CN101745122A - Automatic synthesis anti-cancer drug preparation method based on microfluidic chip - Google Patents
Automatic synthesis anti-cancer drug preparation method based on microfluidic chip Download PDFInfo
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Abstract
An automatic synthesis anti-cancer drug preparation method based on microfluidic chip includes that: biological molecules used as computation medium are used to compute the expression of one cancer-related gene or gene combination through biochemical reaction; when the result meets the requirement, the anti-cancer drug is automatically synthesized and released in targeted manner: the anti-cancer drug is complete suicide gene. The biological molecules used as computation medium are DNA molecules and/or enzyme; the carrier of the preparation method is the microfluidic chip: the chip concretely comprises an input unit, a computation unit and an output unit; the fluid flow channels among the three units are connected sequentially to form directional mobile control of certain substance in electric field effect. The invention firstly proposes an automatic synthesis anti-cancer drug preparation method based on microfluidic chip, synthesizes and releases functional drug on specified conditions, namely the complete suicide gene. The method can be combined with the nanometer technology and micromachining to help improve the targeted therapy of caner.
Description
Technical field
The present invention relates to computer science, molecular biology, medical science and micro-fluidic chip technology.A kind of preparation method of the automatic synthetic cancer therapy drug of energy based on micro-fluidic chip is provided especially.
Background technology
Cancer is a kind of gene disease, diagnoses and treatment is fundamental sum method the most accurately at gene level.Cell carcinogenesis is a process based on the inactivation of the activation of multiple proto-oncogene and antioncogene, therefore, when carrying out gene diagnosis, often needs the expression of several genes is carried out comprehensive judgement.At present, the diagnosis of cancer is carried out external often, by to blood with organize assay etc. to judge, also is far from reaching unicellular level.And in-vitro diagnosis and interior therapeutic are two relatively independent links, and cancer therapy drug often also causes bigger damage to normal cell.How with the specific tumor cell that is discharged into of cancer therapy drug, improving the targeting of treatment, is the difficult problem that biomedical workers endeavour to solve.Up to now, during the targeting that has several different methods and technology to be applied to medicine discharges, as utilize virus (document Cancer Gene Therapy, 2008,15,667-675), nano-particle (document Gene Therapy, 2000,7,1896-1905) and micro-fluidic chip (document Lab On A Chip, 2006,6,1384-1386) grade is carried medicine, but because these systems have lacked " diagnosis " this link, therefore can't accomplish targeting " treatment " the most accurately.
2004, Shapiro group has reported a kind of Biotic molecule computer (document Nature of may command gene expression, 2004,429,423-429), in vitro simulated after small dna computer enters cell, by hybridization, enzyme company, endonuclease reaction repeatedly, carcinoma of prostate and the multiple Expression of Related Genes situation of pulmonary carcinoma are calculated successively, and when diagnosis is set up, discharged short single stranded nucleic acid molecule and treat as medicine.This computer will be diagnosed and treat two relatively independent links first and be connected, and will diagnose and treatment is positioned at unicellular level, improve the targeting of drug release, for the targeted therapy that solves cancer provides new research direction.But this dna computer remains in following some deficiency as will further carrying out in vivo test: 1, all biochemical reactions of this computer carry out in test tube, dna computer will enter functionating in the body, must need a kind of effective carrier carrying dna molecular and enzyme is delivered to corresponding target spot with it, prevent that dna molecular just is degraded before calculating, obviously test tube can not become this effective carrier; 2, the account form that (series connection) carried out in multiple cancer related gene expression employing successively, and a kind of gene of every calculating need be introduced the exogenous molecule of multiple high concentration, might have influence on the normal physiological function of cell; 3, the calculating principle of this computer requires computer must enter ability functionating in the cell, has strengthened operation easier; 4, more of paramount importance, this computer adopts restricted enzyme (FokI enzyme) to carry out endonuclease reaction, restricted enzyme exists only in the unicellular lower eukaryote (as antibacterial, virus etc.), can double chain DNA molecule can be cut off at specific site, therefore might the genome of higher organism be exerted an influence.In sum, in gene diagnosis and the treatment more reasonable calculation method and even more ideal platform must be arranged if dna computer will be applied to.In addition, what discharged in the above-mentioned system is the medicine that existed in advance, a kind of method more flexibly discharge then for synthesizing medicine as required (document Nature Biotechnology, 2003,21,1184-1191).
People expect to obtain a kind of preparation method of the automatic synthetic cancer therapy drug of energy based on micro-fluidic chip.
Summary of the invention
The objective of the invention is to design a kind of preparation method of the automatic synthetic cancer therapy drug of energy based on micro-fluidic chip.
We use dna computer (preferably being carrier with micro-fluidic chip), the several genes expression relevant to cancer calculates simultaneously, and when diagnosis is set up, automatically synthetic complete suicide gene is as effectively cancer therapy drug release, and when diagnosis is false, release only be the medicine of invalid fragmentation.
