Electrical impulses play an important role in cells of the human body. For example, neurons use these impulses to transmit information along their branches and the body also uses them to control the contraction of muscles. The impulses are generated when special channel proteins open in the outer envelope of the cells, allowing charged molecules (ions) to enter or exit the cell. These proteins are referred to as ion channels. Since the 1970s, a method has been available to researchers that enables measurement of the activity of these channels, but until now this method has been used primarily on cells that do not move. Electrical engineers at ETH Zurich and biologists from the University of Bern have now developed the method further, so that they can easily record the activity of moving cells, such as beating cardiac muscle cells in a tissue culture dish.
The existing method involves positioning a glass pipette against the outer membrane of a cell. The opening at the tip of the pipette is so small that it touches only a fraction of the cell surface. Ideally, this tiny patch of cell membrane has exactly one ion channel. The inside of the pipette is filled with a conductive fluid and an electrode, which makes it possible to measure differences in the charge between the outer part of the cell and cell interior (i.e. an electric potential ) and temporary changes in this potential resulting from activity in the ion channels. The method is referred to as the patch-clamp technique because the pipette is used to clamp a patch of the cell membrane.
Atomic force microscope with micro-injection needle
Lead by Tomaso Zambelli, a lecturer at the Institute of Biomedical Engineering at ETH Zurich, and Hugues Abriel, a professor at the Department of Clinical Research at the University of Bern, the researchers have now combined this technique with an atomic force microscope. A sensor tip is seated on a movable mount -- a so called cantilever -- to scan the surface of the microscopic object. Several years ago, the researchers succeeded in producing sensor tips with an internal channel, which allows the computer-controlled injection of molecules into a cell. This technique is now being marketed by the ETH spin-off Cytosurge. However, the scientists continued development of this technique by fitting the micro-injection needle with an electrode to carry out patch-clamp measurements. The researchers have now published the successful results of this venture in the journal Nano Letters.
The patch-clamp technique is not only a central method for basic research in cell biology, it is also used routinely in the development of new drugs. For example, the pharmaceutical industry is legally required as part of the approval process for new drugs to test whether these drugs interact with ion channels. A drug that blocks ion channels may cause severe cardiac dysrhythmia in patients, which should be avoided at all costs.
Longer measurements and automation possible
In the case of the conventional patch-clamp technique, an operator manually positions the pipette against the cell; although automated procedures exists, their applications are limited. Thus, the cells under test must have the same size and shape and must not move (as cardiac cells do).
In the case of the new method, the micro-needle is controlled by a computer using force measurements from the atomic force microscope to hold it at a constant short distance from the cell surface. "This makes the contact between the needle and cell much more stable, which allows us to take measurements over a longer period of time and even test moving cells," explains Zambelli. For the first time, researchers have thus succeeded in measuring electric potential changes in the ion channels of beating cardiac cells. Zambelli says he can also imagine using this as a foundation for development of an automated method for testing any cell, regardless of its shape or size.
