CN104894243A - Nucleic acid aptamer probe and device for cancer cell detection and application of nucleic acid aptamer probe and device for cancer cell detection - Google Patents
Nucleic acid aptamer probe and device for cancer cell detection and application of nucleic acid aptamer probe and device for cancer cell detection Download PDFInfo
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- CN104894243A CN104894243A CN201510238290.8A CN201510238290A CN104894243A CN 104894243 A CN104894243 A CN 104894243A CN 201510238290 A CN201510238290 A CN 201510238290A CN 104894243 A CN104894243 A CN 104894243A
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Abstract
The invention discloses a nucleic acid aptamer probe and device for cancer cell detection and application of the nucleic acid aptamer probe and device for cancer cell detection. The nucleic acid aptamer probe comprises a fixing probe body and an identifying probe body capable of being combined with a cancer cell; the fixing probe body and the identifying probe body are hybridized and complemented; and the device comprises the nucleic acid aptamer probe and a carrier, and the fixing probe body of the nucleic acid aptamer probe is in cross linkage to the surface of the carrier in a covalent or non-covalent manner. The nucleic acid aptamer probe is low in instrument requirement, high in universality, low in cost, simple in operation and easy to popularize, and can be applied to cancer cell detection, tracing and killing.
Description
Technical field
The present invention relates to tumor diagnosis and treatment technical field, particularly relate to a kind of nucleic acid aptamer probe for tumour cell detection, device and application thereof.
Background technology
Tumour be body under the effect of various tumorigenesis factor, the cell of local organization makes it to the normal regulation and control of its growth on gene level, causes clonal abnormality hyperplasia.Clinical tumor can be divided into solid tumor and non-physical knurl, solid tumor and tangible knurl by clinical examination as CT scan, the tangible lump that the Imaging Methods such as B ultrasonic or palpation are laid one's hand on and arrived.Non-physical knurl such as the leukemia in hemopathy just directly cannot be diagnosed by palpation or conventional image method.Tumour not only grows out of control, and arround cell also can locally invade, healthy tissues even transfers to other parts of health via body-internal-circulation system or lymphsystem, serious threat human health.The cancer related mortality of more than 90% is all relevant to the transfer of tumour.The detection of tumour cell, especially splits away off from parent tumor tissue the circulating tumor cell entered in human peripheral blood and tissue, has extremely important meaning for the early diagnosis of cancer, the assessment of result for the treatment of.Circulating tumor cell is the one of the main reasons of cancer metastasis and recurrence.In the past few decades, be developed a variety of method in order to detect tumour cell, as: fluorescent immune method, magnetic immuno method etc.Wherein the CellSearch circulating tumor cell proofing unit of Veridex company is the circulating tumor cell proofing unit that food and medicine Surveillance Authority of China (SFDA) uniquely ratifies to use.But, the expensive equipment needed for these technology, and the spike of tumour cell cannot be realized and kill and wound.
Aptamer (aptamer) is obtained by phyletic evolution technology (SELEX) screening of index concentration part, the single strain oligonucleotide (ssDNA or ssRNA) of energy specific combination target material.Aptamer has the feature of similar antibody to target high specific and high-affinity, and be better than antibody in many aspects, and as: molecular weight is little, non-immunogenicity, target abundant species (comprise ion, amino acid, nucleic acid, polypeptide, protein, complete tumour cell even organize), synthetic method are simple and reproducible, modify flexibly and be convenient to long storage periods and normal temperature transport etc.These characteristics make aptamer obtain as diagnosing tumor probe to pay close attention to widely and approve, therefore become a kind of instrument that can be used for for tumor classification, Diagnosis and Treat just gradually based on aptamer.
At tumour cell conventional at present, especially, there is complicated operation in the detection method of circulating tumor cell and non-physical oncocyte, length consuming time, high in cost of production problem.Nucleic acid aptamer probe can provide a kind of low cost, easy to operate tumour cell diagnostic probe.And due to the characteristic that aptamer is easily modified, not only can diagnose tumour cell, also can realize the tracking of tumour cell and kill and wound.For the tumor therapeuticing method of present stage, mostly need medicine to reach in blood effect that higher concentration just can play treatment.If be fixed on by medicine in a device, make drug release by the stimulation of tumour cell, be expected to the transfer for Tumor suppression, and reduce drug use total amount, the side effect that alleviation heavy dose uses antitumor drug and causes.
Summary of the invention
The technical problem to be solved in the present invention overcomes the deficiencies in the prior art, provide that a kind of instrument requirements is low, universality is strong, cost is low, simple to operate be easy to promote nucleic acid aptamer probe and device, can be applicable to the detection of tumour cell, spike and kill and wound.
For solving the problems of the technologies described above, the technological method that the present invention adopts is:
For the nucleic acid aptamer probe that tumour cell detects, the identification probe comprising stationary probe He can be combined with tumour cell; Described stationary probe and described identification probe portion hybridize complementary.
Above-mentioned nucleic acid aptamer probe, preferably, the nucleotides sequence of described stationary probe is classified as has SEQ ID NO.1, nucleotide sequence described in SEQ ID NO.3; The nucleotides sequence of described identification probe is classified as has SEQ ID NO.2, nucleotide sequence described in SEQ ID NO.4.
Above-mentioned nucleic acid aptamer probe, preferably, described identification probe modification has fluorophor, medicine, photosensitizers or medical science contrast medium, and described stationary probe is modified with vitamin H.