We are example with breast carcinoma, on more than one square centimeters micro-fluidic chip, in the external feasibility of verifying this dna computer principle.Simulated after more microminiaturized micro flow controlling chip DNA computer enters in the body, need not to enter cell, can carry out the process of gene diagnosis and treatment by the Cytoplasm of trace.Molecular species that adopts in the calculating and quantity are all less, and the enzyme that uses is the dna ligase that exists in the higher organism body.Along with the development of nano material and micro-processing technology, be expected to carry out further in vivo test.
Dna computer is based on the new bio computer of dna molecular biochemical reaction.Specifically, the dna molecular of representing certain information exactly carries out " calculating " as " input " through controlled biochemical reactions such as hybridization, degeneration, enzyme company, enzymolysis, and the dna molecular new or that meet certain requirement that the reaction back generates is as " output ".Often need the participation of enzyme in the computational process, as restricted enzyme, dna ligase, exonuclease etc.
According to the implementation of dna computer, dna computer has experienced test tube (document Science, 1994,266,1021-1024), the surface (document Nature, 2000,403,175-179) and chip (document Lab OnAChip, 2005,5,1033-1040) three phases.Dna molecular is fixed on lip-deep account form, can overcome dna molecular in the test tube easily lose and operate difficult, detect difficult characteristics, but a plurality of calculation procedure still must be interrupted under manual intervention and be carried out.Micro-fluidic chip has more remarkable advantages of specific surface, not only certain dna molecular that calculates usefulness can be fixed on the part of chip in the mode of 3 D stereo, and characteristics with Highgrade integration, automatization, miniaturization, microminiaturization, DNA calculates required a plurality of biochemical reactions, all can finish continuously on a small chip.Therefore chip might replace test tube and surface, becomes a kind of ideal platform of dna computer.
Compare with traditional electronic computer, dna computer has advantages such as concurrency height, fast operation, information storage be big, be used to research and solve a complex mathematical difficult problem (document ProceedingsOf The National Academy Of Sciences Of The United Stated States OfAmerica, 2004,101,9960-9965).In addition, because it has biocompatibility, scientists tries hard to apply it in the life science, be used for the diagnosis of cancer and treatment (document ScientificAmerican, 2006,294,44-51).
The preparation method that a kind of automatic synthetic cancer therapy drug of energy based on micro-fluidic chip is provided of the present invention, it is characterized in that: the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip specifically: use biomolecule as computation medium, by biochemical reaction, the expression of one of range gene relevant with cancer or its combination is calculated; Automatically synthetic also targeting discharges effective cancer therapy drug when result of calculation meets the requirements: complete suicide gene;
In above-mentioned computational process, described biomolecule as computation medium specifically is dna molecular and/or enzyme.
The preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip uses micro-fluidic chip as carrier, and described micro-fluidic chip specifically is divided into three functional units: input block, computing unit and output unit; Wherein, computing unit is arranged between input block and the output unit, and liquid flow path connects in proper order between these three unit, and the orientation that can form predetermined substance under electric field action moves control;
The present invention at described cancer therapy drug be have automatic synthesis capability and can be when condition satisfies the cancer therapy drug that discharges of targeting.
In the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip, dna molecular as computation medium is specially: with every kind of corresponding calculating molecule of cancer related gene and medicine sheet segment molecule, and the incomplete suicide gene molecule that has a plurality of breach; The sequence of breach is corresponding with the sequence and the quantity of medicine sheet segment molecule with quantity on the described suicide gene molecule.In the described dna molecular as computation medium, with every kind of corresponding calculating molecule of cancer related gene and medicine sheet segment molecule be single strand dna, the incomplete suicide gene molecule that has breach is a double chain DNA molecule.
In the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip, express when cancer related gene corresponding in the detected object and to exceed normal range when (raise or reduce and all can exceed normal range), the cancer related gene that exceeds part can exchange all or part of medicine sheet segment molecule for getting off for from itself and the matching relationship that calculates molecule are mid-, makes the structure of original calculating molecule and cancer related gene hybridization become new more stable structure;
When the expression of cancer related gene does not meet diagnostic criteria, that is in the medicine sheet segment molecule some or all of not from its with the matching relationship that calculates molecule when being replaced by displacement, have antitumaous effect thereby just can not combine the complete suicide gene molecule of formation with the incomplete suicide gene molecule that has breach.
In the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip: described enzyme as computation medium is the T4DNA ligase;
Described medicine sheet segment molecule is with to calculate molecule structurally complementary, its two can realize hybridization; Particularly, described medicine sheet segment molecule is identical with breach sequence on the incomplete suicide gene molecule; Medicine sheet segment molecule can make it form complete suicide gene the gap fill on the incomplete suicide gene under the effect of T4DNA ligase;
For medicine sheet segment molecule, cancer related gene is with to calculate the complementary element sequence longer, and cancer related gene is easier to and calculates molecular hybridization and medicine sheet segment molecule is replaced to get off.
