JPWO2019202327A5 - - Google Patents

Description

[Technical field]
The present invention relates to detecting cell vitality, that is, determining whether a plurality of cells are alive (vital) or dead (non-vital).
[Background technology]
Conventional methods for detecting the presence of living, or viable, cells in a sample typically involve culturing the sample in a culture medium and determining whether and to what extent the cells are multiplying . It takes. This type of test is common for detecting the presence and amount of bacteria in a sample. As required when culturing a sample, there is a considerable delay before the sample can be cultured, and it may take several days before results are obtained.

Some fluorescent dyes, such as the Sytox® system used in nucleic acid staining methods, can be used to detect multiple cells using susceptible membranes. This is because the dyes are hydrophilic or charged and do not have the routes they would normally use to penetrate lipid membranes. Cells with non-specific pores, e.g. created by electroporation, provide a pathway for the dye to penetrate the lipid membrane, and binding of the dye to the DNA results in the generation of a fluorescent signal. . However, cells with intact membranes do not generate a signal and live cells cannot be detected using these dyes. Therefore, the vitality of a cell can only be expressed by discounting the number of cells that have collapsed.
[Summary of the invention]
According to a first aspect, a method is provided for detecting the vitality of a plurality of cells in a sample, the method comprising:
Add fluorescent dye to the sample,
A part of the sample is placed in the test space between the electrode pair,
applying a voltage between the electrode pair to generate an electric field across a portion of the sample;
Illuminate the test space,
measuring the fluorescence response from the test space over a period of time after applying the voltage;
It involves detecting multiple cells within a sample as alive depending on the change in fluorescence response over time.

The method addresses the problem of how to rapidly and reliably detect living cells within a sample. Compared to conventional techniques that require culturing, this method allows such multiple cells to be detected directly on the target sample in a shorter time.

Electric fields in typical applications may be less than 1 kV/cm. The goal in this case is not to open pores in the cell membrane as in electroporation, but rather to introduce a fluorescent dye into living cells but not into non -living cells. This is to selectively promote this. The electric field may be greater than 100V/cm in typical applications, with example ranges being between about 100V/cm and 800V/cm, or between about 400V/cm and 800V/cm. Preferably, the method does not involve electroporation of a plurality of cells. That is, the electric field is preferably selected such that electroporation of cells does not occur when the voltage is applied.

The method may further detect cells of the sample as non-viable if the fluorescence response decreases or does not increase over the aforementioned time period.

In the detection step, live cells of the sample may be detected if the fluorescence response changes by, for example, more than a factor of two during the aforementioned time period.

The method preferably relates to detecting the vitality or viability of a plurality of bacterial cells, ie the ability of such cells to reproduce. Therefore, the method can be used to detect bacterial strains in a sample by first treating the sample with an antibiotic and then running the method to detect bacteria that are resistant to the antibiotic. Good too. The bacteria may be, for example, coliform bacteria such as E. coli, which is resistant to certain antibiotics, including penicillin and vancomycin.

According to a second aspect, there is provided an apparatus for detecting the vitality of a plurality of cells in a sample, the apparatus comprising:
A sample holder that accommodates a sample and includes a pair of electrodes on opposite sides of a test space;
a voltage source connected to the electrode pair;
a light source arranged to illuminate the test space;
and a photodetector positioned to detect a fluorescent response from the test space when illuminated by the light source.

The method according to the first aspect is preferably at least partially automated by being carried out under the control of a suitably programmed computer controller. Therefore, alternatively, the method may be defined as a method of detecting the vitality of a plurality of cells within a sample, the method comprising:
applying a voltage between a pair of electrodes within a test volume containing the sample and a fluorescent dye to generate an electric field across a portion of the sample;
Illuminate the test space,
measuring the fluorescence response from the test space over a period of time after applying the voltage;
It involves detecting multiple cells within a sample as alive depending on the change in fluorescence response over time.

