CN1235028C - 利用超声波对杂交反应后固体基质进行清洗的方法 - Google Patents
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Abstract
利用超声波对杂交反应后固体基质进行清洗的方法，该方法是将已完成杂交反应的固体基质放入盛有清洗液的容器中，利用超声波发生器产生一定强度的超声并将其传导至清洗液中，利用该超声在清洗液中产生的空化效应进行清洗。由于清洗液中的空化气泡会对固体表面产生强烈射流及局部微冲流，显著减弱液体表面张力及摩擦力，增强液体的流动，从而可以将固体基质表面结合较弱的非特异性吸附及沉淀快速清洗掉。与常规清洗技术相比，可显著缩短清洗时间，提高杂交信号与背景信号的对比度。其装置结构简单，没有运动部件，易于实现集成化和自动化。本发明可应用于Southern、Northern及基因芯片等各种杂交反应体系。
Description
技术领域
本发明涉及一种利用超声波手段促进杂交反应后对固体基质进行清洗的方法及其装置，特别是涉及生物芯片杂交反应后清洗过程的方法及其装置。此方法及装置可应用于临床诊断、生命科学基础研究、农业及环境监测等技术的领域。
背景技术
非特异性吸附是杂交反应中经常遇到的现象，它往往使得生物和医学杂交检测结果难以分析，因此非特异性吸附的清洗是生物和医学分析检测过程中一个非常重要的环节。目前常用的清洗方法是将待清洗的固体基质表面区域浸入清洗液中，通过机械振荡来完成的。这种方法虽然简便，但是清洗效果差，耗时长，背景信号高，均匀性不好，且因为清洗装置存在运动部件，不易实现集成化和微型化。
发明内容
本发明的目的提供一种利用超声波对杂交反应后固体基质进行清洗的方法，以缩短杂交反应后清洗时间、提高杂交信号与背景信号的对比度。
本发明的目的是通过如下技术方案实现的：该方法包括如下步骤：
(1)将已完成杂交反应的固体基质放入盛有清洗液的容器中，使固体基质的待清洗表面与清洗液接触；
(2)在所述的固体基质的上方、下方或四周至少放置一个超声波谐振元件，超声波发生器的谐振元件与清洗液直接接触或间接接触，使超声波发生器产生超声波传导至清洗液中，利用超声波在清洗液中产生的空化效应促进清洗。
实施清洗时，首先将已完成杂交反应的固体基质放入清洗液中，然后开通超声波谐振电路，驱动超声波谐振元件产生超声，利用此超声传导至清洗液中产生的空化效应促进清洗。因为清洗液中的空化气泡会对固体表面产生强烈射流及局部微冲流，显著减弱液体的表面张力及磨擦力，并破坏固-液界面的附面层，增强液体的流动，从而可以将固体基质表面结合较弱的非特异性吸附及沉淀快速清洗掉。
本发明与常规杂交反应清洗技术相比，可以显著缩短清洗时间(实施例结果显示利用超声波可使基因芯片杂交后的清洗时间比常规机械振荡方法缩短5倍)，提高杂交信号与背景信号的对比度。另外，本发明装置结构简单(核心元件仅有超声谐振元件和相应谐振电路及盛装清洗液的容器)，没有运动部件，易于实现集成化和自动化。
附图说明
图1为本发明提出的实施例的装置结构示意图。
图2为本发明提出的实施例中采用压电陶瓷片作为超声波谐振元件的正面视图。
图3为本发明提出的实施例中采用压电陶瓷片作为超声波谐振元件的背面视图。
图4为本发明提出的实施例与常规机械振荡清洗方法所得到的杂交荧光信号比较图。
图5为本发明提出的实施例与常规机械振荡清洗方法得到的背景荧光的比较图。
具体实施方式
下面结合附图进一步说明本发明的具体实施：
实际应用时，首先将已完成杂交反应的固体基质放入清洗液中，使固体基质的待清洗表面与清洗液接触；超声波谐振元件可以与清洗液之间直接接触，也可以通过环氧树脂等粘性胶贴附于盛有清洗液容器的外壁，位置可以是容器的上部、下部或四周；超声波谐振元件的材料可以是压电陶瓷材料，也可以是石英材料。超声波谐振元件的形状可以是圆柱形、锥形、圆筒形、立方形任何有利于产生超声波谐振的元件的形状。超声波谐振元件的体积可以在10立方微米到1000立方厘米之间变化。超声波谐振元件所产生的超声波频率可以在20kHz到100MHz之间变化。超声波发生器的功率可以在1W到200W之间变化。 超声波发生器产生超声波的方式可以连续进行或间断进行。
图1为本发明的一个具体实施例，该实施例利用超声波促进基因芯片的杂交反应后清洗过程。将一直径为2.5厘米频率为1.7MHz的圆形压电陶瓷片2贴附于一尺寸为8.5cm×5cm×2cm的塑料盒1中，压电陶瓷片2通过塑料盒1侧壁所钻小孔引出导线3与超声波谐振电路5相接。超声波谐振电路5的输出功率为20W。然后将导线引出孔用环氧树脂胶密封。压电陶瓷片2边缘套有环形橡皮密封圈4，橡皮密封圈4通过环氧树脂胶7固定于塑料盒1底部，其作用是使焊点与清洗液隔离，防止引起短路。清洗时，待清洗的基因芯片6浸没于塑料盒1中盛装的清洗液8中。
图2和图3为本发明采用压电陶瓷片作为超声波谐振元件的电极排布图。图2为压电陶瓷片正面视图，图3为压电陶瓷片背面视图：9指示超声压电陶瓷片表面所镀金属电极，3指示与超声压电陶瓷片焊接的导线，10指示超声压电陶瓷片背面裸露的压电材料。
