U.S. patent application number 13/811959 was filed with the patent office on 2015-03-19 for measuring cassette and measuring device for the detection of target molecules in a liquid sample by measurement of fluorescence emission after excitation in an evanescent field.
This patent application is currently assigned to DiaSys Diagnostic Systems GmbH. The applicant listed for this patent is Roland SCHENK. Invention is credited to Roland SCHENK.
Application Number | 20150079694 13/811959 |
Document ID | / |
Family ID | 44628734 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150079694 |
Kind Code |
A2 |
SCHENK; Roland |
March 19, 2015 |
MEASURING CASSETTE AND MEASURING DEVICE FOR THE DETECTION OF TARGET
MOLECULES IN A LIQUID SAMPLE BY MEASUREMENT OF FLUORESCENCE
EMISSION AFTER EXCITATION IN AN EVANESCENT FIELD
Abstract
An interchangeable disposable measuring cassette for insertion
into a measuring apparatus for detecting target molecules in a
liquid sample by measuring fluorescence emission has a flow
measurement cell in which an excitation radiation provided by the
measuring apparatus produces an evanescent field in the liquid
sample beyond a boundary layer for the liquid sample and the
measurement cell. To be better able to ensure that no sample liquid
can cross from the measurement cell into the measuring apparatus,
the measuring cassette includes a body including an optically
transparent material and a base in contact with the underside of
the body. The measurement cell is formed by a cutout provided in
the body, the base, or both. The areas on which the body and the
base are on top of one another around this cutout are connected to
one another directly and in fluid-tight fashion by laser
welding.
Inventors: |
SCHENK; Roland; (Kirchheim,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SCHENK; Roland |
Kirchheim |
|
DE |
|
|
Assignee: |
DiaSys Diagnostic Systems
GmbH
Holzheim
DE
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130164857 A1 |
June 27, 2013 |
|
|
Family ID: |
44628734 |
Appl. No.: |
13/811959 |
Filed: |
July 19, 2011 |
PCT Filed: |
July 19, 2011 |
PCT NO: |
PCT/EP2011/062363 PCKC 00 |
371 Date: |
March 13, 2013 |
Current U.S.
Class: |
436/172; 422/554;
422/69 |
Current CPC
Class: |
G01N 2021/056 20130101;
B01L 2400/0487 20130101; G01N 21/648 20130101; B01L 2300/0654
20130101; B01L 2200/04 20130101; B01F 13/1016 20130101; B01F
13/0059 20130101; B01L 3/502715 20130101; B01F 5/0647 20130101;
B01L 3/508 20130101; B01L 2300/0887 20130101; G01N 2021/0346
20130101; G01N 21/05 20130101; B01L 2300/0877 20130101; B01L
2200/027 20130101; B01L 3/502707 20130101; B01L 3/502761 20130101;
B01L 2200/0647 20130101 |
Class at
Publication: |
436/172; 422/554;
422/69 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 26, 2010 |
DE |
10 2010 038 431.3 |
Claims
1. A replaceable disposable measuring cassette for introduction
into a measuring device for detecting target molecules in a liquid
sample by measurement of fluorescence emission, wherein the
measuring cassette has a flow-through measuring cell in which
excitation radiation provided by the measuring device generates an
evanescent field in the liquid sample on the other side of a
boundary surface of the liquid sample and measuring cell, wherein
the measuring cassette comprises a body of an optically transparent
material and a base lying adjacent to the under-side of the body,
wherein the measuring cell is formed by a recess which is provided
in the body, in the base or both in the body and in the base,
wherein the areas on which the body and the base lie on one another
around this recess are bonded to one another directly and in a
fluid-tight manner by laser welding, wherein several different
trapping zones are provided in the measuring cell, in order to be
able to respond to several different analytes at the various
trapping zones using only one light source, and wherein at the
point where the excitation radiation enters into the body the body
forms a converging lens which always directs the beam of the
excitation radiation centrally along the longitudinal axis of the
measuring cell exactly to the desired point of the boundary surface
of the liquid sample and the surface of the transparent material of
the body at which the fluorescence emission to be measured is to be
generated.
2. The measuring cassette according to claim 1, wherein the
measuring cassette has a sample channel which is in fluid contact
with the measuring cell and on which is provided at least one of
the following arrangements a) to c): a) a zone for dissolving a
tracer, b) a sample mixing region and/or c) a liquid detection
region, wherein the sample channel and the arrangements a), b)
and/or c) provided thereon are formed by a recess which is provided
in the body, in the base or both in the body and in the base,
wherein the areas on which the body and the base lie on one another
around this recess are bonded to one another directly and in a
fluid-tight manner by laser welding.
3. The measuring cassette according to claim 1, wherein the body
and/or the base are each produced as a one-component part in an
injection moulding process.
4. The measuring cassette according to claim 1, wherein the
measuring cassette is provided with an electronic memory chip which
can be written to and overwritten through the packaging even after
packing of the measuring cassette.
5. The measuring cassette according to claim 1, wherein a sample
filling opening for introduction of the sample into the measuring
cassette is provided in the body, the sample filling opening being
closable in a pressure-tight manner.
6. The measuring cassette according to claim 1, wherein the sample
channel can be connected to pressure openings via which the sample
can be moved through the sample channel by means of positive or
negative pressure.
7. The measuring cassette according to claim 1, wherein a sample
waste region is provided in the body or in the base at one end of
the sample channel.
