KR20160114752A - Evaluation Method of Singlet Oxygen generated by photosensitizers - Google Patents

Evaluation Method of Singlet Oxygen generated by photosensitizers Download PDF

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KR20160114752A
KR20160114752A KR1020150040519A KR20150040519A KR20160114752A KR 20160114752 A KR20160114752 A KR 20160114752A KR 1020150040519 A KR1020150040519 A KR 1020150040519A KR 20150040519 A KR20150040519 A KR 20150040519A KR 20160114752 A KR20160114752 A KR 20160114752A
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singlet oxygen
oxygen
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최명규
박재명
이보인
김인욱
김주희
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가톨릭대학교 산학협력단
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Abstract

The present invention relates to an evaluation method of singlet oxygen generated by a photosensitizer. According to the present invention, without a measurement reagent, equipment in which measurement equipment, various waves of laser equipment, analysis equipment, and a program are integrated has excellent economic feasibility. Based on a principle in which newly discovered singlet oxygen generates a specific wavelength, the wavelength is directly measured to measure singlet oxygen in real time, and quantitative analysis is possible instead of qualitative analysis for measurement with a measuring value.

Description

{Evaluation Method of Singlet Oxygen generated by photosensitizers}

The present invention relates to a single-element oxygen measurement method that is generated by a photosensitizer.

In the field of photodynamic therapy research, the technique of measuring single-element oxygen generated by a photosensitizer is dependent on an indirect measurement method using the Singlet Oxygen Sensor Green (SOSG) measurement method (Non-Patent Document 1) Has the disadvantage that it is impossible to quantitatively measure the singlet oxygen and it can not be measured in real time. In addition, in order to measure the single-enzyme oxygen generated from the photosensitizer contained in the cell line, it is troublesome to use a large number of cells, a laser generator, and a fluorescence measuring device.

In addition, conventional methods for evaluating photosensitizing agents used a large number of cell lines to determine conditions such as optimal time, optimal path, optimal light wavelength, and amount of cells to be delivered, and then examined the degree of apoptosis in various cancer cell lines This method can be used for 2 to 3 months of time to select optimum conditions, laser equipment to investigate optimal light wavelength, various experiment methods (2-3 months) to compare cell death, It requires at least 3 months time and economic support to equip various equipments.

1. Free Radic Biol Med 27, 873-881 (1999). Chem. Sci., 4, 339-344 (2013)

It is an object of the present invention to provide a method for measuring the singlet oxygen generated by a laser of a specific wavelength by a photosensitizer used in photodynamic therapy.

(1) generating a monoenergetic oxygen by irradiating a laser with a wavelength of 600 to 710 nm to a methanol solution in which a photosensitizer in a white plate is dissolved;

(2) collecting luminescence at a wavelength of 1.22 to 1.32 mu m; And

(3) calculating a singlet oxygen count using the following equation (1): " (1) "

<Formula 1>

Singlet oxygen count = I 1 .27 탆 - average (I 1 .22 탆 & I 1 .32 탆 )

In the above formula 1, I 1 .27 탆 , I 1 .22 탆 and I 1 .32 탆 indicate measured values absorbed at the wavelengths of 1.27 탆, 1.22 탆 and 1.32 탆,

average (I 1 .22 μm & I 1 .32 μm ) represents the background value.

(A) treating cells with a photosensitizer, collecting the cells, and diluting the cells in methanol in a white plate;

(b) irradiating the methanol solution prepared in step (a) with a laser having a wavelength of 600 to 710 nm to generate singlet oxygen;

(c) collecting the luminescence of the singlet oxygen at a wavelength of 1.22 to 1.32 탆; And

(d) calculating a singlet oxygen count using the above-described equation (1).

In the present invention, since a single cell can be measured in a small amount of animal cell lines, it is possible to replace various methods of evaluating photosensitizers that have been developed in the field of photodynamic therapy research.

The present invention overcomes the temporal and economic limitations by using the real-time single-element oxygen measuring device and measuring method, and it is possible to replace the existing optical sensing agent evaluation by continuous research and verification of the measuring method using the corresponding equipment Which can open up new horizons for the developed photoresist evaluation method.

In the diagnosis and treatment of patient-centered cancer, real-time measurement of the individual treatment effect of photodynamic therapy can be used as a selection device for customized therapeutic dosage, and the realization of personalized medicine can be accelerated.

It can be applied not only in the field of medicine but also in various fields requiring active oxygen measurement, and it can be used for the development of active oxygen-related pharmaceuticals and cosmetics, in particular, by continuous research by a qualified expert.

1 is a photograph showing a PMT-based Singlet Oxygen Detection (PMT-based Singlet Oxygen Detection) system (Physical Sciences Inc., MA, USA).
FIG. 2 is a graph showing the result of calculating the measurement value of the singlet oxygen according to the equation 1; FIG.
Figure 3 is a photograph of a single oxygen measurement.
Fig. 4 is a graph showing the results of singlet oxygen measurement at 10 uM PPa diluted in media. Fig.
FIG. 5 is a graph showing the results of single-oxygen measurement at 10 uM PPa dissolved in various solvents.
6 is a graph showing photographs and measurement results of a single anti-oxygen measurement using various containers.
FIG. 7 is a graph showing changes in the single anti-oxygen measurements using a single-oxygen quencher.
Figure 8 is a graph showing the single anti-oxygen max / min measurable concentrations of various photosensitizers.
FIG. 9 shows a method for measuring intracellular single anti-oxygen by treating Che6 using a method for measuring single-element oxygen in a large number of cells.
FIG. 10 is a graph showing the results of measurement of the amount of singlet oxygen generated in a SW480 cell line treated with Che6 in concentration. FIG.
Figure 11 is a graph showing the accuracy of single-enzyme oxygen measurements in small amounts of cells treated with Che6, PPa.
12 is a graph showing the relationship between the measurement amount of monooxygenase and the survival rate of cancer cells.
Fig. 13 is a schematic diagram showing a method for accurately measuring the amount of singlet oxygen generated even in a small number of cells and a method for evaluating the efficacy of a photosensitizer.

Hereinafter, the configuration of the present invention will be described in detail.

(1) generating a monoenergetic oxygen by irradiating a laser having a wavelength of 600 to 710 nm to methanol in which a photosensitizer in a white plate is dissolved;

(2) collecting luminescence at a wavelength of 1.22 to 1.32 mu m; And

(3) calculating a singlet oxygen count using the following equation (1).

<Formula 1>

Singlet oxygen count = I 1 .27 탆 - average (I 1 .22 탆 & I 1 .32 탆 )

In the above formula 1, I 1 .27 탆 , I 1 .22 탆 and I 1 .32 탆 indicate measured values absorbed at the wavelengths of 1.27 탆, 1.22 탆 and 1.32 탆,

average (I 1 .22 μm & I 1 .32 μm ) represents the background value

Step (1) is a step of generating a singlet oxygen by irradiating a laser having a wavelength of 600 to 710 nm to a methanol solution in which a photosensitizer in a white plate is dissolved.

The type of the photosensitizer is not particularly limited and a photosensitizer used in this field can be used. Specifically, PPA (pyropheophorbide-a), ZnPc, ChE6, EtNBS, 5-ALA and the like can be used.

The photosensitizer may be used for photodynamic therapy. Specifically, the photosensitizer generates singlet oxygen or free radicals by light. Such a single-oxygen or free radical can be generated by keeping the normal cells intact in the cell, It can be used for treatment of cancer by inducing destruction by destruction.

Therefore, the measurement of monoenergetic oxygen generated from the photosensitizer can be used to select the dosage of the customized therapeutic agent in real time by measuring the individual treatment effect of the photodynamic therapy in the diagnosis and treatment of cancer, so that the personalized medicine can be realized in realization.

The singlet oxygen can be generated by irradiating a photosensitizer dissolved in methanol with a laser having a wavelength of 600 to 710 nm, a wavelength of 630 to 690 nm, for example, 670 nm. Methanol is a solvent for the determination of the generated singlet oxygen, which is easy to calculate the half life time in a single-oxygen measurement.

The irradiation of the laser can be irradiated by a separate laser generating device. In one embodiment, the methanol containing the photosensitizer may generate singlet oxygen by a diode laser generating a microjoule pulse of 670 nm. In addition, since the laser generating device combines the optical fiber emitting 670 nm and the optical fiber detecting the emission of singlet oxygen, the diode laser has an average power of ~ 230 mW for each pulse, a strategy having a pulse width of 5 us Can operate at a repetition rate of 10 kHz with an energy of 1.15 uJ for each pulse.

