KR20190017083A - Colorectal cancer diagnosis using enzyme-sensitive ratiometric fluorescence dye and antibodyquantum dot conjugates for multiplexed detection - Google Patents

Abstract

The present invention relates to a method for colonoscopic diagnosis of colorectal cancer using multi-fluorescent imaging of an enzyme-sensitive ratiometric fluorescent probe and a biocompatible quantum dot-antibody complex probe. The present invention provides an apparatus for diagnosis of colorectal cancer comprising: organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in colorectal cancer, wherein the organic fluorescent dye has a first fluorescent wavelength and has a second fluorescent wavelength by separation of the functional group; a quantum dot having a third fluorescent wavelength while having an antibody that binds to a second enzyme that is overexpressed in cancer; and an apparatus which fluoresces the organic fluorescent dye and the quantum dot and measures the fluorescence.

Description

[TECHNICAL FIELD] The present invention relates to a method for diagnosing colorectal cancer using colorimetric fluorescent probes and biotin-type quantum dot-antibody complex probes,

The present invention relates to a method for diagnosing colorectal cancer for colonoscopy using multi-fluorescence imaging of an enzyme-responsive quantitative fluorescent probe and a biocompatible quantum dot-antibody complex probe.

Colorectal cancer has recently been on the rise in cancer rates and is the second leading cause of cancer deaths. Although these colon cancers are diagnosed mainly through colonoscopy, there is a possibility that they are not detected because they are detected by the naked eye. If the shape of the polyps does not appear like a hump, they are more likely to miss. ¹ There is also a limit to the number of tissues to be harvested because it takes a long time because the tissue is judged to be cancerous by removing the tissue and performing frozen section pathology examination.

 In order to overcome these limitations, studies on molecular imaging based on fluorescence signals are underway, and organic fluorescent dyes and quantum dots are widely used as fluorescent materials. Fluorescence signal images are more simple than other molecular images and can be used for real - time imaging. The fluorescence wavelength of an organic fluorescent dye is determined according to its molecular structure, and it is possible to combine molecular sieves and other molecular sieves with molecular structure. Quantum dots are semiconducting nanoparticles composed of inorganic materials, which are excellent in photochemical stability and durability, and can introduce various biomolecules onto the surface of the quantum dots.

1. Pasha, S. F .; Leighton, J. A .; Das, A .; Harrison, M. E .; Gurudu, S. R .; Ramirez, F. C .; Fleischer, D. E .; Sharma, V. K., Comparison of the Yield and Missed Bandwidth Imaging and White Light Endoscopy in Patients Undergoing Screening or Surveillance Colonoscopy: A Meta-Analysis. Am J Gastroenterol. 2012, 107, 363-370. 2. Y. Park, Y.M. Ryu, Y. Jung, T. Wang, Y. Baek, Y. Yoon, S.M. Bae, J. Park, S. Hwang, J. Kim, E.J. Do, S.Y. Kim, E. Chung, K.H. Kim, S. Kim, S.J. Myung, Spraying Quantum Dot Conjugates in the Colon of Live Animals Enabled Rapid and Multiplex Cancer Diagnosis Using Endoscopy, ACS Nano, 2014, 8, 8896-8910. 3. Torimoto, T. et al. Remarkable photoluminescence enhancement of ZnS-AgInS2solidsolutionnanoparticlesbypost-synthesistreatment. Chem. Commun. 2010, 46, 2082-2084. 4. Park, J .; Nam, J .; Won, N .; Jin, H .; Jung, S .; Jung, S .; Cho, S.-H., Kim, S., Compact and Stable Quantum Dots with Positive, Negative, or Zwitterionic Surface: Specific Cell Interactions and Non-Specific Adsorptions by the Surface Charges. Adv. Funct. Mater. 2011, 21, 1558-1566.

A problem to be solved by the present invention is to provide a device for diagnosing colon cancer which can obtain a fluorescence image capable of judging colon cancer.

A problem to be solved by the present invention is to provide a method of diagnosing colorectal cancer using a device for diagnosing colorectal cancer which can obtain a fluorescent image capable of judging colon cancer.