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Misukumo ya umeme hutimiza fungu muhimu katika chembe za mwili wa binadamu. Kwa mfano, nyuroni hutumia msukumo huo kuhamisha habari kwenye matawi yao, na mwili pia huutumia kudhibiti misuli. Mlipuko huo hutokea wakati protini maalum za njia zinapofunguka katika kifuniko cha nje cha chembe, na kuruhusu molekuli zenye nishati (ioni) kuingia au kutoka kwenye chembe. Protini hizo huitwa njia za ioni. Tangu miaka ya 1970, mbinu imekuwa inapatikana kwa watafiti ambayo inaruhusu kipimo cha shughuli za njia hizi, lakini hadi sasa mbinu hii imekuwa kutumika hasa juu ya seli ambayo si kusonga. Wataalamu wa ETH Zurich na wanasayansi wa Chuo Kikuu cha Bern wamekuwa wakifanya utafiti wa kutosha kuhusu jinsi ya kuhifadhi chembe za moyo katika chombo cha kukuza tishu. Njia iliyopo inahusisha kuweka pipette ya kioo dhidi ya utando wa nje wa chembe. Ufunguo ulio kwenye ncha ya pipeti ni mdogo sana hivi kwamba unagusa sehemu ndogo tu ya uso wa chembe. Kwa kawaida, sehemu hii ndogo ya utando wa chembe huwa na njia moja tu ya ioni. Pipette ina sehemu ya ndani iliyojaa umeme na electrode, ambayo inawezesha kupima tofauti za malipo kati ya sehemu ya nje ya seli na sehemu ya ndani ya seli. Mabadiliko ya muda katika uwezo huu unaotokana na shughuli katika njia za ion. Mbinu hiyo inaitwa mbinu ya patch-clamp kwa sababu pipette hutumiwa kufunga sehemu ya utando wa chembe. "Michoro ya ""Atomic Force Microscope"" iliyoundwa na Tommaso Zambelli, mhadhiri wa Taasisi ya Uhandisi wa Biomedical katika ETH Zurich, na Hugh Abreu, profesa katika Idara ya Utafiti wa Kliniki katika Chuo Kikuu cha Bern, sasa imeunganisha mbinu hii na darubini ya nguvu ya atomiki." Ncha ya sensorer imeketi kwenye kifaa kinachoweza kuhamishwa - kinachoitwa cantilever - ili kuchunguza uso wa kitu kidogo. Miaka kadhaa iliyopita, watafiti walifanikiwa kutengeneza vidole vya sensorer vyenye njia ya ndani, ambayo inaruhusu kuingiza molekuli ndani ya seli kwa kudhibitiwa na kompyuta. Mbinu hii sasa inazalishwa na ETH spin-off Cytosurge. Hata hivyo, wanasayansi waliendelea kuendeleza mbinu hii kwa kuunganisha sindano ya sindano ndogo na elektroni ili kufanya vipimo vya patch-clamp. Watafiti sasa wamechapisha matokeo ya mafanikio ya mradi huu katika jarida la Nano Letters. Utaratibu wa patch-clamp si njia kuu tu kwa ajili ya utafiti wa msingi katika biolojia ya seli, pia hutumiwa kwa kawaida katika maendeleo ya dawa mpya. Kwa mfano, sekta ya dawa inahitajika kisheria kama sehemu ya mchakato wa idhini ya dawa mpya kupima kama dawa hizi kuingiliana na ion njia. Dawa inayozuia njia za ioni yaweza kusababisha ugonjwa mbaya wa moyo kwa wagonjwa, ambao unapaswa kuepukwa kwa gharama yoyote ile. Kupima kwa muda mrefu na automatisering inawezekana katika kesi ya kawaida patch-clamp mbinu, operator manually nafasi pipette dhidi ya kiini; ingawa utaratibu automatiska ipo, maombi yao ni mdogo. Kwa hiyo, chembe zinazochunguzwa lazima ziwe na ukubwa na umbo lilelile na hazipaswi kuhamia (kama vile chembe za moyo zinavyofanya). Katika kesi ya mbinu mpya, micro-sugua ni kudhibitiwa na kompyuta kwa kutumia nguvu vipimo kutoka atomic nguvu darubini, na kushikilia katika umbali wa muda mfupi kutoka uso wa seli. "Hii hufanya mawasiliano kati ya sindano na seli kuwa imara zaidi, ambayo inaruhusu kufanya vipimo kwa muda mrefu na hata kupima seli zinazozunguka, "Zambelli anaelezea." Kwa mara ya kwanza, watafiti wamefanikiwa kupima mabadiliko ya uwezo wa umeme katika njia za ioni za chembe za moyo zinazopiga. Zambelli anasema anaweza pia kufikiria kutumia hii kama msingi wa maendeleo ya njia ya kiotomatiki ya kupima seli yoyote, bila kujali umbo au ukubwa wake. Namba hii ya ukurasa: