JPS60114763A - Electrochemical discriminating and control method of cell - Google Patents

Abstract

PURPOSE:To perform the mutual discrimination and identification of a cell (microorganism), by bringing cells into contact with an acting electrode and applying scanning potential to said cells while measuring the generated current value. CONSTITUTION:A cell suspension is filtered by a membrane filter 108 and cells 107 are held on the filter 108 while said membrane filter 108 is mounted to an acting electrode 105 and cyclical scanning potential is applied between the electrode 105 and an opposed electrode 104 to measure the generated current. The current-potential curve obtained by this method not only applies a max. current value (a peak current) proportional to a cell concn. but also shows a peak potential value different by the kind of a cell. By the specificity of this curve shape, information sufficient for identification of a microorganism such as bacteria is available.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an electrochemical identification or control method for various cells, and more particularly to a novel control or identification method for cell types such as microorganisms, animals, and plants.

Taxonomic identification or identification of types of F organisms, etc., plays an extremely important role in a wide variety of industrial fields, including the field of clinical testing, but it is carried out using so-called selective media. Since this method relies on colony counting methods, direct observation methods using a microscope, etc., it is difficult, complicated, and takes a long time.

On the other hand, the present inventors previously discovered a phenomenon in which an electric current is obtained when living cells come into direct contact with an electrode, and proposed an electrochemical bacterial count counting method that utilizes this phenomenon (Anal, C1■o,
AcLa, 98+ 25 (1978); Appl.
Environ, Microbiol, 37. 1
1 7 (1979) and Eur, J., Appl, Mi.
crol]io1. BioLecbnol, +10.
125 (1980), but the identification of cell types, mycological properties, etc., as well as their mechanisms, were completely unknown.

In view of the above, the present inventors further conducted extensive research and found that the current obtained by contact between a cell and a working electrode is mainly due to active substances such as coenzyme A (CoA) present in the cell or similar substances present at the electrode. This is due to the exchange of electrons between cells, and the generated current value can be calculated by applying a scanning potential to the cell according to methods such as so-called cyclic voltammetry, differential pulse polarography, phase discrimination AC polarography, or square wave polarography. The present invention was achieved based on the finding that cell types can be identified with extremely high accuracy by measurement. In particular, a gradually increasing DC scanning potential and an appropriate micropotential superimposed thereon are applied to the cells according to methods such as differential pulse polarography, phase discrimination AC polarography, or square wave polarography, and the voltage is enhanced by 4,4'-bipyridine. It has been found that cell types can be identified with extremely high accuracy by measuring the differential current value generated by the cell.

In other words, the current-potential curve obtained by the above method, the potential value (peak potential value) giving the extreme value of the differential current value, the curve pattern, etc. differ depending on the type of cell, and therefore each cell (microorganism) Mutual discrimination and identification can be achieved very clearly.

Furthermore, if cells etc. are gathered at high density on a support such as a membrane filter and brought into contact with the working electrode, an enhanced current value will be observed, so this also applies when the measurement target is a dilute suspension of cells. Clear identification becomes possible by filtering, accumulating on a support (filtering medium), and measuring.

As mentioned above, several types of cells have their own unique peak potentials, but it is surprising that by applying a potential close to that potential from the outside, cellular activities such as respiratory activity can be selectively and effectively suppressed and controlled. This fact has also been discovered, and therefore, the present invention also provides a method for electrochemical control of cells.

Hereinafter, the structure of the present invention will be explained in more detail.

Almost all microorganisms can be measured or controlled, including various microorganisms such as bacteria, actinomycetes, molds, microalgae, and yeast, and various animal and plant cells such as red blood cells, white blood cells, tumor cells, and cultured animal and plant cells. It can be controlled.

Here, according to the method of the present invention, in addition to mycological discrimination and identification of these various microorganisms, for example, classification of Gram-positive bacteria, discrimination of mutation reversion or non-reversion bacterial strains in the so-called Ames test, etc. If appropriate measurement conditions are set, identification and control over an extremely wide range is possible.