Another problem to be solved by the present invention is to provide a combination of phosphors capable of obtaining fluorescence images for diagnosing colon cancer.

Another object of the present invention is to provide a novel organic fluorescent dye capable of diagnosing colorectal cancer.

Another object of the present invention is to provide a method for diagnosing colorectal cancer using a novel organic fluorescent dye.

Another object of the present invention is to provide a novel organic fluorescent dye for cancer diagnosis.

Another object of the present invention is to provide a novel organic fluorescent dye.

Another problem to be solved by the present invention is to provide a new quantum dot for cancer diagnosis.

Another problem to be solved by the present invention is to provide a new quantum dot.

In order to solve the above problems, the present invention provides an organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in colorectal cancer, wherein the organic fluorescent dye has a first fluorescence wavelength, Has a second fluorescence wavelength; A quantum dot having an antibody that binds with a second enzyme overexpressed in cancer and having a third fluorescent wavelength; And an apparatus for fluorescing the organic fluorescent dye and quantum dots and measuring the fluorescence.

In one aspect of the present invention, there is provided an organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in a colon cancer, wherein the organic fluorescent dye has a first fluorescence wavelength, Measuring fluorescence of the organic fluorescent dye by fluorescence treatment with the second fluorescent light; And measuring the fluorescence of the quantum dots by treating the test tissue with a quantum dot having an antibody that binds with a second enzyme to be overexpressed in the cancer and having a third fluorescent wavelength, and measuring fluorescence of the quantum dot .

According to another aspect of the present invention, there is provided an organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in colon cancer, wherein the organic fluorescent dye has a first fluorescence wavelength, Has a fluorescent wavelength; And a quantum dot having an antibody that binds to a second enzyme that is overexpressed in cancer and has a third fluorescent wavelength.

According to another aspect of the present invention, there is provided an organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in colon cancer, wherein the organic fluorescent dye has a first fluorescence wavelength, Has a fluorescent wavelength; And a device for measuring fluorescence of the organic fluorescent dye.

According to another aspect of the present invention, there is provided an organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in a colorectal cancer, wherein the organic fluorescent dye has a first fluorescence wavelength, And a second fluorescent light having a second fluorescence wavelength by the fluorescence of the second fluorescent light.

According to another aspect of the present invention, there is provided an organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in colon cancer, wherein the organic fluorescent dye has a first fluorescence wavelength, A fluorescent dye having a fluorescent wavelength is provided.

In another aspect, the present invention provides an organic fluorescent dye represented by the following formula (1).

In another aspect, the present invention relates to a method of producing an AgInS ₂ Nucleus and ZnS shells are quantum dots that are modified with zwitterionic molecular sieves, have matrix metalloproteinase 14 (MMP14) antibodies, and provide cancer diagnostic kits with 670 to 690 nm fluorescence.

According to another aspect of the present invention, there is provided an organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in cancer, wherein the organic fluorescent dye has a first fluorescent wavelength, Has a fluorescent wavelength; A quantum dot having an antibody that binds with a second enzyme overexpressed in cancer and having a third fluorescent wavelength; And an apparatus for fluorescing the organic fluorescent dye and quantum dots and measuring the fluorescence.

In the present invention, the functional group is a functional group capable of being bonded to an organic fluorescent dye having a second fluorescent wavelength to change the fluorescence wavelength, preferably the peak wavelength, of the fluorescent dye. The change in wavelength by the functional group can be increased or decreased by 10 micrometer or more, preferably 20 micrometer or more, more preferably 30 micrometer or more, and preferably 10 to 100 micrometer, Functional group.

It is preferable that the fluorescent group of the functional group has a similar fluorescence intensity before and after the binding. This similarity is understood to mean that the increase or decrease in the fluorescence of P.L is maintained within the range of ± 20%, preferably within the range of ± 10%.