When cancer related gene with corresponding medicine sheet segment molecule from its with the coupling corresponding relation that calculates molecule replaces after, be substituted the purpose zone (output unit thereafter) that the medicine sheet segment molecule targeting under electric field action that gets off is discharged into correspondence; Thereby with incomplete suicide gene synthetic complete medicine in T4DNA ligase system with antitumaous effect.
When cancer related gene with corresponding medicine sheet segment molecule from its with the coupling corresponding relation that calculates molecule replace after, the medicine sheet segment molecule that comes from the computing unit transmission is transported to specific target area and the hybridization of incomplete suicide gene and carries out even reaction of enzyme under the effect of T4DNA ligase, with corresponding gap fill; Include incomplete suicide gene in the described specific target areas;
When the expression of cancer related gene meets the requirements (being that expression of proto-oncogenes is higher than the normal limitations value and expression of tumor suppressor gene is lower than the normal limitations value), corresponding medicine fragment is all filled up the breach on the incomplete suicide gene, and promptly have complete suicide gene to be synthesized and discharge as cancer therapy drug this moment;
As long as there is a kind of gene expression not meet diagnostic criteria, breach just can not filled up fully, just can not have complete suicide gene to be synthesized accordingly, release only be the incomplete suicide gene that does not have antitumaous effect.
The preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip is implemented according to the carrier micro-fluidic chip, and the structure of described micro-fluidic chip as previously mentioned and can understand with reference to accompanying drawing 2.As the micro-fluidic chip of carrier, material can be quartz, glass, PMMA, PC polymer etc.;
In the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip, calculate molecule and be fixed on the computing unit as the micro-fluidic chip of carrier; Medicine sheet segment molecule is hybridized in advance on the calculating molecule; Incomplete suicide gene and T4DNA ligase system are placed in the output unit in advance.
When proto-oncogene molecule or this two classes cancer related gene of genetic suppressor element with corresponding medicine sheet segment molecule from its with the coupling corresponding relation that calculates molecule when replacing, be substituted the purpose zone (output unit thereafter) that the medicine sheet segment molecule targeting under effect of electric field that gets off is discharged into correspondence; Thereby with incomplete suicide gene in T4DNA ligase system synthetic complete medicine with antitumaous effect (certainly prerequisite be all be substituted the medicine fragment molecular energy that gets off fill up incomplete suicide gene jaggedly make it can form the complete medicine that really has antitumaous effect).
At the output unit of micro-fluidic chip, medicine sheet segment molecule from the computing unit transmission and the hybridization of incomplete suicide gene are also carried out even reaction of enzyme under the effect of T4DNA ligase, with corresponding gap fill;
When the expression of cancer related gene meets the requirements (being that expression of proto-oncogenes raises and expression of tumor suppressor gene reduces), corresponding medicine fragment is all filled up the breach on the incomplete suicide gene, and promptly have complete suicide gene to be synthesized and discharge as cancer therapy drug this moment;
As long as there is a kind of gene expression not meet diagnostic criteria, breach just can not filled up fully, just can not have complete suicide gene to be synthesized accordingly, release only be the incomplete suicide gene that does not have antitumaous effect.
Medicine sheet segment molecule is identical with breach sequence on the incomplete suicide gene, can be with gap fill under the effect of T4DNA ligase.Relation between the molecule sees Fig. 1 for details.
In the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip, be with calculating the detailed process that molecule is fixed on as the computing unit of the micro-fluidic chip of carrier:
In acrylamide solution, final concentration is 5～20 μ m with the end modified calculating molecular melting that acrylamide group or amino group are arranged; Behind uv-exposure, be fixed in the acrylamide hydrogel.(be fixed on calculating molecule in the hydrogel and can keep active, can hybridize, reaction such as degeneration.The acrylamide hydrogel is a kind of transparent substrate, is not having can to play the effect of valve under the electric field action; Under the low voltage electric field effect, can allow dna molecular to pass through.)
In the described micro-fluidic chip as carrier, that input block contains is a plurality of (determines usable ranges according to actual needs, common usable range is 1～300) sample cell, the number of sample cell is decided according to the needs of diagnosis, and different cancer related genes is usually respectively by different sample cell inputs.
Computing unit in the described micro-fluidic chip is specially the parallel channel of the equivalent amount that links to each other with each sample cell respectively; Contain in each passage and corresponding calculating molecule of cancer related gene and medicine sheet segment molecule, respectively heterogeneic expression is calculated and transmit the relative medicine fragment, the calculating of different passages is independently to carry out simultaneously, does not disturb mutually;
Output unit in the described micro-fluidic chip contains a passage and an enzyme connects the pond, and enzyme connects and contains incomplete suicide gene molecule and T4DNA ligase reaction system in the pond; The passage of the medicine sheet segment molecule that comes from the different passage transmission of computing unit by output unit is pooled to enzyme and connects and carry out enzyme the pond and connect reaction;
In the passage of output unit, fixed one section blank hydrogel, it not only can play the effect of valve, and because hydrogel has the effect of concentration of DNA molecule, the medicine sheet segment molecule that comes from the computing unit transmission detect herein than connect at enzyme detect the pond more sensitive, referring to Fig. 2.
The preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip satisfies following two kinds and one of requires:
One is calculating whether expression of proto-oncogenes raises and can be fixed with two sections hydrogels with the measuring in the passage for the computing unit of the segmental micro-fluidic chip of relative medicine of correspondence when it raises; Fixing corresponding calculated molecule only in first section hydrogel; On the fixed calculating molecule of institute, also hybridization has corresponding medicine sheet segment molecule in advance in second section hydrogel;
When proto-oncogene molecule (promptly contain the class in the molecules detected of cancer related gene, another kind of molecules detected is an antioncogene) under the low voltage electric field effect, enter first section hydrogel and corresponding calculated molecular hybridization and caught by it; The concentration of speed of catching and proto-oncogene molecule is dependency;
Prescribe a time limit on normal range when gene expression, through the electrophoresis of certain hour, proto-oncogene is gone to the opposite side edge of first section hydrogel just; And when gene expression raises, because the speed of hybridization is faster, electrophoresis through the identical time, the proto-oncogene of raised portion can pass first section hydrogel, arrives its downstream, and under electric field action, enter second section hydrogel and calculate molecular hybridization, and corresponding medicine sheet segment molecule is replaced to get off, the amount of replacement is corresponding with the amount that gene expression raises, and the medicine sheet segment molecule that replaces further is delivered to output unit under effect of electric field.
They are two years old, whether reduce and replace in the passage of computing unit of the segmental micro-fluidic chip of relative medicine calculating expression of tumor suppressor gene, be fixed with two sections hydrogels: fixing corresponding calculated molecule in first section hydrogel, and hybridization has corresponding medicine sheet segment molecule in advance; Second section hydrogel is blank hydrogel, only plays the effect of valve, do not participate in calculating;
Genetic suppressor element at first enters first section hydrogel under the low voltage electric field effect, corresponding medicine sheet segment molecule is replaced to get off (speed of replacement and the concentration of genetic suppressor element are dependency);
When expression of tumor suppressor gene was prescribed a time limit under normal range, electrophoresis through certain hour, antioncogene is gone to the opposite side edge of first section hydrogel just, and all medicine sheet segment molecules are all replaced, and is substituted the medicine sheet segment molecule that gets off subsequently by reject; When expression of tumor suppressor gene reduced, because replacement speed is slack-off, through the electrophoresis of identical time, still some medicine fragment can not be substituted and be retained in the hydrogel, and the part that replaces is by reject.Be retained in part in the hydrogel when changing deposition condition under the flushing, the amount of this part is corresponding with the amount that gene expression reduces, and further is delivered to output unit under effect of electric field.
In output unit as the micro-fluidic chip of carrier, when expression of proto-oncogenes raises, when reducing, transmitted expression of tumor suppressor gene the corresponding medicine fragment of coming, can hybridize with it in the position of the corresponding breach of incomplete suicide gene molecule, and under the effect of T4DNA ligase medicine sheet segment molecule being connected with slit between the incomplete suicide gene, with corresponding gap fill.When all breach are all filled up, there is not the incomplete suicide gene molecule of active anticancer just to become complete suicide gene molecule, had active anticancer.
In a word, the present invention adopt first micro-fluidic chip as carrier with dna computer be applied to cancer gene diagnosis and the treatment in the middle of, utilize the high-throughout characteristics of micro-fluidic chip, calculate the expression (whether proto-oncogene raises, antioncogene whether reduce) of multiple cancer related gene simultaneously, synthetic also medicine---the complete suicide gene of release function when diagnosis is set up, when diagnosis is false, release only be non-functional medicine fragment.This method combines with nanotechnology and micro-processing technology, helps to improve the targeted therapy of cancer.
Description of drawings
The present invention is further detailed explanation below in conjunction with drawings and the embodiments:
Fig. 1 concerns sketch map between the molecule of this dna computer;
Fig. 2 is the carrier of this dna computer---microfluidic chip structure sketch map (contain and calculate passage and the passage whether the calculating expression of tumor suppressor gene reduces whether expression of proto-oncogenes raises);
One of principle schematic whether Fig. 3 raises for the calculating expression of proto-oncogenes;
Fig. 4 is two of the principle schematic calculating expression of proto-oncogenes and whether raise;
Fig. 5 is three of the principle schematic calculating expression of proto-oncogenes and whether raise;
One of principle schematic whether Fig. 6 reduces for the calculating expression of tumor suppressor gene;
Fig. 7 is two of the principle schematic calculating expression of tumor suppressor gene and whether reduce;
Fig. 8 is three of the principle schematic calculating expression of tumor suppressor gene and whether reduce;
Fig. 9 is functional medicine connection diagram;
Figure 10 is the electrophoresis fluorescence figure of proto-oncogene C-erbB-2 in first section hydrogel of upper limit normal range;
Figure 11 is the result of calculation figure of the proto-oncogene C-erbB-2 of five kinds of variable concentrations;
The eluted electrophoresis fluorescence figure of medicine fragment B when Figure 12 changes deposition condition;
Figure 13 is the result of calculation figure of the antioncogene nm23 of five kinds of variable concentrations;
Figure 14 is one of the synthetic electrophoresis as a result of medicine spectrogram under the different diagnosis situations;
Figure 15 be under the different diagnosis situations the synthetic electrophoresis as a result of medicine spectrogram two;
Figure 16 is the synthetic electrophoresis as a result of the medicine spectrogram system three under the different diagnosis situations;
Figure 17 be under the different diagnosis situations the synthetic electrophoresis as a result of medicine spectrogram four;
Figure 18 be under the different diagnosis situations the synthetic electrophoresis as a result of medicine spectrogram five.
