which the first amino acid was introduced was weighed into 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 four times. 2 mL of 20% (v / v) PIP / DMF was poured into the reaction column, and after 1 minute, 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 with an aspirator was repeated five times to wash the resin.

(C-2) Coupling Fmoc-AA-OH (0.3 mmol each), PyBOP 157 mg (0.3 mmol), DMF 2 mL, DIEA 0.11 mL (0.6 mmol) were added to the reaction column, and the mixture was 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 operation of removing the reaction solution with an aspirator, pouring 2 mL of DMF into the reaction column, shaking lightly, and removing with an aspirator was repeated four times. The amino acids used are shown in Table 1.

(C-3) Kaiser test It was determined whether or not the coupling reaction was completed using a reagent for Kaiser test (code No. 2590077, Domestic Chemistry). Take about 1 mg of the resin with a spatula, put it in a microtube, add 10 μL each of reagents (1) ninhydrin / ethanol, (2) phenol / ethanol, and (3) KCN / pyridine, close the lid, and heat with a dryer for about 1 minute did. When the color of the resin or solution turned blue, unreacted amino groups remained, so the coupling reaction was performed again with the same amino acid. When it became colorless or yellow, it was judged that the reaction was completed, and the above-mentioned 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 into the PD-10 column, gently mix with a spatula, remove 20% PIP / DMF with an aspirator, and remove 2 mL of 20% PIP / DMF. The Fmoc group was deprotected by pouring and shaking for 15 minutes followed by removal. Then pour 2 mL of DMF and remove it with an aspirator 5 times, repeat the operation of pouring t-butyl methyl ether and removing with an aspirator twice, then cover the PD-10 column with aluminum foil and parafilm, and vacuum dry for 3 hours Was.

(D) Cutting (Cleaving and deprotection from resin)
To cleave the peptide from the synthesized resin, a cocktail solution was prepared with the composition shown in Table 2 and excised. A cocktail solution having a thiol-free composition was used (see PE Schneggenburger et al., J. Pept. Sci., 16, 10-14 (2010)) because the azide group is easily decomposed by the reduction action of the thiol.

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 due to heat and light (usually 8 hours at room temperature without light shield) . Two hours later, the lid of the reaction vessel was removed, and the contents of the reaction vessel were dropped into a 15 mL centrifuge tube by applying pressure from the upper port of the vessel, 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, followed by vortexing. Subsequently, after centrifugation at 3500 rpm for 1 minute, the supernatant was removed and the volume was again adjusted to 10 mL with cold diethyl ether. This operation was repeated five times in total. N ₂ gas was blown over the peptide pellet remaining in the centrifuge tube to volatilize the cold diethyl ether (crude peptide).

(E) High performance liquid chromatography (HPLC)
300 μL of AN (acetonitrile) and 100 μL of MilliQ were added to the crude peptide powder remaining in the 15 mL centrifuge tube, 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 insolubles, and collected in a 1.5 mL tube. Again, 400 μL of 75% acetonitrile was added to the centrifuge tube for co-washing, and the tube was 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 the peptide solution (co-washing solution) was injected, and the 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), fractions containing peaks were collected every 15 seconds, and those containing the target peptide were 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 a laser light source. Α-Cyano-4-hydroxycinnamic acid (α-CHCA, Sigma) was used for the matrix. α-CHCA was suspended at 10 mg / mL in 0.1% TFA / AN (3: 2, v / v), irradiated with ultrasonic waves, centrifuged, and the supernatant was used. For calibration, a peptide calibration standard (code No. 206195, Bruker) diluted according to the protocol was used.

  4 μL of the α-CHCA solution and 2 μL each of the fraction solution after HPLC were pipetted in a 2 mL tube, and 2 μL was placed on a MALDI plate and air-dried. The calibration sample was performed in the same manner. Ultraflex ™ was measured in positive ion mode using reflectron mode.