Above-mentioned nucleic acid aptamer probe, preferably, described fluorophor is one or more in fluorescein, rhodamine, Alexa488, Atto550, Atto647, Cy3, Cy5 and Cy5.5; Medicine is one or more in pyropheophorbide-a, uridylic, Zorubicin, camptothecine, taxol, Docetaxel, epirubicin, Rituximab and Herceptin; Described photosensitizers is one or more in pyropheophorbide-a, porphyrin, methylene blue, chlorin e 6, Visudyne, 5-ALA, temoporfin, talaporfin, phthalocyanine and phycobiliprotein; Described medical science contrast medium be pyropheophorbide-a, porphyrin, iofendylate, iohexol, gadolinium-porphyrin complex and
64one or more in Cu-porphyrin complex.
Preferably, fluorescein is FAM and/or FITC.
As a total technical conceive, preferably, present invention also offers a kind of device detected for tumour cell, comprise above-mentioned nucleic acid aptamer probe and carrier, the stationary probe of described nucleic acid aptamer probe is by being covalently or non-covalently crosslinking in described carrier surface.
Above-mentioned device, preferably, described carrier is magnetic particle, micro-fluidic chip, intravascular stent or artificial blood vessel.
Above-mentioned device, preferably, described magnetic particle is the magnetic particle of pan coating Streptavidin.
As a total technical conceive, preferably, present invention also offers above-mentioned nucleic acid aptamer probe or the application of device in tumour cell detects.
As a total technical conceive, preferably, present invention also offers above-mentioned nucleic acid aptamer probe or the application of device in tumour cell spike.
As a total technical conceive, preferably, present invention also offers above-mentioned nucleic acid aptamer probe or the application of device in preparation tumor, preparation killing tumor cells device.
Compared with prior art, the invention has the advantages that:
(1) the invention provides a kind of aptamer of detecting for tumour cell and device thereof, have that instrument requirements is low, universality is strong, cost is low, the advantage being easy to promote simple to operate.
(2) the invention provides a kind of aptamer for tumour cell detection and device thereof, under the hormesis not having tumour cell, stationary probe and identification probe hybridization, identify that probe is in the state of cut out; When tumour cell stimulates, identify that probe is activated, release is incorporated into the surface of tumour cell.Avoiding in the existing detection method fit based on single nucleic acid, for overcoming the loaded down with trivial details washing process that non-specific adsorption signal carries out, shortening detection time.Simultaneously can expansive approach in tumour cell tracking with kill and wound.
(3) the invention provides a kind of aptamer for tumour cell detection and device thereof, fixed nucleic acid aptamers probe unit can adopt the device such as nanometer and micron particle, micro-fluidic chip, artificial blood vessel and intravascular stent according to demand, thus has widened the application of device in tumor diagnosis and treatment that tumour cell stimulates nucleic acid probe release further.
(4) the invention provides a kind of aptamer for tumour cell detection and device thereof, provide a kind of spike of brand-new non-physical oncocyte and kill and wound strategy.By by identifying that contrast medium, medicine that probe carries are fixed on the method for device, avoid contrast medium, medicine direct injection to enter the recycle system.This method is activated by tumour cell and identifies probe, when lower contrast medium, medicine total amount, can realize the tracking of non-physical oncocyte and kill and wound, thus reduces the side effect that heavy dose takes in contrast medium, medicine causes.
Accompanying drawing explanation
For making the object of the embodiment of the present invention, technical scheme and advantage clearly, below in conjunction with the accompanying drawing in the embodiment of the present invention, clear, complete description is carried out to the technical scheme in the embodiment of the present invention.
Fig. 1 is nucleic acid aptamer probe and the apparatus structure schematic diagram of the embodiment of the present invention 1.
Fig. 2 is the nucleic acid aptamer probe of the embodiment of the present invention 1 when being fixed on magnetic particle, for the schematic diagram that tumour cell detects.
Fig. 3 is that the nucleic acid aptamer probe of the embodiment of the present invention 1 and device are at the experimental result picture detecting people's acute lymphoblastic leukemia T lymphocyte (CCRF-CEM).
Fig. 4 is that the nucleic acid aptamer probe of the embodiment of the present invention 1 and device are at detection human B lymphocyte oncocyte (Ramos) control experiment result figure.
Fig. 5 is that the nucleic acid aptamer probe of the embodiment of the present invention 2 and device are at the experimental result picture detecting human liver cancer cell (SMMC-7721).
Fig. 6 is that the nucleic acid aptamer probe of the embodiment of the present invention 2 and device are at the control experiment result figure detecting human liver cancer cell (BEL-7404).
Fig. 7 is the nucleic acid aptamer probe of the embodiment of the present invention 3 and the device schematic diagram for tumour cell spike.
Fig. 8 is the nucleic acid aptamer probe of the embodiment of the present invention 3 and the device experimental result picture in the spike for human liver cancer cell (SMMC-7721).
Fig. 9 is the nucleic acid aptamer probe of the embodiment of the present invention 4 and the device schematic diagram for tumor cytotoxicity.
Figure 10 is that the nucleic acid aptamer probe of the embodiment of the present invention 4 and device are at the experimental result picture killed and wounded for human liver cancer cell (SMMC-7721).
Embodiment
Below in conjunction with Figure of description and concrete preferred embodiment, the invention will be further described, but protection domain not thereby limiting the invention.