The specific embodiment
We are example with breast carcinoma, and the synthetic automatically also feasibility of the preparation method of targeting release anti-cancer medicine based on micro-fluidic chip is described.We have calculated the two kind genes the closest with the breast carcinoma relation, be proto-oncogene C-erbB-2 and antioncogene nm23, express the situation that raises and reduce, and when both expression all met diagnostic criteria, synthetic complete suicide gene HSV-tk was as cancer therapy drug and with its release.
In experiment in vitro, we adopt synthetic short dna molecular to replace long proto-oncogene and antioncogene.Corresponding with proto-oncogene for calculating molecule 1 and medicine Segment A, corresponding with antioncogene for calculating molecule 2 and medicine fragment B.With replacing incomplete suicide gene molecule with medicine Segment A and the complementary template strand of B.Medicine Segment A and B can combine with template strand, form heteroduplex, and connect into complete two strands under the effect of T4DNA ligase, and this complete two strands is represented functional medicine, as shown in Figure 9.For the ease of detecting, at the end fluorophor labelling of gene molecule and medicine sheet segment molecule, the FAM group excites at the 494nm wavelength, and the 522nm wavelength sends green fluorescence when detecting, the TAMRA group excites at the 560nm wavelength, and the 582nm wavelength sends red fluorescence when detecting.The medicine Segment A needs the participation of phosphate groups (PO4-) when being connected with B.Calculating molecular marker has acrylamide group (acrylamide-), can be fixed in the acrylamide hydrogel by covalent bond.The sequence of molecule and the situation of modification see table 1 for details.
Table 1 is the nucleotide sequence and the labelling of dna computer desired molecule
The present embodiment used carrier is a micro-fluidic chip, specifically as shown in Figure 2: be the glass-chip that contains two parallel channels, channel width 200 μ m, dark 80 μ m, the about 50mm of total length (connecting pond 13 to enzyme) from sample cell 1 or 6, fixed every section hydrogel length is 1mm in the passage, and the diameter of liquid pool is 2mm.
To contain in the acrylamide solution injection channel of 10 μ m calculating molecule, when treating to be full of this solution in the passage, cover in black non transparent adhesive plaster other parts chip, the channel part that only will need to form hydrogel exposes, exposure is 5 minutes under uviol lamp, and exposed portions has just formed and has been fixed with the hydrogel that calculates molecule.Other positions are not formed the acrylamide solution of hydrogel, siphon away by the liquid pool of vacuum pump from the hydrogel both sides.Pass through the method, can in the passage whether the calculating proto-oncogene raises, form two sections and contain the hydrogel that calculates molecule 1, in the passage whether the calculating antioncogene reduces, form one section and contain hydrogel and the one section blank hydrogel (not containing any calculating molecule) of calculating molecule 2, in the passage of output unit, form one section blank hydrogel (not containing any calculating molecule).The medicine Segment A is added in the liquid pool 4, between liquid pool 4 and 5, apply 48v voltage, the medicine Segment A enters in the hydrogel and calculates molecule 1 hybridization, behind 5 minutes electrophoresis, hybridization reaches capacity, with the remaining liq reject in liquid pool 4 and 5, medicine fragment B can be hybridized in advance equally and calculate on the molecule 2, all charge into the buffer 1 * TE and the 0.5M NaCl of high salt in each buffer pool.Calculation process detects under fluorescence microscope.
We set 1～2 μ m is the normal range of gene expression.In calculating the passage whether proto-oncogene raise, at first investigate normal range the upper limit promptly the proto-oncogene C-erbB-2 of 2 μ m arrive time of first section hydrogel right side edge, experimental results show that to be 3 minutes, the result as shown in figure 10.Therefore we are decided to be 3 minutes with the electrophoretic time of the first step.Investigate the result of calculation of the C-erbB-2 of 5 kinds of variable concentrations.The C-erbB-2 of variable concentrations input from liquid pool 1 respectively, between liquid pool 1 and 3, apply the voltage of 48v, electrophoresis time is 3 minutes, between liquid pool 3 and 5, apply 48v voltage 4 minutes then, between liquid pool 5 and 13, apply 48v voltage 5 minutes again, the result was as shown in figure 11: through 3 minutes electrophoresis, C-erbB-2 expresses when raising (5 and 10 μ m), pass first section hydrogel, through the second step electrophoresis, enter second block of hydrogel, replaced the medicine Segment A, be substituted the medicine Segment A of getting off and go on foot electrophoresis, process blank hydrogel arrival liquid pool 13 through the 3rd.When the C-erbB-2 expression does not raise (1 and 0.2 μ m), the right side edge through first section hydrogel of first step electrophoresis no show still C-erbB-2 can not occur in the liquid pool 5, should this can not replace the medicine Segment A.Above result verification just has corresponding medicine fragment when having only expression of proto-oncogenes to raise and is passed to output unit.