8. The measuring cassette according to claim 1, wherein in addition
to the body and the base, a cover which at least partly covers the
body is provided, wherein recesses are provided at least in the
region of the beam path of the excitation radiation and the
fluorescence emission, so that the body is not covered by the cover
in this region.
9. The measuring cassette according to claim 1, in which the body
and the base and where appropriate also the cover are produced as
one-component parts in an injection moulding process.
10. The measuring cassette according to claim 7, in which the
connection of the cover to the unit of the body and base is formed
by hot caulking.
11. A measuring device for the detection of target molecules in a
liquid sample by measurement of fluorescence emission after
excitation in an evanescent field, wherein the measuring device has
an insertion shaft for a measuring cassette according to claim 1,
and has arrangements for providing a relative movement of the
measuring cassette with respect to the light source for the
excitation radiation, in order to bring various regions of the
measuring cell into the beam path of the excitation radiation and
to be able to respond to several different analytes in several
different trapping zones provided in the measuring cell using only
one light source, wherein the light source for an excitation
radiation is provided in the measuring device such that the beam of
the excitation radiation meets the measuring cell at an angle of 90
degrees to the direction of the relative movement of the measuring
cassette with respect to the light source.
12. The measuring device according to claim 11, wherein the
measuring device has a pressure source which in the case of a
measuring cassette inserted into the insertion shaft is connected
to the pressure openings of the sample channel so that the sample
can be moved through the sample channel by means of positive or
negative pressure generated by the pressure source.
13. A method for detecting target molecules in a liquid sample by
measurement of fluorescence emission after excitation in an
evanescent field, in which a measuring cassette according to claim
1 is employed in combination with a measuring device and responds
to several different analytes in several different trapping zones
provided in the measuring cell using only one light source.
14. The method according to claim 13, wherein the sample is a body
fluid or an aqueous sample for foodstuffs or environmental
analysis, wherein the sample is optionally treated or not treated
for the detecting of the target molecules.
Description
[0001] The present invention relates to a replaceable disposable
measuring cassette for introduction into a measuring device for
detecting target molecules in a liquid sample by measurement of
fluorescence emission, wherein the measuring cassette has a
flow-through measuring cell in which excitation radiation provided
by the measuring device generates an evanescent field in the liquid
sample on the other side of a boundary surface of the liquid sample
and measuring cell. The present invention moreover relates to a
process for the production of such a measuring cassette, and a
measuring device for the detection of target molecules in a liquid
sample by measurement of fluorescence emission after excitation in
an evanescent field, wherein the measuring device has an insertion
shaft for the abovementioned measuring cassette. The present
invention furthermore relates to a method for detecting target
molecules in a liquid sample by measurement of fluorescence
emission after excitation in the evanescent field, in which the
abovementioned measuring cassette or the abovementioned measuring
device is employed.
[0002] With a measuring cassette or measuring device of the
abovementioned type, target molecules in a liquid sample can be
recorded qualitatively or quantitatively by measurement of
fluorescence emission after excitation in the evanescent field. The
target molecules mentioned either can emit fluorescent radiation
themselves by evanescent field excitation or--if they are not
capable of this--can be rendered appropriately detectable by
coupling with a fluorescent marker.
[0003] In fluorescence excitation in the evanescent field, light of
a suitable wavelength (excitation radiation) is directed on to a
boundary surface between the liquid sample to be analysed and a
surface of an optically transparent material, so that the
excitation radiation is reflected totally with simultaneous
generation of an evanescent field in the liquid sample. The
evanescent field interacts with the liquid sample, and can excite
fluorophores present in this field to fluorescence radiation. This
fluorescence can be recorded with a detector.
[0004] A measuring device with which this type of measurement can
be carried out is already known from the prior art. DE 196 28 002
and DE 197 11 281 describe a device for carrying out fluorescence
immunotests by means of evanescent field excitation, in which a
light beam is directed at an angle suitable for total reflection on
to a boundary surface of a receiving region constructed in the form
of a cuvette, wherein the receiving region is arranged between an
optically transparent base plate and a cover plate. The receiving
region here preferably has a thickness of between 0.001 and 0.5 mm
and is defined by a spacer arranged between the base plate and the
cover plate. In this context, the spacer is preferably a thin foil
provided with an adhesive film on both sides, or a thin adhesive
film which can be stuck on the one hand on the base plate and on
the other hand on the cover plate.
[0005] In the devices from the prior art which are described above,
the excitation radiation is effected through the transparent base
plate, and the fluorescence emission emerges likewise through the
transparent base plate in the downwards direction. The detector and
the excitation source for recording the fluorescence emission are
accordingly arranged underneath the measuring cell formed by the
base plate, spacer and cover plate.
[0006] A disadvantage of the system from the prior art with
adhesive foil or adhesive film is that adhesive foil and adhesive
film have only a limited storage stability, so that during a long
storage period changes in the material may occur, which under
certain circumstances can even lead to non-fluid-tight areas
arising on the measuring cassette, for example due to small cracks
in the adhesive foil or the adhesive film. If such non-fluid-tight
areas arise, liquid sample may possibly leak from the measuring
cell arrangement and enter into the measuring device. In the case
of the devices from the prior art which are described above, the
sample here could, for example, drip on to the fluorescence
emission detector arranged underneath the measuring cell or the
excitation source likewise arranged in this region, which can lead
to an impairment in the fluorescence detection and therefore to an
impairment in all further measurements.
