U.S. patent application number 11/665380 was filed with the patent office on 2009-05-21 for 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.
Invention is credited to Heike Barlag, Siegfried Birkle, Walter Gumbrecht, Daniela Kuhn, Peter Paulicka, Manfred Stanzel.
Application Number | 20090130658 11/665380 |
Document ID | / |
Family ID | 35432485 |
Filed Date | 2009-05-21 |
United States Patent
Application |
20090130658 |
Kind Code |
A1 |
Barlag; Heike ; et
al. |
May 21, 2009 |
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
Abstract
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.
Inventors: |
Barlag; Heike; (Nurnberg,
DE) ; Birkle; Siegfried; (Hochstadt, DE) ;
Gumbrecht; Walter; (Herzogenaurrach, DE) ; Kuhn;
Daniela; (Hemhofen, DE) ; Paulicka; Peter;
(Erlanen, DE) ; Stanzel; Manfred; (Erlangen,
DE) |
Correspondence
Address: |
HARNESS, DICKEY & PIERCE, P.L.C.
P.O.BOX 8910
RESTON
VA
20195
US
|
Family ID: |
35432485 |
Appl. No.: |
11/665380 |
Filed: |
October 17, 2005 |
PCT Filed: |
October 17, 2005 |
PCT NO: |
PCT/EP2005/055303 |
371 Date: |
November 6, 2007 |
Current U.S.
Class: |
435/6.12 ;
435/287.2 |
Current CPC
Class: |
B01L 3/5027 20130101;
Y10T 436/11 20150115; Y10T 436/2575 20150115; B01L 3/502723
20130101; B01L 3/502707 20130101; Y10T 436/25 20150115 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 15, 2004 |
DE |
10 2004 050 576.4 |
Claims
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 .mu.m.
4. The arrangement as claimed in claim 2, wherein the depressions
have a length of ca. 1 mm and a depth of about 100 .mu.m.
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.
Description
PRIORITY STATEMENT
[0001] This application is the national phase under 35 U.S.C.
.sctn. 371 of PCT International Application No. PCT/EP2005/055303
which has an International filing date of Oct. 17, 2005, which
designated the United States of America and which claims priority
on German Patent Application number 10 2004 050 576.4 filed Oct.
15, 2004, the entire contents of which are hereby incorporated
herein by reference.
FIELD
[0002] Embodiments of the invention generally relate to an
arrangement for integrated and automated DNA or protein analysis in
a single-use cartridge. Herein, a flat card in check card format is
described as a cartridge. In addition, embodiments of the invention
generally relate to the production of such a cartridge. Finally,
embodiments of the invention also generally relate to an operating
method for DNA or protein analysis using such a cartridge.
BACKGROUND
[0003] For nucleic acid analysis, for example for the analysis of
white blood cells from whole blood with the aim of answering human
genome questions, firstly, in a first stage as the sample
preparation step, the cells must be broken up and then the DNA thus
liberated must be isolated. In a second stage, a PCR (Polymerase
Chain Reaction) for selective DNA multiplication (amplification) is
performed in order to increase the concentration of the DNA to be
detected sufficiently for it to be detectable in a third stage.
[0004] In the laboratory, the latter component processes are
carried out separately according to the known state of the art. The
three stages mentioned above each include several working steps and
are carried out independently of one another with different
equipment. The individual work steps are largely performed
manually.
[0005] The implementation of these steps is dependent on the
presence of laboratory equipment--such as a cell disintegration
apparatus, a PCR device (so-called thermocycler), possibly a PCR
device which is suitable for quantitative PCR, an electro-phoresis
apparatus, a hybridization stage, an optical reader, so-called
Eppendorf tubes, several pipetting devices and a refrigerated
container for reagents, and must be carried out by trained
personnel with observation of safety procedures with regard to
infection risk, waste disposal or the like. In particular, several
volumetric, i.e. accurate, dispensings (pipettings) of reagent
solutions must be performed. Such work steps are time-consuming and
cost-intensive.
[0006] From the state of the art, devices for biochemical analysis
are known which according to WO 02/073153 A1 make use in particular
of silicon-based measurement modules, which can be integrated into
a chip card. Moreover, according to WO 02/072262 A1, the reagents
used for the analysis are already integrated into the analysis
module in dry-stored form.