As shown in figure 12, when changing deposition condition, voltage direction is switched, buffer changes less salt into by high salt (1 * TE and 0.5M NaCl), and (after 1 * TE), through 6 minutes electrophoresis, medicine fragment B can all be rinsed.
In calculating the passage whether antioncogene reduce, at first investigate normal range lower limit promptly the antioncogene nm23 of 1 μ m arrive time of first section hydrogel right side edge, just the time that medicine fragment B is all replaced, experimental results show that to be 4 minutes.Investigate the result of calculation of 5 kinds of variable concentrations nm23.The nm23 of variable concentrations input from sample cell 6 respectively, between liquid pool 6 and 8, apply the voltage of 48v, electrophoresis time is 4 minutes, then the liquid in the liquid pool 8 is discarded, add buffer 1 * TE and 0.5M NaCl again, change the liquid in the sample cell 6 into 1 * TE, switch the voltage direction between the liquid pool 6 and 8, electrophoresis 6 minutes, and then through the 3rd step electrophoresis (between the liquid pool 8 and 13), the time is 8 minutes.The result was as shown in figure 13: through 4 minutes electrophoresis, nm23 expresses when reducing (0.2 and 0.02 μ m), the right side edge of first section hydrogel of no show still, medicine fragment B can not be replaced fully, the medicine fragment B that keeps through the second step electrophoresis, is rinsed when changing deposition condition, be rinsed the medicine fragment B that gets off and go on foot electrophoresis, arrive liquid pools 13 through two sections blank hydrogel through the 3rd.When nm23 expresses (1 and 0.2 μ m) when not reducing, is replaced also reject of back fully through first step electrophoresis medicine fragment B, therefore, even the change deposition condition does not have medicine fragment B yet and is rinsed.Above result verification just has corresponding medicine fragment when having only expression of tumor suppressor gene to reduce and is passed to output unit.
In liquid pool 13, deposited drug template and T4DNA ligase reaction system in advance,,, connected the enzyme that carries out 30 minutes in the pond at enzyme and connect reaction in (about 25 ℃) under the room temperature through after two passes is all finished three step electrophoresis up and down.Because whether medicine sheet segment molecule connects into complete two strands, only relies on fluorescence to prove, need verify by length.Therefore, enzyme links fruit on another piece cross chip, carries out chip electrophoresis, after enzyme is connected reacted different component and separates, and laser-Induced Fluorescence Detection, the electrophoresis spectrogram is shown in Figure 14～18.B represents medicine fragment B among the figure, the complete functional medicine after AB represents the medicine Segment A and B is connected, and the fluorescence of medicine Segment A labelling is filtered under this experiment testing conditions, therefore detects the peak less than the medicine Segment A.Figure 14 is the electrophoresis spectrogram of standard sample; Figure 15 raises for proto-oncogene and antioncogene synthetic result's of medicine when reducing electrophoresis spectrogram; Figure 16 has only the electrophoresis spectrogram that proto-oncogene raises, medicine synthesized the result when expression of tumor suppressor gene did not reduce; Figure 17 has only the electrophoresis spectrogram that expression of tumor suppressor gene reduces, medicine synthesized the result when proto-oncogene did not raise; Figure 18 does not raise for proto-oncogene and antioncogene synthetic result's of medicine when also not reducing electrophoresis spectrogram.As can be seen from the figure, have only proto-oncogene and expression of tumor suppressor gene all to meet diagnostic criteria, when promptly diagnosis is set up, just have the synthetic and release of functional medicine, as long as a kind of diagnostic criteria that do not meet is arranged, i.e. diagnosis is when being false, and do not have functional medicine synthetic and discharge, release only be non-functional medicine fragment.This illustrates that this dna computer helps the targeted therapy of cancer.
At the four kind gene design relevant with hepatocarcinoma four kinds of corresponding calculated molecules, medicine sheet segment molecules and the suicide gene molecule of four breach is arranged and the micro-fluidic chip that four parallel channels are arranged.According to the calculating principle identical the expression of gene situation is calculated: when MMP-9 and CD34 gene expression raise with embodiment 1, when wild type P53 and Rb gene expression reduce simultaneously, corresponding medicine fragment delivery is arranged to output unit, with the gap fill on the suicide gene, form complete suicide gene; When wherein any gene expression does not meet diagnostic criteria, then there is not complete suicide gene to form and release.
