CN100535666C - Isoelectronic focusing electrophoretic device for inspecting polypeptide agonist - Google Patents
Isoelectronic focusing electrophoretic device for inspecting polypeptide agonist Download PDFInfo
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- CN100535666C CN100535666C CNB200610024274XA CN200610024274A CN100535666C CN 100535666 C CN100535666 C CN 100535666C CN B200610024274X A CNB200610024274X A CN B200610024274XA CN 200610024274 A CN200610024274 A CN 200610024274A CN 100535666 C CN100535666 C CN 100535666C
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Abstract
An isoelectric focusing electrophoresis device for detecting polypeptide analeptic is prepared as connecting anode and cathode to positive and negative electrode of DC power supply, placing anode and cathode in their tubes and connecting anode tube and cathode tube to anode sample tube and cathode sample tube, connecting said sample tubes to two ends of quartz capillary tube free of coating, setting operation platform on box base, placing ultraviolet lamp at two ends of said capillary tube, setting CCD digital imaging instrument at box top and connecting CCD digital imaging instrument to computer through data line .
Description
Technical field
What the present invention relates to is the device in a kind of detection technique field, and specifically, what relate to is a kind of isoelectronic focusing electrophoretic device that peptide excitant detects that is used for.
Background technology
Auxin (hGH) and erythropoietin (hEPO) etc. are former sex hormone in the human body.Owing to can significantly improve sports achievement, the abuse of this class peptide excitant (especially through reorganization r-hGH and r-hEPO) is the trend of obvious rising in the whole world.Mainly contain two class methods at present and be used for the examination of r-hGH and rhEPO abuse, both indirect method and direct method.The indirect method kind is a lot, and preliminary examination foundation can be provided, but conclusive examination foundation can not be provided.Direct detecting method can provide conclusive examination foundation.
Find through the literature search to prior art: Breidbach etc. are in " Clinical Chemistry " (U.S.'s clinical chemistry) (2003, delivered the technical papers of " Detection of Recombinant HumanErythropoietin in Urine by Isoelectric Focusing " (the isoelectric focusing electrophoresis method is measured the recombined human erythropoietin) 49:901-907), this paper has been introduced the basic skills of erythropoietin: 1, urine specimen is carried out ultrafiltration and concentration, 2, urine sample after concentrating is carried out isoelectric focusing electrophoresis separates, 3, separate the back erythropoietin is carried out electrotransfer, 4, erythropoietin after shifting is carried out chemiluminescence handle 5, the CCD digitalized image.But because the on-line preconcentration method of no peptide excitant, exist simultaneously the instability of pH gradient and gel isoelectric focusing electrophoresis-electrotransfer-chemiluminescent time long in IEF, the apparatus and method of adding factors such as content is very low, Breidbach in the short and urine of half life period of peptide excitant can't be applied to actual peptide excitant detection detection.
Summary of the invention
The present invention is directed to the deficiency of prior art existence, a kind of isoelectronic focusing electrophoretic device that peptide excitant detects that is used for is provided, make the speed of its sensitivity that can significantly improve detection, obviously raising detection and flux, raising simultaneously detect stability and repeated.
The present invention is achieved by the following technical solutions, device of the present invention comprises: anode, anode tube, anode sample hose, no external coating quartz capillary, cathode sample pipe, cathode tube, negative electrode, barrier film, direct supply, electrode wires, action pane, door and window, operating platform, support column, cabinet base, casing, case top, uviol lamp, ultraviolet reflector, CCD digital imagery instrument, computing machine, data line.Annexation is: anode is connected with negative pole with the positive pole of direct supply respectively through electrode wires with negative electrode, anode and negative electrode are positioned over respectively in anode tube and the cathode tube, anode tube is connected with the cathode sample pipe with the anode sample hose respectively through barrier film with cathode tube, and the two ends of no external coating quartz capillary connect anode sample hose and cathode sample pipe respectively; By above connection, anode, anode tube, anode sample hose, no external coating quartz capillary, cathode sample pipe, cathode tube, negative electrode and barrier film form the basic separative element of isoelectric focusing electrophoresis; Basic separative element is connected on the operating platform, and operating platform is connected on the cabinet base by support column, and cabinet base is connected with casing, and connecting box top in casing top is provided with an action pane on casing, and action pane connects the door and window that can close unlatching; Uviol lamp is positioned over no external coating quartz capillary both sides, and the side is provided with the ultraviolet light reflector on the uviol lamp, and ultraviolet light shines uniformly on no external coating quartz capillary during imaging; CCD digital imagery instrument is connected on the case top, and CCD digital imagery instrument is connected with computing machine by data line, and CCD digital imagery instrument and computing machine form digitalized image and disposal system.
