WO2008128534A1 - Cuvette for optical analysis of small volumes - Google Patents

Abstract

The aim of the invention is to provide a cuvette for small volumes avoiding the disadvantages of the prior art, particularly allowing measurements directly in a system simply by feeding further samples, and is achieved in that the cuvette for the optical analysis of small volumes comprises a structured layer (support substrate) having at least one channel, wherein at least the underside of the structured layer is closed by a thin, optically transparent layer and the channel has at least two fluid interfaces connected thereto to conduct fluids.

Description

 P2435 Patent and Lawyers Bock Bieber Donath, Hans-Knöll-Str. 1, 07745 Jena

Cuvette for the optical analysis of small volumes

The invention relates to a cuvette for the optical analysis of small volumes.

Cells for optical analysis per se have been known for decades. These are available as conventional cuvettes made of glass or quartz, such as those offered by the companies Hellma or Starna, as well as plastic cuvettes, such as those from Eppendorf or Brand.

A disadvantage of these known cuvettes is that the

Material thickness can not be minimized arbitrarily. That Material thickness of these cuvettes is significantly thicker than that of thin films, so that a higher and disturbing background signal arises when used as intended. This is particularly problematic in the case of the plastic systems offered as disposable cuvettes, since the applicability to the wavelength range above 220 nm, z.T. far above this range is limited. The second disadvantage of the known cuvettes is that their measuring volumes are relatively large and only geometrically simple structures can be realized as a measuring space. That unnecessarily large volumes of samples are necessary for the intended use and the realization of different layer thicknesses for the measurement is not or only partially realizable.

Even with cuvettes that can be used for smaller volumes, the volumes are limited to the range above 50 μl. An exception is the cuvette from Hellma with the name "TrayCell" whose measurement volume is 0.7 to 4 μl or 3 to 5 μl at a layer thickness of 0.2 or 1 mm.

The disadvantage of this system is that it is a glass cuvette that has to be produced in a costly manner by glass structuring and joining of glass elements. The measuring range of the known cuvettes is almost exclusively limited to one layer thickness except for the cuvette of Eppendorf called "UVette", which realizes two thicknesses.Therefore dilutions outside the cuvette and a new measurement are necessary for the acquisition of further measuring ranges.

Another disadvantage is that when measuring several samples, the cuvette must be replaced or cleaned.

If a storage of the measured samples is desired, none of the cuvettes is suitable for a simple, space-saving storage.

The paper by D. Schepers, G. Schulze and W. Frenzel ["Spectrophotometric flow-through gas sensor for the determination of atmospheric nitrogen dioxide" (Analytika Chimica Acta 308 (1995) 109-114] discloses a microfluidic measuring cell, especially for photometer or Spectrometers which are preferably narrow-band, comprising wafer interconnected with each other, are introduced into the microchannels, that in two areas at least two microchannel sections are arranged parallel to each other and they are spatially separated from each other by a selectively selected according to a substance to be analyzed selected membrane , so that an extraction section is formed, wherein in a first wafer at least said first microchannel section and its channel ends detecting inlet and Abströmöffhungen or inlet and outlet channels are introduced for the passage of an analyte, in a second wafer least mentioned second Mikrok and at least one wafer for a measuring light beam used is permeable or provided with a window region which ensures this, and the inlet and outlet openings and inlet and outlet passages for the passage of an extractant (E) are introduced Exit of the measuring light beam is determined by the serving as an extraction channel second micro-channel portion such that, depending on the radiation source used (L), as long as possible optical measuring section is realized. The disadvantage of this solution is that the disclosed system is not an easy-to-use measuring device, such as a cuvette. Furthermore, the system is not suitable for measuring different liquid volumes or in different layer thicknesses.

DE 197 07 044 A1 discloses a micromechanical transmission cell for determining an optical absorption of a sample fluid or as a reactor for exporting an optically detectable chemical reaction. The micromechanical transmission cell has a container for holding the sample fluid, a Lichtdurchlassöffhung for introducing the light into the container and a reflector device which directs the light with respect to the container, that a large part of the light passes through the container without multiple reflections on one of the container walls.

From DE 197 07 044 C1 a microfluidic module for chemical analysis is known in which a rapid sample change and thus inexpensive investigations of fast-running processes time-resolved and with small time constants is made possible and optionally at the same time allows the possibility of performing a scanning calorimetry, wherein the microfluidic module of a first chip, in which an outstretched channel region with a Y-shaped branched input region, to which two input channels connect, is introduced and the first chip is connected to a second chip covering, which is provided on the channel side with at least one thermosensitive thin-film element consists.