[0007] Such a device with an adhesive film furthermore can be
produced only with great manual effort, since the application of
the adhesive film can be automated only with difficulty.
[0008] The adhesive film moreover is conventionally made of a
flexible material, which under certain circumstances relaxes in the
course of time, as a result of which the height of the measuring
cell may change. The systems known from the prior art with adhesive
foil or adhesive film therefore also have the disadvantage that the
height of the measuring cell can vary over time and in dependence
on the amount of the pressure bearing on the base plate and the
cover, which may be associated with undesirable measurement
inaccuracies.
[0009] There was therefore a need for an improvement in the devices
known from the prior art for detecting target molecules in a liquid
sample by measurement of fluorescence emission, in order to ensure
that no sample liquid can arrive at the fluorescence detector from
the region in the measuring cell where the liquid sample is excited
to fluorescence emission.
[0010] The object of the present invention is therefore to provide
a device for detecting target molecules in a liquid sample by
measurement of fluorescence emission, in which it is ensured to a
better degree that no sample liquid from the measuring cell can
enter into the measuring device, in particular cannot arrive at the
fluorescence detector or at the excitation source.
[0011] This object is achieved according to the invention by
providing a measuring cassette of the abovementioned type, wherein
the measuring cassette comprises a body of an optically transparent
material and a base lying adjacent to the under-side of the body,
wherein the measuring cell is formed by a recess which is provided
in the body, in the base or both in the body and in the base,
wherein the areas on which the body and the base lie on one another
around this recess are bonded to one another directly and in a
fluid-tight manner by laser welding.
[0012] In contrast to the measuring cell of the devices known from
the prior art, the measuring cell of the measuring cassette
according to the invention is constructed such that the optically
transparent region is provided on the upper side thereof by the
optically transparent material of the body. The fluorescence
excitation and fluorescence measurement accordingly are effected
from above through the optically transparent material of the body.
The fluorescence detector can accordingly be arranged above the
measuring cell region, which avoids sample liquid leaking from the
region of the measuring cell arriving at the fluorescence detector.
Moreover, if the optical components are arranged underneath the
measuring cell, they are more easily contaminated by dust. If an
optical system operating from above is used, the danger of
contamination of the optical components with dust is significantly
lower.
[0013] In order additionally to ensure that no sample liquid from
the region of the measuring cell can enter into the measuring
device, the present invention moreover provides for the parts of
the measuring cassette which form the measuring cell to be joined
to one another directly and in a fluid-tight manner by laser
welding. Somewhat more precisely, the present invention provides
that the measuring cassette comprises, for the purpose of formation
of a measuring cell, a body of an optically transparent material
and a base lying adjacent to the under-side of the body. In this
context, the measuring cell is formed either by a recess provided
in the transparent material of the body or by a recess provided in
the base. In this context, the recess is covered by the other
particular component (base or body). Alternatively, the recess can
also be provided both in the body and in the base, wherein the
recessed region on the one side (e.g. in the body) and the recessed
region on the other side (e.g. in the base) together define the
recess which forms the area of the measuring cell.
[0014] In each case, the areas of the body and of the base present
lying adjacent to one another around the recess are bonded to one
another directly and in a fluid-tight manner by laser welding.
Bonding by laser welding has the advantage over gluing of the
measuring cell components--as is provided in the abovementioned
prior art--that by this means a direct bond is generated between
the components, without a layer of another material being present
between the components.
[0015] The bonding according to the invention by laser welding
ensures absolutely that the measuring cell is sealed with respect
to the liquid present in the measuring cell. In this context the
absolute seal is also ensured if the liquid in the measuring cell
is under pressure, e.g. if the sample liquid is moved through the
measuring cell with pressure. For example, the measuring cassette
according to the invention ensures absolute fluid-tightness even
under a pressure of up to 300 mbar. This also applies conversely to
a negative pressure situation in the measuring cell. For example,
if a reduced pressure of down to -300 mbar is generated in the
measuring cell, it is ensured that due to the measuring cassette
being closed in a fluid-tight manner, no air can enter into the
measuring cell from the outside.
[0016] Bonding by laser welding moreover has a significantly higher
long-term stability than the bonding via an adhesive foil or an
adhesive film known from the prior art.
[0017] Bonding of the components defining the measuring cell by
laser welding moreover is also of advantage from the production
point of view, since only two components are processed, without a
foil or a layer of adhesive in the required shape also having to be
arranged or applied between these components. It is therefore
possible for the production of the measuring cassette according to
the invention to be automated to a better degree.
[0018] This configuration moreover is more user-friendly, since by
this means cleaning steps which are required in the case of the
corresponding devices from the prior art are omitted, since once
the adhesive film in the known devices has become non-fluid-tight,
all the optical components lying underneath the measuring cell must
be cleaned.
[0019] The term "optically transparent" is used here such that an
optically transparent material is transparent both to the
excitation radiation and to the fluorescence emission. Preferably,
the transparent material of the body is glass or an optically
transparent plastic. In the case of plastics, these can be
selected, for example, from polycarbonates (PC), poly(methyl
methacrylates) (PMMA, Rohaglas) and polyolefins (Topas, COC).
Preferably, in the case of plastic this is a plastic suitable for
laser welding, wherein optically transparent plastics which are
suitable for laser welding are known to the person skilled in the
art.