The functional group is bonded to an organic fluorescent dye having a second fluorescent wavelength by a dehydration condensation reaction to change the fluorescence wavelength of the organic fluorescent dye to a first wavelength and is separated by hydrolysis by a first enzyme overexpressed in a specific cancer And becomes fluorescence at the second wavelength.

In the practice of the present invention, the functional group may be an amino acid, for example, a glutamic acid that can be separated by λ-glutamyltranspeptidase (GGT) that is specifically overexpressed in colorectal cancer.

In the practice of the present invention, the organic fluorescent dye may be a dye having a fluorescent wavelength in the visible light region, and has a peak wavelength of 610 to 650 nm in a state where the functional group is bonded, and has a peak wavelength of 550 to 590 nm The dye having the peak wavelength of < RTI ID = 0.0 >

In a preferred embodiment of the present invention, the organic fluorescent dye may be represented by the following formula (1) in the state where glutamic acid is bound.

In the present invention, the quantum dot may be a heavy metal that affects the human body, for example, a quantum dot free of cadmium or lead. The quantum dot is preferably a quantum dot including a gold component, more preferably AgInS ₂ Lt; / RTI >

In the practice of the present invention, the quantum dot may be in the form of a core-shell in which a shell is formed on the surface to improve the fluorescent property, and preferably has a ZnS shell.

In the practice of the present invention, the quantum dot may be a quantum dot whose surface is modified with an amphoteric ion so as to prevent non-specific binding. The amphoteric ion may be used in the amphoteric molecular sieve disclosed in Korean Patent No. 1276693, which is incorporated herein by reference in its entirety. The zwitterionic molecular sieve may be represented by the following formula (2).

In the practice of the present invention, the surface of the quantum dot may be modified to a -COOH group so that the antibody can easily bind thereto. Preferably, the -COOH group may be formed at the end of the molecular sieve having a moiety capable of binding to the quantum dot. The molecular sieve having the -COOH group may be a molecular sieve as disclosed in Korean Patent Application Publication No. 2016-0046933 filed by POSTECH Cooperation Bureau and the like and incorporated herein by reference in its entirety.

Wherein the molecular sieve comprises an attachment region, a connecting region and a binding region, wherein the attachment region is selected from the group consisting of a thiol group (-SH), a dithiol group (-CS2, -PS2, -CH (SH) (CH2CH2SH) (-NH2, -NH), a phosphonate group (-PO3H), a phosphide group (-P), a phosphine oxide group (-P = O), a carboxy group (-COOH) Imidazole group and diiodo group and the connecting region is selected from amide bond (-CONH-), carbon bond (- (CH2) n-), polyethylene glycol (- (CH2CH2O) n- ), Triazole, and the binding region may be a carboxyl group. Preferably, the molecular sieve having -COOH group can be represented by the following formula (3).

In the present invention, the antibody bound to the quantum dot may be an antibody capable of binding to an extracellular matrix metal metalloproteinase, which is known to play a major role in invasion and metastasis of cancer cells. In the practice of the invention, the antibody is preferably a matrix metalloproteinase 14 (MMP14) antibody that is overexpressed in colon cancer.

In the present invention, the antibody against MMP14 is commercially purchased and used. Commercially available antibodies to MMP14 can be purchased from Abcam, Santa Cruz Biotechnology, and Thermo Fisher Scientific.

In the present invention, the quantum dot has a third fluorescence to be distinguishable from the first fluorescence and the second fluorescence, and preferably has a peak fluorescence in the visible light band of 670 to 690 nm.

In the present invention, the first fluorescence may have a peak wavelength of 550 to 590 nm, the second fluorescence may have a peak wavelength of 610 to 650 nm, and the third fluorescence may have a peak wavelength of 670 to 690 nm.

In the present invention, the fluorescence measurement apparatus may display images of the first fluorescence and the second fluorescence data of the organic fluorescent dye, images of the third fluorescence of the quantum dots, and express the third fluorescence of the quantum dots together with the overlapped image, More accurate diagnosis can be made.

In the present invention, the fluorescence measurement apparatus can measure the ratio of the first fluorescence to the second fluorescence, and it is also possible to measure the ratio of the first fluorescence and the second fluorescence with time. It is preferable to measure the first fluorescence and the second fluorescence with data elapsing more than 30 minutes.