The disadvantage of these solutions is that the disclosed systems are not an easy-to-use measuring device, such as a cuvette. Furthermore, the systems are not suitable for measuring different volumes of liquid or in different layer thicknesses.

The object of the present invention is to provide a cuvette for small volumes, which has the disadvantages of the prior art prevents, in particular directly in a system, only by supplying additional samples, measurements allows.

The object is solved by the characterizing features of the first claim. Advantageous embodiments are covered by the subordinate claims.

The present invention is a chip cuvette for small volume optical analysis.

The essence of the invention is that the chip cuvette has a flat, planar shape, which is characterized in that it has a minimum layer of cuvette material when used as intended in a measuring channel at the measuring points. As a result, the influence of the cuvette material itself (for example, by autofluorescence or self-absorption) is minimized to the measurement. The use of this thin cuvette material, which is preferably in the form of films, overcomes the disadvantages of conventional cuvettes known hitherto and enables measurements even in the VUV range, even in the case where the cuvette material is a plastic polymer.

From the basic structure of the chip cuvette according to the invention is characterized in that it is made up of at least two, preferably three layers, wherein at least one layer is optically transparent. All embodiments have in common that they consist of a structured layer (carrier substrate) with channels or through-holes, which is closed at least from one side, but particularly advantageously both sides with a thin, optically transparent film.

As an alternative embodiment, the structured layer (carrier substrate) and the one film side may be formed in one piece.

The channel system in the structured layer (carrier substrate) serves to supply the sample to the actual, in the structured layer (Carrier substrate) located measuring cell, as a reservoir and as a reaction vessel, where it structurally passes into the measuring cell.

The carrier substrate may also have through holes connected to channels extending in the one outer layer and the two outer layers, respectively.

Due to the flat construction, the precise minimization of the channel structures down to the micrometer range and the reduction of unnecessary dead volumes, significantly smaller volumes can be analyzed by means of the chip cuvette according to the invention compared to the known, commercial cuvettes

The connection of the channel structures to the outside is realized by at least two fluidic interfaces and is used for filling with sample or for taking sample.

These connections are designed in one embodiment of the chip cuvette according to the invention as a simple or conical through holes. The chip cuvette is thereby unproblematic to handle since different pipettes can be used to fill the channel structures via the connections. Furthermore, the automatic filling of the channel structures via the connections, for example, with pipetting robots is possible.

Alternatively, the terminals of the channel structures can be used in a differently shaped fluid connection form, such as, for example, in the form of olives, lures or miniaturized Luer-like connections.

In the intended use, any evaporation problems that occur can be avoided according to the invention simply by covering or masking the Einfullöffhungen the structured layer (carrier substrate), for example. With a film.

The materials used for the chip cuvette according to the invention are a wide variety of plastic polymers, preferably transparent ones Polymers with low optical interferences such as COC, COP, PMMA, PC, etc., but also glass, quartz glass or other crystalline or amorphous materials in combination with thin glass, quartz or other transparent crystalline disks as a lid for use. That is, the chip cuvette may consist exclusively of a plastic, of different plastics or of one of the abovementioned non-plastic materials, which may also be combined with one another, or hybrid constructions of plastics and non-plastics (for example a combination of glass-plastic, Glass - plastic - glass, glass - plastic - glass plus additional connecting layer etc.).

Several channel systems can be integrated simultaneously in the structured layer (carrier substrate) so that several measurements with different samples can be carried out simultaneously or in succession.

By means of a special design of the channel systems of the chip cuvette with a plurality of interconnected measuring cells of different channel depth, it is possible for a sample to be measured simultaneously or consecutively in different measuring ranges.

As a result, the dynamic range for the measurements of the sample increases significantly and expensive dilution series are reduced significantly or become superfluous.

Expansion stages of this platform include the integration of mixers in front of the measuring channels as well as the integration of distribution systems (channel systems) on the chip, in order to carry a sample to different measuring points in which, if necessary, reactions to be tracked can also take place due to the reagents provided. Integrated mixers for creating dilution series are another option for enabling very different measuring ranges, even for the simultaneous acquisition of multiple analytes.