[0020] The material from which the base is made is preferably a
plastic. The plastic is particularly preferably a poly(methyl
methacrylate) (PMMA) or a polyolefin.
[0021] If the material of the body is glass or a plastic which is
not suitable for laser welding, the material of the base must be
made of a plastic which is suitable for laser welding. On the other
hand, if the base is made of a material which is not suitable for
laser welding, the material of the body must be a plastics material
which is suitable for laser welding. Particularly preferably, the
body material and the base material are the same plastics material
which is suitable for laser welding.
[0022] Advantageously, the body and base are each one-component
parts, which are preferably produced in the injection moulding
process. This has the advantage that these components have no seams
or transition points between different materials through which
possibly sample liquid could leak or air could be drawn
in--particularly if a positive or negative pressure is built up in
the system.
[0023] Trapping molecules which capture the molecules of interest
from the sample liquid and fix them on the boundary surface so that
the fluorescence can be excited and measured there are preferably
arranged in the optical region at the boundary surface between the
optically transparent material of the body and the sample liquid.
Various receptor-ligand systems can be employed for this. These
systems include, inter alia, antibody-antigen, lectin-carbohydrate,
DNA- or RNA-complementary nucleic acid, DNA- or RNA-protein,
hormone-receptor, enzyme-enzyme cofactors, protein G- or protein
A-immunoglobulin or avidin-biotin. Preferably, the trapping
molecules are printed on to the boundary surface by means of a
printing process, e.g. by means of a piezo dispensing system.
[0024] In one embodiment of the present invention, the measuring
cassette has a sample channel which is in fluid contact with the
measuring cell and on which is provided at least one of the
following arrangements a) to c): [0025] a) a zone for dissolving a
tracer, [0026] b) a sample mixing region and/or [0027] c) a liquid
detection region, wherein the sample channel and the arrangements
a), b) and/or c) provided thereon are formed by a recess which is
provided in the body, in the base or both in the body and in the
base, wherein the areas on which the body and the base lie on one
another around this recess are bonded to one another directly and
in a fluid-tight manner by laser welding.
[0028] Preferably, the height of the sample channel is not more
than 300 .mu.m. Particularly preferably, the height of the channel
is in the range of from 100 to 300 .mu.m.
[0029] Preferably, one or more liquid detection regions (fluidic
sensor) are provided on the sample channel. Such a liquid detection
region on the sample channel serves to detect undesirable air
bubbles in the sample channel or to establish whether sample liquid
is present at a particular point of the sample channel by
establishing the transition between air and sample in the sample
channel.
[0030] In a preferred embodiment of the invention, the liquid
detection is effected by an optical method in which a light beam is
passed through the sample channel in order to establish with the
aid of the refraction of the light beam whether air or sample
liquid is present in the sample channel at this point. In this
respect, it is expedient and advantageous to embed at least
sections of the sample channel into an optically transparent
material, so that a light beam can be passed through the sample
channel for the purpose of fluid detection. Preferably, the section
of the sample channel in which a liquid detection is to be carried
out is present in the transparent material of the body. If, in such
an embodiment of the invention, the base is made of a material
which is not transparent to the light beam, recesses through which
the light beam can enter or emerge before entry into the sample
channel or after transillumination of the sample channel are
expediently present in the region of the beam path of the light
beam for detection of liquid in the base of the measuring
cassette.
[0031] The liquid detection region can also be used for
determination of the sample volume employed, e.g. via the
parameters of time and pump speed or the position of the sensors
relative to one another.
[0032] In cases where the molecules of interest (target molecules)
in the sample cannot themselves be excited to fluorescence
emission, these are marked with suitable fluorescence markers
(tracers). In a preferred embodiment of the present invention, at
least one zone for dissolving a tracer and optionally at least one
sample mixing region are provided for this on the sample channel
provided in the body for the purpose of reaction/conversion with
the sample in the measuring cassette. In this zone further
reagents, e.g. reagent to adjust the pH of the sample, lysis buffer
or reagents for reducing non-specific binding, can also be
dissolved, and likewise mixed with the sample in the mixing
region.
[0033] In one embodiment of the present invention, the body of the
measuring cassette is produced as a one-component part in the
injection moulding process. In another embodiment, the base is
produced as a one-component part in the injection moulding process.
In yet a further embodiment, both the body and the base are each
produced as one-component parts in the injection moulding
process.
[0034] The fluorescence-marked tracers are preferably printed on by
means of a printing process, e.g. by means of a piezo dispensing
system, into the zone provided for dissolving thereof.
[0035] The sample mixing region is preferably a so-called snake
mixer in which an efficient thorough mixing of the sample can be
achieved through the serpentine-like arrangement of the sample
channel.
[0036] When a measuring cassette according to the invention is
introduced into a suitable measuring device, minimal deviations
from the ideal position may easily occur. Even though these
deviations are only minimal, this can have significant effects on
the precise optical measurement method. In a preferred embodiment
of the invention, at the point where the excitation radiation
enters into the body the body therefore forms a converging lens
with which the beam of the excitation radiation is always directed
reliably to the desired point of the boundary surface of the liquid
sample and the surface of the transparent material of the body.
This converging lens integrated into the body can compensate for
the insertion variations described above. In particular, compared
with systems known from the prior art, by this means
positioning-related variations of the evanescent field (penetration
depth, intensity) can be reduced significantly in that the
alignment of the excitation radiation by the converging lens is
always effected exactly at the region in which the fluorescence
emission which arises there is to be measured, i.e. within the
predetermined limits of the region.
