DE19739119A1 - Microtitration plate for wide application - Google Patents

Abstract

The microtitration plate (1) is opaque, non-fluorescing and resistant to organic solvents. It is thermoformed from e.g. polypropylene. Its frustrated conical sample recesses (2) accept solid and liquid microlitre samples. Preferred features: The thermoplastic includes coloured pigments. Its e.g. side surfaces (3, 4), bulges, steps (6), folds, beads and/or bevels (5), are provided for reinforcement. Scale markings define a grid for sample location, which is embossed during thermoforming. A companion, stabilizing base plate has holes corresponding with the sample recesses of the microtitration plate, into which they are inserted and locked into position, held under friction. This base includes tempering equipment, such as an internal heating spiral, or is in contact with e.g. a fluid bath. A further plate (not shown) which carries ejection pegs, pushes the microtitration plate out of the stabilizing base, the pegs entering the holes from the rear, to press against the bases of the sample recesses.

Description

The invention relates to a microtiter plate, in particular for the treatment of Samples with organic solvents and their analysis using light generating process. It is used in biochemical and immunological research, in medical diagnostics, in the chemical and pharmaceutical industries as well as in biotechnology.

Microtiter plates that contain a plurality of sample vessels in one Coordinate grid of multipipettes corresponding grid, for example 8 × 12 or n × 8 × m × 12, increasingly gain in analytics Meaning. Your advantage is that a variety of the same or different samples, including reference solutions, treated in parallel and can be evaluated. Lately, more and more are coming fluorimetric and luminometric investigations in connection with the Microtiter plate technology, especially because of its high sensitivity, for use.

Such microtiter plates are known per se for many applications [e.g. B. A. Yamaguchi et al., Clin. Chem. 35 (1989) 1962-1964, T. Tuuminen et al., Clin. Chim. Acta 212 (1992) 155-158] and consist in particular of Polystyrene.

In screening examinations using strongly colored samples, blood samples, in particular, lead to a considerable narrowing of the Applicability of optical analysis methods, since the analyzed light intensity is heavily falsified by the sample and therefore becomes a significant one Lowering sensitivity comes. The disturbing can be very often Substances, such as hemoglobin, elegant with organic  Precipitate solvents, d. H. de-protein, and thereby the said disturbances fix or at least greatly reduce. In addition to this type of Screening exams are currently becoming increasingly procedural developed through natural product screening and combinatorial chemistry efficient development of new or improvement of known pharmaceuticals allow. Very often, the samples to be screened must be organic Solvents are treated and then analyzed.

The microtiter plates currently available meet the requirements through high-throughput screening projects (e.g. usage possibility for colored samples, economically justifiable use of expensive, highly standardized reagents and the practical usability of Dry samples on supports such as filter paper die-cuts are not. For those Analysis costs and the microliter range reducing the reagent volume are not solvent-resistant and can be analyzed with optical means Microtiter plates available.

On the one hand, the previously known miniaturized analysis also exist microtiter plates with small cavities, all made of optical media Polystyrene and close because of the intolerance Solvents practically perform an analysis after solvent extraction, in addition, usual filter paper die cuts from on filter paper dried blood samples are not easy and practical in the known Receptacles of the microtiter plate can be introduced.

On the other hand, there are solvent-resistant conventional microtiter plates (e.g. from Greiner) relatively expensive, not because of its transparency properties Suitable for all optical analysis methods and also require large ones Sample volumes and thus reagent quantities. These are microtiter plates due to the expected high costs for screening examinations also not suitable.

The invention is therefore based on the object, one with little Effort to create and easy to handle microtiter plate, which does not have the aforementioned disadvantages as a whole and therefore for one Many different screening tests, especially with Use of fluorescence and luminescence measurements, universally applicable is. The microtiter plate should be despite the small volumes of the analysis vessels without problems, e.g. B. with filter paper die cuts.

According to the invention, a microtiter plate is proposed which consists of opaque, non-fluorescent and against organic solvents resistant resilient thermoplastics, such as thermally deformable Polypropylene, and in which the, preferably by thermal Deformed, sample vessels, frusto-conical for samples volumes are formed in the microliter range.

In the subclaims 2 to 8 expedient design features of the Invention, such as means for increasing the mechanical stability, for Expansion of application options and improvement of Handling, described.

The microtiter plate can be very easily and therefore very economically, for example by thermal molding from a matrix (master) are manufactured and is due to their material and shape properties the Sample vessels for at least a multitude of applications Suitable for sample treatment and sample evaluation. These applications also include optical evaluation methods without the Material properties of the microtiter plate, the sensitivity of the evaluation affect. Due to the frustoconical shape of the sample vessels ensures a good and practicable loading of the sample vessels, whereby also particles, such as filter paper die cuts etc., despite the small vessel volumes  are also included. Allow the small volume sizes also a very inexpensive sample evaluation.

All in all, the microtiter plate specifically combines individual advantages for certain applications of already known microtiter plates for a wide and universal applicability among different Conditions of use.

The invention will be explained below with reference to the drawing. Show it:

Fig. 1 perspective view of the microtiter plate with stabilizing plate

Fig. 2 microtiter plate in plan view

Fig. 3 basic arrangement of the microtiter plate with stabilizing plate in side sectional views

Fig. 4 Principle arrangement of the microtiter plate with stabilizing plate and ejection plate in side sectional views

Fig. 5 Supernatants of blood-containing samples dried on filter paper after de-proteinization in the microtiter plate with various organic solvents

Fig. 6 precision of the fluorescence measurement in the microtiter plate

Fig. 7 Imprecision of a miniaturized newborn screening test for galactose-1-phosphate uridyltransferase deficiency

Fig. 8 Signal level and imprecision of a miniaturized newborn screening test for galactose-1-phosphate-uridyltransferase deficiency using various de-proteinizing agents.

In Fig. 1 and 2, a microtiter plate 1 is shown in perspective view and plan view in a per se known xy grid 8 × 12 96 sample containers 2 has for receiving and treating samples with volumes in the microliter range. The microtiter plate 1 consists of polypropylene LM-885/30-T1480-160 and is produced by thermal deformation (molding from a die) from a foil-like plate. In this molding process, the 96 wells for the sample vessels 2 were simultaneously stamped into the board and shape stabilizing elements of side surfaces 3 and 4 , corner bevels 5 and gradations 6 were produced. The embossed depressions for the sample vessels 2 are frustoconical, so that their upper diameter 7 is larger than their lower diameter 8 at the bottom of the analysis vessels 2 and side walls 9 are conical (cf. FIG. 2). With the frustoconical design of the sample vessels 2 in the µl sample volume range, good and practical loading (including filter paper die-cuts) is possible compared to a known trough or cone shape. The small cavities in the sample vessels 2 make these examinations, which therefore require only small amounts of sample, very advantageous and economical in every respect.

The material of the microtiter plate 1 is resistant to organic solvents, such as, in particular, acetone, acetonitrile, methanol, butanol, hexane and mixtures thereof, which also makes them suitable for numerous applications (e.g. de-proteinization in biochemical and clinical-chemical analysis, natural product screening , combinatorial chemistry etc.) is given.

Furthermore, this material of the microtiter plate 1 is opaque and non-fluorescent, so that it can be used for optical analysis processes without the vessel material restricting or influencing the sample evaluation.

On the surface of the microtiter plate 1 (see FIG. 2), a coordinate identifier 10 for the grid of the sample vessels 2 in the form of letters and numbers of the corresponding coordinate of the 8 × 12 sample grid is visible. This can, for example, be printed on (screen printing or the like) or can also be embossed when the microtiter plate 1 is thermally molded.