SUMMARY
[0007] At least one embodiment of the invention is directed to the
implementation of an inexpensive, simply manageable, complete DNA
or protein analysis process in a miniaturized cartridge. In
particular in at least one embodiment, the following improvements
compared to the laboratory method should be implemented: [0008]
complete integration of all substances (possibly except for water)
in a closed, single-use cartridge; [0009] preparation of the
reagents in a form stable on storage at room temperature; [0010]
automatic implementation of all processes in the cartridge; [0011]
no manual working steps, apart from injection of the sample to be
analyzed, e.g. blood; [0012] no direct contact with substances
hazardous to health (blood and reagent wastes remain in the
cartridge); [0013] cartridge geometry allows efficient and rapid
thermocycling; [0014] all detection processes should operate
electrically and be easy to read; [0015] the cartridge used is
small and inexpensive to produce.
[0016] At least one embodiment of the invention is in particular
based on WO 02/072262 A1 and the further state of the art mentioned
there. Therein is described an analytical device with reagents
stable at room temperature, dry-stored in fluid channels, which are
brought into solution through the introduction of water shortly
before their use as intended. At least one embodiment of the
invention is further based on the unpublished DE 10 2004 021780 A1
and the unpublished DE 10 2004 021822 A1. In addition, the specific
use of electrically readable detection modules is also known.
[0017] In contrast to this, an object of at least one embodiment of
the invention is such a single-use cartridge with a system of
microchannels and/or microcavities for a predefined process
sequence after sample uptake, wherein the cartridge has structures
to accommodate the dry reagents and devices for the implementation
both of the cell disintegration on the one hand and of the PCR on
the other, but also of the electrochemical detection, are allocated
to these structures. In particular, the channels therein have
different, problem-adapted structures. Specifically, the
disintegration channel advantageously has stepped cross-sections
for optimal wetting with the dry reagent, while the PCR chamber and
the Elisa reagent channels have pot-shaped depressions.
[0018] It can thus be achieved that the introduction and
preparation of the sample, the DNA amplification and the actual
detection of the DNA is possible in the course of one process.
[0019] By way of the system of geometric structures in the
micro-channels or microcavities to accommodate dry reagents
according to at least one embodiment of the invention, suitable
conditions are obtained for DNA analysis on the one hand and
protein analysis on the other. In at least one embodiment, the
following features and measures are essential: [0020] the reagents
introduced into the microchannel or into the microcavity are
dryable substances with negligible vapor pressure. As the
substances are stable at room temperature, their properties for
cell disintegration and/or PCR and/or detection are retained. In
addition, mixtures of the substances with additives can form thin
films, and the mixtures can be water-tightly covered with thin
layers of paraffin wax.
[0021] In at least one embodiment of the invention, the reagents
and additives are already introduced as dry substances into
depressions of the cartridge channels during production. The
following advantages result from this: [0022] simple and precise
application of the reagents during the production of the cartridge;
[0023] protection of the reagents during the filling of the reagent
channels, i.e. the reagents are not washed away during a so-called
water flow, but are retained during the filling of the whole
channel. Only after the filling of the channel do the reagent spots
dissolve by diffusion processes and a homogeneous solution is
produced.
[0024] In a further example embodiment, the depressions are located
at predefined intervals along the reagent channel. Here, the
intervals can be equidistant or particularly advantageously can be
arranged in variable spacing patterns.
[0025] The depressions can advantageously be filled with variable
quantities of dry reagent. Through the combination of different
amounts of dry reagent and spacing patterns of the depressions, the
desired concentration profiles of the finished reagent solutions
can be established.
[0026] For certain functions, such as for example cell
disintegration in the presence of magnetic beads and lysis
reagents, an even distribution of the insoluble components, i.e.
the beads, in the dry reagent is necessary. For this, the magnetic
beads are dispensed into the lysis channel as a suspension. On
evaporation of the solvent, it is observed that the beads are drawn
back into the edge region of the lysis channel and an even
distribution does not result from this. Through stepped structuring
of the lysis channel cross section, the magnetic beads distribute
themselves across the steps and an even distribution is
achieved.
[0027] In order that a cell disintegration, a PCR and a so-called
DNA/protein ELISA test can equally be performed with the cartridge
according to the invention, it is advantageous that substrates with
DNA-binding properties, in particular the DNA-binding magnetic
beads, be present in the microchannels or microcavities. Here, the
lysis reagents and the magnetic beads can be contained together in
a single dry matrix. Further, the reagents for an ELISA assay are
also present in the card. In particular, for the ELISA assay two
reagents are needed, i.e. a labeling enzyme as the first reagent
and an enzyme substrate as the second reagent.
[0028] In particular, a detection module for the electrical
detection of the hybridization processes is arranged in the
cartridge. The detection module advantageously consists of a noble
metal/plastic composite or a semiconductor-processed silicon chip
with noble metal electrodes. Especially suitable for electric
detection here are electrochemical, magnetic or piezoelectric
measurement procedures.