The synthesized two-branched 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 substance had a high purity of 98% or more. Also, error between the calculated value in the analysis result of the MALDI-TOF MS is not less than 0.1% was confirmed to be the desired product (Exact Mass: 1486.94, calculated [M ⁺ H] + 1487.95, measured [M + H] ⁺ 1487.95; calculated [M + Na] ⁺ 1509.84, measured [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 determined 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 tetrafluoroborate) is synthesized. First, the terminal alkyne is cross-coupled with the aryl halide by coupling, thereby obtaining an alkynylated aryl (aromatic acetylene) (see S. Anderson, Chem. Eur. J. 7, 4706-4714 (2001)). ). Palladium, copper, and a base were used as a catalyst. Further, the amino group was diazoniumized to perform electrolytic grafting.

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

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

Celite filtration A filter paper was placed on a Kiriyama funnel, celite was spread over the funnel, and hexane was absorbed while sucking with a pump. The reaction solution in the flask was filtered and washed with hexane. Reaction solids that could not be removed from the wall were dissolved or suspended in hexane by sonication and filtered. The eggplant flask containing the filtrate was placed in an evaporator, and the filtrate was concentrated (about 2 mL).

Silica gel column chromatography The developing solvent used was hexane: ethyl acetate = 10: 1. 75 cc of silica (Silica gel 60, Merck) was dispersed in a developing solvent and packed in a column. The concentrated filtrate was gently and uniformly added to sea sand, and the cock was opened to collect 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. A solution obtained by dissolving iodoaniline (R _f = 0.29) as a raw material in dichloromethane on a TLC plate (TLC silica gel 60 F ₂₅₄ , (105714, Merck Millipore)), and a solution before silica gel column chromatography are respectively spotted and developed. After that, it was confirmed by UV irradiation. Next, the solution in the test tube was spotted and spread on a TLC plate in order, and a test tube sample in a range where a reaction product (R _f = 0.39) was 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 dried under vacuum 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. Put the eggplant flask containing the TIPS-Eth-Ar-NH ₂ (1.1 g, 4.0 mmol) obtained in (A) into a bath containing methanol (MeOH) and liquid nitrogen, and the temperature in the flask is −5 ° C. Chilled down. After adding 4 mL of H ₂ O and mixing well with a stirrer until dispersed, 3.36 mL of HBF4 (16 mmol, d = 1.4 g / cm ³ )
Was added. ₂ mL of a pre-cooled aqueous solution of NaNO ₂ was added to the flask to adjust the temperature of the flask 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.
Table 4 shows the reagents used.

Suction filtration
₄ mg (36.4 nmol) of NaBF ₄ was dissolved in 80 mL of H ₂ O to prepare a 5% NaBF ₄ aqueous solution. The 5% aqueous NaBF ₄ solution, MeOH, diethyl ether, and H ₂ O used as the washing solution were previously cooled at 4 ° C. The filter paper was placed on the Kiriyama funnel, and the filter paper was mixed with H ₂ O while sucking. The reaction solution was all filtered, washed with H ₂ O. Washing was performed with a 5% NaBF ₄ solution, then MeOH, and diethyl ether in that order, and the remaining reactants were collected by sonication. After drying the powder on the filter paper 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, the target compound was able to be synthesized because the proton ratio of each peak of TIPS-Eth-Ar-NH ₂ was consistent. 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. Fabrication 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 the doping trimethylborane in the raw material was 0.3% w / w. Surface morphology was characterized using a scanning electron microscope. The quality of the thin film was confirmed by Raman spectroscopy. The BDD electrode thus manufactured was used. The details will be described below.

Preparation of conductive diamond thin film by vapor phase synthesis (A) Pretreatment of Si substrate Si substrate (diameter: 5 cm, thickness: 1 mm) is placed on a dish containing diamond powder so that the mirror surface faces downward, and the Si substrate is placed for 20 minutes. Was rotated by hand to scratch the substrate surface. Thereafter, the Si substrate was immersed in a beaker containing 2-propanol, and washed by irradiating ultrasonic waves for 20 minutes. Finally, the solvent was evaporated and dried with N ₂ gas.