Described basic separative element, its quantity between 1-960, parallel arranged each other.
Described no external coating quartz capillary, length is at 5-30 centimetre, the internal diameter carrier ampholyte and the 0.1-10% antiseptic of even distribution 0.5-5% difference pH gradients in coating 0.5-10% bifunctional material, the pipe in 10-200 micron, the no polymer coating in surface, pipe.
Described direct supply, the adjustable voltage of its output between 1-5000 volt, the scalable electric current between the 1-200 milliampere, the controllable time is between 1 second-2 hours.
Described operating platform, support column, cabinet base, casing, case top, action pane, door and window are formed can close unlatching mechanical support unit, offers action pane in the casing front.
When the present invention works, at first urine sample is carried out fluorescent dye/mark.To dye afterwards/the good urine sample of mark is with regulating with anolyte and catholyte respectively, and the urine sample that regulates adds anode and cathode sample pipe respectively.Prefabricated no external coating contain ampholyte linear polyacrylamide gel quartz capillary pipe be connected with the cathode sample pipe with the anode sample hose, again anolyte and catholyte are added anode tube and cathode tube respectively.Connect anode tube, anode sample hose, quartz capillary, cathode sample pipe and cathode tube successively, apply DC voltage and carry out enrichment, desalination and IEF and separate.Separate finish after, carry out UV-irradiation, peptide excitant fluorescence developing, CCD digitalized image after separating.Machine changes into chromatogram image after handling as calculated, carries out etiologic diagnosis and quantitative Analysis simultaneously.
The present invention has following advantage and effect:
The first, greatly improve detection sensitivity, solve the problem of peptide excitant detection sensitivity.Peptide excitant is fast the human body metabolism, and the half life period is short, and content is extremely low in the urine, therefore is difficult to detect.Owing to adopt the bilateral large volume sample injection, the multiple of quartz capillary volume sampling volume can reach thousand times relatively in the present invention, even ten thousand times.This can bring up to detection sensitivity more than thousand times.
The second, significantly improve detection speed, greatly shorten the time of analyzing and testing.The separation times such as traditional cellulose acetate membrane electrophoresis, PAGE, gel IEF are long, and the time of separating operations such as the back is fixing, dyeing, decolouring is longer.Therefore the speed that detects is slow, flux is low.Even quartz capillary IEF also exists similar problem, especially flux problems.The present invention gives full play to the quick advantage of quartz capillary IEF, adopts the real-time fluorescence imaging, has avoided above problem to a great extent, and the analyzing and testing time can finish in very short time.Parallel quartz capillary synchronous detection has improved the flux that detects greatly.
Three, improve the stable and repeated of IEF.The used electrolyte of the present invention designs according to the MCRB method, can carry out stable example enrichment, desalination and IEF enrichment.The problems such as instability that this has solved conventional I EF have to a great extent improved the stable and repeated of IEF.
Four, significantly reduced the use cost of IEF.The gel use amount of conventional I EF is big, and especially Ang Gui carrier ampholyte consumption is very big.The present invention reduced to a great extent expensive ampholyte use amount, reduced use cost.
Description of drawings
Fig. 1 is the structural representation of apparatus of the present invention.
Among Fig. 1,1 be anode, 2 for anode tube, 3 for the anode sample hose, 4 for no external coating quartz capillary, 5 for the cathode sample pipe, 6 for cathode tube, 7 for negative electrode, 8 for barrier film, 9 for direct supply, 10 for electrode wires, 11 for action pane, 12 for door and window, 13 for operating platform, 14 for support column, 15 for base, 16 for casing, 17 for case top, 18 for uviol lamp, 19 for the ultraviolet reflector, 20 for ultraviolet light, 21 for CCD digital imagery instrument, 22 be that computing machine, 23 is a data line.
Embodiment
As shown in Figure 1, the present invention includes: anode 1, anode tube 2, anode sample hose 3, no external coating quartz capillary 4, cathode sample pipe 5, cathode tube 6, negative electrode 7, barrier film 8, direct supply 9, electrode wires 10, action pane 11, door and window 12, operating platform 13, support column 14, cabinet base 15, casing 16, case top 17, uviol lamp 18, ultraviolet reflector 19, CCD digital imagery instrument 21, computing machine 22, data line 23.Annexation is: anode 1 is connected with negative pole with the positive pole of direct supply 9 respectively through electrode wires 10 with negative electrode 7, anode 1 and negative electrode 7 are positioned over respectively in anode tube 2 and the cathode tube 6, anode tube 2 is connected with cathode sample pipe 5 with anode sample hose 3 respectively through barrier film 8 with cathode tube 6, and the two ends of no external coating quartz capillary 4 connect anode sample hose 3 and cathode sample pipe 5 respectively; Operating platform 13 is connected on the cabinet base 15 by support column 14, and cabinet base 15 is connected with casing 16, and connecting box top, casing 16 top 17 is provided with an action pane 11 on casing 16, and action pane 11 connects the door and window 12 that can close unlatching; Uviol lamp 18 is positioned over no external coating quartz capillary 4 both sides, and the side is provided with ultraviolet light reflector 19 on the uviol lamp 18, and ultraviolet light 20 shines uniformly on no external coating quartz capillary 4 during imaging; CCD digital imagery instrument 21 is connected on the case top 17, and CCD digital imagery instrument 21 is connected with computing machine 22 by data line 23, and CCD digital imagery instrument 21 and computing machine 22 form digitalized image and disposal system.
Described anode 1, anode tube 2, anode sample hose 3, no external coating quartz capillary 4, cathode sample pipe 5, cathode tube 6, negative electrode 7 and barrier film 8 constitute the basic separative element of isoelectric focusing electrophoresis; Basic separative element is connected on the operating platform 13.
Described basic separative element quantity between 1-960, parallel arranged each other.
Described no external coating quartz capillary 4, length is at 5-30 centimetre, the internal diameter carrier ampholyte and the 0.1-10% antiseptic of even distribution 0.5-5% difference pH gradients in coating 0.5-10% bifunctional material, the pipe in 10-200 micron, the no organic polymer coating in surface, pipe.
Described direct supply 9, the adjustable voltage of its output between 1-5000 volt, the scalable electric current between the 1-200 milliampere, the controllable time is between 1 second-2 hours.
Described operating platform 13, support column 14, cabinet base 15, casing 16, case top 17, action pane 11, door and window 12 are formed can close unlatching mechanical support unit, offers action pane 11 in casing 16 fronts.
Claims (8)
1, a kind of isoelectronic focusing electrophoretic device that is used for the peptide excitant detection, comprise: anode (1), anode tube (2), anode sample hose (3), no external coating quartz capillary (4), cathode sample pipe (5), cathode tube (6), negative electrode (7), barrier film (8), direct supply (9), electrode wires (10), action pane (11), door and window (12), operating platform (13), support column (14), cabinet base (15), casing (16), case top (17), uviol lamp (18), ultraviolet reflector (19), CCD digital imagery instrument (21), computing machine (22), data line (23), it is characterized in that: anode (1) is connected with negative pole with the positive pole of direct supply (9) respectively through electrode wires (10) with negative electrode (7), anode (1) and negative electrode (7) are positioned over respectively in anode tube (2) and the cathode tube (6), anode tube (2) is connected with cathode sample pipe (5) with anode sample hose (3) respectively through barrier film (8) with cathode tube (6), the two ends of no external coating quartz capillary (4) connect anode sample hose (3) and cathode sample pipe (5) respectively, operating platform (13) is connected on the cabinet base (15) by support column (14), cabinet base (15) is connected with casing (16), connecting box top, casing (16) top (17), on casing (16), be provided with an action pane (11), action pane (11) connects door and window (12), uviol lamp (18) is positioned over no external coating quartz capillary (4) both sides, uviol lamp (18) is gone up the side and is provided with ultraviolet light reflector (19), ultraviolet light during imaging (20) shines uniformly on no external coating quartz capillary (4), CCD digital imagery instrument (21) is connected on the case top (17), and CCD digital imagery instrument (21) is connected with computing machine (22) by data line (23).
2, the isoelectronic focusing electrophoretic device that is used for the peptide excitant detection according to claim 1, it is characterized in that, anode (1), anode tube (2), anode sample hose (3), no external coating quartz capillary (4), cathode sample pipe (5), cathode tube (6), negative electrode (7) and barrier film (8) constitute the basic separative element of isoelectric focusing electrophoresis, and basic separative element is connected on the operating platform (13).
3, according to claim 1ly be used for the isoelectronic focusing electrophoretic device that peptide excitant detects, it is characterized in that, described no external coating quartz capillary (4), length at 5-30 centimetre, internal diameter at the 10-200 micron.
4, according to claim 1 or the 2 or 3 described isoelectronic focusing electrophoretic devices that are used for the peptide excitant detection, it is characterized in that described no external coating quartz capillary (4), coating 0.5-10% bifunctional material in the pipe.
5, according to claim 1 or the 2 or 3 described isoelectronic focusing electrophoretic devices that are used for the peptide excitant detection, it is characterized in that, described no external coating quartz capillary (4), the carrier ampholyte and the 0.1-10% antiseptic of even distribution 0.5-5%pH gradient in the pipe.
6, the isoelectronic focusing electrophoretic device that is used for the peptide excitant detection according to claim 1, it is characterized in that, described operating platform (13), support column (14), cabinet base (15), casing (16), case top (17), action pane (11), door and window (12) are formed the mechanical support unit, offer action pane (11) in casing (16) front.
7, the isoelectronic focusing electrophoretic device that is used for the peptide excitant detection according to claim 1, it is characterized in that, described direct supply (9), the regulation voltage of its output between 1-5000 volt, regulate electric current between the 1-200 milliampere, the control time is between 1 second-2 hours.
8, the isoelectronic focusing electrophoretic device that is used for the peptide excitant detection according to claim 1 is characterized in that, described CCD digital imagery instrument (21) and computing machine (22) form digitalized image and disposal system.
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| CNB200610024274XA CN100535666C (en) | 2006-03-02 | 2006-03-02 | Isoelectronic focusing electrophoretic device for inspecting polypeptide agonist |
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| CNB200610024274XA CN100535666C (en) | 2006-03-02 | 2006-03-02 | Isoelectronic focusing electrophoretic device for inspecting polypeptide agonist |
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| CN102120754B (en) * | 2010-11-30 | 2013-09-11 | 上海交通大学 | Miniature capillary array device for isoelectric focusing electrophoresis |
| CN104034789A (en) * | 2013-04-02 | 2014-09-10 | 上海交通大学 | Protein isoelectric focusing electrophoresis device |
| CN106932459A (en) * | 2015-12-31 | 2017-07-07 | 深圳翰宇药业股份有限公司 | A kind of polypeptide polymer capillary isoelectric focusing analysis method |
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Non-Patent Citations (4)
| Title |
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| 毛细管等电聚焦电泳技术进展. 杨春等.色谱，第21卷第2期. 2003 |
| 毛细管等电聚焦电泳技术进展. 杨春等.色谱，第21卷第2期. 2003 * |
| 激光诱导荧光检测芯片毛细管电泳分离蛋白质和多肽的研究. 唐敏.中国优秀博硕士学位论文全文数据库. 2005 |
| 激光诱导荧光检测芯片毛细管电泳分离蛋白质和多肽的研究. 唐敏.中国优秀博硕士学位论文全文数据库. 2005 * |
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Effective date of registration: 20190107 Address after: 201108 room 2, 6 building, 4299 Jin Du Road, Minhang District, Shanghai, C8 Co-patentee after: Cao Chengxi Patentee after: Shanghai Jiaotong University Intellectual Property Management Co., Ltd. Address before: 200240 No. 800, Dongchuan Road, Shanghai, Minhang District Patentee before: Shanghai Jiao Tong University |