At the six kind gene design relevant with carcinoma of prostate six kinds of corresponding calculated molecules, medicine sheet segment molecules and the suicide gene molecule of six breach is arranged and the micro-fluidic chip that six parallel channels are arranged.According to the calculating principle identical the expression of gene situation is calculated: when PSMA, Pim-1, KLK11, PSA express rising with embodiment 1, IGF1 and P27 express when reducing simultaneously, corresponding medicine fragment delivery is arranged to output unit, with the gap fill on the suicide gene, form complete suicide gene; When wherein any gene expression does not meet diagnostic criteria, then there is not complete suicide gene to form and release.
4 one kinds of preparation methoies of embodiment based on the automatic synthetic cancer therapy drug of energy of micro-fluidic chip
Present embodiment and embodiment 1 content are basic identical, and its difference mainly is:
1) we at least a in the following proto-oncogene (C-erbB-2, EGFR, c-myc, ras, int-2, bcl-2, BAG-1, BCSG-2, survivin) relevant with breast carcinoma and with the relevant antioncogene (P53, nm23, PTEN, Rb, P16, P21, CHEK2, BRCA1, BRCA2) of breast carcinoma at least a as design considerations, carry out the design work in advance and the corresponding preparation process of medicine sheet segment molecule.
2) we are at 1) in selected medicine fragment MOLECULE DESIGN have the incomplete suicide gene molecule of breach, be specially the double chain DNA molecule of the breach that has the medicine sheet segment molecule corresponding identical sequence selected with it.
3) as the computing unit of the micro-fluidic chip of carrier and calculate passage according to above-mentioned 1), 2) concrete condition of process selects for use, test parameterss etc. also match.
Claims (10)
1. based on the preparation method of the automatic synthetic cancer therapy drug of energy of micro-fluidic chip, it is characterized in that:
The preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip is: use the biomolecule as computation medium, by biochemical reaction, the expression of one of range gene relevant with cancer or its combination is calculated; Automatically synthetic also targeting discharges effective cancer therapy drug when result of calculation meets the requirements: complete suicide gene;
In above-mentioned computational process, described biomolecule as computation medium specifically is dna molecular and/or enzyme;
The preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip is carrier with the micro-fluidic chip, and described micro-fluidic chip specifically is divided into three functional units: input block, computing unit and output unit; Wherein, computing unit is arranged between input block and the output unit, and liquid flow path connects in proper order between these three unit, and the orientation that can form predetermined substance under electric field action moves control.
2. according to the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip of claim 1, it is characterized in that: in the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip, dna molecular as computation medium is specially: with every kind of corresponding calculating molecule of cancer related gene and medicine sheet segment molecule, and the incomplete suicide gene molecule that has a plurality of breach; The sequence of breach is corresponding with the sequence and the quantity of medicine sheet segment molecule with quantity on the described suicide gene molecule.
3. according to the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip of claim 2, it is characterized in that: in the described dna molecular as computation medium, with every kind of corresponding calculating molecule of cancer related gene and medicine sheet segment molecule be single strand dna, the incomplete suicide gene molecule that has breach is a double chain DNA molecule.
4. according to the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip of claim 3, it is characterized in that: in the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip, when cancer related gene expression corresponding in the detected object exceeds normal range, the cancer related gene that exceeds part can exchange all or part of medicine sheet segment molecule for getting off for from itself and the matching relationship that calculates molecule are mid-, makes the structure of original calculating molecule and cancer related gene hybridization become new more stable structure;
When the expression of cancer related gene does not meet diagnostic criteria, that is in the medicine sheet segment molecule some or all of not from its with the matching relationship that calculates molecule when being replaced by displacement, have antitumaous effect thereby just can not combine the complete suicide gene molecule of formation with the incomplete suicide gene molecule that has breach.
5. according to the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip of claim 4, it is characterized in that: in the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip: described enzyme as computation medium is the T4DNA ligase;
Described medicine sheet segment molecule is with to calculate molecule structurally complementary, its two can realize hybridization; Particularly, described medicine sheet segment molecule is identical with breach sequence on the incomplete suicide gene molecule; Medicine sheet segment molecule can make it form complete suicide gene the gap fill on the incomplete suicide gene under the effect of T4DNA ligase;
For medicine sheet segment molecule, cancer related gene is with to calculate the complementary element sequence longer, and cancer related gene is easier to and calculates molecular hybridization and medicine sheet segment molecule is replaced to get off.
6. according to the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip of claim 5, it is characterized in that: when cancer related gene with corresponding medicine sheet segment molecule from its with the coupling corresponding relation that calculates molecule replaces after, be substituted the purpose zone that the medicine sheet segment molecule targeting under electric field action that gets off is discharged into correspondence; Thereby with incomplete suicide gene synthetic complete medicine in T4DNA ligase system with antitumaous effect.