By integrating optical elements into the chip cell, the input and output of light can be optimally designed, the scattered light significantly reduced and thus also sensitive fluorescence measurements are possible.

The chip cuvette in its design as a flat cuvette with several channel systems accommodates different samples or sample series and thus offers the possibility of a very simple and space-saving later storage. Possibly. The chip cuvettes can also be frozen and thus made accessible to later analyzes. For archiving, labeling is possible via permanent markers as well as barcodes or RFID tags.

The channels of the chip cuvette according to the invention can, for example, be designed such that, with the same measuring arrangement, measurements can be carried out simultaneously or successively in different measuring ranges. Thus, it is possible to measure in two or more different optical regions, since two or more layer thicknesses (different channel depths or complete apertures in the material) can be realized by the different channel depth.

An embodiment of the chip cuvette according to the invention may, for example, be designed in such a way that a small channel is arranged on one side and a larger channel on the other side of the carrier substrate and the ends of which are connected by a hole or collide at the ends by their depths and so already make a breakthrough.

The chip cuvette may be provided with a depression above the channel to minimize the material thickness between sample and excitation light source as well as detector.

The chip cuvette can have a three-layer structure in which the connection of the small measuring volume with the large measuring volume is realized by a small through-channel or a small through-hole in the chip. In the case of the chip cuvette according to the invention, the inputs and outputs of the respective measuring elements can have different fluidic interfaces, for example bores with different diameters, in order to realize different possibilities of fluid supply (eg in order to inject or remove samples with different sized pipette tips.

These interfaces can be designed in the form of conical inlet and outlet nozzles, which can be designed differently in order to be able to use the widest possible spectrum of different pipette tips with the chip cuvette.

The conical inlet nozzle according to the invention ensure a good seal against pipette tips and at the same time prevent closing of the channel by the pipette tip.

The fluidic interfaces may be provided with sealing rings to allow a good seal, for example. With respect to a fluidic equipment connection. By means of these directly integrated seals a liquid and / or gas-tight termination of the chip cell to other components, such as. A control gear, can be realized.

In the case of the chip cuvette according to the invention, numerous, preferably parallel, channels can be provided for the measurement of different samples, which can be assigned to a measuring unit. Through different channel widths different measurement volumes can be realized.

The chip cuvette may have the format of a slide, possibly with a smaller or larger thickness, wherein the fluidic interfaces are arranged such that they can be filled with common pipetting robots, i. have the well spacing of microtiter plates (18 mm, 9 mm, 4.5 mm, 2.25 mm ...).

The chip cuvette may also have the format of a titer plate, wherein the fluidic interfaces are arranged such that they are common Pipetting robots can be filled, ie the well spacing of microtiter plates (18 mm, 9 mm, 4.5 mm, 2.25 mm ...) have.

The chip cuvette according to the invention can be provided with integrated optical elements for the use of total reflection, in order to obtain the longest possible optical path through the channel or to enable coupling and / or decoupling of the light. The chip cuvette according to the invention can be designed with different measuring elements, for example different volumes, channel geometries, etc., in order to be able to measure various analytical tasks with a sample on the chip.

In the case of the chip cuvette according to the invention, the carrier substrate can be sealed after infestation with a foil (for example self-adhesive, self-adhesive or tight-fitting foil or aluminum foil) or a plane-lying or geometrically adapted cover, in order, for example, to be able to carry out long-term measurements without evaporation effects. In order to subsequently remove the sample, embodiments of the chip cuvette with subsequently removable or pierceable films can be provided.

The chip cuvette according to the invention may, for example, be widened by a mixer which is integrated in the channel within the carrier substrate in order to mix certain substances before the measurement or to dilute certain substances on the chip cuvette either simultaneously or in one or more dilution stages offset to be able to measure.

The chip cuvettes according to the invention may also have a distribution system (channel system) in order to guide the sample to different measuring points in which, if appropriate, reactions can be carried out and monitored by reagents presented.

In contrast to the well-known commercial glass or quartz cuvettes especially the "TrayCell" Hellma the chip cuvette is a robust and inexpensive measuring means. Designed as a disposable component, the chip cuvette according to the invention, as well as commercially available disposable plastic cuvettes, has no contamination problems.

