CN107356756A - A kind of fluorescence probe and its synthetic method and the application in circulating tumor cell detection - Google Patents
A kind of fluorescence probe and its synthetic method and the application in circulating tumor cell detection Download PDFInfo
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- CN107356756A CN107356756A CN201710438776.5A CN201710438776A CN107356756A CN 107356756 A CN107356756 A CN 107356756A CN 201710438776 A CN201710438776 A CN 201710438776A CN 107356756 A CN107356756 A CN 107356756A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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Abstract
A kind of application the invention discloses fluorescence probe and its synthetic method and in circulating tumor cell detection.Fluorescence probe of the present invention includes fluorophor, the substrate and folate molecule of cathepsin B, and the substrate of fluorophor and cathepsin B from degraded key connection, then with folate molecule by being connected;Fluorophor excites in the free state light that can be excited, and is quenched after being connected with other molecule covalents.Because fluorescence probe of the present invention is the switching mode fluorescence probe with spontaneous cascade amplification function, for the complicated operating procedure such as antibody or detection of nucleic acids can be avoided in circulating tumor cell detection process, it is used to automate in Biochemical Analyzer as matched reagent, the automatic detection of tumour cell is may advantageously facilitate, there is simple and convenient, the time-consuming advantage such as short, low to instrument requirements.Fluorescence probe of the present invention can be also used for the positioning of microscopic tumor cells form in clinical operation, tubercle, have broad application prospects and huge market value.
Description
Technical field
The invention belongs to technical field of biological chemistry detection, more specifically to one kind using cathepsin B as target spot
Fluorescence probe and its synthetic method and the application in lesion detection.
Background technology
Tumour is global the first fatal disease, and its morbidity and mortality occupy each disease forefront always, and in recent years
To there is the trend of rapid growth.Because tumor invasion compares concealment, typically all shifted when patient there are clinical symptoms, according to
Count the tumor patient more than 90% and die from metastases.
Peripheral blood tumour cell quantity is few, and the interference of a large amount of normal cells be present, and detection is more difficult.It is currently used
Detection technique mainly has conventional cytology dyeing detection, immunocytochemical technique.This kind of method mostly by human peripheral blood,
The tumour cell of abdominal cavity or tumor region is collected, centrifuged, and the hematoxylin eosin staining method of the routine such as use is dissociated
The detection of tumour cell, process is numerous and diverse, and positive rate is very low.The new diagnostic techniques to grow up in recent years has fluidic cell inspection
Survey technology, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction, immunomagnetic beads and based on gross tumor volume and deformability
And the filter membrane filtration method and micro-fluidic detection technology designed.The detection method advantage of PCR-based is that sensitivity is high, can be detected
Several or even single tumour cells.But tumour cell is difficult to find very specific mRNA marks, some are normal thin
Born of the same parents also have the expression of extremely low tumor marker gene, and highly sensitive PCR, which often expresses these backgrounds, to be expanded, and causes vacation
Positive result.And normal dyeing agent SABC operating process is cumbersome, sensitivity is relatively low, and tumour cell is in operation
It is easily lost, the problems such as causing false negative.
Recent years with the development of material and engineering science, there is very big development based on micro-fluidic Cell Measurement Technique,
There is more novelty and easily detection technique and device.Microflow control technique has sensitive, convenient and sample in terms of lesion detection
The few advantage of this amount, but these methods are still based on Standard PCR or immunofluorescence technique, to tumor-marker gene or tumour antigen
There is very big dependence, and detection process needs multiple elution steps, causes the loss of tumour cell, therefore accuracy and reliability
Still need to further verify.
Above-mentioned several detection methods all rely on laboratory manual operation, and human factor has a great influence, therefore automation or half
The diagnostic instrments of automation is more suitable for the use of the units such as hospital.Most general automatic detection system is that the U.S. is strong in the world at present
The detection of the circulating tumor cell based on immuno magnetic cell separation and analysis system (CellSearch System) that raw company releases.
The equipment is also international first and obtains FDA's (FDA) certification and by its validity of a large amount of clinical research confirmations
And the equipment of reliability.Using cancer cell conventional sign thing and haemocyte conventional sign thing to the tumour cell in blood during detection
Sorting positively and negatively is carried out, then specific oncoprotein label is marked and dyed on the cell to obtaining, most
Pass through semi-automated microscope scanning and counting afterwards.The advantage of this method is that detection can realize semi-automation, high sensitivity, but it is detected
Somewhat expensive, single are charged 5000-8000 yuans, and larger financial burden is brought to patient.