In one embodiment, the methanol in which the photosensitizer is dissolved can be placed in a white plate, specifically a white culture plate, followed by laser irradiation. In the case of a transparent container, the influence of the surrounding environment can be minimized by using a white container because there is a possibility of affecting other containers when the laser is irradiated.

In one embodiment, the content of the methanol solution is not particularly limited, and may be 10 μl to 10 ml. Particularly, in the present invention, it is easy to measure the singlet oxygen even with a small amount of solution of 10 to 1 ml or 100 to 200 ul.

Further, in one embodiment, the concentration of the photosensitizer dissolved in methanol is not particularly limited, and can be measured at a concentration of 10 nM to 10 uM. Particularly, in the present invention, it is possible to measure a single oxygen even at a low concentration of 10 nM to 1 uM or 100 to 200 nM.

In addition, the content of the photosensitizer dissolved in methanol in one embodiment is not particularly limited, and may be 0.005 ng / ul to 0.005 μg / ul. Particularly, in the present invention, it is possible to measure single-element oxygen even with a small amount of sample 0.005 ng / ul to 0.5 ng / ul.

In the present invention, step (2) is a step of collecting luminescence at a wavelength of 1.22 to 1.32 mu m of the singlet oxygen generated in step (1).

The singlet oxygen generates a wavelength of 1.27 mu m and a single oxygen can be measured by collecting a wavelength of 1.22 to 1.32 mu m.

In one embodiment, the measurement of singlet oxygen can be measured using a PMT detector. The PMT measuring instrument includes a slide (BP) for controlling a filter of each wavelength band by a computer, so that it can collect singlet oxygen luminescence.

In the present invention, step (3) is a step of calculating a singlet oxygen count using the following equation (1).

<Formula 1>

Singlet oxygen count = I 1 .27 탆 - average (I 1 .22 탆 & I 1 .32 탆 )

In the following Equation 1, I 1 .27 탆 , I 1 .22 탆 and I 1 .32 탆 indicate measured values absorbed at wavelengths of 1.27 탆, 1.22 탆 and 1.32 탆,

average (I 1 .22 탆 & I 1 .32 탆 ) may be a background value.

The monoenergetic oxygen emission near 1.27 탆 collected in step (2) is the emission value at 1.22 탆 and 1.32 탆 (out-of-band wavelength) including the remaining light emission wavelength of the photosensitizer and other substances as the background value And can be calculated through Equation (1).

The calculation of Equation (1) can be measured through a separate computer.

In the present invention, the measurement value of a single oxygen may be 1 X 10 3 to 2 X 10 6 .

In the present invention, the steps (1) to (3) described above can be performed through one system. In one embodiment, the measurement of the singlet oxygen can be performed through a PMT-based singlet oxygen detection system (PMT-SOD system). The PMT-SOD system consists of a laser system that generates a laser with a wavelength of 670 nm, a PMT-based detector that measures the singlet oxygen generated, and a computer that calculates the measured and measured values of each instrument for measurement Lt; / RTI &gt;

(A) treating cells with a photosensitizer, collecting the cells, and diluting the cells in methanol in a white plate;

(b) irradiating the methanol solution prepared in step (a) with a laser having a wavelength of 600 to 710 nm to generate singlet oxygen;

(c) collecting the luminescence of the singlet oxygen at a wavelength of 1.22 to 1.32 탆; And

 (d) calculating a Singlet oxygen count using the following equation (1): &quot; (1) &quot;

In the present invention, it is possible to measure a single anti-oxygen in a cell by using the above-described single-element oxygen measuring method.

In one embodiment, the cells treated with the photosensitizer may be diluted in methanol and placed in a white plate, specifically a white culture plate, followed by laser irradiation. In the case of a transparent container, the influence of the surrounding environment can be minimized by using a white container because there is a possibility of affecting other containers when the laser is irradiated.

In one embodiment, the content of the methanol solution containing the diluted cells, that is, the methanol solution prepared in (a) is not particularly limited and may be 10 μl to 10 ml. Particularly, in the present invention, it is easy to measure the singlet oxygen even with a small amount of solution of 10 to 1 ml or 100 to 200 ul.

In addition, in one embodiment, the concentration of the photosensitizer to be treated on the cells is not particularly limited, and may be treated at a concentration of 10 nM to 10 uM. Particularly, in the present invention, even if the cells are treated at a low concentration of 10 nM to 1 uM or 100 to 200 nM, it is possible to measure the monoenergetic oxygen through the steps described below.