In order to increase the mechanical dimensional stability of the microtiter plate 1 (especially for very thin-walled and sheet-like flexible plate designs), a stabilizing plate 11 (base body) is provided on which the microtiter plate 1 is placed. For this purpose, the stabilization plate 11 has corresponding depressions 12 for receiving the sample vessels 2 (see FIG. 1). Fig. 3 shows the basic arrangement of the microtiter plate 1 with stabilizing plate 11 in side sectional views (separated and attached state). The representation corresponds to the sectional plane AB indicated in FIG. 2. A thin-walled and thus compliant design of the sample containers 2 (film-like side walls 9 ) supports a positive and non-positive fit of the sample containers 2 in the recesses 12 , which is for example a close thermal contact when the sample containers 2 are tempered via the stabilizing plate 11 (in the drawing) not shown) is relevant.

In order to enable a practical separation of the microtiter plate 1 from the stabilization plate 11 , an ejection plate 13 is provided which has two ejection pins 14 . The stabilizing plate 11 has two holes 15 corresponding to it, through which the ejection pins 14 extend and touch the microtiter plate 1 for ejection on its underside. The sectional view of FIG. 4 corresponds to the sectional plane CD indicated in FIG. 2.

In order to demonstrate the universal applicability of the microtiter plate 1 according to the invention for the most varied of sample evaluations, four application examples are to be presented with the drawings 5-8.

Application example 1 Precipitation of hemoglobin with organic solvents

A 3 mm platelet is placed in each sample vessel 2 of the microtiter plate 1 and with 20 ul 100 mM triethanolamine / HCl, pH 7.8 with 0.133% (w / v) digitonin, 0.9 mM EDTA, 0.63 mM Dithiothreitol and 0.02% NaN ₃ (1 and 2 in Fig. 5) and H ₂ O (3 and 4 in Fig. 5) eluted at 37 ° C for two hours. The microtiter plate is then cooled to room temperature and pipetted into two sample vessels identically 40 µl (1 and 3 in Fig. 5) and 60 µl (2 and 4 in Fig. 5) of the following solvents: (A) acetone, (B ) Acetonitrile, (C) methanol, (D) ethanol, (E) propanol, (F) isopropanol, (G) butanol, 1/1 (v / v) mixtures of (H) ethanol / acetone, (I) methanol / Acetone, (J) butanol / acetone, (K) methanol / ethanol, (L) methanol / propanol, (M) propanol / acetone, (N) methanol / isopropanol, (O) methanol / butanol and (P) H ₂ O.

Platelet eluate and solvent are mixed on a microtiter plate shaker and centrifuged at 4000 g for 5 min. 5 µl of the supernatant are pipetted onto MN818 and the presence of hemoglobin on the filter paper is assessed visually. Fig. 5 shows that under all conditions only acetone, acetonitrile, propanol and methanol and 1/1 mixtures of methanol / acetone produce complete de-proteinization.

Example of use 2 Fluorescence measurement in the microtiter plate

The fluorescence of all 96 sample vessels 2 of the microtiter plate 1 are empty, or filled with 80, 90 or 100 μl of a buffer without or with NADH or methylumbelliferone at 460 nm (excitation 355 nm) in a fluorimeter reader known per se. In FIG. 6, signal-ups and imprecision of these measurements are summarized.

The microtiter plate according to the invention thus has a conventional one White or black microtiter plates used for fluorimetry Polystyrene has comparable precision properties.

Example of use 3 Miniaturized newborn screening test for galactose-1-phosphate-Uri dyltransferase activity

20 μl of the test mixture are pipetted into one blood platelet (3 mm 3) per sample vessel 2 of the microtiter plate 1 , which contains normal blood from an adult voluntary donor or corresponding dilutions in the patient's own plasma. The test mixture contains 100 mM triethanolamine / HCl, pH 7.8, 0.133% (w / v) digitonin, 0.9 mM EDTA, 0.63 mM dithiothreitol, 0.02% NaN3 (w / v), 6.48 mM Galactose-1-phosphate, 4.4 mM UDP-glucose, 4.82 mM NADP. Individual blank values are determined in parallel with the same test mixture, but without galactose-1-phosphate.