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

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

As a result, a peak of boron-boron bond was observed around 520 cm ⁻¹ . At around 1333 cm ^-1 , a Raman peak of carbon having sp ³ structure was observed. On the other hand, near 1560 cm ⁻¹ , no Raman peak of carbon having the sp ² structure was observed. From this, it was determined that a highly pure 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 table with silicon grease (Shin-Etsu Chemical Co., Ltd.). The accelerating voltage was set at 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) The scheme of presentation of alkynyl groups by electrolytic grafting is shown below.

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

(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. The entire amount was placed in a cell. The control PC was set as shown in Table 8 and cyclic voltammetry (electrolytic grafting) was performed five times at -0.7 to +0.6 V.

(C) Deprotection of triisopropylsilyl (TIPS) group After the measurement is completed, first add 9.5 mL of THF to the cell, then add 0.5 mL of THF solution (1 mol / L) of TBAF, and allow to stand for 20 minutes to remove TIPS group. 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 ultrasonically treated 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 (the amount of the peptide charged was 100 times), and further a reaction solution was prepared by diluting it with MeOH: H ₂ O = 1: 1 to 0.1 nM. (Peptide preparation amount 0.01 times).

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

For 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 three cycles to confirm the surface condition. In addition, CV measurement was also performed in PBS, which is a solvent for HA and IFV, in three cycles, and the change in background was confirmed. In addition, the contact angle was observed with these electrodes, and the surface wetting characteristics were examined.

4. Detection of hemagglutinin protein (HA) and influenza virus (IFV) using boron-doped diamond (BDD) electrode Cyclic voltammetry (CV) assay Measurement of HA protein using unmodified and peptide-modified BDD electrodes The interaction was followed by a CV measurement. 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 (prepared amount of peptide × 0.01), and the background was measured for 3 cycles with PBS. Thereafter, about 60 μL of 500 nM HA / PBS was added into the cell, and the cell was adjusted so that the electrode portion was immersed, and allowed to interact for 30 minutes. Thirty minutes later, the HA solution was removed, and the cells were rinsed three times with PBS. The cells were filled with PBS, and three cycles of cyclic voltammetry were performed. These operations were performed again while changing the location on the electrode, and the interaction was observed at a total of two locations. The measurement conditions used in the following table were used.

  In the same manner as above, a 50 to 500 nM HA solution diluted with PBS was allowed to interact with each other for 30 minutes. Interaction was performed in ascending order of concentration, and CV measurement was performed for three cycles while washing the electrode with PBS before changing the concentration. The measurement conditions were as shown in Table 10.

  The results are shown in FIG. FIG. 20 left shows HA measurement in a solution when a diamond electrode not modified with a peptide is used. An increase in current density was observed with the 500 nM HA solution. FIG. 20 right shows the case where a diamond electrode modified with a peptide was used. At the first cycle, a remarkable increase in the current density was observed compared to the unmodified diamond electrode, and HA could be detected. From the second cycle onward, the current density was not so different from that of the case with only PBS, indicating that most of the HA protein was detected in the first cycle.

Measurement of IFV Using Unmodified and Peptide-Modified BDD Electrodes A cell was assembled with a peptide-immobilized electrode (prepared amount of peptide × 0.01), and the background of PBS was measured at three places for three cycles. Thereafter, 1 mL (200 pfu) of a 200 pfu / mL virus solution was added to adjust so that the electrode portion was immersed, and allowed to interact for 15 minutes. After 15 minutes, the virus solution was removed, and the cells were rinsed three times with PBS. The cells were filled with PBS, and cyclic voltammetry measurements were performed at three places at three locations, each for three cycles.

  The results are shown in FIG. FIG. 21 left shows the IFV measurement in a solution when a diamond electrode not modified with a peptide was used. An increase in current density was observed with the 200 pfu IFV solution. The right side of FIG. 21 shows the results obtained using the peptide-modified diamond electrode. A remarkable increase in the current density was observed as compared with the unmodified diamond electrode, and IFV could be detected. Thus, IFV could be detected with high sensitivity.