Also, in one embodiment, the content of cells diluted in methanol is not particularly limited, and may be 0.005 ng / ul to 0.005 ug / ul. Particularly, in the present invention, it is possible to measure single-element oxygen even with a small amount of sample 0.005 ng / ul to 0.5 ng / ul.

The present invention does not require a measurement reagent, and is an economical merit of a measuring instrument, a laser instrument of various wavelengths, an analysis instrument and a program, and it is also excellent in that the newly discovered single oxygen generates a specific wavelength Based on the theory, it is possible to measure single oxygen in real time by directly measuring this wavelength band, and it is possible to perform quantitative analysis instead of qualitative analysis by comparison of comparison values.

Example

One. PMT - SOD  System-based Singlet  How to measure oxygen

The singlet oxygen measurement measured the emission of singlet oxygen at 1270 nm corresponding to the singlet anti-triplet transition state by means of detection.

1 is a photograph of a PMT-based Singlet Oxygen Detection (PMT-SOD) system (Physical Sciences Inc., MA, USA), which is a detection means of the present invention. The PMT- , A PMT based detector for measuring the generated singlet oxygen, and a computer for controlling the measurement of each instrument for measuring and computing the detected measurement value.

The solution containing the photosensitizer to be measured generates a singlet oxygen by a diode laser generating a microjoule pulse of 670 nm in the PMT-SOD system. The fiber of the system is composed of an optical fiber emitting 670 nm and an optical fiber detecting the emission of singlet oxygen. The diode laser has an average output power of ~ 230 mW for each pulse, a pulse of 5 μs Operating at a repetition rate of 10 kHz with a width of 1.15 μJ.

In order to measure the wavelength of 1.22, 1.27 and 1.32 μm in the entire wavelength band generated by the laser by the laser, each wavelength band filter was combined with a PMT detector (R5509-42; Hamamatsu Corp., Bridgewater, NJ). This filter separates the emission of near singlet oxygen at a wavelength of 1.27 μm from the long wavelength spectral background signal, and the emission of singlet oxygen at 1.27 μm separates the remaining light of the photosensitizer and other substances Using the emissions from 1.22 and 1.32 μm (out-of-band wavelengths), including wavelengths, as background values, we can calculate the measurement of singlet oxygen in a computer using Figure 2 and Equation 1.

<Formula 1>

Figure pat00001

In the graph on the left side of FIG. 2, the green color is 1.32 .mu.m, the blue color is 1.22 .mu.m, and the red color is 1.27 .mu.m. The graph on the upper right graph shows the measurement value of the single-element oxygen calculated by Equation 1. It can be confirmed that the measurement value of the single-element oxygen decreases after the laser of 670 nm is shut off. The lower right graph shows that the cumulative total of the measurements of the monoenergetic oxygen, which is reduced after laser interception, is calculated as the measurement of the final singlet oxygen.

2. Various cell lines and In the photosensitizer  Occurring Singlet  Establishment of oxygen measurement method and verification of reproducibility

(1) Solvent selection for measuring a single oxygen

(a) Experimental method

(Gibco, USA), acetone, methanol and PBS buffer (PBS) containing 2 ml of 10% FBS were added to each well of a solution containing 10% FBS (Pyropheophorbide-a, PPa; Frontier Scientific, (1 μM), respectively, and then they were placed in a brown glass container to measure the oxygen concentration (FIG. 3).

(b) Experimental results

A graph of the result measured by the experimental method of (a) is shown in Figs. 4 and 5. Fig. FIG. 4 is a graph showing the results of single-element oxygen measurement of PPa diluted with media, and FIG. 5 is a graph showing the results of singlet oxygen measurement of PPa diluted in acetone, methanol and PBS buffer.

In the PPa dissolved in media (RPMI 1640), the singlet oxygen count value was measured as 1,241, but no measurable value was obtained to measure the half life time (FIG. 4).

However, when PPa was diluted in acetone, methanol and PBS, Singlet oxygen count values were measured as 509,406 (methanol), 1,827,661 (acetone) and 22,873 (PBS), respectively. , Half life time of PBS was 0.2 ~ 0.4 us due to photon bleaching effect.

Since acetone has the disadvantage of being volatilized during the measurement process, it is preferable to use methanol as a solvent for experiments using cell lines.