[0029] For the application of at least one embodiment of the
invention, in particular an input port for a whole blood sample is
present in the cartridge according to at least one embodiment of
the invention. Moreover, device(s) for the addition of water are
present, for example inlet ports for connection to an external
water supply or an integrated water reservoir. In the microchannels
or microcavities, dry buffer substances have defined ionic strength
after the addition of water.
[0030] In the application of at least one embodiment of the
invention for the analysis of white blood cells from whole blood,
device(s) for mixing of a whole blood sample with water or a buffer
solution are advantageously present. At the same time, device(s)
for passing blood or blood/water mixtures or blood/buffer mixtures
through the micro-channel or microcavity coated with
lysis/bead/reagent are present.
[0031] Further, for the PCR to be performed in the cartridge
according to at least one embodiment of the invention during use
specifically for DNA analysis, device(s) for generation of a
magnetic field for immobilization of the DNA/magnetic bead complex
in a PCR cavity are present. For this purpose, the PCR cavity must
be capable of being suitably sealed and device(s) for thermocycling
must be present.
[0032] Finally, in the cartridge according to at least one
embodiment of the invention, it is essential that device(s) for
storage of used sample material and used reagents be present, which
constitute waste reservoirs. At the same time, the device(s) must
be suitable for the germproof, cell-free and particle-free venting
of at least one waste reservoir. Finally, for the reading of the
cartridge in a reader device, which is not an object of at least
one embodiment of the invention, device(s) for the immobilization
of the cartridge must be present.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Further details and advantages of embodiments of the
invention follow from the following descriptions of the diagrams of
practical examples on the basis of the drawings in combination with
the patent claims. In diagrammatic form, respectively:
[0034] FIG. 1 shows a cartridge with an overview of individual
microchannel/microcavity systems with the relevant function
designations,
[0035] FIG. 2 shows a top view of a cell disintegration
channel,
[0036] FIG. 3 shows the cross section through the cell
disintegration channel according to FIG. 5,
[0037] FIG. 4 shows two alternatives for the throughflow channel
cross section, shown enlarged,
[0038] FIG. 5 shows the top view of the PCR chamber in FIG. 1,
[0039] FIGS. 6 and 7 show the cross section through the PCR chamber
according to FIG. 5,
[0040] FIG. 8 shows the top view of an ELISA reagent channel in
FIG. 1,
[0041] FIGS. 9 and 10 show the cross section of the ELISA reagent
channel according to FIG. 8 and
[0042] FIGS. 11 to 23 show the top view of the cartridge according
to FIG. 1 in various process states during an automated
evaluation.
[0043] In the figures, the same or similarly operating components
have the same reference symbols. In particular, FIGS. 1 to 10 are
described together, and FIG. 11 to are described together.
DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS
[0044] FIG. 1 shows a cartridge 100 for an ELISA ("Enzyme Linked
Immuno Sorbent ASSAY") test with a front view of the micro-channel
or microcavity system present therein, wherein for clarity the
relevant function designations are also shown. In detail, the
cartridge 100 consists of a plastic base 101 with fluidic
structures incorporated therein, which are covered by a plastic
film. The structures are further described below on the basis of
FIGS. 2 to 10.
[0045] In the top view according to FIG. 1, there can be seen a
sample port 102 with a dispensing section 105 connected thereto,
through which liquid samples in particular for nucleic acid
analysis, for example for the analysis of white blood cells from
whole blood, for answering human genome questions can be introduced
in a defined manner. This is connected to a channel area 110 for
the cell disintegration of the sample and moreover specifically for
a DNA analysis an area 120 for a PCR (Polymerase Chain Reaction)
for selective DNA multiplication (amplification) in order to
increase the concentration of the DNA to be detected sufficiently
for it to be detectable in a third stage. The actual PCR chamber is
sealable by valves 122 and 122'. The detection of the samples thus
prepared, in particular according to the ELISA method, then takes
place in the area 130.
[0046] Also visible in FIG. 1 are water ports 103 to 103'''.
Through these, water can be introduced into the cartridge 100 as a
transport agent and solvent during the preparation of a sample.
Further, venting ports 104 to 104''' are present.
[0047] As mentioned, the cartridge 100 has in particular an input
port 102 for a whole blood sample. In addition, at least one device
for the introduction of water is present. An inlet port for
connection to an external water source can be present, or the feed
port can be connected to an integrated water reservoir.
[0048] In the normal case, the microchannels or microcavities 101
to 131 are filled with dry buffer substances which ensure a defined
ionic strength after the introduction of water. For blood analysis,
at least one device for mixing of whole blood samples and water or
the buffer solution and/or at least one device for passing a flow
of blood or blood-water or blood buffer mixture through a
micro-channel coated with a lysis bead reagent or the microcavity
are present.