FIG. 1 is a schematic diagram of an example of an apparatus for measuring the vitality of a plurality of cells within a sample. FIG. 2 is a schematic plan view of a comb-shaped electrode array used in the apparatus of FIG. FIG. 3 is a micrograph of multiple cell samples after application of an electric field in the presence of a fluorescent dye. Figure 4a is an image of fluorescence emission from multiple cells during application of an electric field at different times. Figure 4b is an image of fluorescence emission from multiple cells during application of an electric field for different times. Figure 4c is an image of fluorescence emission from multiple cells during application of an electric field at different times. FIG. 5 is a graph of the relative fluorescence response that occurred over time when an electric field was applied to two selected cells within a sample. FIG. 6 is another graph of the relative fluorescence response that occurred over time when an electric field was applied to two selected cells within the sample. FIG. 7 is a schematic diagram of another example of an apparatus for measuring the vitality of a plurality of cells within a sample. FIG. 8 is a schematic diagram of another alternative example apparatus for measuring the vitality of a plurality of cells within a sample. FIG. 9 is a schematic flowchart diagram illustrating a method for detecting the vitality of a plurality of cells within a sample. FIG. 10 is a schematic diagram of an example of an apparatus for measuring the vitality of a plurality of cells within a sample. FIG. 11 is a schematic diagram of the circuit configuration of the device of FIG. 10. FIG. 12 is a schematic cross-sectional view of an example optical assembly of the apparatus of FIG. 10. FIG. 13 is a plan view of an example of the electrode layout of the sample holder. FIG. 14 is a plan view of a portion of the example electrode layout of FIG. 13. FIG. 15 is a schematic diagram of the configuration of a sample holder having the electrode layout of FIG. 13.

When cells are alive, the opening of potassium channels results in hyperpolarization of the cell membrane, because living cells store large amounts of potassium in their cytoplasm. In dead cells , there is limited or no metabolism, so there is no metabolic energy available to reverse the potassium ion gradient. This means that when a potassium channel opens upon stimulation with an electrical pulse, the membrane potential collapses, or depolarizes. Therefore, by adding a fluorescent dye that responds to changes in membrane polarization, the survival or death of a plurality of cells may be determined based on how the dye reacts with the cell membrane exposed to an external electric field. Thus, hyperpolarization may be manifested as an increase in fluorescence emission when using Nernstian dyes, or as a decrease in fluorescence signal with other types of dyes. Fluorescent dyes, such as FluoVolt®, decrease their fluorescent signal when hyperpolarized. Other fluorescent indicators for membrane potential, such as di-4-ANEPPS dye, change their spectra depending on membrane polarity.

Although the main mechanism involved in the introduction of molecules is considered to be the opening of selective ion channels by an applied electric field, other mechanisms are also at play. For example, electrical pulses can temporarily alter the ion concentration of the extracellular medium, altering the ionic gradient within the cell membrane and causing efflux, resulting in transport rather than changes in the state of the transport machinery. There are gradient changes that promote This response may also be due in part to induced stress within multiple cells, which may also trigger the opening of ion channels, particularly potassium channels. This stress may be caused by electrical signals or chemicals that are electrochemically generated during the application of electrical pulses. This multi- cell reaction may also be due in part to the generation of reactive species through electrochemical reactions caused by the flow of current between the electrodes. These species may also modulate channels or induce stress responses. Such species may be reactive oxygen species or oxidizing chlorine compounds. All of the above-mentioned mechanisms have the common property that they result in modulating the conductivity of ion channels in the cell membrane and the rate of ion transport, thereby regulating the movement of molecules into the cell.

FIG. 1 illustrates an example apparatus 100 for measuring the vitality of a plurality of cells within a sample. A sample 101 is placed inside a sample box 102 on the surface of a substrate 103 on which electrodes (not shown in FIG. 1) are provided. The sample 101 contains a medium, and the medium contains a plurality of cells and a fluorescent dye that can be absorbed through the cell membrane. The medium may be in the form of a liquid or gel medium, for example.