Embodiment
The material adopted in following examples and instrument are commercially available.Its chips SU-8 anode membrane is the snakelike micro-fluidic chip anode membrane of Dalian Chemistry and Physics Institute processing; Sygard 184 solidifying agent is purchased from Dow corning company.
embodiment 1
See Fig. 1: a kind of device detected for tumour cell of the present invention, comprise aptamer probe and carrier, wherein nucleic acid aptamer probe comprises stationary probe, identifies probe, stationary probe 3 ' is terminal modified has vitamin H, 5 ' terminal modifiedly to have quenching group BHQ1, and identifying that probe 3 ' is terminal modified has fluorophor FAM; Carrier is that bag is by the superparamagnetic magnetic particle of Streptavidin.
The nucleotides sequence of stationary probe is classified as the nucleotide sequence had described in SEQ ID NO.1; Be specially:
Stationary probe-CEM:5 '-BHQ1-TCT AAC CGT ATT TTT TTT TT-biotin-3 '.
Identify that the nucleotides sequence of probe is classified as the nucleotide sequence had described in SEQ ID NO.2; Be specially:
Identify probe-CEM:5 '-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-FAM-3 '.
The TCT AAC CGT A fragment of stationary probe-CEM is connected with the TA CGG TTA GA fragment hybridize complementary identified in probe-CEM.Streptavidin specific effect on the vitamin H that wherein in nucleic acid aptamer probe, stationary probe is end modified and superparamagnetic magnetic particle, makes stationary probe be fixed on superparamagnetic magnetic particle surface.
With reference to Fig. 2: the device being used for tumour cell detection is detected tumour cell, when there is not target tumor in determinand, identify probe process closing condition; When there is target tumor in determinand, target tumor is combined with identification probe, forms hairpin structure, and departs from stationary probe.Then it is separated magnetic particle to utilize Magneto separate, utilizes and identifies that the fluorophor of probe modification detects tumour cell.
A kind of application of device in people's acute lymphoblastic leukemia T lymphocyte (CCRF-CEM) detects detected for tumour cell of embodiment 1, adopt people's acute lymphoblastic leukemia T lymphocyte (CCRF-CEM) as target cell, detect with the device that the embodiment of the present invention 1 provides.The present embodiment is the interference avoiding false positive signal, determining the terminal modified quenching group BHQ1 of probe 5 '.Design simultaneously can be hybridized with stationary probe, but can not identify the sequence probe in contrast of target cell, for differentiating positive signal.
Contrast probe nucleotides sequence be classified as the nucleotide sequence had described in SEQ ID NO.5; Be specially:
Contrast probe-CEM:5 '-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTA CGG TTA GA-FAM-3 '.
Its application method specifically comprises the following steps:
(1) material prepares:
Hybridization buffer: with Dulbecco ' s PBS for solvent, comprise the MgCl of 5 mM
2.
Binding buffer solution: with Dulbecco ' s PBS for solvent, comprises 5 mM MgCl
2, 4.5g/L glucose, 0.1mg/mL yeast tRNA, 1mg/mL bovine serum albumin.
Bag by the superparamagnetic magnetic particle of Streptavidin: by concentration be 10 mg/mL Streptavidin bag by magnetic particle obtain bag by the superparamagnetic magnetic particle of Streptavidin, (wrapping by the superparamagnetic magnetic particle of Streptavidin in the application is commercial goods, be purchased from Invitrogen, specification is Dynabeads MyOne Streptavidin C1.).
(2) nucleic acid aptamer probe is prepared: be 1.5 mM stationary probe-CEM by final concentration with final concentration be that 1.0 mM identify probe-CEM, hybridize in hybridization buffer, after 95 DEG C of sex change 5min, it is for subsequent use to be slowly down to ambient temperature overnight, as the positive nucleic acid aptamer probe needed for the present embodiment.
(3) preparation contrast probe: be 1.5 mM stationary probe-CEM by final concentration be that 1.0 mM contrast probe-CEM with final concentration, hybridize in hybridization buffer, after 95 DEG C of sex change 5min, it is for subsequent use to be slowly down to ambient temperature overnight, as the negative control probe needed for the present embodiment.
(4) get 30 mL bags by the superparamagnetic magnetic particle of Streptavidin, wash three times with hybridization buffer, be then divided into six parts.The three parts of bags got wherein are at room temperature carried out with the positive nucleic acid aptamer probe of 50 mL hatching (hatching buffered soln is hybridization buffer) by the superparamagnetic magnetic particle of Streptavidin respectively.After hatching 1 hour, suck supernatant by Magneto separate, remaining magnetic particle cleans three times with hybridization buffer again, obtains the device detected for tumour cell.Three parts of negative control probe treatment processs are with positive nucleic acid aptamer probe.
(5) by CCRF-CEM cell dispersal in the binding buffer liquid of 200 mL, make CCRF-CEM cell concn be 10
6individual/mL, then joins in the solution of the device that three parts are detected for tumour cell, respectively at 4 DEG C, 20 DEG C, hatches 30 minutes under 37 DEG C of conditions.After having hatched, Magneto separate 5 minutes, draws cell conditioned medium liquid, and precipitation adds 200 mL binding buffer solution washing magnetic beads once, Magneto separate 5 minutes again again, and Aspirate supernatant also merges, for the detection of flow cytometer with the cell conditioned medium liquid of last time.