[0049] In the channel system, wide regions 106, 107, 108 and 109
are provided as reservoirs to accommodate waste. Apart from this, a
region with channels 131 or 131' to accommodate different ELISA
reagents is present.
[0050] In each of FIGS. 2 to 4, reference symbols 101 again
indicate the cartridge base. The base contains a throughflow
channel 111 shaped in a particular way especially for cell
disintegration ("lysis") with step-shaped depressions 112 formed by
the side edges to accommodate reagents. Here the depressions 112
have several steps with step heights from 10 to 500 .mu.m and have
an extent of ca. 1 mm and a depth of about 100 .mu.m.
[0051] Specifically in the presentation according to FIG. 4a, there
arises the alternative option, with a throughflow channel with no
additional depressions, of providing for the accommodation of the
lysis reagents only in the region 113 of the edges of the
throughflow channel 111. On the other hand, in FIG. 4b, such
reagents, which in particular also contain magnetic beads for the
binding of the DNA liberated, are evenly distributed between the
steps 112 across the throughflow channel 111. Magnetic beads have
DNA- and protein-binding properties, if they have been
appropriately pretreated. They can be coated with DNA-binding
properties and if necessary also with antibodies. For the
introduction of dry substances as a matrix with lysis reagent and
magnetic beads, reference is in particular made to the applicant's
prior DE 10 2004 021780 A1 and prior DE 10 2004 021822.
[0052] FIGS. 5 to 7 show the structure of a PCR chamber 120 in the
cartridge base 101 with a flow channel 111. The valve arrangement
for closure of the PCR chamber during use as intended is not shown
here. It is essential that circular cylindrical depressions 124 and
124' are present in the PCR chamber 120 to accommodate specific
reagents 127 and 127', which are needed in the implementation of
the PCR. Specifically in FIG. 7, it is also shown that a dry,
storable PCR reagent 127 or 127', storable at room temperature, is
firstly covered with a paraffin wax layer 128 or 128'.
[0053] The correct implementation of the PCR with valve-controlled
thermocycling within a cartridge is described in detail in the
applicant's parallel applications DE 10 2004 050576.4 and DE 10
2004 050510.1 with the same application priority, to which in the
present connection reference is expressly made ("Incorporation by
Reference"). In particular, the use of magnetic beads for DNA
binding and concentration of the magnetic beads with the DNA in the
PCR chamber 120 by way of controllable magnetic fields is described
therein, concerning which no more detailed description will be
given here.
[0054] FIGS. 8 to 10 show the design and the structure of the ELISA
reagent channels 131 and 131' of FIG. 1. Dish-shaped depressions
132 to 132.sup.6' respectively are present, which are suitable to
accommodate pre-dispensed and pre-apportioned quantities of
reagents for the ELISA process according to FIG. 9. This has
already been described in detail in WO 02/072262 A1, mentioned at
the outset as state of the art, to which in the present connection
reference is also expressly made ("Incorporation by Reference"). In
FIG. 10, the circular cylindrical depressions 132 to 132.sup.6' are
shown filled with dry reagents 133 to 133.sup.6'. Here a first
reagent embodies a labeling enzyme and a second reagent an enzyme
substrate, such as is known to be needed in the hybridization of
the sample, also prepared by a PCR if necessary, with specific
capture probes. In the detection zone 130, shown only
schematically, different sensors for detection of biochemical
reactions can be located in a module of a noble metal/plastic
composite. Especially during electrochemical measurements with
semiconductor-processed chips, i.e. in particular silicon-based
sensors, the signals can be detected electrically and immediately
further processed. Apart from the electrochemical measurement
methods, magnetic and/or piezo-electric measurement methods with
corresponding sensors are also possible.
[0055] In each of FIGS. 11 to 23, the cartridge 100 according to
FIG. 1 is shown in top view, the zone of the cartridge 100 active
in the analytical process being marked in each: for this the
cartridge 100 is inserted into an analytical device, which is not
shown in detail in the diagrams and is not an object of the present
patent application.
[0056] The evaluation is now illustrated on the basis of eleven
concrete component process steps a) to m), after the cartridge has
been inserted into an evaluation device with at least one device
for accommodating the cartridge, and the evaluation device with the
cartridge immobilized therein has been activated. In detail, with
reference to FIG. 1, the following component steps are involved:
[0057] a) Ca. 10 .mu.l of blood are introduced as the measurement
sample. 1 .mu.l is automatically dispensed via the dispensing
capillary 105. [0058] b) The excess blood is washed into the cavity
106 (waste 1). [0059] c) Next, 1 .mu.l of blood sample is diluted
with water and transferred to the cell disintegration channel 110.
