DE19960398B4 - Method and electrochemical sensor with heated electrodes for biochemical analysis, in particular for the investigation of hybridization processes of the nucleic acids - Google Patents

Abstract

Method for biochemical analysis, in particular for hybridizing the nucleic acids by means of an electrochemical sensor, characterized in that
a) the electrochemical sensor on its surface or in the electrode body is modified with nucleic acid molecules or fragments thereof, which serve as probe sequences for recognizing the target sequences,
b) the electrochemical sensor is heated directly by means of alternating current without the electrochemical measurements taking place at the same time being significantly impaired, and
c) only a very thin layer of the examined solution is heated near the electrode surface, but the majority of the solution remains at an almost unchanged temperature

Description

The invention relates to a method and an electrochemical sensor with heated electrodes for biochemical Analytics, in particular for the investigation of hybridization processes of the Nucleic acids.

Nucleic acids, especially the different ones Forms of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), but also synthetically produced like peptide nucleic acids (PNA) in large Scope for the analysis of biologically active substances and the identification of individual ones Organisms used.

The basis is usually the process of hybridization (Dangler, CA, ed., Nucleic acid analysis: Principles and bioapplications. Wiley-Liss, New York 1996; Ross, J .; Nucleic acid hybridization: Essential techniques ₁ Wiley, New York 1997).

A certain fragment of a nucleic acid is considered Probe (probe sequence or probe nucleic acid sequence) for detection another nucleic acid used, which usually is in the form of short or long fragments and the so-called Target sequence (or target nucleic acid sequence) forms. The latter is made up of the double helix structure normally present won by denaturation. In this state, the molecules combine the probe sequence and the target sequence, i.e. hybridize if their molecular structure is strictly complementary and therefore precise coverage of certain parts of the molecule, the "bases", becomes possible (Guanine only with cytosine and adenine only with thymine). Ribonucleic acid strands (e.g. from gene expression) also occur as a target sequence. Another form of hybridization is the formation of a triplex from double-stranded and single-stranded nucleic acid molecules.

The successful hybridization delivers highly selective detection of individual target sequences and thus individual genes or biological species.

A known embodiment of electrochemical sensors of the type mentioned form electrode surfaces where probe sequences have been immobilized (Mikkelsen, S.R., Electroanalysis 8 (1996) 15; Wang, J. et al., Anal. Chim. Acta 347 (1997)).

The successful hybridization will recognizable by electrochemical effects, which in many cases also can be used for a quantitative analysis of the amount of target sequences are. Such electrochemical processes represent a further development and simplification of routine methods for nucleic acid analysis represents.

In most known methods for nucleic acid analysis, where hybridization is important, temperature increases are used to the double or triple strands of nucleic acids or to separate the hybrid from fragments of nucleic acid. In the case of probe sequences, immobilized on electrode surfaces are e.g. a temporary Temperature increase up to serve just below the boiling point of the water Target Molecule free from the hybrid with the probe sequence and thereby the electrode surface for the next recognition process free. Typical for Electrochemical sensors for nucleic acid analysis are therefore often repeated temperature changes between an upper temperature at which the complementary probe and target sequences are separated, and a lower temperature (usually close to room temperature) at which hybridization is possible. Both limit temperatures must with high precision can be set.

Another process in nucleic acid analysis, at the temperature increases play a role is the release of natural DNA or RNA from biological Cells.

The necessary thermostat processes for the above temperature changes in electrochemical measuring cells are associated with a high expenditure of time and work. Such investigations can also be carried out generally only in an appropriately equipped laboratory carried out and not e.g. in a remote area of the environment. A such decentralized diagnostics would be however desirable since the transportation of organisms or substances from them in far remote laboratories is not always possible. Another disadvantage The known method is that the total volume of the examined Solution the temperature changes is exposed, causing an unnecessary thermal load on the biologically important substances present there. Lots these substances are particularly at risk from elevated temperatures.

The present invention lies the problem underlying a process and an electrochemical Sensor for hybridization of the nucleic acids to realize the desired temperature changes generated directly on the electrode surface by the electrode is itself heated by means of electrical alternating current. The electrochemical Measurement should be done both during as well as before or after the heating process. That's why guarantee, that for Nucleic acid analysis typical small measuring currents not by many orders of magnitude stronger Heating current disturbed become.