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

First, a solution of a redox substance was prepared. 0.164 g (0.5 mmol) of K ₃ [Fe (CN) ₆ ] and 0.211 g (0.5 mmol) of K ₄ [Fe (CN) ₆ ] were separately weighed into a volumetric flask, dissolved in 50 mL of PBS, and dissolved in 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 obtain 5 mM [Fe (CN) ₆ ] ^{3- / 4-} / PBS.

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

As in the case of the CV measurement, HA solutions at various concentrations were allowed to interact with each other using a three-electrode method (working electrode: conductive diamond electrode, counter electrode: Pt, reference electrode: Ag / AgCl). First, a cell was assembled with a peptide-immobilized electrode (prepared amount of peptide × 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, adjusted so that the electrode portion was immersed, and allowed to interact for 15 minutes. After 15 minutes, the solution was removed, and the cells were rinsed three times with PBS. The cells were filled with 5 mM [Fe (CN) ₆ ] ^{3- / 4-} / PBS and subjected to three cycles of measurement. This operation was performed using an HA solution (5 to 500 nM, each 1 to 100 μg / mL), an IFV solution (1 to 140 pfu), and a bovine serum albumin (BSA) solution (5 to 500 nM).

  EIS measurement results of HA 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 concentration. From these results, HA was detected with high sensitivity and specificity.

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

  From the above, 20 pfu of influenza virus could be detected using a conductive diamond electrode having an element that recognizes a protein or a 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, which is 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 a pathogen immobilized on the surface. Further, the device of the present invention includes the cartridge, and can detect a protein or a pathogen by electrochemical measurement.

Reference Signs List 100 Cartridge 101 Working electrode 102 Counter electrode 103 Reference electrode 104 Supporting electrolyte solution 105 Container 106 Counter electrode insertion port 107 Metal electrode insertion port 110 of reference electrode Conductive diamond electrode 111 Outside air introduction port 112 Introducing filter 113 Outside air introduction side ventilation port 120 Bubble generating filter 121 Bubble 123 Reference electrode liquid junction 124 Internal electrolyte solution 125 Member for holding internal electrolyte solution 130 Liquid surface 140 Working electrode 145 on outside air inlet port Working electrode 150 on outside air outlet port side Outside air outlet side Vent 155 Outside air outlet 200 Cartridge 201 with liquid level up / down movement mechanism Liquid level up / down movement mechanism 202 Air inlet 230 for liquid level up / down movement mechanism Liquid level 240 Working electrode 245 on outside air introduction port side Outside air outlet port side Working electrode 300 Detector 301 Working electrode element is fixed Surface 401 Memory-reproducible element 402 Substrate 403 Wiring 500 Apparatus 501 with cartridge 100 of the present invention Electrochemical measurement circuit 505 External air intake port 506 External air intake pump 507 External air exhaust port 508 External 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 Switch 600 Device with cartridge 100 and outside air tank 610 Outside air tank 620 Circulation 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 up-and-down moving mechanism Inlet 703 for pump of 701 Exhaust port for pump of 701

Claims (21)