The enzyme-active quantitative fluorescent probe and the quantum dot-antibody probe according to the present invention can diagnose colorectal cancer more precisely by comparing fluorescent signals of two probes operating with different mechanisms to diagnose colon cancer. This can reduce diagnosis errors that can occur when only one probe is used, and can detect colon cancer with high reliability. In addition, it is possible to detect a flat cancer that is difficult to detect with the naked eye, or a fluorescent signal that is not yet rapidly progressing.

Fig. 1 shows the molecular structure of the synthesized CV-Glu probe and the molecular structure of Cresyl violet and respective fluorescent images (A). (B) and fluorescence (C) spectra of the probe when treated with GGT enzyme in a CV-Glu probe.
FIG. 2 shows the expression level of GGT enzyme in A. coli normal cells (CCD 841 CoTr) and various colorectal cancer cells (LOVO, HT29, HCT116, SW480, SW620) by Western blotting. B. Process ratio metering units in colonic normal cells (CCD 841 CoTr) and various colorectal cancer cells (LOVO, HT29, HCT116, SW480, SW620). Transmission image (first line), 580-600 nm fluorescence image (second line), 615-640 nm fluorescence image (third line), fluorescence ratio image (fourth line).
Figure 3 verifies the ratio metering unit ex vivo for colon and mouse model mouse colon tissues of normal rats. The fluorescence ratio image (A) at 10 min and 30 min after the ratio metering unit treatment, and the H & E staining image (B) of the selected portion of the colon tissues.
Figure 4 (A) Fluorescence signal ratio image over time after CV-Glu probe treatment in colon cancer tissue (left) and normal tissue (right).
Fig. 5 (B) Fluorescence signal spectrum at colon cancer tissue (left) and normal tissue (right) at each time. Fig.
Figure 6 (C) Fluorescence signal ratio image according to tissue depth.
7 shows (A) the absorption and fluorescence spectra of AgInS _{2 /} ZnS quantum dots. (B) Transmission electron microscope image of AgInS _{2 /} ZnS quantum dots. (C) Large intestine photograph of mouse colon cancer model in white light (left) and fluorescent signal image of MMP14 quantum dot probe (right). For the control experiment, immunoglobulin antibody probe (IgG-QD) was applied to the two intestinal tissues, and the MMP14 quantum dot probe was applied to the three intestinal tissues, followed by washing and observing the fluorescence signal.
FIG. 8 is a schematic diagram of a method for diagnosing cancer in the colon tissue of a rat using (A) a ratio measuring fluorescent probe and a biocompatible quantum dot-antibody complex probe.
9 is a fluorescent signal image of (B) a quantum dot-antibody complex probe.
FIG. 10 is an image of autofluorescence and quantum dot fluorescence measured by two-photon microscopy in a mouse colorectal cancer tissue after (C) quantum dot probe treatment. FIG.
Figure 11 (D) Fluorescence signal ratio image after CV-Glu probe treatment in the same tissue.
12 shows (E) an overlay image of two probe fluorescence signals.
13 shows (F) two-photon fluorescence image over time after CV-Gluv probe treatment.
Figure 14 shows (G) two-photon fluorescence images with depths in the same tissue location of the two probes.
FIG. 15 is a graph showing the fluorescence signal ratio and fluorescence signal intensity after CV-Glu probe treatment in the same tissue as the fluorescence signal image and the fluorescence signal average value (B) of the quantum dot-antibody complex probe in the patient's normal colon tissue and colon cancer tissue, (C) An image expressed in pseudo color on two probes and an image overlaid on it. (D) H & E images of organizations.
16: (A) Cell viability according to various concentrations and treatment time of CV-Glu probe and biocompatible quantum dots in HCT116 cells (human colorectal cancer cells). (B) Toxicity assessment and hematology marker analysis in mice treated with CV-Glu fluorescence-modulated probes or biomimetic quantum dots and PBS alone. This makes it suitable for medical applications.

Hereinafter, the present invention will be described by way of examples. An embodiment of the present invention discloses a method for diagnosing colorectal cancer by developing a ratio-metering fluorescence probe responsive to enzyme activity and marking multiple colon tumors using a fluorescence signal together with a pair of quantum dot-antibody complexes. The probe was synthesized by binding an amino acid to an organic fluorescent dye. The probe was designed to change the fluorescence wavelength by breaking the amide bond of the amino acid of the probe due to the enzyme activity that is specifically overexpressed in the colon cancer. By measuring the degree of activity of the enzyme according to the ratio of the fluorescence signal at the two fluorescence wavelengths, the colon cancer can be discriminated by detecting the overexpressed enzyme in the colon cancer. The quantum dots probe was modified to have a low specific binding on the surface using AgInS ₂ quantum dots composed of biocompatible elements that do not contain heavy metals such as cadmium and lead and was found to specifically bind to proteins known to be overexpressed in colon cancer Antibody was introduced to synthesize a quantum dot-antibody complex. Using these two fluorescence probes, colonoscopy can be used to label cancer in colorectal cancer models and human colorectal adenomas in rats.

< Example  1> Enzyme susceptibility ratio Weighing fluorescence Of the probe  Development

 We have developed a ratio - metering fluorescent dye probe that specifically modulates the fluorescence wavelength in a cancerous environment. A CV-Glu fluorescence-modulated probe was synthesized by binding an amino acid called glutamic acid (Glu) to a Cresyl violet (CV) fluorescent dye and sensitive to λ-glutamyltranspeptidase (GGT) enzyme overexpressed in the cancerous environment. The probe has a fluorescence of 580 nm, and the amide bond between the glutamic acid and the fluorescent dye is broken by GGT, resulting in fluorescence of the original CV fluorescence of 620 nm (FIG. Therefore, the degree of activity of GGT can be determined by measuring the ratio of CV-Glu and CV fluorescence signals. The synthesized CV-Glu probe was treated with GGT enzyme in the liquid phase to measure the absorption and fluorescence spectra (Fig. 1B, C)

Example 2 Confirmation of Cancer Cell Marking Ability of CV-Glu Probe in Human Colorectal Cancer Cells and Normal Cells

GGT expression levels of the human colon cancer cells (LOVO, HT29, HCT116, SW480, SW620) and normal colon cells (CCD841CoTr) were measured by Western blot method. (Fig. 2. A). GGT expression in the normal cells was the least, and the GGT expression level was high in the colon cancer cell lines and relatively low in the HT-29 cells. The same cell lines were treated with CV-Glu probe and fluorescent signals were observed at 620 nm and 580 nm after 30 minutes. In addition, the ratio image (620/580 nm) showed a ratio image (FIG. 2. B). In cancer cells, fluorescence signals in the 580-600 nm region were hardly visible, and fluorescence signals at 615-640 nm were strong. In addition, the fluorescence ratio (I ₆₂₀ / I ₅₈₀ ) image of the ratio metering unit showed a low fluorescence ratio in normal cells but a high fluorescence ratio in cancer cells. Therefore, it has been verified that the ratio metering fluorescent probe is able to label colon cancer cells well at the cell level.

Example 3 Confirmation of Cancer-Marking Ability of CV-Glu Ratio Fluorescence Probe in Normal Colonic and Cancerous Tissues of Rats.

 Colonies of normal mouse colon and colon cancer mouse model were excised. Cut it long so that the inside of the colon comes up. Fluorescence ratio images were obtained at 10 and 30 minutes after treatment of the CV-Glu ratio measuring fluorescent probe into the colon tissue. The CV-Glu probe labeled the cancer within 10 min in the rat colon tissue. (1, 2, 3) and almost no signal (n) with H & E staining with a high ratio in the fluorescence ratio image, (N) was judged to be a normal tissue, and all the remaining tissues were judged to be cancer tissues, so that the ratio measuring unit can discriminate the cancer tissue part well from the normal tissues, However, there is a difference in fluorescence ratio between cancer tissues, which is thought to be due to cancer heterogeneity.

Example 4 Confirmation of Intra-Tissue Fluorescence Imaging and Permeation Depth of Anti-CV-Glu Probe Using Two-Photon Microscope

 Using a two-photon microscope, fluorescence images were measured over time at high magnifications in the tissues of CV-Glu probes. We also measured the penetration depth of CV-Glu by measuring fluorescence imaging according to tissue depth. The CV-Glu probe confirmed that the ratio of the fluorescence signal at two wavelengths gradually increased with time in the colorectal cancer tissue, and no change in the fluorescence signal ratio was observed in the normal colon tissue (Fig. It was confirmed that when the fluorescence signal of the tissue was measured by spectroscopy and the spectrum of the fluorescence signal was measured, a spectrum corresponding to the change from CV-Glu to CV was observed in cancer tissue (Fig. 5.B). Also, the change of fluorescence signal with time of CV-Glu was observed according to the depth of colon cancer tissue (Fig. 6.C). Reaching up to 40 μm in a short period of time, and a strong fluorescence signal at 10-20 μm.

Example  5> Living body Conforming type AgInS ₂  base Qdot - Antibody Of the probe  Synthesis and Rat In cancer tissue  Check the cancer marking ability.

We have developed a quantum dot-antibody probe, and in the previous research, a fluorescent probe for colonoscopy was prepared using CdSe quantum dots. ² AgInS _{2 The} quantum dot is synthesized as follows. Silver nitrate, indium nitrate hydrate, zinc hydrate nitrate and sodium diethyldithiocarbamate trihydrate were stirred in deionized water for 1 hour. The resulting product was filtered and dried. Then, 50 mg of this solid and 3 ml of oleylamine solution Followed by stirring for 30 minutes. In this way made AgInS to coat the ZnS shells on _two quantum dots into the thioacetamide and zinc acetateoleylamine the oleylamine solution 110 ℃, after stirring for 1 hour in vacuum, synthesized AgInS stirred for 1 hour at _second quantum dot and 150 ℃ temperature while injecting the nitrogen mixing do. This synthesis method is referred to in Non-Patent Document 3. In this patent, ZnS shells were stacked on AgInS ₂ quantum dots composed of biocompatible elements to synthesize biocompatible quantum dots with excellent photosensitivity that fluoresce at 680 nm, which is 6.1 nm in average size. On the surface of ³ quantum dots, The surface was modified using a molecule having a carboxyl group (Fig. 7.A, B). The reason for using zwitterionic molecules is to minimize the non-specific binding of the quantum dot surface to selectively label the cancer target substance by the antibody, and the reason why the molecule having a carboxyl group is used is that the surface of the quantum dot Antibody, Ab) is introduced to form a complex. ^{4 The} probes were synthesized by conjugating the antibody to the quantum dots to identify matrix metalloproteinase 14 (MMP14), which is known to be highly expressed in cancer cells at surface-modified quantum dots. Immunoglobulin G (IgG), an antibody that has the same species specificity as the antibody but does not selectively label cancer, was conjugated to each quantum dot for the control experiment. Quantum dots fluoresce at 680 nm, so they have fluorescence in a different region than CV-Glu probes, so they can distinguish between the two probes. In order to confirm the cancer marking ability of the QT-antibody probe in the colon cancer mouse model, a QT-antibody probe was applied inside the extracted colon tissue, and after 30 minutes, the fluorescence signal was observed (FIG. In the case of the MMP14 quantum dot-antibody complex probe, the tissue portion which is clearly seen as cancer under the white light shows a strong fluorescence signal as compared with the immunoglobulin G (IgG-QD) probe as a control experiment (t, t ' , t "). In addition, it is difficult to judge cancer by visual examination. However, the portion showing strong fluorescence signal (1, 2 in Fig. 7.C) was proved to be adenoma through histopathological examination. Therefore, it is expected that not only cancer tissues which can be judged by general colonoscopy but also early cancer tissues which can not be classified into external cancer tissues can be molecularly imaged. On the other hand, there was a part of the tissue in which the fluorescent signal of the QD probe was insufficient in the part visible to the naked eye (Fig. 7.C m). This shows that it is difficult to accurately diagnose cancer with only one quantum dot probe.