Detected result as shown in Figure 3, at 4 DEG C, 20 DEG C, under 37 DEG C of conditions, identify probe can under the stimulation of tumour cell with Cell binding, and with contrast probe in conjunction with situation difference to some extent.Therefore, the device detected for tumour cell in the present embodiment can at different temperatures, detect target cell CCRF-CEM.
Embodiment 1 is only the preferred embodiments of the present invention, in the present invention, identify that the terminal modified fluorophor of probe 3 ' can also replace with in fluorescein, rhodamine, Alexa488, Atto550, Atto647, Cy3, Cy5 or Cy5.5 one or more, and reach same or analogous technique effect.
comparative example 1
A kind of application of device in human B lymphocyte oncocyte (Ramos) detects detected for tumour cell of embodiment 1, adopt human B lymphocyte oncocyte (Ramos) as target cell, detect with the nucleic acid aptamer probe that the embodiment of the present invention 1 provides.Detection method is identical with embodiment 1.
Detected result as shown in Figure 4, at 4 DEG C, 20 DEG C, under 37 DEG C of conditions, identifies probe and control tumor Cell binding and indifference, illustrate two kinds of probes all not with control tumor Cell binding.
embodiment 2
A kind of device detected for tumour cell of the present invention, comprises nucleic acid aptamer probe and carrier, and wherein nucleic acid aptamer probe comprises stationary probe and identifies probe, and stationary probe 3 ' is terminal modified vitamin H, and identifying that probe 5 ' is terminal modified has fluorophor FAM; Carrier is that bag is by the superparamagnetic magnetic particle of Streptavidin.
The nucleotides sequence of stationary probe is classified as the nucleotide sequence had described in SEQ ID NO.3; Be specially:
Stationary probe-7721:5 '-CGC GCG CTT TTT TTT TTT-biotin-3 '.
Identify that the nucleotides sequence of probe is classified as the nucleotide sequence had described in SEQ ID NO.4; Be specially:
Identify probe-7721:5 '-FAM-AAA GCG CGC GCG CGC ATA GCG CGC TGA GCT GAA GAT CGT ACC GTG AGC GCG CT-3 '.
The CGC GCG CTT T fragment of stationary probe is connected with the AAA GCG CGC G fragment hybridize complementary identified in probe, Streptavidin specific effect on the vitamin H that stationary probe is end modified and carrier, makes stationary probe be fixed on superparamagnetic magnetic particle surface.
When there is not target tumor in determinand, identify probe process closing condition; When there is target tumor in determinand, target tumor is combined with identification probe, forms hairpin structure, and departs from stationary probe.Then it is separated magnetic particle to utilize Magneto separate, utilizes and identifies that the fluorophor FAM of probe modification detects tumour cell.
Design simultaneously can be hybridized with stationary probe, but can not identify the sequence probe in contrast of target cell, for differentiating positive signal.
Contrast probe nucleotides sequence be classified as the nucleotide sequence had described in SEQ ID NO.6; Be specially:
Contrast probe-7721:5 '-FAM-AAA GCG CGC GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TT-3 '.
Embodiment 2 for tumour cell detect device at human liver cancer cell (SMMC-7721)) detect in an application, concrete application method is:
(1) material prepares:
Hybridization buffer: with Dulbecco ' s PBS for solvent, comprise the MgCl of 5 mM
2.
Binding buffer solution: with Dulbecco ' s PBS for solvent, comprises 5 mM MgCl
2, 4.5g/L glucose, 0.1mg/mL yeast tRNA, 1mg/mL bovine serum albumin.
Bag by the superparamagnetic magnetic particle of Streptavidin: by concentration be 10 mg/mL Streptavidin bag by magnetic particle obtain bag by the superparamagnetic magnetic particle of Streptavidin, (wrapping by the superparamagnetic magnetic particle of Streptavidin in the application is commercial goods, be purchased from Invitrogen, specification is Dynabeads MyOne Streptavidin C1.).
(2) nucleic acid aptamer probe is prepared: be 1.5 mM stationary probes-7721 by final concentration be that 1.0 mM identify probe-7721 with final concentration, hybridize in hybridization buffer, after 95 DEG C of sex change 5min, it is for subsequent use to be slowly down to ambient temperature overnight, as the positive nucleic acid aptamer probe needed for the present embodiment.
(3) preparation contrast probe: be 1.5 mM stationary probes-7721 by final concentration be that 1.0 mM contrast probe-7721 with final concentration, hybridize in hybridization buffer, after 95 DEG C of sex change 5min, it is for subsequent use to be slowly down to ambient temperature overnight, as the negative control probe needed for the present embodiment.
(4) get 30 mL bags by the superparamagnetic magnetic particle of Streptavidin, wash three times with hybridization buffer, be then divided into six parts.The three parts of bags got wherein are at room temperature carried out with the positive nucleic acid aptamer probe of 50 mL hatching (hatching buffered soln is hybridization buffer) by the superparamagnetic magnetic particle of Streptavidin respectively.After hatching 1 hour, suck supernatant by Magneto separate, remaining magnetic particle cleans three times with hybridization buffer again, obtains the device detected for tumour cell.Three parts of negative control probe treatment processs are with positive nucleic acid aptamer probe.
