Arrangement for integrated and automated dna or protein analysis in a single-use cartridge, method for producing such a cartridge and operating method for dna or protein analysis using such a cartridge
A cartridge (card) having a system of microchannels and/or microcavities is used for automated DNA or protein analysis. In at least one embodiment, the microchannels or microcavities include geometrical structures for receiving dry reagents. For the purpose of industrial production, the cartridge is produced from a flat card support, e.g., by injection moulding. The reagents are spotted into the open channels, dried and then the channels are sealed by way of a film. A finished cartridge can thus be provided with a test sample and the fully automated measuring sequence can be initiated by inserting said cartridge into a read-out device.
1 . An arrangement for the integrated and automated DNA or protein analysis of a measurement sample in a single-use cartridge filled with dried reagents, the arrangement comprising:
a system of at least one of microchannels and microcavities for microfluidic process technology, present in the cartridge,
the at least one of microchannels and microcavities including predefined geometric structures to accommodate reagents, wherein
the reagents are stored ready in a storage-stable form at defined sites in the at least one of microchannels and microcavities of the cartridge; and
means for making the dry-stored reagents available for the relevant component process in suitable form.
2 . The arrangement as claimed in claim 1 , wherein the structures include depressions to accommodate the dry, storage-stable reagents.
3 . The arrangement as claimed in claim 2 , wherein the depressions include at least one step with step heights of 10 to 500 μm.
4 . The arrangement as claimed in claim 2 , wherein the depressions have a length of ca. 1 mm and a depth of about 100 μm.
5 . The arrangement as claimed in claim 3 , wherein the depressions are cylindrical, and wherein the length represents the diameter.
6 . The arrangement as claimed in claim 1 , wherein the reagents introduced into the at least one of micro-channels and microcavities include the following properties:
they are dryable substances with negligible vapor pressure, which are stable at room temperature, so that the properties remain unchanged for one cell disintegration or one PCR or for the detection of biochemical quantities.
7 . The arrangement as claimed in claim 1 , wherein that mixtures of the particular substance with additives form thin films which adhere to the walls.
8 . The arrangement as claimed in claim 7 , wherein the substances or mixtures introduced into parts of the at least one of micro-channels and microcavities are watertightly covered with thin paraffin wax layers.
9 . The arrangement as claimed in claim 1 , wherein the substances introduced into parts of the at least one of microchannels and microcavities include at least one of -DNA- and protein-binding properties.
10 . The arrangement as claimed in claim 1 , wherein the substances introduced into parts of the at least one of microchannels and microcavities are magnetic beads with specific binding properties.
11 . The arrangement as claimed in claim 10 , wherein the magnetic beads are coated with antibodies.
12 . The arrangement as claimed in claim 10 , wherein the magnetic beads are coated with DNA-binding substances.
13 . The arrangement as claimed in claim 1 , wherein lysis reagents and magnetic beads are simultaneously present, and wherein the lysis reagents and the magnetic beads are contained in a single dry matrix.
14 . The arrangement as claimed in claim 1 , wherein a so-called DNA ELISA assay or protein ELISA assay is performable, and wherein a label enzyme and an enzyme substrate are present as reagents for the ELISA assay.
15 . The arrangement as claimed in claim 1 , wherein a detection module for the electrical detection of the hybridization processes is present.
16 . The arrangement as claimed in claim 15 , wherein the detection module consists of a noble metal/plastic composite.
17 . The arrangement as claimed in claim 15 , wherein the detection module consists of a semiconductor-processed silicon chip with noble metal electrodes.
18 . The arrangement as claimed in claim 15 , wherein at least one of electrochemical, magnetic and piezoelectric measurement methods are used by the module for the electrical detection.
19 . The arrangement as claimed in claim 1 , wherein the cartridge includes an input port for a whole blood sample.
20 . The arrangement as claimed in claim 1 , further comprising means for the introduction of water.
21 . The arrangement as claimed in claim 20 , further comprising an inlet port for connection to an external water source.
22 . The arrangement as claimed in claim 21 , wherein the inlet port is connected to an integrated water reservoir.
23 . The arrangement as claimed in claim 1 , wherein the at least one of microchannels and microcavities are filled with dry buffer substances of defined ionic strength after addition of water.
24 . The arrangement as claimed in claim 1 , further comprising means for the mixing of whole blood samples and water or the buffer solution.
25 . The arrangement as claimed in claim 1 , further comprising means for passing at least one of blood, blood-water and blood-buffer mixture through the at least one of the microchannel and microcavity coated with lysis bead reagent.
26 . The arrangement as claimed claim 1 , further comprising means for generating a magnetic field for the purpose of immobilizing at least one of the DNA/magnetic bead and protein/magnetic bead complex.
27 . The arrangement as claimed in claim 1 , further comprising means for generating a magnetic field for the purpose of immobilizing the DNA/magnetic bead complex in a PCR cavity.
28 . The arrangement as claimed in claim 21 , claim 1 , further comprising means for closure of the PCR cavity.
29 . The arrangement as claimed in claim 1 , further comprising means for the thermocycling of the sample are present.
30 . The arrangement as claimed in claim 1 , further comprising, in the cartridge, means for the storage of at least one of used sample material and used reagent.
31 . The arrangement as claimed in claim 30 , wherein the means for the storage of at least one of used sample material and used reagents constitute waste reservoirs.
32 . The arrangement as claimed in claim 1 , further comprising means for at least one of the germproof, particle- and cell-free venting of the waste reservoirs.
33 . The arrangement as claimed in claim 1 , further comprising means for the immobilization of the cartridge in a reading device.
34 . A method for the production of a cartridge comprising:
making from polymer, a cartridge base with at least one of channels and cavities;
spotting reagents into open channels and drying the reagents there; and
closing the at least one of channels and cavities with a film.
35 . A production method as claimed in claim 34 , wherein the card base is produced by injection molding technology.
36 . The production method as claimed in claim 34 , wherein special materials are applied onto the card body.
37 . The production method as claimed in claim 34 , wherein, before the sealing of the card body, a detection module with measurement devices is introduced.
38 . An operating method for DNA analysis in an arrangement as claimed in claim 1 , the method comprising:
introducing the sample into the cartridge;
inserting the cartridge into the reading device; and
starting a fully automatic assay.
39 . The operating method as claimed in claim 38 , comprising the following steps during operation of the fully automatic assay:
sample dispensing via a dispensing section;
washing the dispensing section;
diluting the measurement sample and introducing it into the lysis channel;
having a cell disintegration take place by residence in the lysis channel;
carrying the DNA-bead complex formed into the PCR chamber by a liquid flow and holding it in the PCR chamber via a bead collector;
washing of the DNA-bead complex with water;
closing the PCR chamber is closed;
performing the PCR;
transporting, after completion of the PCR, the PCR product into the detection chamber;
having hybridization processes, with specific capture probes, take place in the detection chamber;
flushing the detection chamber with labeling enzyme;
flushing the detection chamber with enzyme substrate;
performing the electrochemical measurement; and
performing the electrochemical measurements at various temperatures and various flow rates of the enzyme-substrate solution.
40 . The operating method as claimed in claim 39 , wherein, during the PCR, the ELISA reagent channels are filled with water.
41 . The operating method as claimed in claim 39 , wherein, after the hybridization, both ELISA channels are vented, that next the detection chamber is firstly flushed gas bubble-free with the first ELISA reagent and then flushed gas bubble-free with the second ELISA reagent, and that the electrochemical measurement is then performed.
42 . The operating method for protein analysis in an arrangement as claimed in claim 1 , the method comprising:
introducing the sample into the cartridge;
inserting the cartridge into the reading device; and
starting the fully automatic assay.
43 . The operating method as claimed in claim 42 , with the following steps during operation of the fully automatic assay:
sample dispensing via a dispensing section;
washing the dispensing section;
diluting the measurement sample and transporting it into the detection chamber by a liquid flow;
having binding processes between the proteins of the measurement sample and specific capture antibodies or capture proteins take place in the detection chamber;
flushing the detection chamber with an antibody solution bearing an enzyme label (ELISA reagent 1);
flushing the detection chamber with enzyme substrate (ELISA reagent 2); and
performing the electrochemical measurements.
44 . The operating method as claimed in claim 43 , wherein, after the hybridization, both ELISA channels are vented, that next the detection chamber is firstly flushed gas bubble-free with the first ELISA reagent and then flushed gas bubble-free with the second ELISA reagent, and that the electrochemical measurement is then performed.
45 . The arrangement as claimed in claim 1 , wherein the dry-stored reagents are made available for the relevant component process as a liquid reagent.
46 . The arrangement as claimed in claim 20 , wherein the means for the introduction of water includes an inlet port.
47 . The arrangement as claimed in claim 1 , further comprising an inlet port for the introduction of water.
48 . The production method as claimed in claim 36 , wherein special materials include at least one of sealing membranes and venting membranes.