A cartridge for detecting a protein or a 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 having the same or different kind, a conductive diamond electrode having an element for recognizing a protein or a pathogen immobilized on the surface thereof,
The working electrode, the counter electrode, and the reference electrode are configured so that electrical conduction with the outside is possible,
The working electrode, the counter electrode, and the liquid junction of the reference electrode are arranged in the container so that the supporting electrolyte solution can be contacted when the supporting electrolyte solution is present in the container,
The container has an outside air inlet, an outside air outlet side vent and an outlet,
The inlet and the vent on the outside air outlet side are configured so that the outside air introduced from the inlet passes through the supporting electrolyte solution held in the container and is led to the vent on the outside air outlet side. The outside air that has passed through the outside air outlet side vent is configured to be further led out to the outlet of the cartridge,
The container and the working electrode are configured so that the outside air can contact one or more working electrodes when passing through the supporting electrolyte solution, and when the outside air is in contact with one or more working electrodes, The counter electrode and the reference electrode are configured such that the liquid junction of the counter electrode and the reference electrode are always in contact with the supporting electrolyte solution,
It is configured to be able to inject the supporting electrolyte solution and the internal electrolyte solution of the reference electrode into the container,
When the outside air is brought into contact with the working electrode and when performing electrochemical measurement with the working electrode, the surface on which the element of the working electrode is fixed is configured to face downward,
The outside air inlet, outside air outlet, and vent on the outside air outlet side are located higher than the highest liquid level of the supporting electrolyte solution when the outside air is brought into contact with the working electrode and when the electrochemical measurement is performed by the working electrode. Have been
A cartridge for detecting the protein or pathogen.   2. The cartridge according to claim 1, wherein the ventilation port on the side of the outside air introduction port includes a bubble generation filter that converts outside air into bubbles. 3.   When the cartridge has 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 connected to the outside air. The cartridge according to claim 1, wherein the cartridge is arranged at a position higher than the working electrode on the inlet side. 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 raising and lowering mechanism capable of raising and lowering the liquid level, a protein or a pathogen. A cartridge for detecting,
The one or more working electrodes, each having the same or different kind, a conductive diamond electrode having an element for recognizing a protein or a pathogen immobilized on the surface thereof,
The working electrode, the counter electrode, and the reference electrode are configured so that electrical conduction with the outside is possible,
The container includes an outside air inlet, an outside air outlet side vent and an outlet, and further includes an air inlet 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 level of the supporting electrolyte solution 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 more working electrodes,
The vent on the outside air outlet side is arranged so that the outside air blown to one or a plurality of working electrodes can pass therethrough, and the outside air passing through the vent on the outside air outlet side is further led to the outlet of the cartridge. Is composed of
When adsorbing proteins or pathogens 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 liquid 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 level of the supporting electrolyte solution is raised, and the liquid junctions of one or more working, counter, and reference electrodes are in contact with the supporting electrolyte solution. Has been
It is configured to be able to inject the supporting electrolyte solution and the internal electrolyte solution of the reference electrode into the container,
When performing the electrochemical measurement by time and a working electrode contacting the outside air to the working electrode, the surface of the element of the working electrode is fixed is configured to be a downward,
The outside air inlet, outside air outlet, and vent on the outside air outlet side are located higher than the highest liquid level of the supporting electrolyte solution when the outside air is brought into contact with the working electrode and when the electrochemical measurement is performed by the working electrode. Have been
A cartridge for detecting the protein or pathogen.   When the cartridge is provided with a plurality of working electrodes, the working electrode on the outside air inlet side is arranged at a position higher than the other working electrodes on the outlet side, and the other working electrode on the outlet side is connected to the outside air. The cartridge according to claim 4, wherein the cartridge is arranged at a position lower than the working electrode on the inlet side.   The cartridge according to claim 4, wherein the air inlet for the liquid level up / down mechanism is higher than the highest liquid level of the supporting electrolyte solution.   The cartridge according to any one of claims 1 to 6, wherein the supporting electrolyte solution is injected from a counter electrode insertion port, and the container is configured to have a sealed structure by attaching the counter electrode.   8. The container according to claim 1, wherein an internal electrolyte solution for the reference electrode is injected from a metal electrode insertion opening of the reference electrode, and the container is made to have a sealed structure by attaching the metal electrode. 9. The cartridge according to item.   The cartridge according to any one of claims 1 to 8, further comprising an element capable of storing and reproducing.   The cartridge according to claim 9, wherein the element capable of storing and reproducing data can be recorded and reproduced from outside the cartridge via a substrate.   The cartridge according to claim 1, further comprising an introduction filter immediately after the outside air introduction port. An apparatus for detecting a protein or a pathogen, comprising one or more cartridges according to any one of claims 1 to 11,