The microtiter plate is briefly shaken on a microtiter plate shaker, covered with an adhesive film and incubated for two hours at 38 ° C. Then the de-proteinization is carried out by adding 60 μl of methanol / acetone (1/1, v / v), shaking and centrifuging at 4000 g for 5 min. The fluorescence of the solution in the sample vessels 2 is measured directly in the microtiter plate 1 , which is pressed into the stabilization plate 11 , at 460 nm (excitation 355 nm) in a fluorimeter. The signals (scale divisions) and imprecisions of the test obtained are shown in FIG. 7.

The signal levels exceed those of conventional tests larger volumes can be achieved by 5-10 times. They reached Precisions make the test well suited for screening purposes.

Example of use 4 Test for galactose-1-phosphate uridyl transferase activity using various deicing agents

The miniaturized test is carried out as in application example 3, but different solvents are used as deicing agents with a volume of 40 or 60 μl: 6 samples are treated equally in a series.
60 µl test buffer: acetone / methanol (1/1), ethanol, methanol, acetone, propanol, acetonitrile;
40 µl ethanol.

The fluorescence and imprecision achieved are shown in Fig. 8.

It can be seen that the solvents methanol and acetone / methanol (1/1), which completely deproteinize (see FIG. 5), also enable the highest test fluorescence signals with good test precision. Propanol, acetonitrile and acetone lead to a reduction in NADPH fluorescence. Test buffers and ethanol do not remove the interfering hemoglobin or only partially.

Reference list

1

Microtiter plate

2nd

Sample vessel

3rd

,

4th

Side surface

5

Corner bevel

6

gradation

7

,

8th

diameter

9

Side wall

10th

Coordinate identifier

11

Stabilizing plate

12th

deepening

13

Ejection plate

14

Ejection pin

15

hole

Claims (8)

1. Microtiter plate, in particular for the treatment of samples with organic solvents and for analysis by means of light-generating methods, with sample vessels arranged in a grid known per se, for example n × 8 × m × 12, characterized in that the microtiter plate ( 1 ) is made of opaque , non-fluorescent and resistant to organic solvents, resilient thermoplastics, such as thermally deformable polypropylene, and that the sample vessels ( 2 ), preferably produced by thermal deformation, are frustoconical in shape to accommodate solid and liquid samples in the microliter volume range. 2. Microtiter plate according to claim 1, characterized in that the Thermoplastic is provided with colored pigments. 3. Microtiter plate according to claim 1, characterized in that the microtiter plate ( 1 ) means ( 3 , 4 , 5 , 6 ) to increase the dimensional stability, such as side surfaces, beads, steps, folds, beads, bevels and the like. are provided. 4. Microtiter plate according to claim 1, characterized in that on the surface of the microtiter plate ( 1 ) a coordinate identifier ( 10 ) for the grid of the sample vessels ( 2 ) is specified. 5. Microtiter plate according to claim 4, characterized in that the coordinate identifier ( 10 ) is also impressed by thermal deformation in the surface of the microtiter plate ( 1 ). 6. A microtiter plate according to claim 1, characterized in that a stabilizing plate ( 11 ) is provided which has corresponding receiving recesses ( 12 ) in which the sample vessels ( 2 ) of the microtiter plate correspond to the sample vessels ( 2 ) formed as depressions of the microtiter plate ( 1 ) ( 1 ) be used positively and non-positively. 7. Microtiter plate according to claim 6, characterized in that the stabilizing plate ( 11 ) has means for temperature control, for. B. an internal electric heating coil, or is connected to such, z. B. to a liquid bath. 8. Microtiter plate according to claim 1, characterized in that an ejection plate ( 13 ) is provided which has at least two ejection pins ( 14 ) which for ejecting the microtiter plate ( 1 ) from the stabilizing plate ( 11 ) through corresponding holes ( 12 ) in the stabilizing plate Reach through ( 11 ) and touch the microtiter plate ( 1 ) from the bottom.