[0037] Preferably, the excitation radiation is aligned by the
converging lens on the region in which the fluorescence emission to
be measured is to be generated, i.e. centrally along the
longitudinal axis of the measuring cell. Preferably, the area of
the excitation region is circular or elliptical. Particularly
preferably, the area of the excitation region is elliptical having
dimensions in the region of about 0.7.times.0.25 mm.
[0038] The measurement cassette has a sample filling opening for
filling the measuring cassette with the liquid sample. Preferably,
the sample filling opening is provided on the upper side of the
body of the measuring cassette. In one embodiment of the invention,
the sample filling opening can be closed in a pressure-tight
manner. Preferably, the sample filling opening can be closed and be
pressure-tight up to a pressure or reduced pressure of +/-100 mbar.
Preferably, pressure seals, which particularly preferably are made
of polypropylene, are provided for this. In a particularly
preferred embodiment, the closure for closing the sample filling
opening is a slide valve which can be pushed from an opened
position into a closed position after filling of the sample,
wherein the slide valve is pressed on to a pressure seal provided
on the sample filling opening due to the geometry of the slide
guide.
[0039] In order to be able to generate a positive or negative
pressure in a sample channel of a measuring cassette according to
the invention, in preferred embodiments of the invention at least
one pressure opening is provided which can be connected to a
pressure source and is in ("pressure") contact with the sample
channel. Positive or negative pressure can be built up in the
sample channel in this manner, by means of which the sample can be
moved through the sample channel. Preferably, the pressure openings
are configured such that a pressure of at least +/-100 mbar can be
applied. In these embodiments, the sample channel is also
expediently configured such that it withstands this pressure
without problems.
[0040] In a particular embodiment, a sample waste region in which
sample liquid which has already been analysed can be accommodated
is provided in the measuring cassette at the end of the sample
channel. This has the advantage that the sample liquid which has
already been analysed does not leave the measuring cassette after
flowing through the measuring cell, but remains in the measuring
cassette. The sample waste region can be provided either in the
body or in the base of the measuring cassette.
[0041] In one embodiment of the present invention, in addition to
the body and the base, a cover which at least partly covers the
body is provided on the measuring cassette, wherein recesses are
provided at least in the region of the beam path of the excitation
radiation and the fluorescence emission, so that the body is not
covered by the cover in this region. The cover is firmly connected
to the unit of the body and base. Preferably, the connection of the
cover to the body and base is effected by hot caulking which
extends through the cover, body and base. In the embodiments with
the cover which have in their sample channel a liquid detection
region for optical detection of liquid, a recess in the cover is
provided in the region of the beam path of a light beam for
detection of liquid, so that the optical detection of liquid is
possible in spite of the cover.
[0042] For each measuring cassette batch, it is necessary also to
supply batch-specific calibration data. In known systems, this is
effected by a storage medium, such as e.g. a barcode or a memory
chip (EEPROM). Ideally, such a medium is to be present on every
measuring cassette, so that all the information for identification
of a measuring cassette and in particular the associated batch
calibration data are applied directly to this in the production
process and can be recorded and verified by the measuring
instrument when measuring with such a measuring cassette. In
principle, however, the batch calibration data can always be
determined only after the production of a batch has been concluded
completely (i.e. including packaging). The period of time between
the production of the first parts of a batch and the determination
of said calibration data can be several hours to days. To maintain
the stability of the reagents in such a measuring cassette, this
requires that this is packed air- and light-tight. In order to
apply said medium with the calibration data to the measuring
cassette, an additional, cost- and time-intensive unpacking and
packing step is therefore necessary, during which damage to the
measuring cassette may additionally occur. In the case of many
known systems, attempts are made to avoid this step by enclosing
one of these media separately in a pack unit. The user must then
either insert a memory chip with said information or read a barcode
into the apparatus before the first use.
[0043] In order to bypass this cumbersome and not necessarily
error-free process, a preferred embodiment of the measuring
cassette according to the invention is provided with an electronic
memory chip which can be written to and overwritten through the
packaging even after packing of the measuring cassette. Preferably,
this electronic memory chip is an RFID label. The RFID label can be
provided either with or without additionally visually readable
information.
[0044] The electronic memory chip can be applied to each measuring
cassette continuously during the production process of a batch.
Each measuring cassette therefore receives an individual,
electronic number. Each measuring cassette can be packed in the
desired packaging, for example a suitable primary packaging,
directly after assembly thereof and stored under the required
storage conditions. The application of the subsequently determined
batch-specific information, e.g. the calibration data, can then be
carried out in a contact-free manner through the packaging at any
time, even e.g. in a refrigerated environment. A further advantage
of this process is that in the context of regular production
monitoring over the life cycle of a batch, a necessary
recalibration can be carried out even after several months without
impairment of the product integrity.
[0045] According to a further aspect of the present invention, a
process for the production of a measuring cassette of the type
described above is provided, wherein in this process the body and
the base are produced as one-component parts in the injection
moulding process. Both the body and the base are therefore in each
case a one-piece component which can be produced from one material
each. In the measuring cassette embodiments which also have a cover
in addition to the body and base, the cover is also preferably
produced as a one-component part in the injection moulding process.
In the embodiments in which the measuring cassette has a closure
for closing the sample filling opening, this closure is preferably
an integral constituent of the one-component part on which the
closure is provided.