The shape of the peak potential or current-potential curve between a cell and an electrode in cyclic voltammetry or the like for cell identification or determination is due to the characteristics of the cell wall or cell membrane of the cell used for measurement. epi
E scl+er has a peak potential in der+n1dis and a cell wall structure more complex than the Gram-negative bacteria mentioned above.
icl+ 1acoli and Sal+nonella
A clear difference can be seen between the peak potential and the current-potential curve in Gram-negative bacteria such as typl+i+auriu+n.

Therefore, by measuring the peak potential between cells and electrodes by voltammetry, it is possible to identify a wide variety of cells.

Furthermore, as mentioned above, the generation of redox potential or current-potential curve between cells and electrodes is based on electrochemically active substances such as CoA present in the cell wall, so when measuring peak potential etc. Identification is possible not only by using cells as they are, but also by using, for example, cell wall fragments obtained by subjecting living cells to ultra-A wave destruction treatment.

Measurement Conditions and Field of Application Although various conventional voltammetry devices can be used as the device, FIG. 1 is a schematic explanatory diagram of one example of a cyclic voltammetry device. That is, the exemplary device has a working electrode 1. t=J pole 2 and 5SC
An electrolytic cell 4 comprising a reference electrode 3 such as E. Potentiostat 5. It consists of a linear scanning or sweeping current source 6 and an XY recorder or synchroscope 7. Here, the electrodes are ordinary platinum, gold, and silver.

Electrodes such as charcoal and various modified electrodes such as coating these with polymers etc. can be used, and if the potential of the counter electrode 2 is stable and unchanged, a circuit configuration equivalent to a normal polarograph lacking the reference electrode 3 is sufficient. .

Regarding the electrolytic cell, in the method of the present invention, as shown in FIG.
An electrode system is made up of a working electrode (105) equipped with a support or filter (108) that holds the electrode, a counter electrode (104), and a reference electrode (106) separated by a glass tube, immersed in a phosphate buffer solution. A suspension of JI7 cells is filtered through a membrane filter, the cells are retained on the filter, the membrane filter is attached to the working electrode, and a periodic scanning (sweep) potential is applied between the electrodes to measure the generated current. However, the potential scan is usually a so-called linear scan (L) that changes the potential in proportion to time.
inearSIlleep) is preferably adopted.

The current-potential curve obtained in this way (VolLa+
nmoHram) not only provides a maximum current value (peak current) that is proportional to the cell concentration, but also the specificity of its curve shape, such as the peak potential value that differs depending on the cell type, as will be detailed later. It also provides sufficient information. That is, when compared with the prior art, the method of the present invention significantly shortens the response time and measurement time, and because it uses a scanning potential, it eliminates various disturbing factors in the electrode reaction, allowing for more accurate measurements. This provides many practical advantages, such as being able to identify not only the cell concentration (number) but also the type of cells at the same time. Therefore, the present invention can be said to be extremely useful as a sensor means in a wide variety of fields, such as a sensor for real-time control of fermentation processes, measurement of the degree of microbial contamination of water, and measurement of red blood cell and white blood cell counts.

On the other hand, a schematic illustration of an example of a differential pulse polarography device particularly useful for cell identification is shown in FIG. 2.

That is, the exemplary device is a BPG (Basal P!an
e PyrolyLicC; ral+l+ i Lr
e), a working electrode 11 and a counter electrode 12 made of carbon such as HPG (high purity carbon for spectroscopic analysis), platinum, gold, silver, etc., and a reference electrode 13 such as 5SCE (saturated chloride electrode). cell 14, pulse sequencer 15, potential programmer 16, potentiostat 17, drop mecca 1g, i/e converter 19, sample hole l'(τ) 110 and sample hole l'(τ') 1
11, a D7T lens amplifier amplifier 112, and a refuder 113.

In this case, the measurement is performed by attaching a membrane filter holding cells to the working electrode in the cell 14, applying an incrementally increasing scanning (sweep) potential with a superimposed micropotential between the electrodes, and measuring the generated current. However, the potential scan is usually a so-called linear scan (L) that changes the potential in proportion to time.
inear Su+eep) is preferably adopted.

