The invention relates to a method and an apparatus for determining analytes by electronic detection using a microfluidic support.
In recent years, the technology of receptor arrays immobilized on a support, for example DNA chips, has established a valuable means which enables complex analyte determination methods to be carried out rapidly and in a highly parallel manner. The biophysical principle on which the receptor arrays are based is that of the interaction of a specific immobilized receptor with an analyte present in a liquid phase, for example via nucleic acid hybridization, the support being provided with a multiplicity of receptors, for example hybridization probes, which bind specifically to analytes present in the sample, for example complementary nucleic acid analytes.
A binding event between immobilized receptor and analyte is usually detected via detection of a marker group which is bound to the analyte. A support and a method for analyte determination, which allow an integrated synthesis of receptors and analysis, are described, for example, in WO 00/13018. However, such supports and methods have the disadvantage that binding of analytes without marker group to the receptor cannot be readily detected.
DE 199 01 761, DE 199 21 940 and DE 199 26 457 relate to methods for the electrochemical or electronic detection of nucleic acid hybridization events. In this connection, single-stranded hybridization probes whose one end is bound to a support surface and whose other, free end is linked to a redox active unit serve as hybridization matrix. Hybridization of a nucleic acid analyte increases the originally nonexistent or only weak electric communication between the conductive surface area of the support and the redox active unit. Thus it is possible to detect a hybridization event by electrochemical methods such as voltammetry, amperometry or conductivity measurement. In this connection, photo-inducible or chemically inducible redox active units may be used.
Further methods for electrochemical or electronic detection of hybridization events are described in WO 93/20230, WO 95/12808, WO 97/41425, WO 98/30893, WO 98/51819, WO 00/11473, WO 99/37819, WO 96/40712, U.S. Pat. No. 5,968,745, U.S. Pat. No. 5,952,172 and JP-A-92 88 080.
It was the object of the present invention to provide an integrated system which allows highly parallel in situ preparation of complex populations of receptors, immobilized in microstructures, for the detection of analytes.
The present invention therefore relates to a method for determining analytes, which comprises the following steps:
The invention further relates to an apparatus for determining analytes, which comprises
The present invention is distinguished in particular by the fact that the detection system for analyte determination combines a light source matrix, a microfluidic support and an electronic detection matrix in an at least partly integrated structure. Said detection system may be used for integrated synthesis and analysis, in particular for the construction of complex supports, for example biochips, and for the analysis of complex samples, for example for genome, gene expression or proteome analysis.
In a particularly preferred embodiment, the receptors are synthesized in situ on the support, for example by directing fluid containing receptor synthesis building blocks over the support, immobilizing said building blocks location- or/and time-specifically at in each case predetermined areas on the support and repeating these steps until the desired receptors have been synthesized at the in each case predetermined areas on the support. Said receptor synthesis preferably comprises at least one illumination step initiated by the light source matrix or/and a process step mediated by the electronic detection matrix and also on-line process monitoring, for example by using the electronic detection matrix. It is possible here to use for the receptor synthesis electronically removable protective groups such as, for example, p-nitrobenzyloxycarbonyl, 2-(p-nitrophenyl)ethyloxycarbonyl, 2,4-dinitrobenzyl oxycarbonyl or/and 2,4(p-dinitrophenyl)ethyl oxycarbonyl.
The light source matrix is preferably a programmable light source matrix, for example selected from the group consisting of a light valve matrix, a mirror array, a UV-laser array and a UV-LED (diode) array.
The support is a flow cell or a microflow cell, i.e. a microfluidic support having channels, preferably closed channels, which contain the predetermined positions with the in each case differently immobilized receptors. The channels preferably have diameters in the range from 10 to 10,000 μm, particularly preferably from 50 to 250 μm, and may in principle be designed in any form, for example having round, oval, square or rectangular cross sections.