[0046] In a preferred process, in the production of a measuring
cassette with a cover connecting of the cover to the unit of the
body and base is carried out by hot caulking. For this, the cover
has appropriate pins of plastic which are passed through bores at
the corresponding points in the body and base, and a
positive-locking connection of the cover to the body is established
at the base by thermoforming.
[0047] A further aspect of the present invention relates to a
measuring device for the detection of target molecules in a liquid
sample by measurement of fluorescence emission after excitation in
an evanescent field, wherein the measuring device has an insertion
shaft for a corresponding measuring cassette. In the insertion
shaft of the measuring device, the measuring cassette is arranged
in at least one position such that the light source for the
excitation radiation arranged in the measuring device can provide
an excitation radiation which meets the boundary surface of the
liquid sample and measuring cell such that total reflection of the
excitation radiation to form an evanescent field in the liquid
sample is effected. The penetration depth and the intensity of the
evanescent field can be influenced via the choice of the angle of
the incident excitation beam. Preferably, the angle with which the
excitation radiation meets the boundary surface is about 8
degrees.
[0048] A measuring device of the abovementioned type which has an
insertion shaft for a measuring cassette according to the present
invention is claimed in particular. Inter alia, the complete system
of a measuring device of the abovementioned type and one or more
measuring cassettes according to the present invention is also
claimed. A measuring device of the abovementioned type with a
measuring cassette according to the present invention inserted into
the insertion shaft is claimed in particular.
[0049] In a preferred embodiment of the measuring device according
to the invention, this also comprises arrangements with which a
relative movement of the measuring cassette with respect to the
light source for the excitation radiation can be performed, in
order thus to bring various measuring cell regions into the beam
path of the excitation radiation. In this manner, several different
trapping zones can be provided in the measuring cell, in order to
respond to several different analytes at the various trapping zones
using only one light source.
[0050] In one alternative to the abovementioned embodiment, the
measuring cell in the insertion shaft is moved optionally
continuously or in a stepwise manner along an axis of movement from
a first position into at least a second position, in order to bring
various measuring cell regions into the beam path of the excitation
radiation. As an alternative to this, the measuring device
according to the invention can also be configured such that the
light source for the excitation radiation can be moved continuously
or in a stepwise manner along an axis of movement such that it
aligns its excitation beam to various regions of the measuring
cell.
[0051] Regardless of the alternative chosen, the light source for
the excitation radiation is preferably provided in the measuring
device such that the beam of the excitation radiation meets the
measuring cell at an angle of 90 degrees to the direction of
movement of the relative movement of the measuring cassette with
respect to the light source. Inter alia, the path of the excitation
beam through the body is minimized in this manner. Furthermore,
always only a small section of the optically transparent material
is illuminated. The path of the excitation beam through the body
furthermore is always the same over the entire region traveled
through by the excitation beam. Moreover, a better local resolution
of the excitation light within the measuring cell is achieved
overall. This advantage of the measuring device according to the
invention means that a better signal to noise ratio is
achieved.
[0052] In one embodiment of the measuring device according to the
invention, this is distinguished in that it has a pressure source
which, in the case of a measuring cassette which is inserted into
the insertion shaft and has a pressure opening on the sample
channel, is connected to the pressure openings of the sample
channel such that a sample present in the sample channel can be
moved through the sample channel by means of positive or negative
pressure generated by the pressure source. Preferably, the pressure
source and the connections of the pressure source to the pressure
openings are configured such that a pressure of at least +/-100
mbar can be applied.
[0053] In a further aspect of the present invention, a method for
detecting target molecules in a liquid sample by measurement of
fluorescence emission after excitation in an evanescent field is
described, wherein a measuring cassette according to the invention
in combination with a measuring device according to the invention
are employed in the method. All methods of the abovementioned type
in which aqueous samples are analysed in a corresponding manner,
for example also corresponding methods in environmental and
foodstuffs analysis, are possible here. The method according to the
invention is preferably immunoassays or DNA binding assays.
However, the present invention is in no way limited to these, but
includes all methods which can be realized in this connection in
which target molecules are recorded qualitatively and/or
quantitatively in a liquid sample by measurement of fluorescence
emission after excitation in an evanescent field.
[0054] In one embodiment of the method according to the invention,
the sample upon which the measurement is carried out is a treated
or untreated aqueous sample for foodstuffs or environmental
analysis. In connection with the present invention, a treated
sample is to be understood as meaning a sample which has been taken
from a sample source and has been treated by measures known to the
person skilled in the art for the purpose of carrying out the
measurement in order to facilitate or to improve the measurement
procedure. One possibility for treatment of a sample is, for
example, prior removal from the sample of components which possibly
interfere in the measurement.
[0055] In an alternative embodiment of the method according to the
invention, the sample upon which the measurement is carried out is
a treated or untreated body fluid. The body fluid here can be
selected, for example, from blood, urine, saliva etc. Treated body
fluids can be, for example, blood plasma and serum. In the
embodiments of the method according to the invention in which the
sample is whole blood, the circumstance that under laminar flow in
the measuring cell centralization of the blood corpuscles takes
place is utilized, so that the evanescent field excitation can be
effected in a region of the whole blood in which no impairment of
the excitation or radiation emission by blood corpuscles is
effected.