(5) will go down to posterity more than 24 hours, and be in the SMMC-7721 cell of logarithmic phase, with cell dissociation buffer digestion, use binding buffer solution washing, being separated into concentration is 10
6the SMMC-7721 cell dispersal liquid of individual/mL.Get 200 mL SMMC-7721 cell dispersal liquid to join respectively in every part of device for tumour cell detection, respectively at 4 DEG C, 20 DEG C, under 37 DEG C of conditions, hatch 30 minutes.After having hatched, Magneto separate 5 minutes, draws cell conditioned medium liquid, and precipitation adds 200 mL binding buffer solution washing magnetic beads once, Magneto separate 5 minutes again again, and Aspirate supernatant also merges, for the detection of flow cytometer with the cell conditioned medium liquid of last time.
Detected result as shown in Figure 5, at 4 DEG C, 20 DEG C, under 37 DEG C of conditions, identify probe can under the stimulation of tumour cell with Cell binding, and with contrast probe in conjunction with situation difference to some extent.Therefore, the device detected for tumour cell in the present embodiment can at different temperatures, detect target cell SMMC-7721.
Embodiment 2 is only the preferred embodiments of the present invention, in the present invention, identify that the terminal modified fluorophor of probe 5 ' can also replace with in fluorescein, rhodamine, Alexa488, Atto550, Atto647, Cy3, Cy5 or Cy5.5 one or more, and reach same or analogous technique effect.
comparative example 2
The application of device in human liver cancer cell (BEL-7404) detects detected for tumour cell of embodiment 2, adopts human liver cancer cell (BEL-7404) as target cell, detects with the device that the embodiment of the present invention 2 provides.Detection method is identical with embodiment 2.
Detected result as shown in Figure 6, at 4 DEG C, 20 DEG C, under 37 DEG C of conditions, identifies probe and control tumor Cell binding and indifference, illustrate two kinds of probes all not with control tumor Cell binding.
embodiment 3
See Fig. 7: a kind of device detected for tumour cell of the present invention, comprise nucleic acid aptamer probe and carrier, wherein nucleic acid aptamer probe comprises stationary probe and identifies probe, and stationary probe 3 ' is terminal modified vitamin H, identifies that probe carries medical science contrast medium; Carrier is micro-fluidic chip (carrier can also be other microchannels such as artificial blood vessel, intravascular stent).The medical science contrast medium that the present embodiment adopts is pyropheophorbide-a, and micro-fluidic chip is the micro-fluidic chip of PDMS polymkeric substance and bond glass.
The nucleotides sequence of stationary probe is classified as the nucleotide sequence had described in SEQ ID NO.3; Be specially:
Stationary probe-7721:5 '-CGC GCG CTT TTT TTT TTT-biotin-3 '.
Identify that the nucleotides sequence of probe is classified as the nucleotide sequence had described in SEQ ID NO.4; Be specially:
Identify probe-7721:5 '-FAM-AAA GCG CGC GCG CGC ATA GCG CGC TGA GCT GAA GAT CGT ACC GTG AGC GCG CT-3 '.
The CGC GCG CTT T fragment of stationary probe is connected with the AAA GCG CGC G fragment hybridize complementary identified in probe, and the avidin effect on the vitamin H that stationary probe is end modified and carrier, makes stationary probe be fixed on micro-fluidic chip surface.
During target tumor flow channel, target tumor is combined with identification probe, and the identification probe probe that no longer continues to be fixed is fixed; During target cell flow pass, by identifying that probe carries medical science contrast medium, for the tracking of tumour cell.This device can be used as tumor cell in vitro proofing unit and interior tumor cell follow-up mechanism.
Design simultaneously can be hybridized with stationary probe, but can not identify the sequence probe in contrast of target cell, for differentiating positive signal.
Contrast probe nucleotides sequence be classified as the nucleotide sequence had described in SEQ ID NO.6; Be specially:
Contrast probe-7721:5 '-FAM-AAA GCG CGC GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TT-3 '.
The application of nucleic acid aptamer probe in human liver cancer cell (SMMC-7721) spike for embodiment 3, concrete application method is:
(1) material prepares:
Hybridization buffer: with Dulbecco ' s PBS for solvent, comprise the MgCl of 5 mM
2.
Binding buffer solution: with Dulbecco ' s PBS for solvent, comprises 5 mM MgCl
2, 4.5g/L glucose, 0.1mg/mL yeast tRNA, 1mg/mL bovine serum albumin.
(2) nucleic acid aptamer probe is prepared: be 60 mM stationary probes-7721 by final concentration be that 40 mM identify probe-7721 with final concentration, hybridize in hybridization buffer, after 95 DEG C of sex change 5min, it is for subsequent use to be slowly down to ambient temperature overnight, as the positive nucleic acid aptamer probe needed for the present embodiment.
(3) preparation contrast probe: be 60 mM stationary probes-7721 by final concentration be that 40 mM contrast probe-7721 with final concentration, hybridize in hybridization buffer, after 95 DEG C of sex change 5min, it is for subsequent use to be slowly down to ambient temperature overnight, as the negative control probe needed for the present embodiment.
(4) micro-fluidic chip is prepared: mixed in 10: 1 ratios with sygard 184 solidifying agent (Dow corning company) by aggressiveness before PDMS, after stirring and evenly mixing, be placed in vacuum chamber and remove bubble, then at SU-8 force plate upper, gauge control 6 mm.Then be placed in 80 DEG C of curing ovens after about 2 hours, carefully PDMS substrate peeled off from template, make at two ends, microchannel injection port and outlet position perforating by punching device the aperture that diameter is 2 mm.Dry by the glass substrate deionized water rinsing of 50mm × 30mm and in nitrogen.Finally, PDMS chip and clean glass substrate directly carry out reversible laminating.