As the minute potential to be superimposed on the scanning potential, one having an appropriate waveform and period capable of giving a differential current value as described in No. 111 may be selected and used as appropriate.

The current-potential curve obtained in this way (V o l
La+++mo8ram) not only makes it possible to identify distinct peak potential values that differ depending on the type of cell, but also provides information on the electrochemical activity of microorganisms such as bacteria by analyzing the peak waveform. Become something. In other words, when compared with the conventional technology, the method of the present invention allows identification and identification of various types of bacteria to be achieved in an extremely short time and easily.

Here, 4,4'-bipyridine (4,4'
The usage conditions of -bil+yridiuc:BP) are summarized as follows.

That is, in the present invention, BP becomes involved in the cell-electrode reaction by being directly added to the solution, or by being fixed on a nitrocellulose membrane or the like and attached to the electrode. Note that the concentration of Bl) in the buffer solution varies depending on the type of target cells, but is generally about several + nM to 10 (L + M).

As shown in the later Jl experimental example, in the method of the present invention, BP
By performing voltammetry under coexistence, the peak current value of various cells is enhanced by about 1.5 to 2.5 times, the waveform becomes clearer, and the peak of the differential current value becomes sharper. becomes.

Mechanism of electro)gae production Electron exchange between cells and carbon electrodes is thought to be closely related to the production of coenzymes. Therefore, it is presumed that the peak current value is related to the metabolic pathway. So S, ce
suspension of A. revisiae (2,4X108 cells
・+J') and the metabolic inhibitor rotinone ('7, 6
+11M), antimycin (5,7+nAL
When cyanide (11) (0+nM) was added and the current value of the cyclink voltammogram was measured, the peak current value did not decrease with the addition of rotinone, antimycin, and cyanide, whereas arsenite A decrease of 4.8 μA to 3.7 μA was observed with the addition of . Rotinone specifically inhibits electron transfer in NAD H dehydrogenase, and antimycin inhibits cytochrome 1
], 0, and cyanide inhibits the electron transfer between cytochrome oxyguse and 02. On the other hand, arsenite 1 is known to inhibit pyruvate dehydrodenase. Therefore, it is determined that the generation of peak current is related to pyruvate dehydrokenase and the citric acid cycle.

In addition, as shown in Experimental Example 1 in the second stage, when the peak current value of the cells and their eluate was measured after eluting the compounds present in the cell wall by ultrasonication, the peak current value from the cells decreased; The peak current value (peak potential value: t, G 5 vs, 5SCE) of the solution gradually increased. Since the number of cells was not affected by sonication, the above results indicate that electrochemically active substances in the cell wall were eluted by sonication and detected electrochemically. The absorption at 2 Ci (ln1n) of the eluate is related to the adenine ring, and increases with the peak current value when the cell suspension is treated with ultra-1'' J waves. l) H,NA
DP)-1, I)"MNII, and CoA can be electrochemically oxidized, but NAD I-1 and N
A I') The absorption at 340 ntn corresponding to P l-1 and the absorption at 4115-450 ntn corresponding to FMNH2 and F A D l-1 were less common than the eluate.

About carbon electrodes N A D H and N A D P l
-1 experimental half-wave potential is 0.35-0.75V vs.
It was within the scope of SCE. FMNII2.

and F7~l) l-1, -f), 4V vs SC
It has been reported that E is oxidized electrochemically. BP(i(Basal Plane PyrolyL
NA using ic (rapl + ite) electrodes
Cyclic portammogram of D H and CoA
;) As a result, the peak current value of N A D I-1 is
The peak current values of 0.35 V vs. 5SCE and CoA were observed at l), G 5 V vs. 5SCE, respectively.

Since the peak potential value of CoA is similar to that of the eluate from cells treated with ultra-η wave treatment, the CoA in the eluate was enzymatically quantified by the method of SLadman eL al, and it was 3.610M Co. A was detected.