< Example  6> Ratio Weighing Fluorescence Probe and  Living body Conforming type Qdot - antibody complex The probe  Identification of Cancer Marking Ability in Combined Rat Colon Tissue.

 A more accurate diagnosis was possible by continuously processing and imaging the CV-Glu probe and the MMP14 quantum dot probe (Fig. 8.A). When the MMP14 quantum dot probe was processed to obtain a fluorescence signal, a large fluorescence signal was observed in the cancer tissue portion (arrows 1-4) as shown in FIG. 9.B, and a portion (n) Normal tissue. The autofluorescence signal of the tissue and the fluorescence signal of the quantum dot were obtained from a two-photon microscope image of a colorectal cancer tissue of a rat treated with a quantum dot (Fig. 10.C). The CV-Glu probe was treated in the same tissue and the fluorescence signal ratio image was obtained 5 minutes and 30 minutes later. And showed a slightly increased fluorescence ratio at 30 minutes (Fig. 11.D). We have seen that the overlaid images of two probes detect cancer independently of each other, and by comparing and detecting two different targets, it is likely that more accurate diagnosis of the cancer site is possible (Fig. 12.E). When CV-Glu was processed and observed with a two-photon microscope in the same tissue section where the quantum dot signal was confirmed, it was found that the ratio of the fluorescence signal was different from that of the quantum dot, and these two probes were distributed, It is likely that the respective target proteins are differentially expressed in cancer tissues (Fig. 13.F). Also, the two probes showed the largest fluorescence signal at 20 μm, and the penetration depth was more deeply transmitted through the quantum dots (FIG. 14G)

< Example  7> Ratio Weighing Fluorescence Probe and  Living body Conforming type Qdot - antibody complex Pro Identification of Cancer Marking Ability in Patients with Colorectal Cancer Patients.

Quantum-antibody-probe and ratio-measuring fluorescent probes were successively treated in three cancer tissues cut out from different parts of normal colon tissue obtained from patients with colorectal cancer (Fig. 15). In the quantum dot fluorescence image, nonspecific binding was seen in the normal tissue, but the fluorescence signal was large in the cancer tissue, which showed a nearly 2-fold difference in the five repeated experiments (Fig. When the same tissue was treated with a CV-Glu fluorescent probe, it was confirmed that the fluorescence ratio was high in the cancer tissue as compared with the normal tissue (Fig. 15B). When comparing the two probes by overlaying the quantum dots in red and the ratio metering unit in green, it was found that the degree of predominance of the probes holding different targets was different depending on the tissues even in cancer tissues extracted from the same patient 15.C). Therefore, it was confirmed that the accuracy of diagnosis can be improved by using a probe targeting two different biomolecules rather than using only one probe. Each tissue was determined to be normal tissue and cancer tissue by histological examination (Fig. 15.D).

< Example  8> Ratio Weighing Fluorescence Probe and  Living body Conforming type Quantum dot  Cytotoxicity and In small animals  Toxicity test

 Each probe was treated with HCT116 cells (human colorectal cancer cells) at various concentrations and treatment times and cytotoxicity was measured (Fig. 16.A). The cytotoxicity was confirmed by treatment with cancer cells at a high concentration for a long time in order to confirm that the ratio measuring fluorescent probe developed was stable to the cells. It was confirmed that 80% or more of the cells were alive at a concentration of 100 μM until 24 hours, indicating that the cytotoxicity was very low. In the case of biocompatible quantum dots, it was confirmed that more than 80% of the cells were alive at a concentration of 500 nM up to 24 hours. A Balb / c mouse was used to apply a high-concentration CV-Glu probe and a biocompatible quantum dot to the inside of the anus in comparison with the optical density of the probe, and washed 30 minutes later. Seven days later, a specimen was obtained and a toxicity test was conducted (Fig. 16B). No apparent abnormality was found in the rats of the comparative group as a factor relating to various blood factors, liver and kidney (Fig. 16B). It is expected that medical applications will be appropriate.