A controller 108 is connected to the voltage source 105, the light source 107, and the optical device 104, and controls the voltage source 105 to acquire images from the optical device 104 over a specified period of time during which the voltage signal is applied to the sample 101. is configured to do so. The controller 108 may, for example, obtain a first image at the beginning of the time period before the voltage signal is applied, and then obtain a second image at the end of the time period during which the voltage signal was applied, and combine the first image with the first image. A third image may be calculated and output based on the difference between the two images, such as a difference in brightness level, and a plurality of cells that showed a change in fluorescence emission during this time may be determined.

In one experimental setup, bacterial cells were inoculated onto an agarose pad and placed on an electrode surface, allowing the cells to be monitored with single- cell resolution under a microscope. Thioflavin T (ThT), a fluorescent Nernst indicator dye that has been widely used with bacteria, was used to monitor membrane potential dynamics in multiple cells.

A series of electrical pulses (±1.5 V AC, 0.1 kHz, 2.5 seconds) are applied to multiple E. coli (K12 strain) cells placed in the gap between the electrodes, and the pulses cause the intracellular activation of multiple living cells. caused hyperpolarization (as indicated by an increase in the fluorescence signal of ThT). FIG. 3 shows the result of applying an electric field into a sample 304 as seen between opposing electrode pairs 301, 302 of a device of the type illustrated in FIG. Prior to application of the electric field, a portion of the sample was exposed to sufficient ultraviolet light to kill cells within a defined area 303 of the sample holder. An electric field was then applied and the displayed image represented the difference in fluorescence emission over time after the electric field was applied. As shown, the change in fluorescence emission within the UV-exposed region 303 can be distinguished from the change in fluorescence emission in the rest of the sample 304. Areas of the sample that were not affected by UV radiation showed an increase in fluorescence emission, whereas areas 303 that were affected by UV radiation did not. This clearly shows that the change in fluorescence emission is an indication of the vitality of multiple cells within the sample.

Figures 4a-4c show microscopic images during the application of an electric field to a sample of Bacillus subtilis at different times. A signal of 3 V amplitude was applied to the sample held between electrodes with a spacing of 50 μm, giving a peak field strength between 400 V/cm and 800 V/cm. This is comparable to cell electroporation, which may use at least 3,000-24,000 V/cm. Figure 4a shows a sample with an electric field applied for less than 1 second, where one bacterium 401 is fluorescent. Figure 4b shows the sample after a further 1 second application, and it can be seen that two more bacteria 402 are now fluorescent, but the fluorescence intensity of the first bacteria 401 has decreased. . Figure 4c shows the sample after an additional 2 seconds of application, and the two bacteria are now strongly fluorescent, but the fluorescence from the original bacteria remains low. This example shows that measurements of the vitality of multiple cells can be obtained in just a few seconds, and the electric field application time required before vitality measurements are possible is likely less than 10 seconds, and less than 5 seconds. Furthermore, it has been demonstrated that it can take less than 3 seconds.

Living cells accumulate Nernst dye at levels much higher than the surrounding medium. This action is selective for Nernst dyes that are already capable of penetrating cell membranes, and also excludes dyes such as Sytox® that do not cross living cell membranes. This action does not result in the transport of dye by electroporation of multiple cells, which is only possible at considerably high electric field levels, but instead by increasing the transport of dye across living cell membranes at existing cell potentials. transportation occurs.

FIG. 5 shows the results of selective ultraviolet treatment on a Bacillus subtilis sample followed by treatment by applying an electric field. The sample (shown in Figure 3) was exposed to an AC pulse of 3V at a frequency of 100Hz for 2.5 seconds. The average response 501 of 10 cells treated with ultraviolet rays is shown in FIG. 5 alongside the average response 502 of 10 cells not treated with ultraviolet rays. The cell viability index, expressed as the logarithm of the ratio of current to initial fluorescence emission, indicates the relative increase or decrease in fluorescence emission, and indicates the relative increase or decrease in fluorescence emission between living and dead cells . It clearly shows the difference in reactions between the two. In this case, living cells showed a significant increase, while dead cells showed a significant decrease.

Figure 6 shows the logarithm of the fluorescence response of multiple cells of two species, B. subtilis and E. coli, in the presence of vancomycin. Vancomycin is inactive against coliform bacteria such as E. coli, but targets nearly all other species of bacteria. In mixed cultures, vancomycin-treated Bacillus subtilis cells exhibited depolarization 601, while E. coli cells exhibited hyperpolarization 602. This indicates that this effect can selectively show the vitality of coliform bacteria in mixed culture. Therefore, the presence of E. coli or other coliform bacteria can be confirmed by fluorescence analysis of samples treated with selective antibiotics such as vancomycin.

FIG. 7 shows an apparatus 700 that is another example of an apparatus for detecting the vitality of a plurality of cells within a sample. In this example, the apparatus includes a test space 701 that accommodates a sample between a transparent lid 702 and an optical fiber flat plate 702. Electrodes are provided on one or both of surfaces 703 and 704 of each of the lid 702 and the optical fiber flat plate 702, which define the opposing surfaces of the test space 701. The electrodes may, for example, be in the form of electrode pairs on one of the plates, or a comb-shaped electrode pattern, or they may be provided in the form of transparent electrodes on both plates. Here, the gap between both plates defines the electric field in the sample.

A photodetector 706 is provided on the opposite side of the optical fiber flat plate 702. The photodetector 706 may be in the form of an image sensor such as a CCD. By interposing the optical fiber plate 702 between the image sensor 706 and the test space 701, it is possible for the sensor 706 to directly image multiple cells in the test space without the need for placing a lens between them. Become.

A controller 708 is connected to the voltage source 705, the light source 706, and the image sensor 706, and controls the voltage source 705 to capture images from the image sensor 706 over a specified period of time during which the voltage signal is applied to the sample 701. is configured to retrieve. The controller 708 may, for example, acquire a first image at the beginning of the time before the voltage signal is applied, and then acquire a second image at the end of the time that the voltage signal was applied, and combine the first image and the second image. A third image may be calculated and output based on the difference between the two images, such as a difference in brightness level, and a plurality of cells that showed a change in fluorescence emission during this time may be determined.

FIG. 8 shows yet another apparatus 800 for detecting the vitality of a plurality of cells within a sample. The apparatus includes a channel-shaped sample holder 801 within a microfluidic device 802, and the holder 801 is arranged to allow the sample to flow from an inlet 803 to an outlet 804. Channel 801 may be dimensioned to allow multiple cells to pass through device 802 one at a time so that fluorescence measurements can be made for each individual cell. Opposite walls of the channel may be provided with pairs of electrodes, each connected to a voltage source 805.

The excitation light may be provided by a 405 nm laser diode, for example, and the laser beam may be focused into the channel 801 of the microfluidic device 802 with a 10-20 μm spot. When cells are present, fluorescent light is emitted and follows the excitation light path back through the fiber towards the light source, but instead of being reflected back towards the light source, the fluorescent light passes through a dichrome mirror. It heads toward the photodetector 809a. Using two identical configurations, it is possible to compare the fluorescence emission when a cell passes through the first part of the channel and the fluorescence emission when the same cell then passes through the second part of the channel. Quantify the fluorescence emission of the cells by: If the speed at which a plurality of cells pass through the channel is known, it becomes possible to calculate the measured value of the relative change in fluorescence emission on a cell-by-cell basis. Each photodetector 809a, 809b may be, for example, a photomultiplier tube or an avalanche photodiode.

FIG. 9 shows a schematic flowchart diagram illustrating a method for detecting the vitality of a plurality of cells within a sample. The method begins by providing a sample comprising a plurality of cells and a fluorescent dye (step 901). A sample, or a portion thereof, is placed within a test space between a pair of electrodes (step 902). A voltage is then applied between the electrode pair (step 903) to generate an electric field across a portion of the sample within the test volume. The test space is illuminated (step 904) and the fluorescence response is measured over time after applying a voltage between the electrodes (step 905). If the fluorescence response increases during the measurement time, multiple cells in the sample are detected as alive (step 906). At least steps 903 to 906 may be performed automatically by a suitably programmed controller.

FIG. 10 illustrates an example apparatus 1000 for measuring the vitality of a plurality of cells within a sample. The sample is housed in a holder 1001 attached to a moving stage 1002, and the moving stage 1002 can move the sample holder 1001 in two directions within the XY plane, similar to the moving stage of a conventional microscope. Preferably, the stage 1002 is configured such that a sample slide (not shown) attached to the holder 1001 can be moved to perform sequential readings at two or more separate positions on the slide. An exemplary slide 1501 and holder 1001 are shown in detail in FIG. 15, and in this example, slide 1501 has three locations where a sample may be placed. Slide 1501 is inserted into holder 1001 and has an electrode layout with edge terminals 1502 that attach to PCB edge connectors 1503.

A detailed view of an example optical assembly for illuminating the sample is shown in FIG. A light source 1201, such as an LED, outputs light incident on the sample 1202, and the incident light passes through a lens 1203, a filter 1204, and a light guide 1205 before reaching the sample 1202. Light from the sample is received through lens assembly 1206, which focuses the incident light onto the image sensor, as described above. In a typical embodiment, a sample test space containing a plurality of cells and a fluorescent dye is illuminated with light from a light source 1201 that is incident on the sample at an oblique angle to the sample holder plane. Incident light may be directed to the sample through light guide 1205. An advantage of this configuration is that it reduces or minimizes any stray light emitted from the light source 1201 and incident on the lens assembly.

Claims (19)

A method for detecting the vitality of a plurality of cells in a sample, the method comprising:
Prepare a sample containing multiple cells and a fluorescent dye that responds to changes in membrane potential ,
A part of the sample containing the plurality of cells and a liquid or gel medium containing the fluorescent dye is placed in a test space between the electrode pair,
applying a voltage between the electrode pair to generate an electric field across the portion of the sample;
illuminating the test space;
measuring a fluorescence response from the test space over a period of time after applying a voltage between the electrode pair;
A method of detecting a plurality of cells within said sample as alive depending on a change in fluorescence response over said time period. 2. The method of claim 1, wherein the change in fluorescence response is an increase in fluorescence response during the time period. 3. A method according to claim 1 or 2, wherein the electric field is less than 1 kV/cm. 4. The method of claim 3, wherein the electric field is greater than 100 V/cm. 2. The method of claim 1, wherein the electric field is alternating current. 2. The method of claim 1, wherein the voltage is applied for a period of time less than 10 seconds. 6. The method of claim 5, wherein the voltage is applied for a period of time greater than 0.1 seconds. 8. A method according to claim 6 or 7, wherein the voltage is applied for a time shorter than 5 seconds. 2. The method of claim 1, wherein cells of the sample are detected as non-viable if the fluorescence response decreases over the time period. 2. The method of claim 1, wherein the time period is less than 60 seconds. 11. The method of claim 10, wherein the time period is less than 10 seconds. 12. A method according to claim 10 or 11, wherein the time period is longer than 0.1 seconds. 2. The method of claim 1, wherein the fluorescent dye is a Nernstian dye. The sample flows within the test space from a first point to a second point in the test space during the time period, and the step of illuminating the test space with light and measuring a fluorescence reaction is performed. , repeated at the first and second locations. 2. The method of claim 1, wherein the detecting step detects a plurality of live cells of the sample if the fluorescence response changes by more than a predetermined amount during the time period. 16. The method of claim 15, wherein the predetermined amount is greater than 50%. A method for detecting bacterial strains in a sample, the method comprising:
treating the sample with an antibiotic;
A method of detecting bacteria resistant to the antibiotic by carrying out the method according to any one of claims 1 to 16. 18. The method of claim 17, wherein the bacterium is E. coli. 19. The method of claim 18, wherein the antibiotic is vancomycin.