A working electrode, a counter electrode, and a reference electrode on the outer surface of the cartridge, which are configured to allow electrical communication with the outside, are electrically connected to an apparatus for detecting the protein or the pathogen;
When the cartridge includes an element capable of storage and reproduction, the element capable of storage and reproduction is electrically connected to the detection device,
Equipped with an electrochemical measurement circuit capable of performing cyclic voltammetry measurement and / or electrochemical impedance measurement,
A display circuit that displays the measurement result, or an output terminal that outputs or transmits the measurement result,
Power supply circuit,
An external air intake port for sucking external air into the detection device is provided, and the external air intake port is connected to an external air inlet of the cartridge,
An outside air exhaust port for discharging outside air from the detection device is provided, and the outside air exhaust port is connected to an outside air outlet of the cartridge,
It has an outside air suction pump connected to the outside air intake port,
Equipped with a control circuit,
An apparatus for detecting the protein or pathogen.   The cartridge according to any one of claims 4 to 11, further comprising a liquid level up / down movement mechanism, wherein the detection device includes a pump for the liquid level up / down movement mechanism, and the pump is provided in a control circuit of the detection device. 13. The device of claim 12, wherein the device is connected.   14. The apparatus according to claim 12, 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.   Apparatus 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 for recording / reproducing data to / from the element when the cartridge includes an element capable of storing / reproducing.   The apparatus according to any one of claims 12 to 16, further comprising an outside air filter between an outside air intake port of the apparatus and an outside air inlet of the cartridge.   The air tank according to any one of claims 12 to 17, further comprising an outside air tank and a circulation pump, and 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 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 device according to any one of claims 12 to 18. If the cartridge is equipped with elements that can be stored and played back, from each element, information on the protein or pathogen to be measured by one or more working electrodes, the initial characteristics of one or more working electrodes, and already measured For the cartridge used for, the time used for the measurement, the step of reading the information of the detected numerical value,
A step of operating the outside air suction pump and sending the sucked outside air into the cartridge;
An adsorption step in which the inhaled outside air is blown onto a surface on which the element of one or more working electrodes is fixed, or is brought into contact with one or more working electrodes as bubbles in a supporting electrolyte solution.
An exhaust process in which the outside air after the adsorption step is passed through a vent on the outside air outlet side of the cartridge and discharged from the outside air outlet and the outside air exhaust port of the device;
Stopping the outside air suction pump, while sequentially switching one or more working electrodes of one or more cartridges, an electrochemical measurement step of performing cyclic voltammetry measurement or electrochemical impedance measurement,
A step of recording the measurement result in the element of each cartridge when the cartridge has an element capable of storing and reproducing, and communicating the recording information to the outside if 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:
If the cartridge is equipped with elements that can be stored and played back, from each element, information on the protein or pathogen to be measured by one or more working electrodes, the initial characteristics of one or more working electrodes, and already measured For the cartridge used for, the time used for the measurement, the step of reading the information of the detected numerical value,
Opening the outside air tank exhaust valve, activating the outside air suction pump, and introducing unmeasured outside air into the outside air tank;
Closing the outside air tank exhaust valve and stopping the outside air suction pump,
Activating the circulation pump to send the outside air in the outside air tank into the cartridge,
An adsorption step in which the inhaled outside air is blown onto a surface on which one or more working electrode elements are fixed, or is brought into contact with one or more working electrodes as bubbles in a supporting electrolyte.
A step of passing outside air after the adsorption step through a vent on the outside air outlet side of the cartridge and exhausting the outside air from the outside air outlet to an outside air tank,
Stopping the circulation pump, while sequentially switching one or more working electrodes of one or more cartridges, an electrochemical measurement step of performing cyclic voltammetry measurement or electrochemical impedance measurement,
A step of recording the measurement result in the element of each cartridge when the cartridge includes an element capable of storing and reproducing, and
A step of communicating the recorded information to the outside if the device has a wireless or wired communication circuit,
A method for detecting a protein or a pathogen, comprising:

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