In general, the CTC diagnostic methods clinically used both at home and abroad at present must the following steps:1. human peripheral blood is carried out
Separation and enrichment;2. extraction nucleic acid is expanded or tumour antigen is marked;3. electrophoresis, sequencing detection DNA or microscope
Observation counts.This these method all refers to multiple elutions and recycling step, the Loss generally existing of tumour cell, artificial behaviour
Making the increase of process also causes increasing for detection error factor, and many tumour cells do not express or low expression some are normal
Marker molecule is advised, therefore also largely influences CTC recall rate.The most important is that these technologies are difficult to showing
The full-automation for having detection device to be implemented in combination with CTC diagnosis, it is unfavorable for detecting the raising of stability and accuracy.
In view of this, it is necessory to provide a kind of high sensitivity, simple and efficient to handle, lower-cost tumour cell detects
Method.
The content of the invention
It is an object of the invention to:Overcome existing tumour cell detect present in cost height, complex operation, accuracy compared with
The problems such as poor, there is provided a kind of high sensitivity, simple and efficient to handle, lower-cost fluorescence probe and its synthetic method and application.
In order to realize foregoing invention purpose, the present invention provides a kind of fluorescence probe, and it includes fluorophor, cathepsin
The substrate of B substrate and folate molecule, fluorophor and cathepsin B from degraded key connection, then with folate molecule by connecting
Connect;Fluorophor excites in the free state light that can be excited, and is quenched after being connected with other molecule covalents.Because quantum yield is very low,
Excited so the fluorescence probe is difficult the light that is excited under Substrate Protection state, can not send fluorescence under Substrate Protection or only send out
Emitter-base bandgap grading hypofluorescence.
One kind as fluorescence probe of the present invention is improved, and the substrate of the cathepsin B is Phe-Lys.
One kind as fluorescence probe of the present invention is improved, and the degraded key certainly is by least two skeleton units head and the tail connection shape
Into (amino of a skeleton unit is connected with the hydroxyl of next skeleton unit), skeleton unit is by aminobenzene methanol, aminobenzene
Dimethanol connects composition with the methanol of aminobenzene three.
One kind as fluorescence probe of the present invention is improved, and the substrate of a cathepsin B can combine several fluorescent bases
Group, is single times of fluorescence probe when one fluorophor of Binding Capacity of cathepsin B;When combining two fluorophors,
Amplify fluorescence probes for 2 times, by that analogy.The multiple of fluorescence probe is higher, and its detection sensitivity is also higher.
In order to realize foregoing invention purpose, the present invention also provides the synthetic method of above-mentioned fluorescence probe, comprised the following steps:
(1) reacted, obtained with the phenylalanine ester of benzyloxycarbonyl group protection and the lysine of di-tert-butyl dicarbonate (PG1) protection
To lysyl phenylalanine ester;
(2) lysyl phenylalanine ester and isobutyl chlorocarbonate are reacted, obtains acid anhydrides intermediate;
(3) acid anhydrides intermediate reacts with fluorophor-lysyl phenylalanine ester, obtains being connected with the centre of fluorophor
Body;
(4) the Boc protection groups being connected with the intermediate of fluorophor are sloughed using trifluoroacetic acid, then with folic acid in acyl
Condensation reaction occurs under amination reagent system, makes folic acid and be connected with the intermediate of fluorophor to be coupled, then sloughs through hydrazinolysis bad
Protection group PG1 on propylhomoserin, obtains fluorescence probe.
In order to realize foregoing invention purpose, present invention also offers a kind of circulating tumor cell detection method, the detection side
Method is completed based on the fluorescence probe.
One kind as circulating tumor cell detection method of the present invention is improved, in the detection method, the fluorescence probe
Incubation detection is directly mixed with tumor sample, or the fluorescence probe is prepared into liposome or is wrapped in nano material, then
Incubation detection is mixed with tumor sample.
One kind as circulating tumor cell detection method of the present invention is improved, and the tumor sample includes blood sample, group
Knit internal test serum in sample or operation.
One kind as circulating tumor cell detection method of the present invention is improved, when the tumor sample is blood sample or group
When knitting sample, the fluorescence probe described in buffer solution, by the fluorescence probe and blood sample or tissue samples at 37 DEG C
5min~1h is incubated, then fluorescence intensity;
When internal test serum during the tumor sample is operation, the fluorescence probe is coated directly onto to be measured in vivo
Tissue, ocal resection tissue is carried out under the guiding of specific LED irradiation.
In order to realize foregoing invention purpose, present invention also offers a kind of circulating tumor cell detection kit, it includes
The fluorescence probe and reaction buffer.
One kind as circulating tumor cell detection kit of the present invention is improved, wherein, the fluorescence probe can be made in advance
It is standby into liposome or to be wrapped in nano material.
The a kind of of purification process as toxin protein of the present invention improves, and in step (4), the mixing stands the temperature of reaction
Spend for 25 DEG C.
The a kind of of purification process as toxin protein of the present invention improves, and in step (4), the purifying is the affine layer of albumen
Analysis purifying.
Compared with prior art, the present invention has the advantages that:
1) fluorescence probe of the present invention does not fluoresce or only emitter stage hypofluorescence, but in cathepsin B under Substrate Protection
The cleavable thing that breaks off the base, causes fluorophor to discharge under effect, produces the free fluorescence molecule that can be inspired high fluorescence;Make
When detecting tumour cell with fluorescence probe of the present invention, without carrying out the operation such as separating to tumour cell, there is simple and fast, take
The advantages of short, low to instrument requirements, beneficial to automatic detection;
2) fluorescence probe of the present invention is the switching mode fluorescence probe that function is expanded with spontaneous cascade, is examined for tumour cell
Survey, operation complicated in antibody and detection of nucleic acids can be avoided, be alternatively arranged as matched reagent and be used in automation Biochemical Analyzer,
Be advantageous to tumour cell/CTC automatic detection, not only increase sensitivity, also add detection flux and convenience, make up
The deficiency of existing detection method;
3) fluorescence probe of the present invention is cancellation state under non-digestion state, therefore be can also be used for small swollen in clinical operation
The positioning of oncocyte or tubercle, the supplementary means as operation;
4) the tumor staining liquid phase ratio being rinsed with needs such as traditional methyl blue, Lugol's iodine solutions, fluorescence of the present invention
Probe has big advantage in terms of ease of handling, specificity or sensitivity, has a good application prospect and economic valency
Value.
Brief description of the drawings
With reference to the accompanying drawings and detailed description, to fluorescence probe of the present invention, its synthetic method and application and beneficial
Effect is described in detail.
Fig. 1 is put for the fluorescence probe structures of 2 times of amplifications in embodiment 1 by taking Tokyo Green fluorophors as an example and 4 times
Big fluorescence probe structural representation, wherein, (1) is the structure of Tokyo Green fluorophors, and (b) is the fluorescence of 2 times of amplifications
Probe structure, (c) are the fluorescence probe structure of 4 times of amplifications.
Fig. 2 is the fluorescence probe synthetic route in embodiment 1.
Fig. 3 is CTC testing result figures in embodiment 2.
Fig. 4 is the colour developing figure of mouse experiment in vivo in embodiment 3.
Embodiment
In order that goal of the invention, technical scheme and the advantageous effects of the present invention become apparent from, with reference to embodiments,
The present invention will be described in further detail.It should be appreciated that the embodiment described in this specification is just for the sake of explanation
The present invention, being not intended to limit the present invention, the formula of embodiment, ratio etc. can suit measures to local conditions to make a choice and have no reality to result
Matter influences.
The synthesis of 2 times of amplification fluorescence probes exemplified by the Tokyo Green fluorophors of embodiment 1
The fluorescence probe of twice of amplification mainly includes four parts:Phe-Lys protection group, from the key, glimmering of degrading
Light group Tokyo Green and the folate molecule for playing targeting CTC effects.
The phenylalanine ester (1a) of benzyloxycarbonyl group protection and the lysine (1b) of di-tert-butyl dicarbonate (PG1) protection first
Reaction, obtains product 1c lysyl phenylalanine esters.1d dimethyl chlorides
1c reacts to form acid anhydrides intermediate with dimethyl chloride, is then reacted with 1f fluorophors-lysyl phenylalanine ester,
Obtain connecting 2 fluorophor Tokyo Green intermediate 1g.
The Boc protection groups in compound 1g are sloughed using trifluoroacetic acid, then again with folic acid in amidation reagent such as EDCI/
Condensation reaction occurs under HOBt systems and forms 1g and folacin coupled product, the product sloughs the protection group on lysine through hydrazinolysis
PG1, obtain the fluorescence probe (Probe 1) of 2 times of final amplifications.
The CTC of embodiment 2 is detected
8 pipe 5mL normal person's anticoagulations are taken, different number of colon cancer cell is separately added into and mixes, 1500rpm centrifugations 5min
After abandon supernatant, the buffer solution that 1mL contains fluorescence probe made from 0.1% embodiment 1 is added, with glimmering after 37 degree incubation 10-20min
Light spectrophotometer detects, as a result as shown in Figure 3.
The mouse peritoneal tumor imaging of embodiment 3
Take 0.5mL to contain the buffer solution of fluorescence probe made from 0.1% embodiment 1, be injected into mouse peritoneal, after 20min
That anesthetized mice of barbital, after fixing limbs, abdominal cavity is cut off with eye scissors, with observing tumour (figure under the LED of 488 exciting lights
4)。
The announcement and teaching of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out appropriate change and modification.Therefore, the invention is not limited in embodiment disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In used some specific terms, but these terms are merely for convenience of description, do not form any restrictions to the present invention.
Claims (10)
1. a kind of fluorescence probe, it is characterised in that substrate and folate molecule including fluorophor, cathepsin B, fluorescent base
The substrate of group and cathepsin B is connected by degraded key connection certainly, then with folate molecule;Fluorophor is in free state energy quilt
Excitation, it is quenched after being connected with other molecule covalents.
2. fluorescence probe according to claim 1, it is characterised in that the substrate of the cathepsin B be phenylalanine-
Lysine;Described to be formed from degraded key by the head and the tail connection of at least two skeleton units, skeleton unit is by aminobenzene methanol, aminobenzene
Dimethanol connects composition with the methanol of aminobenzene three.
3. fluorescence probe according to claim 1, it is characterised in that the fluorophor is several.
4. the synthetic method of fluorescence probe described in any one claim in claims 1 to 3, it is characterised in that including such as
Lower step:
(1) reacted with the phenylalanine ester of benzyloxycarbonyl group protection and the lysine of di-tert-butyl dicarbonate protection, obtain lysyl
Phenylalanine ester;
(2) lysyl phenylalanine ester and isobutyl chlorocarbonate are reacted, obtains acid anhydrides intermediate;
(3) acid anhydrides intermediate reacts with fluorophor-lysyl phenylalanine ester, obtains being connected with the intermediate of fluorophor;
(4) the Boc protection groups being connected with the intermediate of fluorophor are sloughed using trifluoroacetic acid, then with folic acid in amidatioon
Condensation reaction occurs under reagent system, makes folic acid and is connected with the intermediate coupling of fluorophor, then lysine is sloughed through hydrazinolysis
On protection group PG1, obtain fluorescence probe.
5. a kind of circulating tumor cell detection method, it is characterised in that the detection method is based on any in claims 1 to 3
Fluorescence probe described in one claim is completed.
6. circulating tumor cell detection method according to claim 5, it is characterised in that described in the detection method
Fluorescence probe directly mixes incubation detection with tumor sample, or the fluorescence probe is prepared into liposome or is wrapped in a nanometer material
In material, then incubation detection is mixed with tumor sample.
7. circulating tumor cell detection method according to claim 5, it is characterised in that the tumor sample includes blood
Internal test serum in sample, tissue samples or operation.
8. circulating tumor cell detection method according to claim 7, is further characterized in that, when the tumor sample is blood
When liquid sample or tissue samples, with the fluorescence probe described in any one claim in buffer solution Claims 1 to 4,
The fluorescence probe and blood sample or tissue samples are incubated 5min~1h at 37 DEG C, then fluorescence intensity;
When internal test serum during the tumor sample is operation, by described in any one claim in Claims 1 to 4
Fluorescence probe be coated directly onto in internal test serum, specific LED irradiation guiding under carry out ocal resection group
Knit.
9. a kind of circulating tumor cell detection kit, it is characterised in that will including any one right in Claims 1 to 4
Ask described fluorescence probe and reaction buffer.
10. circulating tumor cell detection kit according to claim 9, it is characterised in that by the fluorescence probe system
It is standby into liposome or to be wrapped in nano material.
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN108546682A (en) * | 2018-05-14 | 2018-09-18 | 东南大学 | A kind of mixing photon crystal composite material and application based on modified with folic acid |
CN108956565A (en) * | 2018-06-28 | 2018-12-07 | 中山大学 | A kind of fluorescence probe and the application in detection SIRT2 enzymatic activity |
CN111172235A (en) * | 2020-01-15 | 2020-05-19 | 山东师范大学 | Biosensor for detecting cathepsin B and detection method and application thereof |
CN113552367A (en) * | 2021-07-19 | 2021-10-26 | 上海思路迪生物医学科技有限公司 | Marker combination and kit for detecting tumor cells in body fluid sample |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108546682A (en) * | 2018-05-14 | 2018-09-18 | 东南大学 | A kind of mixing photon crystal composite material and application based on modified with folic acid |
CN108546682B (en) * | 2018-05-14 | 2019-10-11 | 东南大学 | A kind of mixing photon crystal composite material and application based on modified with folic acid |
CN108956565A (en) * | 2018-06-28 | 2018-12-07 | 中山大学 | A kind of fluorescence probe and the application in detection SIRT2 enzymatic activity |
CN111172235A (en) * | 2020-01-15 | 2020-05-19 | 山东师范大学 | Biosensor for detecting cathepsin B and detection method and application thereof |
CN111172235B (en) * | 2020-01-15 | 2023-03-21 | 山东师范大学 | Biosensor for detecting cathepsin B and detection method and application thereof |
CN113552367A (en) * | 2021-07-19 | 2021-10-26 | 上海思路迪生物医学科技有限公司 | Marker combination and kit for detecting tumor cells in body fluid sample |
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Application publication date: 20171117 |