[0056] For the purpose of the original disclosure, it is pointed
out that all the features such as are revealed to a person skilled
in the art from the present description, the drawings and the
claims, including if they have been described specifically only in
connection with certain further features or represented in the
subsequent figures, both individually and in any desired
combinations, can be combined with other features or feature groups
disclosed by the description or representation, as long as this has
not been expressly ruled out or technical circumstances make such
combinations impossible or senseless. Comprehensive, explicit
explanation or representation of all conceivable combinations of
features is dispensed with here merely for brevity and readability
of the description.
[0057] Further individual features and combinations of features of
the invention and further advantages of individual features and
combinations of features of the invention emerge from the figures
attached to this application and the following description thereof.
It is pointed out in this connection that it goes without saying
for the person skilled in the art that the embodiments represented
in the figures merely serve to indicate by way of example features
and combinations of features on the basis of possible embodiments
of the present invention. The person skilled in the art will
readily understand that beyond the embodiments represented in the
figures, all other embodiments which have the features or
combinations of features according to the invention mentioned in
the claims and the description lie within the scope of protection
of the invention. Comprehensive, explicit explanation or
representation of all conceivable embodiments is dispensed with
here merely for brevity and readability of the description.
[0058] The attached figures show in detail:
[0059] FIG. 1: plan view of an embodiment of a measuring cassette
according to the invention, comprising a transparent body and a
base,
[0060] FIG. 2: plan view from underneath of the base-facing side of
the body of an embodiment of a measuring cassette according to the
invention (without showing the base lying on top),
[0061] FIG. 3: plan view from the top of the base-facing side of
the body of an embodiment of a measuring cassette according to the
invention (without showing the body lying on top),
[0062] FIG. 4: diagram of the principles of a fluidic sensor in an
embodiment of a measuring cassette according to the invention in
the case of a sample channel filled with air (a) and in the case of
a sample channel filled with sample liquid (b),
[0063] FIG. 5: diagram of a section through the region of an
embodiment of a measuring cassette according to the invention in
which the measuring cell is arranged,
[0064] FIG. 6: diagram of a section through the region of an
alternative embodiment of a measuring cassette according to the
invention in which the measuring cell is arranged, wherein a
converging lens for the excitation radiation is provided in the
region, and
[0065] FIG. 7: diagram of an embodiment of a measuring device
according to the invention.
[0066] FIG. 1 shows a plan view of a measuring cassette according
to the invention, comprising a transparent body (1) and a base (9)
which are bonded to one another by a laser welding process. The
body (1) and base (9) are produced as one-component parts in the
injection moulding process.
[0067] FIG. 2 shows a plan view from underneath of the base-facing
side of the body of a measuring cassette according to the invention
(without showing the base lying on the body on this side). The body
part comprises a sample filling opening (3) for filling with
sample, connected directly and without branches to a microchannel
structure (sample channel), having a mixing region (4), a zone for
dissolving the tracer (5) and a further mixing region (6), an
optical region (2) and a subsequent sample waste region (7). The
channel ends (8a) and (8b) are configured such that these are
connected to the base (9) at the pressure connection openings (11a)
and (11b) present there.
[0068] In the plan view from the top of the base-facing side of the
body of a measuring cassette according to the invention of FIG. 3
(without showing the body lying on top), the pressure connection
openings (11a) and (11b) which are present in the base (9) and are
mentioned in the description of FIG. 2 are to be seen.
[0069] In this context, the corresponding measuring device is to be
configured such that the measuring cassette can be drawn in
completely automatically with the aid of a toothed structure (13)
and after conclusion of a measuring operation is ejected again
completely automatically. The drawing in moreover is configured
such that user errors during insertion of the measuring cassette
into the corresponding measuring device are ruled out.
[0070] The embodiment of the measuring cassette according to the
invention shown in FIGS. 2 and 3 is configured such that the
filled-in sample can be moved by a positive or negative pressure
between the sample and pressure connection openings (11a) or (11b)
from the sample filling opening (3) through the fluid microchannels
(4-6) and through the measuring cell (12). To generate the required
pressure, the embodiment of the measuring device according to the
invention into which the measuring cassette for carrying out the
analysis is introduced expediently has a pressure source, such as
e.g. a syringe, and connection possibilities on the pressure
connection opening (11a) or (11b) of the measuring cassette,
wherein the connection between the pressure source and pressure
connection openings preferably is configured such that a pressure
of at least .+-.100 mbar can be used. If positive/negative pressure
is used via port 11a, the sample inlet must be closed. If port 11b
is used, on the other hand, closure of the sample inlet is not
necessary.
[0071] In the embodiment shown here, the microchannels (4-6) in the
body part (1) are characterized in that there are no junctions at
which air bubbles can be trapped, and the maximum difference in
cross-section between the smallest and largest channel
cross-section is a factor of 2, so that membrane formation and
therefore a disturbance in the fluidics cannot occur.
[0072] During production of the measuring cassette,
fluorescence-marked tracers are printed into the microchannel
region (5) between the mixing regions (4, 6) by means of a printing
process, e.g. a piezo dispensing system. Trapping molecules (21)
are printed on to the boundary surface (19) of the optical element
(2) within the region of the measuring cell (12) by means of the
same process.