The invention will be explained in more detail below with reference to an exemplary embodiment. Show it:

1 is a schematic representation of a first embodiment of the cuvette according to the invention, Fig. Ia is a longitudinal section through the cuvette of FIG. 1,

2 is a schematic representation of a second embodiment of the cuvette according to the invention,

2a is a longitudinal section through the cuvette of FIG. 2,

3 is a schematic representation of a third embodiment of the cuvette according to the invention,

3a shows a longitudinal section through the cuvette of FIG. 3,

FIG. 3b shows a cross section through the deep measuring chamber of the chip cuvette according to FIG. 3 with representation of the measuring light geometry and FIG. 3c shows a cross section through the flat measuring chamber of the chip cuvette according to FIG. 3 with representation of the measuring light geometry.

In order to guarantee a good and largely accepted handling in the laboratory, the variants of the cuvette according to the invention shown in Figures 1 to 3 in the slide format (75 x 25mm) are executed.

The embodiments illustrated in FIGS. 1 to 3 consist of a series of channels which are introduced at a spacing of 4.5 mm corresponding to the grid dimension of a 384er titer plate into a flat carrier substrate (1), which has a cover film (2, 3) along the open channel course ) are closed.

Depending on the application and measuring principle, this version can accommodate a maximum of 14 - 16 channels. The channels are numbered with numbers. In principle, larger chip board formats with multi-row arrangement of the channels are possible. Glass, quartz or polymers are used as carrier and film material, which have good transparency with regard to their spectroscopic properties for the UV-VIS range. The polymers are at present special TOPAS ^® COC types show a high transparency up to the UV-range, the drawn films exhibit compared to the granule again shifted in the UV region absorption edge and are therefore preferably selected. This polymer material also shows good chemical resistance to a number of frequently used in the analysis of solvents such as dilute mineral acids but also organic solvents such as ethanol, acetonitrile or dimethyl sulfoxide. What is also important for an application in the diagnostic field is the biocompatibility and sterilizability of this polymer material. As shown in the longitudinal sections (FIGS. 1a, 2a, 3a) of all the mentioned exemplary embodiments, all channels have two measuring chambers (6, 7), the larger chamber (6) extending over the entire carrier thickness.

The channel sections (10) located in front of and between the two measuring chambers are likewise very shallow and serve as an evaporation barrier. In the illustrated three embodiments, the channel sections (10) have the same depth as the flat measuring chamber, which results on the one hand from the selected thickness of the chip carrier and on the other hand manufacturing advantage. For the thickness of the chip cell carrier (1) and thus the maximum layer thickness is selected 1 mm and for the small layer thickness a measuring channel height of 0.1, which corresponds to exactly one order of magnitude. This is advantageous for the user because of the easy conversion (factor 10) and, in terms of the small layer thickness, is within a good tolerance range in terms of production.

The channel shape is trapezoidal in the beam direction both along (Fig. 1a, 2a, 3a) and transversely (Fig. 3b, c) and thus adapted to the opening angle of the measuring beam (17). The ratio of measuring chamber length to width is generally common elongated (rectangular) input gap of the spectral optical system adapted.

For the smallest currently used lOOμm coupling fibers (14) and a 500μm decoupling fiber, a channel width of 600 μm is determined as the optimum. This results in a necessary sample volume of 0.3 .mu.l for a filling from the side of the small measuring chamber and a necessary sample volume of 2 .mu.l for a filling from the side of the large measuring chamber. The capacity with complete filling of the entire channel up to the channel openings is approx. 4μl.

The two ends of each channel are led upwards via cones (4, 5), which can be used as fluidic interfaces for commercial pipettes. The tips used on the market for volumes of 0.1-200μl can be divided into two size groups according to their suction conical shape, for each of which an optimized conical shape could be found. The smaller cone is located on the side of the smaller measuring chamber and the larger one on the side of the large measuring chamber. Thus, leakage and bubble-free filling as well as removal of sample liquid with crystal tips from one side (5) and with 200μl tips from the other side (4) can be realized.

To realize the handling of multipipettes, the chip design shown in FIG. 2 is provided. It consists of raised edge areas (11) at the ends of the channel where the cones (4, 5) are located. These serve to accommodate the funnel-shaped attachments (12) which adjoin the cones (4, 5) and compensate for the tolerances in the position of the tips of the multipipettes to each other when inserted into the sealing Koni (4, 5).

The channel design according to the invention shown in FIGS. 1 to 3 allows both a good filling and on the other hand has no air bubble formation during filling, since the air bubbles are reliably outside the measuring window.