(5) micro-fluidic chip of positive aptamer probe modification is prepared: the avidin solution taking the concentration of hybridization buffer preparation as 1 mg/mL, pass into chip and be full of chip, at 37 DEG C, hatch 1 hour, then use hybridization buffer irrigation channel three times.Continue to pass into positive nucleic acid aptamer probe, hatch 1 hour in 7 DEG C, then obtain the micro-fluidic chip of positive aptamer probe modification with hybridization buffer irrigation channel for three times.The micro-fluidic chip operation that negative control probe is modified is identical with the micro-fluidic chip of positive aptamer probe modification.
(6) will go down to posterity more than 24 hours, and be in the SMMC-7721 cell of logarithmic phase, with cell dissociation buffer digestion, use binding buffer solution washing, being separated into concentration is 10
6the SMMC-7721 cell dispersal liquid of individual/mL.The SMMC-7721 cell dispersal liquid of 200 mL is passed in the passage of the micro-fluidic chip of positive aptamer probe modification, collects the cell flowed out from passage.Again with binding buffer solution irrigation channel twice, merge the outflow cell of effluent liquid and collection.Adopt flow cytometer, cell is analyzed.
Detected result as shown in Figure 8, under 37 DEG C of conditions, positive nucleic acid aptamer probe can under the stimulation of tumour cell with SMMC-7721 Cell binding, and with contrast probe in conjunction with situation difference to some extent.Therefore, this device can be used for the spike of target cell SMMC-7721.
Embodiment 3 is only the preferred embodiments of the present invention, in the present invention, identify the medical science contrast medium that carries of probe can also replace with porphyrin, iofendylate, iohexol, gadolinium-porphyrin complex,
64cu-porphyrin complex, and reach same or analogous technique effect.
embodiment 4
See Fig. 9: a kind of device detected for tumour cell of the present invention, comprises nucleic acid aptamer probe and carrier, and wherein nucleic acid aptamer probe comprises stationary probe and identifies probe, and stationary probe 3 ' is terminal modified vitamin H, identifies that probe carries photosensitizers.The photosensitizers that the present embodiment adopts is pyropheophorbide-a (Pa, CAS:15664-29-6); Carrier is micro-fluidic chip (can also be other microchannels such as artificial blood vessel, intravascular stent).
The nucleotides sequence of stationary probe is classified as the nucleotide sequence had described in SEQ ID NO.3; Be specially:
Stationary probe-7721:5 '-CGC GCG CTT TTT TTT TTT-biotin-3 '.
The nucleotides sequence killing and wounding probe is classified as the nucleotide sequence had described in SEQ ID NO.4; Be specially:
Kill and wound probe-7721:5 '-Pa-AAA GCG CGC GCG CGC ATA GCG CGC TGA GCT GAA GAT CGT ACC GTG AGC GCG CT-3 '.
The CGC GCG CTT T fragment of stationary probe is connected with the AAA GCG CGC G fragment hybridize complementary killed and wounded in probe, and the avidin effect on the vitamin H that stationary probe is end modified and carrier, makes stationary probe be fixed on micro-fluidic chip surface.
During target cell flow channel, target tumor is combined with identification probe, and the identification probe probe that no longer continues to be fixed is fixed; During target cell flow pass, identify that probe and entrained photosensitizers combine the surface with tumour cell.Under the effect of illumination, photosensitizers produces singlet oxygen, destroys cellularstructure and impels apoptosis.This device can develop into embedded type device, after target cell is identified probe identification, can impel under light illumination when flowing through peripheral vascular and identify that the target cell death that probe is combined designs simultaneously and can hybridize with stationary probe, but the sequence probe in contrast of target cell can not be identified, for differentiating positive signal.
The application of nucleic acid aptamer probe in human liver cancer cell (SMMC-7721) kills and wounds for embodiment 4, concrete application method is:
(1) material prepares:
Hybridization buffer: with Dulbecco ' s PBS for solvent, comprise the MgCl of 5 mM
2.
Binding buffer solution: with Dulbecco ' s PBS for solvent, comprises 5 mM MgCl
2, 4.5g/L glucose, 0.1mg/mL yeast tRNA, 1mg/mL bovine serum albumin.
(2) nucleic acid aptamer probe is prepared: be 60 mM stationary probes-7721 by final concentration be that 40 mM identify probe-7721 with final concentration, hybridize in hybridization buffer, after 95 DEG C of sex change 5min, it is for subsequent use to be slowly down to ambient temperature overnight, as the positive nucleic acid aptamer probe needed for the present embodiment.
(3) micro-fluidic chip is prepared: mixed in 10: 1 ratios with sygard 184 solidifying agent (Dow corning company) by aggressiveness before PDMS, after stirring and evenly mixing, be placed in vacuum chamber and remove bubble, then at SU-8 force plate upper, gauge control 6 mm.Then be placed in 80 DEG C of curing ovens after about 2 hours, carefully PDMS substrate peeled off from template, make at two ends, microchannel injection port and outlet position perforating by punching device the aperture that diameter is 2 mm.Dry by the glass substrate deionized water rinsing of 50mm × 30mm and in nitrogen.Finally, PDMS chip and clean glass substrate directly carry out reversible laminating.
