Methods and apparatuses for electronic determination of analytes

The invention relates to a method and an apparatus for determining analytes by electronic detection using a microfluidic support.

The present invention therefore relates to a method for determining analytes, which comprises the following steps: (a) providing an apparatus comprising (i) a light source matrix, (ii) a microfluidic support having channels which contain a plurality of predetermined areas at which in each case different receptors are immobilized on the support, (iii) means for supplying fluids to the support and for discharging fluids from the support and (iv) an electronic detection matrix having a plurality of electrodes assigned to the predetermined areas containing immobilized receptors on the support, (b) contacting the support with a sample containing analytes and (c) determining the analytes by electronic detection via binding thereof to the receptors immobilized on the support. The invention further relates to an apparatus for determining analytes, which comprises (i) a light source matrix, (ii) a support having channels which contain a plurality of predetermined areas at which in each case different receptors are immobilized on the support, (iii) means for supplying fluids to the support and for discharging fluids from the support and (iv) an electronic detection matrix having a plurality of electrodes assigned to the predetermined areas containing immobilized receptors on the support. 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 &mgr;m, particularly preferably from 50 to 250 &mgr;m, 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 &mgr;m 2 , particularly preferably in the range from 15 to 2,500 &mgr;m 2 . 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: FIG. 1 shows the basic structure of an electronic integrated synthesis-analysis (eISA) system. The system shown in FIG. 1A contains 3 layers, a light source matrix ( 2 ), a microfluidic support ( 4 ) and an electronic detection matrix ( 6 ). The apparatus shown in FIG. 1B consists of two layers, namely the light source matrix ( 2 a ) and a microfluidic support with integrated electronic detection matrix ( 4 a ). FIG. 2 shows different embodiments for immobilizing receptors, for example a DNA oligomer strand, on the electrode structure. According to FIG. 2 A, an electrically conducting layer ( 12 ) and above it a permeation layer ( 14 ) are provided, to which the receptor, for example a DNA oligomer ( 16 ), is bound covalently or noncovalently via a spacer ( 18 ). According to FIG. 2 B, the receptor ( 16 a ) is directly bound covalently or noncovalently via a spacer ( 18 a ) to the electrically conducting layer ( 12 a ). According to FIG. 3 , the receptor is bound directly on the electrically conductive layer ( 22 ). The surface of the microfluidic support alternately comprises insulating ( 24 ) and electrically conductive ( 26 ) areas, with the receptor ( 28 ) being bound to an electrically conducting area via a spacer ( 30 ). FIG. 4 is a detailed representation of the binding of a DNA oligonucleotide strand to an electrically conductive area (electrode) of the support via a spacer. FIG. 5A shows a microfluidic reaction support ( 32 ) with a microchannel ( 34 ) in the interior of the support and inlet orifices ( 36 ) and outlet orifices ( 38 ) for fluid. FIG. 5B shows the pattern of an electrode structure ( 40 ) with electrically conductive connections ( 40 a ) in connection with a section of the channel structure ( 34 a ) of the support shown in FIG. 5A . FIG. 6 A and FIG. 6B show an alternative electrode structure ( 42 ) in connection with a channel structure ( 44 ) of the support ( 46 ). The electrically conductive connections ( 42 a ) shown in FIG. 6B run from the electrodes ( 42 ) to an edge of the support. FIG. 7 A and FIG. 7B show a projection of the light source matrix through the microfluidic support onto the electronic detection matrix. The support ( 50 ) contains a light source matrix ( 52 ) with active pixels ( 52 a ) and nonactive pixels ( 52 b ), a fluidic area ( 54 ) with one or more channels ( 54 a ) and structural areas of the support matrix ( 54 b ) and electronic detection matrix ( 56 ) with a plurality of electrodes ( 56 a ) and non-electrode areas ( 56 b ). Receptors ( 58 ) are immobilized on the electrodes. The electrodes furthermore have an electrically conductive connection ( 60 ). Active pixels of the light source matrix ( 52 a ) and of the electronic detection matrix ( 56 a ) are preferably arranged directly above one another. FIG. 8 shows variants of the connection technique for measuring an electronic detection signal, e.g. a glass, e.g. Pyrex/metal, e.g. a silicon/glass, e.g. Pyrex sandwich structure. In the embodiment of the support ( 70 ) shown in FIG. 8 A, electrodes, preferably transparent electrodes, are arranged in the form of columns ( 72 ) and rows ( 74 ) on the top and bottom sides of the fluid channel ( 76 ). In the embodiment shown in FIG. 8 B, the support structure ( 80 ) has a sandwich-like arrangement, with two cover layers ( 82 a , 82 b ) being arranged above and below, respectively, a structural layer ( 84 ) containing the fluidic system. The cover layers ( 82 a , 82 b ) are preferably, at least in the area of the microchannels ( 84 a ), optically transparent, for example made of glass. The intermediate layer ( 84 ) consists at least partially of a conductive material, for example of metal, e.g. silicon. Conducting sublayers ( 84 b ) which provide the electrodes may be provided on the walls ( 86 , 88 ) of the structural layer ( 84 ) surrounding a microchannel ( 84 a ). The support structure ( 90 ) shown in FIG. 8C is constructed similarly to the support structure according to FIG. 8B . It contains 2 , preferably optically transparent, cover layers ( 92 a , 92 b ) and in between a structured layer ( 94 ), for example a metal layer such as, for example, silicon, with microchannels ( 94 a ). The walls of the structural layer ( 94 ) which are adjacent to the microchannel contain, at least partially, an electrically conductive sublayer ( 96 ), for example a positively charged layer. Opposite electrodes, preferably transparent opposite electrodes ( 98 ), are arranged on the top or/and bottom side of the microchannel ( 94 ). Whereas the embodiments shown in FIG. 8 are suitable in particular for supports working according to the transmitted-light principle, FIG. 9 shows an embodiment for back light. The support structure ( 100 ) contains an optically transparent cover layer ( 102 ) through which the light of the light source matrix (not shown) can be introduced and reflected again. Furthermore, a structural layer ( 104 ) is provided which preferably consists of metal or another fully or partially conductive material, for example a doped plastic material. The material of the structural layer is particularly preferably silicon. Microchannels ( 104 a , 104 b , 104 c ) are provided in the structural layer ( 104 ). In microchannel ( 104 a ), an electrode (−) on the bottom of the microchannel and an external opposite pole (&plus;) are provided. In microchannel ( 104 b ), an electrode (−) on the bottom and opposite poles (&plus;) on the wall are provided. In microchannel 104 c , an electrode (−) on the bottom and an internal opposite pole (&plus;) at the top, for example a transparent electrode as described above, are provided. FIG. 9B is a plan view of the apparatus depicted in FIG. 9 A and shows the support structure ( 100 ) with the microchannel ( 110 ) and electrodes ( 112 ) arranged along the microchannel. FIG. 10 finally shows preferred nucleotide building blocks for the electronically controlled in situ nucleic acid synthesis. Py is an electronically removable protective group, for example p-nitrobenzyloxycarbonyl, 2-(p-nitrophenyl)ethyl oxycarbonyl, 2,4-dinitrobenzyloxycarbonyl or 2,4-(p-dinitrophenyl)ethyloxycarbonyl.