(2) Selection of a single anti-oxygen measuring vessel to measure single-enzyme oxygen even in small amounts of solutions and cell lines

(a) Experimental method

In order to develop a method capable of measuring small amounts of solutions and cell counts, chlorin E6 (Che6; Frontier Scientific, Inc., UA) was prepared in methanol by using 96 well culture plates (clear / white) The oxygen values were compared with the standard curve and the R 2 values were compared.

(B) was added to 200 μl of a white 96-well culture plate, and (C) 200 μl of a clear 96-well culture plate was added to the methanol solution containing Che6 diluted twice from 10 μM Respectively.

(b) Experimental results

A graph of the result measured by the experimental method of (a) is shown in Fig.

6 (a) and 6 (b), the values of R 2 , A, B and C were all similar to each other. It can be concluded that measurement is possible. However, in case (C), it is preferable to use a (B) container, that is, a white 96 well culture plate, which can minimize the influence of light because the wavelength of the laser may affect an adjacent well.

(3) Experiments to determine if the actual singlet oxygen is being measured

(a) Experimental method

10 μM of Che6 or PPa was diluted in methanol, and the concentration of sodium azide, which is a quencher (quencher) that inhibits the emission of singlet oxygen, The measurement of oxygen and the decrease of half life time were measured.

(b) Experimental results

The results measured by the experimental method of (a) are shown in Fig.

In the present invention, FIG. 7 is a graph showing a change in a single oxygen measurement value using a quencher.

In the methanol solution containing sodium azide in Che6 and PPa, the singlet oxygen and half life time were decreased with the concentration of sodium azide, respectively.

Therefore, it can be confirmed that the measurement of the singlet oxygen according to the present invention measures the singlet oxygen occurring in the actual photosensitizer.

(4) Single anti-oxygen measurements and data accumulation in various photosensitizers

(a) Experimental method

In the case of high concentration of photosensitizers (PPa, Chlorin E6 and ZnPC), dilution was performed twice from methanol at 10 μM, diluted twice from 1000 nM at low concentration, and 200 μl per well in a white 96 well culture plate The singlet oxygen was measured.

After repeated at least four times the experiment, maximum measured concentration of the measured values of the singlet oxygen has been selected for the maximum concentration out a linear (R 2> 9.5) by using a regression analysis, the minimum measured density by performing the ANOVA test P <0.01 was selected as the minimum level of significance.

(b) Experimental results

The results measured by the experimental method of (a) are shown in Fig.

Figure 8 shows the mono-oxygen max / min measurable concentrations of various photosensitizers, which can be measured up to a concentration of at least 15 nM of photosensitizer and a maximum measurable range of up to 6 uM photo sensitizer concentration .

(5) Establishment of measurement method of singlet oxygen generated from intracellular photosensitizer (conventional method)

(a) Experimental method

SW480 (colorectal cancer cell line) was cultured in a 100 mm culture dish at 1 × 10 6 cells. After 24 hours, Che6 was treated with 10 μM, 50 μM and 100 μM. After 24 hours of incubation, the cells were washed three times with PBS, and then the cells were harvested and suspended in 1 ml of methanol. The turbid solution was transferred to a brown bottle to measure the singlet oxygen (Fig. 9). (Conventional measurement method A)

(b) Experimental results

The results measured by the experimental method of (a) are shown in Fig.

FIG. 10 is a graph showing the amount of unipolar oxygen generated by the concentration in the Che4-treated SW480 cell line. As a result, it can be seen that the measured amount of intracellular single-enzyme oxygen increases according to the concentration of Che6. This is a result of the accumulation of photosensitizer intracellular accumulation by a single oxygen measurement.

(6) Establishment of a single anti-oxygen measurement method that occurs in the intracellular intracellular photosensitizer in a small number of cells

(a) Experimental method

1 x 10 4 cell counts of four cancer cell lines (AsPC-1, MiaPaCa-2, HT29 and SW480) were cultured in 96-well culture plates (white), and after 24 hours, 1 μM of PPa and 10 μM of Che6 And diluted 2-fold. After culturing for 24 hours, the culture solution was removed, washed three times with PBS, and then 200 μl of methanol was added to measure the amount of single oxygen generated in each well.

In addition, PPa (excitation / emission = 565 nm / 670 nm) and Che6 (excitation / emission = 500 nm / 670 nm) were measured using an FL-meter in order to compare the amount of singlet oxygen generated with the actual scale of the intracellular photosensitizer. nm). The measured values were calculated as the concentration of the photosensitizer using the standard cuvette of uniaxial oxygen and FL-meter, respectively, and the final concentration value (x and y axis: FL & PMT) was determined as Correlation coefficient r> 0.95 Respectively.