图4和图5为本发明提出的实施例与常规清洗方法所得到的杂交荧光信号及背景荧光强度的比较图。如图4和图5所示实施例的清洗条件1为用机械振荡清洗固液杂交体系15分钟，(振荡频率：80转/分钟，振幅：15mm)，清洗条件2为以1.7MHz的压电陶瓷片在20W功率下清洗同样的固液杂交体系3分钟。清洗时，基因芯片置于压电陶瓷片上方约2mm处，有探针的一面背对压电陶瓷片，并加入清洗液浸没基因芯片，清洗液体积为10mL，清洗液成份为0.2％SDS。
点阵1和点阵2所用的探针、杂交样品及杂交条件均相同。探针为35个碱基的寡核苷酸，通过共价方式连接于醛基修饰的玻片上，样品长度为1045bp的DNA，其中一段与探针完全匹配，样品DNA链上带有Cy5荧光标记分子，杂交条件为65℃、90分钟，检测方法为激光诱导荧光检测，所用仪器为ScanArray 4000，检测器光电倍增管(PMT)的设置值为仪器最大值的75％，激光光源功率均设定为仪器最大值的80％。
从图4和图5中的信号值可以看出，超声辅助条件下清洗3分钟可达到与常规清洗相同的清洗效果，其杂交荧光信号(图4)及背景荧光强度值(图5)均无显著差异。此实验已重复8-9次，实验表明利用超声的空化效应可在较短的时间达到与机械清洗相同的清洗效果，从而可以加快整个检测的速度。
Claims (1)
1.一种利用超声波对杂交反应后的固体基质进行清洗的方法，该方法包括如下步骤：
(1)将已完成杂交反应的固体基质放入盛有清洗液的容器中，使固体基质的待清洗表面与清洗液接触；
(2)在所述的固体基质的上方、下方或四周至少放置一个超声波谐振元件，超声波发生器的谐振元件与清洗液直接接触或间接接触，所述的超声波发生器产生超声波的方式采用连续进行或间断进行，使超声波发生器产生超声波传导至清洗液中，利用超声波在清洗液中产生的空化效应促进清洗；所述超声波发生器的功率在1W～20W之间变化：所述超声波谐振元件所产生的超声波频率在1.7MHz～100MHz之间变化。
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| CNB021288607A CN1235028C (zh) | 2002-08-16 | 2002-08-16 | 利用超声波对杂交反应后固体基质进行清洗的方法 |
| CA002495643A CA2495643A1 (en) | 2002-08-16 | 2002-12-10 | Method and apparatus for washing solid substrate with ultrasonic wave after hybridization reaction |
| AU2002354071A AU2002354071A1 (en) | 2002-08-16 | 2002-12-10 | Method and apparatus for washing solid substrate with ultrasonic wave after hybridization reaction |
| EP02787326A EP1548124A4 (en) | 2002-08-16 | 2002-12-10 | METHOD AND APPARATUS FOR WASHING A SOLID SUBSTRATE USING AN ULTRASONIC WAVE AFTER A HYBRIDIZATION REACTION |
| US10/524,903 US20060124152A1 (en) | 2002-08-16 | 2002-12-10 | Method and apparatus for washing solid substrate with ultrasonic wave after hybridization reaction |
| JP2004528249A JP2005535891A (ja) | 2002-08-16 | 2002-12-10 | ハイブリダイゼーション反応後に、超音波を用いて固体基板を洗浄するための方法および装置 |
| PCT/CN2002/000877 WO2004016808A1 (fr) | 2002-08-16 | 2002-12-10 | Procede et appareil permettant de laver un substrat solide a l'aide d'une onde ultrasonore apres une reaction d'hybridation |
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| CA1296604C (en) * | 1986-03-20 | 1992-03-03 | Kathleen Murphy | Method for releasing rna and dna from cells |
| US5085982A (en) * | 1986-06-12 | 1992-02-04 | City Of Hope | Method of detecting specific substances by selective growth of living cells |