When cells are sonicated, the concentration of Co A increases with the peak current value of the eluate, so the increase in the peak current value obtained from the eluate is due to the increase in the concentration of Co A in the TIJ fluid. On the other hand, since the CoA present in the cell wall is slowly released into the Ij fluid, the peak current value obtained from the cell decreases.

The above results indicate that CoA present in the cell wall mediates electron transfer between the cell and the carbon electrode. The peak potential value obtained from the eluate is similar to that of CoA and different from that obtained from the whole cell.

The peak potential value is determined by the PH, and therefore the above phenomenon is ascribed to the difference in PH between the buffer and the environment surrounding CoA in the cell wall.

Electrochemical control of cell activity As shown in the experimental examples below, cell activities such as respiratory activity can be selectively controlled by applying a peak potential value specific to the cell. is cell suspension δ
It is sufficient to bring the accumulated cells into contact with the working electrode and apply a periodic scanning potential or a constant electric current tIy based on a predetermined peak potential for a predetermined period of time.

Therefore, this control method can be an effective means in various fields such as alcoholic engineering and genetic engineering in terms of selectively controlling the activity of microorganisms.

Garrison U.

1, ri)ly-r-X 4g1 e')hepui> ]8
+ K1-1,1-'(-)40.5B+Mg5O,・
Medium containing 7H200,'2B] (,1f-I
J (P 117°0) S, ccrcvisiae 3
After culturing under bf aerobic conditions ('I) for 18 h at o'c,
℃, 81) OOX, and centrifuged the collected bacterial cells in a 0.1 M phosphoric acid solution (PI-1'7.0).
Washed twice with

This was mixed with 0.1M 17 phosphoric acid mild N+j solution (P l-17, (
+ ) + t,l Jnijl cloudy cell concentration: 9×1
0F' pieces/+nN). Next, add 3 cultured bacterial cells.
The eluate was obtained by sonication for 0 min and centrifugation at 5 °C at 180 (', 10 × g), and the cells were collected and resuspended in buffer.

This cell and its eluate were used as a sample.

(on electrolysis), the volume is approximately 25+nl, and the potentiostat (
11okut.

Electric Works Model I-4A 3 (11), linear scanning power supply (Hokuto Electric Works 84oJe I-I B I
U 4) and the XY recorder (RIKEN F35)
The circuit was constructed as shown in the figure and used as a cyclic voltammetry device. The working electrode constituting the electrolytic cell is made of BPG (Basal Plane Pyrolyte).
c Grapl+1Le) electrode, a platinum wire electrode was used as the moon electrode, and a sodium chloride saturated-moon electrode (SSCE) was used as the reference electrode.

Each of the samples was injected into the electrolytic cell of the Iclink voltammetry device described in Section 2, and a cyclic portamogram was obtained at 25±2° C., and the peak current value of each sample was measured over time.

The results are shown in FIG. 4 (vertical axis: peak current value (μA); horizontal axis: two hours (minutes)).

In the figure, curve U is the value of the eluate and the 11 cells of the same sample.

As is clear from the figure, it is recognized that the electrochemically active substance exists in the cell wall and can be easily eluted by ultrasonication. In addition, the peak potential is the cell (Wl+ole Ce
l l)0.74 V vs.

5SCE, output fLo, G5\'vs, 5SCE.

2, then the method of SLadLman et al.

J, 13iol, CI+em,, 191 367 (]
! When the active substance in the eluate was assayed using J51)), it was confirmed that it was a substance similar to cO/\Jp, so: When this was done, the value of the potential was 0.65 to 'v9%5SCL: ((1).

Experimental Example 2 FIG. 3 shows an example of an electrode system used for cell identification according to the present invention. This electrode system has a surface area of 0, 17, which is equipped with membrane fillers (08) to support cells (107) that are not being measured.
2B P G electrode (4o5L counter electrode consisting of platinum wire (104L potassium chloride saturated 11 water reference electrode (SCE)
) (106), potentiostat (llokul, o
Electric model llA3 (11) (102), scanning power supply (1-1oku 1.o electrician model l113th) 4)
@(1(,+1), and Rub with a polishing cloth soaked with a water suspension.