(4) micro-fluidic chip of positive aptamer probe modification is prepared: the avidin solution taking the concentration of hybridization buffer preparation as 1 mg/mL, pass into chip and be full of chip, at 37 DEG C, hatch 1 hour, then use hybridization buffer irrigation channel three times.Continue to pass into positive nucleic acid aptamer probe, hatch 1 hour in 7 DEG C, then obtain the micro-fluidic chip of positive aptamer probe modification with hybridization buffer irrigation channel for three times.
(5) to modify separately the chip of stationary probe, as the compared with control cells chip of this experiment.
(6) will go down to posterity more than 24 hours, and be in the SMMC-7721 cell of logarithmic phase, with cell dissociation buffer digestion, use binding buffer solution washing, being separated into concentration is 10
6the SMMC-7721 cell dispersal liquid of individual/mL.The SMMC-7721 cell dispersal liquid of 200 mL is passed in the passage of the micro-fluidic chip of positive aptamer probe modification, collects the cell flowed out from passage.Again with the binding buffer solution irrigation channel twice of 400 mL, merge the outflow cell of effluent liquid and collection.
By the cell application of sample of collection in 96 orifice plates, every hole adds 50mL cell suspension (namely cell suspension flows out the cell collected and obtain from micro-fluidic chip passage), add 10 holes, (cell culture fluid composition is that RPMI 1640 trains base to add the cell culture fluid of 200 mL again, adding final concentration is 15% newborn calf serum, and final concentration is 100 IU/mL Streptomycin sulphates and 100 IU/mL penicillin).Wherein illumination is not carried out in five holes, the LED light source of other five hole 10W, illumination 10 min.From modifying the cell collected the chip of stationary probe, application of sample is in 96 orifice plates, and every hole adds 50mL cell suspension, adds 5 holes, then adds the cell culture fluid of 200 mL, is 100% as supposition survival rate.96 orifice plates are placed in cell culture incubator 37 DEG C, 5% CO
2after cultivating 24 h under condition, every hole adds 20 μ L MTS solution, continues to hatch 4 h, stops cultivating.10 min are hatched in concussion, and crystallisate is fully dissolved.Selected absorbing wavelength is 570 nm, microplate reader is used to measure the absorbance in each hole, wherein be assumed to 100% from the porocyte survival rate of modifying the cell that contrast probe chip flows out, the porocyte survival rate not adding cell is assumed to 0, and each identical treatment group does 5 holes.With following formulae discovery cell survival rate, the results are shown in Figure 10.
As seen from Figure 10, under the effect of illumination, target cell death nearly 50%, significantly difference and the cell not having illumination.Therefore, illustrate that this device has the characteristic of specific killing target cell.
Embodiment 4 is only the preferred embodiments of the present invention, in the present invention, identify that the photosensitizers that carries of probe can also replace with in pyropheophorbide-a, porphyrin, methylene blue, chlorin e 6, Visudyne, 5-ALA, temoporfin, talaporfin, phthalocyanine and phycobiliprotein one or more, and reach same or analogous technique effect.
Above embodiment is only the preferred embodiments of the present invention, in the present invention, identification probe in aptamer or device also can modified medicaments, and medicine is one or more in pyropheophorbide-a, uridylic, Zorubicin, camptothecine, taxol, Docetaxel, epirubicin, Rituximab and Herceptin.When identifying modified medicaments in probe, can be applicable to prepare tumor.
The above is only preferred embodiment of the present invention, not does any pro forma restriction to the present invention.Although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention.Any those of ordinary skill in the art, when not departing from spirit of the present invention and technical scheme, the Method and Technology content of above-mentioned announcement all can be utilized to make many possible variations and modification to technical solution of the present invention, or be revised as the Equivalent embodiments of equivalent variations.Therefore, every content not departing from technical solution of the present invention, according to technical spirit of the present invention to any simple modification made for any of the above embodiments, equivalent replacement, equivalence change and modification, all still belongs in the scope of technical solution of the present invention protection.
<110> Hunan University
<120> is used for nucleic acid aptamer probe and the application thereof of tumour cell detection
<130> without
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(20)
<223> experimentally requires and designs, for the stationary probe-CEM of nucleic acid aptamer probe
<400> 1
tctaaccgta tttttttttt 20
<210> 2
<211> 41
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(41)
<223> experimentally requires and designs, as the identification probe-CEM of nucleic acid aptamer probe
<400> 2
atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41
<210> 3
<211> 18
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(18)
<223> experimentally requires and designs, as the stationary probe-7721 of nucleic acid aptamer probe
<400> 3
cgcgcgcttt tttttttt 18
<210> 4
<211> 53
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(53)
<223> experimentally requires and designs, as the identification probe-7721 of nucleic acid aptamer probe
<400> 4
aaagcgcgcg cgcgcatagc gcgctgagct gaagatcgta ccgtgagcgc gct 53
<210> 5
<211> 41
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(41)
<223> experimentally requires and designs, for the contrast probe-CEM as nucleic acid aptamer probe
<400> 5
tttttttttt tttttttttt tttttttttt ttacggttag a 41
<210> 6
<211> 53
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(53)
<223> experimentally requires and designs, as the contrast probe-7721 of nucleic acid aptamer probe
<400> 6
aaagcgcgcg tttttttttt tttttttttt tttttttttt tttttttttt ttt 53
Claims (10)
1., for the nucleic acid aptamer probe that tumour cell detects, it is characterized in that, the identification probe comprising stationary probe He can be combined with tumour cell; Described stationary probe and described identification probe portion hybridize complementary.