7. according to the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip of claim 6, it is characterized in that: when cancer related gene with corresponding medicine sheet segment molecule from its with the coupling corresponding relation that calculates molecule replace after, medicine sheet segment molecule is transported to specific target area and the hybridization of incomplete suicide gene and carries out enzyme under the effect of T4DNA ligase and connects reaction, with corresponding gap fill; Include incomplete suicide gene in the described specific target areas;
When the expression of cancer related gene met the requirements, corresponding medicine fragment was all filled up the breach on the incomplete suicide gene, and promptly have complete suicide gene to be synthesized and discharge as cancer therapy drug this moment;
As long as there is a kind of gene expression not meet diagnostic criteria, breach just can not filled up fully, just can not have complete suicide gene to be synthesized accordingly, release only be the incomplete suicide gene that does not have antitumaous effect.
8. according to the preparation method of one of them described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip of claim 1～7, it is characterized in that:
In the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip, calculate molecule and be fixed on the computing unit as the micro-fluidic chip of carrier; Medicine sheet segment molecule is hybridized in advance on the calculating molecule; Incomplete suicide gene and T4DNA ligase system are placed in the output unit in advance.
9. according to the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip of claim 8, it is characterized in that:
With calculating the detailed process that molecule is fixed on as the computing unit of the micro-fluidic chip of carrier be: in acrylamide solution, final concentration is 5～20 μ m with the end modified calculating molecular melting that acrylamide group or amino group are arranged; Behind uv-exposure, be fixed in the acrylamide hydrogel;
In the described micro-fluidic chip as carrier, input block contains 1～300 sample cell, and different cancer related genes is respectively by different sample cell inputs;
Computing unit in the described micro-fluidic chip is specially the parallel channel of the equivalent amount that links to each other with each sample cell respectively; Contain in each passage and corresponding calculating molecule of cancer related gene and medicine sheet segment molecule, respectively heterogeneic expression is calculated and transmit the relative medicine fragment, the calculating of different passages is independently to carry out simultaneously, does not disturb mutually;
Output unit in the described micro-fluidic chip contains a passage and an enzyme connects the pond, and enzyme connects and contains incomplete suicide gene molecule and T4DNA ligase reaction system in the pond; The passage of the medicine sheet segment molecule that comes from the different passage transmission of computing unit by output unit is pooled to enzyme and connects and carry out enzyme the pond and connect reaction;
In the passage of output unit, fixed one section blank hydrogel, it not only can play the effect of valve, and because hydrogel has the effect of concentration of DNA molecule, the medicine sheet segment molecule that comes from the computing unit transmission detect herein than connect at enzyme detect the pond more sensitive.
10. according to the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip of claim 9, it is characterized in that: the preparation method of the described automatic synthetic cancer therapy drug of energy based on micro-fluidic chip also satisfies following two kinds and one of requires:
One is calculating whether expression of proto-oncogenes raises and can be fixed with two sections hydrogels with the measuring in the passage for the computing unit of the segmental micro-fluidic chip of relative medicine of correspondence when it raises; Fixing corresponding calculated molecule only in first section hydrogel; On the fixed calculating molecule of institute, also hybridization has corresponding medicine sheet segment molecule in advance in second section hydrogel;
When the proto-oncogene molecule under the low voltage electric field effect, enter first section hydrogel and corresponding calculated molecular hybridization and caught by it; The concentration of speed of catching and proto-oncogene molecule is dependency;
Prescribe a time limit on normal range when gene expression, through the electrophoresis of certain hour, proto-oncogene is gone to the opposite side edge of first section hydrogel just; And when gene expression raises, because the speed of hybridization is faster, electrophoresis through the identical time, the proto-oncogene of raised portion can pass first section hydrogel, arrives its downstream, and under electric field action, enter second section hydrogel and calculate molecular hybridization, and corresponding medicine sheet segment molecule is replaced to get off, the amount of replacement is corresponding with the amount that gene expression raises, and the medicine sheet segment molecule that replaces further is delivered to output unit under effect of electric field.
They are two years old, whether reduce and replace in the passage of computing unit of the segmental micro-fluidic chip of relative medicine calculating expression of tumor suppressor gene, be fixed with two sections hydrogels: fixing corresponding calculated molecule in first section hydrogel, and hybridization has corresponding medicine sheet segment molecule in advance; Second section hydrogel is blank hydrogel, only plays the effect of valve, do not participate in calculating;
Genetic suppressor element at first enters first section hydrogel under the low voltage electric field effect, corresponding medicine sheet segment molecule is replaced to get off;
When expression of tumor suppressor gene was prescribed a time limit under normal range, electrophoresis through certain hour, antioncogene is gone to the opposite side edge of first section hydrogel just, and all medicine sheet segment molecules are all replaced, and is substituted the medicine sheet segment molecule that gets off subsequently by reject; When expression of tumor suppressor gene reduced, because replacement speed is slack-off, through the electrophoresis of identical time, still some medicine fragment can not be substituted and be retained in the hydrogel, and the part that replaces is by reject.The part that is retained in the hydrogel is rinsed when changing deposition condition down, and the amount of this part is corresponding with the amount that gene expression reduces, and further is delivered to output unit under effect of electric field.
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