Reference 11 using a glass measurement cell with the three electrodes mentioned above.
t) The electrodes are isolated from the main part of the cell by penetrating a glass tube whose tip consists of 7 liters of sintered glass. IE 5eller i-chia co
li was 0.1 after culturing on agar medium for 37°012 hours.
It was suspended in M phosphate buffer (PH7,0). Membrane filter (Toyo membrane filter, TM-2 type, nitrocellulose, pore size 0.45μ+11.diameter 25+
7115 ml of the suspension of the above-mentioned cultured cells was dropped on top of the cell suspension with slight suction. Next, by bringing this membrane filter into contact with the surface of the carbon electrode, the cultured cells held on 7.Lua were also brought into contact with the carbon electrode through the filter. The electrode system was inserted into phosphate buffer and cyclic portamograms were obtained at 25±2°C.

anode wave (anodic wave) at 0.72V (vs, 5CE)
u+ave) appeared.

No corresponding decrease peak was observed during the reverse scan.

Therefore, the cell's electrode reaction is irreversible. 0.1
~1.9 is determined from the peak current value of cyclic voltammetry.The minimum detectable number of E. coli cells is 5 x 107 cells.
ls-mN-' (in terms of FJ suspension).

Experimental Example 3 Using the same electrode system as Experimental Example 2, Bacillus
s 5ubL mountain is (gram positive) + Lactoba
cillus fermcntum (gram positive)!
5LrepLococcus 5aHuis (Gram positive L S Lapbylococcus' el+1
der+n1dis (Gram positive)1. Escl+er
icl+ia coli (gram negative) ISal+II
onella Lypl + imurium (Gram negative) each onto the same membrane filter as in the previous example at 6X10.
Eight pieces were held and the potential at which the peak current value appeared was measured by cyclic voltammetry. The results are shown in Table 1 below.

Table 1 Peak potential (V vs 5CE) 5ubLilis+L, fermentum
The plasma membrane of Gram-positive bacteria, such as Treptococcus nidis, is surrounded by a cell wall (typically 250 cells thick) composed of peptidoglycan and ticoyl acid. E. coli and S. typl+i
The structure of the cell wall and cell membrane of Gram-negative bacteria such as B. nurium is more complex, and the plasma membrane is surrounded by a 30A thick peptidoglycan wall, which is made up of proteins,
lipids.

and is covered by the outer part III of 80A, which is a mosaic of lipopolysaccharide. The above results indicate that the structure of the cell wall influences the peak potential value of cyclic voltammetry.

The peak current of Gram-negative bacteria appears at a potential of 1111 more positive than that of Gram-positive bacteria.

Experimental example 4 ``Li1is IFO3009, Takashi 1±jus st city
was cultured in Nutrient BroLh (beef extract 1%, peptone 1%), harvested at logarithmic phase, and cultured at 0.1 M'j.
After washing with acid buffer (PH7, 0), suspend in the same buffer to obtain a cell suspension (1, 2X 109 cells cIo-3
) was prepared.

Similarly, 5acal+aro+IIyccs cer
evisiae (YRD medium: yeast extract 1%, polypepsin 2%, glucose 2%), LacLol+a
cillus fermeutum IFO3071(
NutrienLBrotl+), Leuconost
oc +nesinteroidyq I F 03
8 3 2 (Toloato Juice BroL
l+: ) Lipton 1%, yeast 1 extract 1%, tomato juice 20%) and P, scl+ericl+ia
E. coli (Nutrient Brotb) was cultured, collected in the logarithmic phase, and suspended in the buffer solution until the viable cell number concentration was 1.
．． 03X109.5', OX 1. (+8.1.3
X109 and 1, OX11)” cells
Each suspension of can-3 was prepared.