Claims (22)

An organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in colon cancer, wherein the organic fluorescent dye has a first fluorescence wavelength and has a second fluorescence wavelength by separation of a functional group;
A quantum dot having an antibody that binds with a second enzyme overexpressed in cancer and having a third fluorescent wavelength; And
And an apparatus for fluorescing the organic fluorescent dye and quantum dots to measure the fluorescence. [Claim 2] The colon cancer diagnostic apparatus according to claim 1, wherein the first enzyme is an enzyme of lambda-glutamyltranspeptidase (GGT). 3. The colon cancer diagnostic apparatus according to claim 1 or 2, wherein the functional group is an amino acid. The colon cancer diagnostic apparatus according to claim 1 or 2, wherein the functional group is glutamic acid. The colon cancer diagnostic apparatus according to claim 1 or 2, wherein the organic fluorescent dye is expressed by the following formula (1).

The apparatus for diagnosing colorectal cancer according to claim 1 or 2, wherein the quantum dots are capnium or lead-free quantum dots. 3. The method according to claim 1 or 2, wherein the quantum dot is AgInS ₂ The colorectal cancer diagnosing apparatus comprising: The apparatus for diagnosing colorectal cancer according to claim 1 or 2, wherein the quantum dots are surface-modified with amphoteric ions. The diagnostic device of claim 1 or 2, wherein the antibody is a matrix metalloproteinase 14 (MMP14) antibody. The method of claim 1 or 2, wherein the first fluorescence has a peak wavelength of 550 to 590 nm, the second fluorescence has a peak wavelength of 610 to 650 nm, the third fluorescence has a peak wavelength of 670 to 690 nm Wherein the colorectal cancer diagnosing apparatus comprises:  The apparatus for diagnosing colorectal cancer according to claim 1 or 2, wherein the device for measuring fluorescence is a graph in which the fluorescence of the organic fluorescent dye and the fluorescence of the quantum dot are overlapped. An organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in a colon cancer, wherein the organic fluorescent dye has a first fluorescence wavelength and has a second fluorescence wavelength by separation of a functional group ; Fluorescence and measuring the fluorescence of the organic fluorescent dye; And
Measuring the fluorescence of the quantum dots by treating the test tissue with a quantum dot having an antibody that binds to a second enzyme that is overexpressed in cancer and having a third fluorescent wavelength;
/ RTI &gt; The diagnostic method for colon cancer according to claim 12, wherein the data of fluorescence of the organic fluorescent dye and the data of quantum dot fluorescence are superimposed and expressed. An organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in colon cancer, wherein the organic fluorescent dye has a first fluorescence wavelength and has a second fluorescence wavelength by separation of a functional group; And
A quantum dot having an antibody that binds to a second enzyme that is overexpressed in cancer and has a third fluorescent wavelength. An organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in colon cancer, wherein the organic fluorescent dye has a first fluorescence wavelength and has a second fluorescence wavelength by separation of a functional group; And
And an apparatus for measuring fluorescence of the organic fluorescent dye. An organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in a colon cancer, wherein the organic fluorescent dye has a first fluorescence wavelength and has a second fluorescence wavelength by separation of a functional group ; And the fluorescence is measured to diagnose colon cancer. An organic fluorescent dye having a functional group separated by a first enzyme specifically overexpressed in colorectal cancer, wherein the organic fluorescent dye has a first fluorescent wavelength and has a second fluorescent wavelength by separation of a functional group. 18. The fluorescent dye according to claim 17, wherein the first enzyme is? -Glutamyltranspeptidase (GGT). The fluorescent dye according to claim 17 or 18, wherein the functional group is an amino acid. The fluorescent dye according to claim 17 or 18, wherein the functional group is glutamic acid. The fluorescent dye according to claim 17 or 18, wherein the organic fluorescent dye is represented by the following formula (1).

An organic fluorescent dye represented by the following formula (1).

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