[0073] FIG. 4 illustrates the principle of a fluidic sensor for
fluid detection in one embodiment of the measuring cassette
according to the invention. In this context, fluid detection is
effected by coupling light (15), e.g. by means of an LED, wherein
it can be seen from the refraction of the coupled light beam
whether or not sample liquid is present in this channel region. If
the channel is empty, in this embodiment the difference in the
refractive index between the body and air is selected such that the
incident beam is refracted only to the extent that it meets a
detector (16) lying opposite, e.g. a photodiode, whereas the
difference in refractive index when the channel is filled means
that the incident beam is refracted such that it is reflected at
the body-base boundary surface and does not reach the detector (16)
lying opposite. So that the light beam can reach said detector in
the case of an empty channel, recesses (14) are provided in the
base (9) at the appropriate points.
[0074] FIG. 5 shows a diagram of a section through the region of an
embodiment of a measuring cassette according to the invention in
which the measuring cell is arranged.
[0075] The measuring cell (12) can be filled with the sample to be
analysed via a microchannel structure, such as is shown, for
example, in FIGS. 2 and 3. The measuring cell (12) has an optically
transparent region (2) which is a constituent of the body (1) lying
on the base (9). This optically transparent region (2) has a light
entry area (18) and a boundary surface of total reflection (19).
The material of the optically transparent region (2) expediently
has a higher refractive index than the samples to be measured.
[0076] When light (preferably monochromatic light) from a light
source (17) in a measuring device in which the measuring cassette
of FIG. 5 is arranged enters as a beam having a particular geometry
(preferably elliptical or circular) via the light entry area (18a)
into the optically transparent region (2) at a suitable angle, the
light beam meeting the boundary surface (19) undergoes total
reflection.
[0077] As a result of the total reflection generated at the
boundary surface (19), an evanescent field arises on the reverse
thereof. The penetration depth and the intensity of said evanescent
field can be influenced via the choice of the angle of the incident
light beam.
[0078] In the embodiment shown here, a recess is provided in the
base part (9), which in combination with the boundary surface (19)
forms the measuring cell (12) through which the sample, for example
coming from the mixing region (6), is moved by means of positive or
negative pressure, preferably with a constant, homogeneous flow
rate. This measuring cell (12) is characterized here by a
particularly low height compared with the other channels of an
optionally upstream and/or downstream microfluidic structure. As a
result, the sample volume is utilized to the optimum, since a
longest possible measurement time at a highest possible flow rate
is rendered possible. A preferred height of the measuring cell (12)
is .ltoreq.300 .mu.m.
[0079] At the boundary surface (19) in the measuring cell (12), at
least one trapping zone (21) is provided, in which trapping
molecules are arranged, which capture the molecules of interest
from the sample liquid and fix them on the boundary surface so that
the fluorescence can be excited and measured there are
arranged.
[0080] For the measurement of the fluorescence, a detector (20) for
measurement of a fluorescence intensity which changes with respect
to time (e.g. PMT or CCD camera) is provided in a measuring device
in which the measuring cassette of FIG. 5 is arranged.
[0081] In the embodiment of FIG. 5, the light entry area is a plane
(18a). In the alternative embodiment of a measuring cassette
according to the invention which is shown in FIG. 6, for the
excitation radiation the light entry area is configured as a
converging lens (18b). The converging lens (18b) ensures that in
this embodiment the light beam from the light source (17) always
meets the boundary surface (19) centrally within the region of the
measuring cell (12) and the effects of variations in insertion on
an evanescent field forming under total reflection are therefore
compensated. This compensation reduces positioning-related
variations in the evanescent field, i.e. the penetration depth or
intensity thereof, compared with known systems.
[0082] FIG. 7 is diagram of an embodiment of a measuring device
according to the invention. The beam path of the excitation light
from the light source (17) and of the fluorescence light from the
measuring cell to the detector (20), inter alia, are shown.
[0083] The light source (17) and the detector (20) are connected to
one another mechanically in the measuring apparatus in one plane to
form a unit. This unit and the measuring cassette of body (1) and
base (9) for the measurement are moved relative to one another. In
this context, the light source (17) is arranged transversely to the
direction of movement, so that the light beam radiates on to the
boundary surface (19) transversely to the direction of movement.
With this configuration, it has been possible to avoid essential
disadvantages of the known system, since (i) the path of said light
beam through the optically transparent region (2) is minimized,
(ii) always only a small section of the optically transparent
region (2) is illuminated, (iii) the path of the excitation light
through said optically transparent region (2) is always the same
length over the entire detection region and (iv) a better local
resolution is achieved.
[0084] The thickness of the optically transparent region (2) here
is to be chosen independently of the number of trapping zones (21).
A large number of analytes can therefore be determined with one
measurement at the various trapping zones (21) using only one light
source (17) and only one fluorescent dyestuff for all the analytes,
the same conditions for excitation and detection can be created
over the entire scanning region and the effect of the intrinsic
autofluorescence of the plastics can be reduced significantly and
the signal-noise ratio therefore improved significantly.
LIST OF REFERENCE SYMBOLS
[0085] 1 Body [0086] 2 Optical region [0087] 3 Sample filling
opening [0088] 4 Mixing region [0089] 5 Zone for dissolving the
tracer [0090] 6 Second mixing region [0091] 7 Sample waste region
[0092] 8 Channel end [0093] 9 Base [0094] 11 Pressure connection
opening [0095] 12 Measuring cell [0096] 13 Toothed structure [0097]
14 Recesses [0098] 15 Coupling of light [0099] 16 Detector [0100]
17 Light source [0101] 18 Light entry area [0102] 19 Boundary
surface [0103] 20 Fluorescence detector [0104] 21 Trapping zone
* * * * *