There the cell disintegration (lysis) of the blood cells and the
binding of the liberated DNA to the magnetic beads take place.
[0060] d) Next, the magnetic beads are transferred to the PCR
chamber and collected there. A washing process takes place, the
wash solution being collected in the cavity 107 (waste 2). [0061]
e) The washing process is now concluded. [0062] f) Next, the PCR
chamber valves 122 and 122' are closed and the PCR is performed.
[0063] g) During the PCR, the ELISA reagent channel 131 which
contains the enzyme substrate is simultaneously filled with water.
[0064] h) Simultaneously during the PCR reaction, the ELISA reagent
channel 131' which contains the label enzyme is filled with water.
[0065] i) After the PCR, the PCR chamber valves 122 and 122' are
opened and the PCR product is passed via the detection module 130
where the hybridization with the specific capture probes takes
place (into waste 3, channel 108). [0066] j) The enzyme substrate
channel is vented into the waste channel 108 (waste 3). [0067] k)
The label enzyme is vented into the waste channel 108 (waste 3).
[0068] l) The label enzyme solution flows via the detection module
130 for the labeling into the waste zone 109 (waste 4). [0069] m)
The enzyme substrate solution flows via the detection module 130
for the enzymatic-electrochemical detection of the hybridization
into the waste zone 109 (waste 4).
[0070] Thereby the analytical process is concluded. In particular
in the case of electrochemical detection, the signals arising can
be read electrically and evaluated using a processor in accordance
with a preset program.
[0071] The cartridge described in detail with channels and cavities
in FIG. 1 is produced from a polymeric material, such as for
example polycarbonate, for example by injection molding technology.
During this, the card base 101 with structures open upwards is
first produced, and the reagents are spotted into the initially
open channels or cavities and then dried. The detection module is
introduced in a suitable manner, in particular glued, into the
cartridge. In conclusion, the channels and the cavities are fitted
for example with an elastic film as the upper covering and is thus
sealed for use as directed.
[0072] It is also possible to apply certain special fittings, for
example as sealing materials and/or venting materials onto the open
card base 101 before the closing and finishing of the cartridge on
the cover side.
[0073] The specific measurement method is illustrated on the basis
of FIGS. 11 to 23 for one specific case of DNA analysis of a sample
of whole blood. In general, the use of the cartridge described is
envisaged for DNA analysis on the one hand and/or protein analysis
on the other, wherein, as already mentioned above, an appropriate
reading device and corresponding evaluation algorithm are utilized.
Defined operating methods follow from this, which for the first
time render the cartridge described on the basis of the examples
suitable in practice for decentralized use in the context of a
medical "Point of Care" application.
[0074] In conclusion, specifically for DNA analysis, the integrated
operating method for the cartridge described in detail above is
once again summarized as a combination or in the sequence of the
individual component steps: [0075] introduction of the sample into
the cartridge [0076] insertion of the cartridge into the reading
device [0077] starting of the completely automatic assay [0078]
sample dispensing via dispensing section [0079] washing of the
dispensing section [0080] dilution of the sample and introduction
into lysis channel [0081] residence in the lysis channel [0082]
collection of the DNA-bead complex by bead collectors in the PCR
chamber [0083] washing of the DNA-bead complex with water [0084]
closure of the PCR chamber [0085] implementation of the PCR [0086]
during the PCR: filling of both ELISA reagent channels with water
[0087] opening of the PCR chamber [0088] transport of the PCR
product into detection chamber [0089] hybridization in the
detection chamber [0090] venting of both ELISA reagent channels
[0091] filling and rinsing of the detection chamber with ELISA
reagent 1 [0092] filling and rinsing of the detection chamber with
ELISA reagent 2 [0093] implementation of the electrochemical
measurements.
[0094] In the electrochemical measurements, firstly the rinsing of
the detection chamber with an antibody solution bearing an enzyme
label (ELISA reagent 1) takes place. Then the rinsing of the
detection chamber with enzyme substrate (ELISA reagent 2) takes
place. The electrochemical measurements are performed in a manner
in itself known at predefinable, different temperatures and
variable flow rates of the enzyme-substrate solution.
[0095] For the protein analysis, corresponding procedures are used,
but in this case the PCR is not used.
[0096] Example embodiments being thus described, it will be obvious
that the same may be varied in many ways. Such variations are not
to be regarded as a departure from the spirit and scope of the
present invention, and all such modifications as would be obvious
to one skilled in the art are intended to be included within the
scope of the following claims.
* * * * *