According to the invention, this problem is solved by that in the claims 1 to 5 listed Features solved.

The electrochemical sensors required to carry out the method can each contain a working electrode, for example in the form of thin fibers or layers, and conductive strands of pasty mixtures. Preferred materials are carbon, carbon-containing masses or conductive organic polymers, as well as metals. The working electrode is heated directly with alternating current on a symmetrically designed electrode arrangement in a manner known per se. (Comp. DE-OS 195 43 060 A1 Such an electrochemical sensor ensures that the alternating current used for heating does not interfere with the electrochemical measurement process.

If the electrochemical measurements however not during of the heating process, but e.g. before or after are provided is also a simpler asymmetrical arrangement of the working electrode usable.

The fixation or immobilization naturally or more artificial nucleic acids or their molecular fragments as a probe sequence can be carried out according to all previously known methods, so e.g. by mixing with a pasty conductive electrode material electrostatic fixation or by forming a homeopolar Bond over corresponding functional groups.

Since the known method for direct electrode heating ( DE-OS 195 43 060 A1 ) the precise knowledge and precise dosing of temperature and heat spread enables the requirements of biochemical analysis, in particular hybridization techniques with nucleic acids, to be met even without the use of external thermostatting.

The method according to the invention therefore has the Advantage that the surface temperature the small working electrode modified with the probe sequences precise is checked without the arbitrarily caused temperature change over a spatial small area, i.e. a very thin layer in the micrometer range goes directly to the working electrode surface. This will an impairment in the solution Avoid existing sensitive substances. The required temperature changes can brought about at high speed as temperature jumps and can be repeated any number of times. The process is therefore easy to automate. The energy expenditure for that The process of producing the upper limit temperature is so low that the entire apparatus operated without problems with batteries and as portable Device can. This makes it an off-road application far from human Settlements possible. The electrochemical sensors used are process-related small. In contrast to previous practice, the procedure requires not closed vessels, but can be placed anywhere, e.g. even in tight, inaccessible tubes, in flowing Stream or can even be used in living organisms.

The invention will follow an embodiment explained in more detail.

A known electrochemical sensor for the detection of the hybridization, which on its Au working electrode via -SH contains immobilized probe sequences is designed such that Electric heating and electrochemical at the same time Measurement possible is. This is achieved by using a thin gold wire (25 μm diameter) is used, which contacts at both ends in a suitable manner is. Can continue corresponding gold layers on different substrates (glass, Plastic) can be used.

Claims (5)

Process for biochemical analysis, in particular for hybridizing the nucleic acids by means of an electrochemical sensor, characterized in that a) the electrochemical sensor is modified on its surface or in the electrode body with nucleic acid molecules or fragments thereof, which serve as probe sequences for recognizing the target sequences , b) the electrochemical sensor is heated directly by means of alternating current without the electrochemical measurements taking place at the same time being significantly impaired, and c) only a very thin layer of the investigated solution is heated near the electrode surface, but the majority of the solution remains at an almost unchanged temperature Electrochemical sensor for performing the The method of claim 1, wherein a) electrochemical sensor on its surface or in the electrode body immobilized nucleic acid molecules or fragments of which contains which can recognize a target sequence as a probe sequence, and b) electrochemical sensor with alternating current within an electrochemical The measuring cell can be heated, so that it can run simultaneously or in close proximity with the heating process a measurement of the electrochemical changes as a result of biochemical processes Parameters can take place. Electrochemical sensor according to claim 2, characterized characterized that the working electrode of the sensor made of carbon Materials, especially carbon pastes or layers of carbon with them brought in or superficial adsorbed probe nucleic acid sequences consists. Electrochemical sensor according to claim 2, characterized characterized that the working electrode of the sensor made of metal exists and the probe nucleic acid sequences via suitable Atoms or groups of atoms are bound to its surface. Electrochemical sensor according to claim 2, characterized in that the working electrode the sensor is coated with layers of conductive polymers to which the probe nucleic acid sequences are chemically bound.