2. nucleic acid aptamer probe according to claim 1, is characterized in that, the nucleotides sequence of described stationary probe is classified as has SEQ ID NO.1 or the nucleotide sequence described in SEQ ID NO.3; The nucleotides sequence of described identification probe is classified as has SEQ ID NO.2 or the nucleotide sequence described in SEQ ID NO.4.
3. nucleic acid aptamer probe according to claim 1 and 2, is characterized in that, described identification probe modification has fluorophor, medicine, photosensitizers or medical science contrast medium, and described stationary probe is modified with vitamin H.
4. nucleic acid aptamer probe according to claim 3, is characterized in that, described fluorophor is one or more in fluorescein, rhodamine, Alexa488, Atto550, Atto647, Cy3, Cy5 or Cy5.5; Medicine is one or more in pyropheophorbide-a, uridylic, Zorubicin, camptothecine, taxol, Docetaxel, epirubicin, Rituximab and Herceptin; Described photosensitizers is one or more in pyropheophorbide-a, porphyrin, methylene blue, chlorin e 6, Visudyne, 5-ALA, temoporfin, talaporfin, phthalocyanine and phycobiliprotein; Described medical science contrast medium be pyropheophorbide-a, porphyrin, iofendylate, iohexol, gadolinium-porphyrin complex and
64one or more in Cu-porphyrin complex.
5. for the device that tumour cell detects, it is characterized in that, comprise the nucleic acid aptamer probe according to any one of Claims 1-4 and carrier, the stationary probe of described nucleic acid aptamer probe is by being covalently or non-covalently crosslinking in described carrier surface.
6. device according to claim 5, is characterized in that, described carrier is magnetic particle, micro-fluidic chip, intravascular stent or artificial blood vessel.
7. device according to claim 6, is characterized in that, described magnetic particle is the magnetic particle of pan coating Streptavidin.
8. the nucleic acid aptamer probe according to any one of a Claims 1-4 or the application of the device according to any one of claim 5 to 7 in tumour cell detects.
9. the nucleic acid aptamer probe according to any one of a Claims 1-4 or the application of device in tumour cell spike according to any one of claim 5 to 7.
10. the nucleic acid aptamer probe according to any one of a Claims 1-4 or the application of the device according to any one of claim 5 to 7 in preparation tumor, preparation killing tumor cells device.
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| CN105181422A (en) * | 2015-09-29 | 2015-12-23 | 武汉大学 | Kit for benign lesion identification and malignant lesion detection of oral mucosa and detection method thereof |
| CN105259163A (en) * | 2015-10-26 | 2016-01-20 | 深圳华迈兴微医疗科技有限公司 | Magnetic particle direct chemiluminiscence microfluidic chip for whole blood sample testing |
| CN106267219A (en) * | 2016-08-08 | 2017-01-04 | 湖南大学 | The application of the application of targeting vector, targeted drug and targeted drug, targeted probes and targeted probes |
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| CN101812528A (en) * | 2010-04-26 | 2010-08-25 | 湖南大学 | Switch mode aptamer probe and application thereof in tumor living cell and vital detection |
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| CN101812528A (en) * | 2010-04-26 | 2010-08-25 | 湖南大学 | Switch mode aptamer probe and application thereof in tumor living cell and vital detection |
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| CN105181422A (en) * | 2015-09-29 | 2015-12-23 | 武汉大学 | Kit for benign lesion identification and malignant lesion detection of oral mucosa and detection method thereof |
| CN105181422B (en) * | 2015-09-29 | 2016-08-17 | 美泰克斯商贸（北京）有限公司 | Optimum disease damage investigation and malignant change detection kit and the detection method thereof of a kind of oral mucosa |
| CN105259163A (en) * | 2015-10-26 | 2016-01-20 | 深圳华迈兴微医疗科技有限公司 | Magnetic particle direct chemiluminiscence microfluidic chip for whole blood sample testing |
| CN105259163B (en) * | 2015-10-26 | 2018-05-04 | 深圳华迈兴微医疗科技有限公司 | The direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection |
| CN110049817A (en) * | 2016-06-30 | 2019-07-23 | 通用自动化实验技术公司 | The high resolution system, kit, device and method using magnetic bead for high-flux microorganism application |
| CN106267219A (en) * | 2016-08-08 | 2017-01-04 | 湖南大学 | The application of the application of targeting vector, targeted drug and targeted drug, targeted probes and targeted probes |
| CN106267219B (en) * | 2016-08-08 | 2019-03-29 | 湖南大学 | Targeting vector, the application of targeted drug and targeted drug, the application of targeted probes and targeted probes |
| CN107389919A (en) * | 2017-07-27 | 2017-11-24 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of label-free fluorescence aptamer sensor and its preparation method and application |
| CN107389919B (en) * | 2017-07-27 | 2020-05-26 | 广东省微生物研究所（广东省微生物分析检测中心） | Label-free fluorescent aptamer sensor and preparation method and application thereof |
| CN108467866A (en) * | 2017-12-18 | 2018-08-31 | 广州医科大学附属第医院 | A kind of aptamer and its application with the specific binding of (1,3)-callose |
| CN111250177A (en) * | 2018-11-30 | 2020-06-09 | 山东大学 | Biomolecule detection method |
| CN111250177B (en) * | 2018-11-30 | 2022-06-24 | 山东大学 | Biomolecule detection method |
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