(b) Experimental results

The results measured by the experimental method of (a) are shown in Fig.

FIG. 11 (a) is a graph showing the results obtained by measuring single-oxygen and FL (fluorescence) in four cell lines treated with Che6 and PPa, (Che6; r = 0.9553, PPa; r = 0.9448), respectively. From the above results, it can be confirmed that the single-oxygen generated in a small number of cells reflects the intracellular accumulation of the photosensitizer.

3. Optic  Efficacy evaluation

(1) The relationship between the measured amount of monooxygenase and survival rate of cancer cells

(a) Experimental method

1 × 10 4 cell counts of four cancer cell lines (AsPC-1, MiaPaCa-2, HT29 and SW480) were cultured in 96-well culture plates (white / clear), and then photosensitizer (Che6 10 μM, PPa 500 nM Diluted by 2 folds) was treated by concentration.

After 6 hours, 670 nm laser wavelength was irradiated to clear 96 well culture plate at 6 J / cm 2 , and cell viability was measured by MTT assay after 24 hours.

At the same time, white 96-well culture plates were washed three times with PBS, and then 200 μl of methanol was added to each well to measure the amount of single-enzyme oxygen produced and compared with cell viability results.

(b) Experimental results

The results measured by the experimental method of (a) are shown in Fig.

FIG. 12 is a graph showing the relationship between the measured amount of monooxygenase and the survival rate of cancer cells. It can be seen that the single enzyme oxygen generated in a small number of cells reflects the cell viability result.

(2) Development and establishment of a single anti-oxygen measurement method for evaluating the efficacy of various cell lines or various photosensitizers

As a result of the above experiments, we have developed a method for accurately measuring the amount of singlet oxygen generated in a small number of cells and a method for evaluating the efficacy of a photosensitizer.

The evaluation method is described in Fig.

Claims (9)

(1) generating a monoenergetic oxygen by irradiating a laser with a wavelength of 600 to 710 nm to a methanol solution in which a photosensitizer in a white plate is dissolved;
(2) collecting luminescence at a wavelength of 1.22 to 1.32 mu m; And
(3) calculating a singlet oxygen count using the following equation 1: < EMI ID =
<Formula 1>
Singlet oxygen count = I 1 .27 탆 - average (I 1 .22㎛ & I 1 .32㎛)
In the above formula 1, I 1 .27 탆 , I 1 .22 탆 and I 1 .32 탆 indicate measured values absorbed at the wavelengths of 1.27 탆, 1.22 탆 and 1.32 탆,
average (I 1 .22 μm & I 1 .32 μm ) represents the background value.
The method according to claim 1,
Wherein the content of the methanol solution is 10 to 10 ml.
The method according to claim 1,
Wherein the content of the methanol solution is 10 μl to 1 ml.
The method according to claim 1,
Wherein the concentration of the photosensitizer in the methanol solution is from 10 nM to 10 uM.
The method according to claim 1,
Wherein the concentration of the photosensitizer in the methanol solution is 10 nM to 1 uM.
The method according to claim 1,
Wherein the singlet oxygen count is 1 x 10 3 to 2 x 10 6 .
(a) treating cells with a photosensitizer, collecting the cells and diluting them in methanol in a white plate;
(b) irradiating the methanol solution prepared in step (a) with a laser having a wavelength of 600 to 710 nm to generate singlet oxygen;
(c) collecting luminescence at a wavelength of 1.22 to 1.32 mu m; And
(d) calculating a Singlet oxygen count using the following formula 1: < EMI ID =
<Formula 1>
Singlet oxygen count = I 1 .27 탆 - average (I 1 .22 탆 & I 1 .32 탆 )
In the above Equation 1, I 1 .27 탆 , I 1 .22 탆 and I 1 .32 탆 are 1.27 탆, 1.22 탆 and 1.32 탆 Represents a measurement value absorbed at a wavelength,
average (I 1 .22 μm & I 1 .32 μm ) represents the background value.
8. The method of claim 7,
wherein the content of the methanol solution prepared in step (a) is 10 μl to 1 ml.
8. The method of claim 7,
Wherein the concentration of the photosensitizer to be treated on the cell is 10 nM to 10 uM.
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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
1. Free Radic Biol Med 27, 873-881 (1999). Chem. Sci., 4, 339-344 (2013)

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