These advantages are also achieved when using polymeric materials in the event that a mold insert pair is ready is, which can be positioned in the corresponding injection molding tool with respect to the tolerances micrometer accurate to each other.

The resulting very low surface roughness in the channels reliably prevents the formation of air bubbles at the Meßkammerstellen. In order to obtain as little as possible disturbing influences above the small measuring chamber (7) by the material of the chip carrier (1), in particular in polymer materials, the material thickness is minimized by introducing a depression in the form of an optically polished measuring window (8). For manual positioning of the chip cuvette according to the invention in a control gear notches are selected which are arranged on both longitudinal sides in each case centrally to the channel. The design variant shown in FIG. 2 is designed for automated handling in an operating device with XY positioning device and orientation and orientation of the chip as well as the determination of the position by an automatic calibration.

In order to simultaneously carry out both transmission and emission spectroscopic examinations with one and the same sample in one channel, a groove with a total reflection surface (13) is introduced next to each measuring chamber, as shown in FIGS. 3, 3a-c. This allows emission through a fiber light guide for the emission excitation light (14), lateral, 90 ° to the fiber optic of the transmission and emission detection system excitation of the emission, allowing a significant reduction of the excitation scattered light compared to a frontal excitation.

In the case of the small measuring chambers (see FIG. 3c), the optically flat surfaces of the depression (8) and of the film (3) likewise act as total reflection surfaces.

All features shown in the description can be essential to the invention both individually and in any combination with one another. LIST OF REFERENCE NUMBERS

1 carrier substrate (chip carrier)

2 cover foil (upper)

3 cover foil (lower)

4 big cone (fluidic interface)

5 small cone (fluidic interface)

6 larger measuring chamber chamber

7 small measuring chamber

8 measuring windows (recess)

9 notches

10 channel sections

11 elevated edge area

12 attachments (funnels)

13 total reflection surface (groove)

14 input fibers (emission excitation light)

15 Beam path of the emission excitation light

16 input fibers (transmission measuring light)

17 Measuring beam (beam profile profile of the transmission measuring light

18 fiber optic cable to the detector system for

Transmission and emission

Claims

claims 1. cuvette for the optical analysis of small volumes consisting of a structured carrier substrate (1), which at least one Channel (10) is provided, characterized in that the structured carrier substrate (1) is planar and optically transmissive and the channel (10) has at least two measuring chambers (6; 7) with different channel depths, wherein one side of the structured carrier substrate (1) with a thin, optically transparent film (2, 3) is closed, which has at least two fluidic interfaces (4, 5), which are liquid-conductively connected to the channel (10). 2. Cuvette for the optical analysis of small volumes consisting of a structured carrier substrate (1) which is provided with at least one channel (10), characterized in that the structured carrier substrate (1) is planar and optically transparent and the channel (10) at least two measuring chambers (6; 7) with different channel depths, wherein one side of the structured carrier substrate (1) is closed with a thin, optically transparent film (2; 3) which has at least two fluidic interfaces (4; which are liquid-conductively connected to the channel (10), and the other side of the structured carrier substrate (1) is likewise closed by a thin, optically transparent film (2; 3). 3. A small volume optical analysis cuvette according to claim 1 or 2, characterized in that the structured carrier substrate (1) is provided with integrated optical elements for utilizing the total reflection. 4. cuvette for the optical analysis of small volumes according to claim 1, 2 or 3, characterized in that the structured carrier substrate (1) is a film. 5. A cuvette for the optical analysis of small volumes according to claim 1, 2, 3 or 4, characterized in that the structured carrier substrate (1) and the thin film (2; 3) consist of plastic. 6. A cuvette for small volume optical analysis according to claim 1, 2, 3 or 4, characterized in that the structured carrier substrate (1) and the thin film (2; 3) of glass or quartz or of combinations of glass and quartz glass consist. 7. A small volume optical analysis cuvette according to claim 1, 2, 3 or 4, characterized in that the structured carrier substrate (1) and the thin film (2; 3) are made of different amorphous or crystalline materials. 8. A cuvette for optical analysis of small volumes according to claim 1, 2, 3 or 4, characterized in that the structured carrier substrate (1) and the thin film (2; 3) of a combination of glass - plastic, glass - plastic - Glass or glass - plastic glass with additional bonding layer. 9. Use of a cuvette for the optical analysis of small volumes according to one or more of the preceding claims as a disposable component.