| US4886741A (en) * | 1987-12-09 | 1989-12-12 | Microprobe Corporation | Use of volume exclusion agents for the enhancement of in situ hybridization |
| DE4113157A1 (de) * | 1990-09-06 | 1992-03-12 | Bayer Ag | Hochverzweigte polyphosphonate auf melamin-basis |
| DE4121304A1 (de) * | 1991-06-27 | 1993-01-14 | Siemens Ag | Reinigungsmittel und -verfahren zum entfernen von pasten der dickschicht-hybridtechnologie von substraten |
| JPH0751644A (ja) * | 1993-08-13 | 1995-02-28 | Dainippon Ink & Chem Inc | 電子写真感光体用基体の洗浄方法 |
| US6287850B1 (en) * | 1995-06-07 | 2001-09-11 | Affymetrix, Inc. | Bioarray chip reaction apparatus and its manufacture |
| JPH09327671A (ja) * | 1996-06-12 | 1997-12-22 | Kaijo Corp | 超音波洗浄機 |
| US6291180B1 (en) * | 1999-09-29 | 2001-09-18 | American Registry Of Pathology | Ultrasound-mediated high-speed biological reaction and tissue processing |
| CN1314494A (zh) * | 2000-03-20 | 2001-09-26 | 上海博德基因开发有限公司 | 一种基因芯片荧光标记物的洗涤方法及其专用洗脱液 |
| CN1321890A (zh) * | 2000-04-30 | 2001-11-14 | 南京益来基因医学有限公司 | 核酸扩增基因芯片杂交检测仪 |
| EP1281966A3 (en) * | 2001-07-30 | 2003-06-18 | Fuji Photo Film Co., Ltd. | Method and apparatus for conducting a receptor-ligand reaction |
| CN2495656Y (zh) * | 2001-09-03 | 2002-06-19 | 赵雨杰 | 核酸扩增基因芯片杂交智能化并行检测仪 |
-
2002
- 2002-08-16 CN CNB021288607A patent/CN1235028C/zh not_active Expired - Fee Related
- 2002-12-10 CA CA002495643A patent/CA2495643A1/en not_active Abandoned
- 2002-12-10 WO PCT/CN2002/000877 patent/WO2004016808A1/zh active Application Filing
- 2002-12-10 JP JP2004528249A patent/JP2005535891A/ja active Pending
- 2002-12-10 AU AU2002354071A patent/AU2002354071A1/en not_active Abandoned
- 2002-12-10 EP EP02787326A patent/EP1548124A4/en not_active Withdrawn
- 2002-12-10 US US10/524,903 patent/US20060124152A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| JP2005535891A (ja) | 2005-11-24 |
| US20060124152A1 (en) | 2006-06-15 |
| CN1397790A (zh) | 2003-02-19 |
| EP1548124A1 (en) | 2005-06-29 |
| AU2002354071A1 (en) | 2004-03-03 |
| CA2495643A1 (en) | 2004-02-26 |
| EP1548124A4 (en) | 2006-08-23 |
| WO2004016808A1 (fr) | 2004-02-26 |
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