The electronic detection matrix contains a plurality of electrodes which are assigned to those areas of the support on which receptors are immobilized. Preference is given to assigning to an area with in each case identical receptors a separate electrode which may be surrounded, for example, by an insulator area. The electrodes of the electronic detection matrix contain a conductive material such as, for example, a metal, for example silicon, a conductive polymer or a conductive glass. The electrodes preferably form an integral part of the microfluidic support and may form, for example, part of the walls of the microchannels of the support. Furthermore, the support is preferably at least partly optically transparent, in particular on the side facing the light source matrix. However, it is not necessary for the support to be optically transparent on both sides. The electrode areas are preferably in the range from 15 to 250,000 μm2, particularly preferably in the range from 15 to 2,500 μm2.
Electronic detection may be carried out according to known techniques (see, for example, the abovementioned documents), for example by measuring parameters which change in a detectable manner, owing to binding of an analyte to the receptor. Examples of such parameters are conductivity, impedance, voltage or/and current, all of which can be determined via the electrodes using a suitable electronic detector. Depending on the structure of the analytical apparatus, the measurement may comprise a potentiometric measurement, a cyclovoltametric measurement, an amperometric measurement, a chronopotentiometric measurement or another suitable principle of measurement.
In a particularly preferred embodiment, the detection comprises a light source matrix-initiated redox process which correlates with the binding of analytes, for example by hybridization, to the receptors immobilized on the support.
The receptors are preferably selected from biopolymers which may be synthesized in situ on the support from the appropriate synthesis building blocks by light-controlled or/and chemical processes, for example nucleic acids such as DNA, RNA, nucleic acid analogs such as peptide nucleic acids (PNA), proteins, peptides and carbohydrates. Particular preference is given to selecting the receptors from the group consisting of nucleic acids and nucleic acid analogs, and binding of the analytes comprises a hybridization.
The analyte determination of the invention preferably comprises parallel determination of a plurality of analytes, i.e. a support is provided which contains a plurality of different receptors which can react with in each case different analytes in a single sample. Preference is given to the method of the invention determining at least 50, preferably at least 100 and particularly preferably at least 200, analytes in parallel.
The receptors may be immobilized to the support by covalent binding, noncovalent self assembly, charge interaction or combinations thereof. Covalent binding preferably comprises providing a support surface having a chemically reactive group to which the starting building blocks for receptor synthesis can be bound, preferably via a spacer or linker. Noncovalent self assembly may take place, for example, on a noble metal surface, for example a gold surface, by means of thiol groups, preferably via a spacer or linker.
The apparatus of the invention may be used for the electronically controlled in situ synthesis of nucleic acids, for example DNA/RNA oligomers, it being possible to use as temporary protective groups electronically removable protective groups such as, for example, p-nitrobenzyloxycarbonyl, 2-(p-nitrophenyl)ethyloxy carbonyl, 2,4-dinitrobenzyloxycarbonyl or/and 2,4-(p-dinitrophenyl)ethyloxycarbonyl. It is also possible, where appropriate, to use combinations of photoactivatable protective groups, chemical protective groups or/and electronic protective groups. The location- or/and time-resolved receptor synthesis may be carried out by specifically addressing the electrodes of the detection matrix, by specifically supplying fluids to defined areas or area groups on the support or/and by specific illumination via the light source matrix.
The present invention makes possible considerable improvements compared with known analyte determination methods, for example by providing an integrated electronic system for receptor synthesis and for analyte detection without movable parts. The detection may be varied via different designs of the electrode structures. An improved on-line process control may also be achieved by combining light, fluid supply and electronic detection.
Furthermore, the following figures are intended to illustrate the present invention:
According to
In the embodiment shown in
The support structure (90) shown in
Whereas the embodiments shown in
Number | Date | Country | Kind |
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101 20 663.1 | Apr 2001 | DE | national |
101 56 433.3 | Nov 2001 | DE | national |
Number | Date | Country | |
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Parent | 10132167 | Apr 2002 | US |
Child | 10895981 | Jul 2004 | US |