Next, each of these cell suspensions was placed on the same membrane filter as in the previous example. Working electrode B was dropped with slight suction, and this membrane filter was attached to the surface of the working electrode B.
Scanning potential 0 to 1, Ov (vs, S S CE
), sampling time 2 (l ms, modulation voltage 5
0mV and 100+nV, potential single sweep 0.5mV/s,
Differential pulse voltammetry was performed at a measurement temperature of 25°C.

The device used was "Polarograph Model 312" manufactured by Fuso Seisakusho. Figure 7 shows the voltamodalum diagram of B.
CE) has a very clear peak potential (in the figure, symbols (a) and (1,) indicate the modulation voltage 1 (1 mv per month, 5
0 mv).

The peak potential values of each bacteria are summarized in Table 2 below.

As is clear from the table, the method of the present invention allows bacteria to be distinguished from each other very clearly and easily. are clearly distinguished 1! J-Kotoha Note 1
” is worthy of this.

Table 2 B, 5ubLilis o, 68 S, ccrevisiae O, 74Lace, fe
rme+mountain 0.75Lcuco, +nescn
Leroides O, 80+-Concave ring-0,85 Tile I! 1, microorganism S, cerevisiae, glucose 4g + polypeptone IgyKH2PO4 (1,5g and 1/Mg5O
,+78200.2g) medium (F'
H7,0) L: Aerobic 1. :, 30”012 hours, Sa
l+l1onella Lyl+Iyamaouriu+o
T A 100 was grown aerobically for 30'C in a 100+aN medium containing 1 g of polypeptone and meat extract, Escberichia coli K 12 was
Glucose 0.1 To Bacto Tryptone Ig+ Yeast Extract o, s.

Lactate culture medium (PH7, IJ) containing 5 g of NaCl and Lactate was incubated aerobically at 30°C for 12 hours.
obacillus fermenLuu+ ATCC
9338 is triptych case 18. trizo)・-su l)
, 3g.

Acid m extract 0.5gr KH2PO4O,3B+ K2
1-IPO40,3g+ (Nl-L)21-1-ci
LraLe (1,2g + glucose 2g + Tween 80 0.1g, cysteine-hydrochloride 0.0
2g, and [70,5a+N) solution (M2SO4・711
2o.

11.5%, FeSO4・7. H2O0.68%, Mn
10 (l I
Anaerobically at 37°C for 16 hours in medium (PII6.8) + Bacillus st city Li115M1112
is K2HPO411.4g + KH2po410.6
g(NH<)2sO+ 0.2g+ Na:+ ciL
raLe l-1,16+ Kasami 7 acid 0.5g+
100-degree medium (PH7,0) containing 0.5 g of glucose and MH8O4.7H2O at 30°C aerobically.
5 hours.

Each was cultured.

2.4.4'-bipyridine modified electrode 4.1.56 g of 4'-bipyridine was added to 100 tn (!(
7) Dissolve in a solution of 100ml and adjust the concentration of the solution to 100ml.
It was set as M. A BPG electrode (
0.1'7 cm2) was immersed in the above solution and gently rocked for 1 minute.

As described above, the BPG electrode was also modified with 4.4'-bipyridine extralayer before each operation.

3.Measurement method Potentiostat (Hokuto Electric Works, Model HA
301).

Scanning power supply (Hokuto Electric Works, Model 1-IB104
), and using XY Record it (RIKEN DENSHI, F35),
Cyclic voltammetry was performed on the 4,4'-bipyridine modified carbon electrode. The cyclic voltammetry cell is a glass cell with a volume of approximately 25 mN and has a modified double electrode system.

The counter electrode is a platinum wire and the reference electrode is a saturated Amagi electrode. The reference electrode is isolated from the main part of the cell by immersing the tip in a glass tube consisting of 7 liters of sintered glass. 1.1 x 109 cells of each type to be measured were held on a membrane filter (Toyo Membrane Filter, type TM-2, nitrocellulose, pore size 0.45μ "0 diameter 25 + 11111), and this membrane filter was - deviceed on a bipyridine-modified carbon electrode.

Polarograph (Fuso Seisakusho, Model 312) and X
Using a Y recorder, record a differential pulse polarogram at 1 iJ under the same conditions as the cyclic portamogram.

Figure 6 shows SacclIaromyc at pH 7.0.
3 shows cyclic and differential pulse portamograms for E. cerevisiac. In the figure, (A) shows a cyclic portamogram, (B) shows a differential pulse portammogram, (,) is related to the 4゜4゛-bipyridine-modified electrode, and (1]) is 4,4'-bipyridine. This relates to an unmodified electrode. Measurements were performed under standard conditions. Cyclic portamograms were obtained at a scanning rate of 1 (JmV/s), and differential pulse portamograms were obtained at a scanning rate of 10 mV/s and a wave height of 1
110 IIIV and a pulse width of 20+ns.

During the first scan in the positive direction, an anode wave (ano wave) was applied at 0.74 V vs.
dic u+avc) was observed. The peak potential value of the cyclic voltammogram and the differential pulse voltammogram coincide. The peak current values obtained from the 4゜4゛-bipyridine modified electrode were higher than the peak current values obtained from the unmodified electrode. In the absence of cells, no peak current was generated.

When the peak potential was measured for the various microorganism samples, E, coli+ (1,72V+ S, typh
iIIluriu+n, (1,70V, B.

5ul) Lilis+ 0.68V, L, fertn
enLutn, 0.68V vs SCE.

Experimental Example 6 (Method) B, 5ubtilis Ml 112 Arg-15,
leu B8thi5 r-to-rec C4 is 5p
izizen Mediu+n 10 (h(1°B,
5ubti, lis Ml 112 (PTLI 2)
is 1 μg/mn of Tmp (trimeLI+opri+
5pizizen Med: utnl O containing n)
The cells were inoculated into 0 tnfl and cultured aerobically at 37°C for 112 hours. After collecting bacteria, add 0.1hi + J acid buffer (PH7,
0), and the bacterial cells were attached to the surface of the BPG electrode using a membrane filter. A 20+nRH type cell was used for the measurement, BPG (0,17c+02) was used as the working electrode, a platinum wire was used as the counter electrode, and a saturated well water Calomero electrode was used as the reference electrode, and the electrochemical behavior was measured by cyclic voltammetry. A current-potential curve was drawn. Potential scanning speed is 10+oV
- The measurement was performed at 25° C. with a setting of 5eQ=.

(Results) B, 5ubtilis Ml 112 (Arg-15
+ leu'B8 thi5 r'-III-rec
C4B, sul+Li1is NN 112 (Pl-L
As a result of calculating the current-potential curve of 12), a peak current was obtained at a specific potential in each cell as shown in Table 3 below.

Peak potential (\' vs SCE) plasmids-f), 62 typhi+nuriu+n, Proteus vu
lgaris on Rogosa agar at 37°C.
The cells were incubated statically in an aerobic manner for an hour. Scrape each colony and suspend it in 0.1M phosphate buffer (PH7.0),
This was dropped onto a membrane filter, and bacteria were placed on the filter. Then, connect this to the B, P, and G electrodes (Basa
l plane pyroliLicgraph i
te) as the working electrode, a platinum wire as the lunar pole, and a saturated Amagi electrode (S, C, E,) as the reference electrode.
Electrochemical behavior was measured by cyclic voltammetry using an H-type cell containing 10 Iol of 0.1 MIJ acid buffer (PH7,0), and an i-5 curve was determined.

The measurement results of the two potentials of each sample are shown in the fourth section below.
Shown in the table.

m4 Table S. cerevisiae cultured aerobically for 12 hours
After collecting the bacteria, suspend them in 0.1M phosphate buffer solution (PH7,0), immobilize them on a membrane filter in a single layer of bacteria, and then attach the bacteria on the BPG electrode (working electrode). did. 2SmffH type cell with 0.1M phosphate buffer t(P)1
7.0), a platinum wire was used as the counter electrode, and a saturated Amagi electrode (S, C6E,) was used as the reference electrode, and potential scanning was performed by cyclic voltammetry.
Specific current peaks were obtained near 74 V vs.'S, C, and E. Therefore, in order to react with the electrode active material (CoA) attached to the cell surface, 0 to IV vs.
When potential scanning was repeated at S, C, and E, respiratory activity was inhibited by about 40% with one cyclic cycle, 960% with two cycles, and about 70% with 3 to 10 cycles. Next, when we changed the scanning potential variously and performed cyclic cycles three times each, the results ranged from 0 to 0.
, 7 V or 0.8 V, the respiratory activity was significantly inhibited, and the activity decreased to about 40%. From the above results, the peak potential of cyclic voltammetry (0
, 74V vs. s, C, E, M; was suggested to inhibit respiratory activity. Therefore, when constant potential electrolysis was performed at this potential and the relationship between electrolysis time and inhibition of respiratory activity was investigated, the activity was inhibited with time, and the activity decreased to 75% in 1 minute. Therefore, we performed constant potential electrolysis for 10 minutes at various potentials and investigated the relationship between potential and activity inhibition.
( ), 7-0.75V vs. S, C, E, the respiratory activity was most efficiently inhibited. PH of this temporary electrode surface
When measured, it was found to be neutral. This indicates that this inhibition of respiratory activity is not due to a change in PH on the electrode surface. Further, when the electrode surface was covered with a dialysis membrane or the cells were converted to protoplast F so that no electrode active substance was attached to the cell surface, almost no inhibition of respiratory activity was observed. These facts indicate that inhibition of respiratory activity does not occur only when the electrode active material attached to the surface of the cell migrates to the electrode surface, but also because the electrode active material reacts with the electrode, which affects respiratory activity. It became clear. Also, Bacillus
sul]tilis (Gram-positive bacterium), EsC1+e
When ricl+ia coli (Gram-negative bacteria) was subjected to constant potential electrolysis in a similar manner, respiratory activity was most efficiently inhibited near the peak potential of each cyclink voltammetry. Therefore, at a specific potential, a specific 4
It is also possible to selectively control only :I cell types.

Experimental Example 9 Bacillus 5ubtilis IFO3009
, Saccbaromyces cerevisiae+
Lactobacillus fer+oentu+
n I F○ 3071Leuconostoc ++
+esenteroides I F○ 3832.
and Escbericl+ia coli were cultured under the same conditions as in Experimental Example 3, and the bacteria were collected in the logarithmic phase to prepare each sample with a viable cell count of 1.3×10 9 cells/filter.

Next, differential pulse voltammetry was performed on each of these samples held on the filter under the same conditions as in Experimental Example 4 using the 4,4'-bipyridine-modified BPG electrode used in Experimental Example 5 above.

The peak potential values of each sample are summarized in Table 5.

As is clear from the same table, according to the method of the present invention, 411
Bacteria can be distinguished from each other very clearly and easily.

Table 5B, 5ubtilis (1,68 S, cerevisiae O,74Lact, f
ermenLum O,75Leuco, +nese
nteroides 0.8 (IE, coli 0.
85

[Brief explanation of drawings]

Figures 1 to 3 are schematic explanatory diagrams of the apparatus used in the examples of the present invention, and Figures 4 to 7 are explanatory diagrams of the same experiment. 4... Electrolytic cell, 5... Potentiostat, 6
...Linear sweep power supply, 7.XY recorder or synchroscope, 11.Working electrode, 12.
. . . Counter electrode, 14 . Patent applicant: Matsunaga Figure 1 Figure 2 5 Figure 3 Figure 4 ^) Figure 5 0 0.5 1.0 (y vs SCE) Figure 6 0 0.5 1.0 (V vs Se5 ) Figure 7 (V VS 5SCE)

Claims (2)

[Claims] (1) A method for electrochemical identification of cells, which comprises bringing cells or cell wall fragments accumulated on a support into contact with a working electrode, applying a scanning potential, and measuring the generated current value. (2) A method for electrochemical control of cell activity, which comprises applying a predetermined potential to the cell based on its peak potential.