METERING DEVICE

Abstract

The invention relates to a dispensing device comprising a housing having at least one pressure chamber, having a supply opening for the supply of liquid into the pressure chamber and having a multiplicity of conduits between the pressure chamber and an external side of the housing, there being situated in each of the conduits a tube, the first end of which protrudes into the pressure chamber and the second end of which protrudes out of the housing on the external side.

Claims

1. A dispensing device comprising a housing having at least one pressure chamber, having a supply opening for the supply of liquid into the pressure chamber and having a multiplicity of conduits between the pressure chamber and an external side of the housing, there being situated in each of the conduits a tube, the first end of which protrudes into the pressure chamber and the second end of which protrudes out of the housing on the external side.

2. The dispensing device as claimed in claim 1, characterized in that the pressure chamber has a larger extent in one spatial dimension than in the other two spatial dimensions and the longitudinal axis of the pressure chamber runs in the direction of the larger extent and the longitudinal axis of the pressure chamber runs in parallel to the external side of the housing.

3. The dispensing device as claimed in claim 1 or 2, characterized in that the conduits are situated in one of the walls of the pressure chamber, which runs in parallel to the longitudinal axis of the pressure chamber, and the conduits are arranged in parallel to one another.

4. The dispensing device as claimed in any of claims 1 to 3, characterized in that the pressure chamber is cylindrical or cuboid.

5. The dispensing device as claimed in any of claims 1 to 4, characterized in that at least two conduits are present.

6. The dispensing device as claimed in any of claims 1 to 5, characterized in that the conduits are arranged in one or more rows in parallel to the longitudinal axis of the pressure chamber.

7. The dispensing device as claimed in any of claims 1 to 6, characterized in that there are at least 8, preferably 24 or 48, conduits per row.

8. The dispensing device as claimed in any of claims 1 to 7, characterized in that the tubes are capillaries.

9. The dispensing device as claimed in any of claims 1 to 8, characterized in that the tubes are made of metal.

10. The dispensing device as claimed in any of claims 1 to 9, characterized in that the tubes have an inner diameter within the range from 0.1 mm to 0.8 mm, preferably 0.2 mm to 0.6 mm.

11. The dispensing device as claimed in any of claims 1 to 10, characterized in that the tubes are arranged such that they are substantially perpendicular (90) to the external side of the housing or are inclined at an angle to the external side of the housing within the range from 40 to <90.

12. The dispensing device as claimed in any of claims 1 to 11, characterized in that the sections of the tubes which are situated on the external side of the housing are sheathed by sleeves, preferably of plastic.

13. The dispensing device as claimed in any of claims 1 to 12, characterized in that the supply opening is situated in one of the walls arranged perpendicularly to the longitudinal axis of the pressure chamber.

14. The dispensing device as claimed in any of claims 1 to 13, characterized in that the housing comprises a vent opening.

15. The dispensing device as claimed in any of claims 1 to 14, characterized in that the vent opening is situated in one of the walls arranged perpendicularly to the longitudinal axis of the pressure chamber.

16. The dispensing device as claimed in any of claims 1 to 15, characterized in that the pressure chamber has a cross-sectional area from 60 mm2 to 300 mm2 or a diameter from 4 mm to 10 mm, preferably from 5.5 mm to 6.5 mm.

17. The dispensing device as claimed in any of claims 1 to 16, characterized in that the housing comprises two, three or four pressure chambers, the longitudinal axes of which run in parallel, wherein belonging to each pressure chamber is a separate supply opening for the supply of liquid.

18. A dispensing system comprising a dispensing device as claimed in any of claims 1 to 17, and a liquid reservoir which is connected to the supply opening via a line.

19. The dispensing system as claimed in claim 18, characterized in that a valve is situated between the liquid reservoir and the supply opening.

20. The use of the dispensing system as claimed in claim 18 or 19, characterized in that the liquid from the liquid reservoir rests against the valve under a pressure within the range from 0.5 to 2 bar, preferably within the range from 0.5 to 0.85 bar, and the valve has a switching time within the range from 5 ms to 200 ms, preferably within the range from 5 ms to 50 ms.

21. The use of the dispensing system as claimed in claim 18 or 19, characterized in that the addition volume during the opening time of the valve is within the range between 0.3 l per capillary and 300 l per capillary, preferably between 1 l per capillary and 30 l per capillary.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The invention will now be explained in reference to the enclosed drawings, which show advantageous example embodiments:

[0032] FIG. 1 is a perspective view of two dispensing devices with two pressure chambers;

[0033] FIG. 2 is a perspective view of the inner structure of the dispensing device with two pressure chambers;

[0034] FIG. 3 is a rear view of the dispensing device with two pressure chambers;

[0035] FIG. 4 is a side view of the dispensing device;

[0036] FIG. 5 is a side view (valve-side) of an enlarged section of the dispensing device;

[0037] FIG. 6 shows the dispensing system with liquid reservoir;

[0038] FIG. 7 shows the imaging measurement instrument (one-channel detection);

[0039] FIG. 8 shows the imaging measurement instrument (two-channel detection);

[0040] FIG. 9 shows the false-color image of the emission of a microtiter plate with 1536 assay wells; and

[0041] FIG. 10 A-C show the measurement of the kinetics of the binding of ANS to BSA using the imaging measurement instrument.

DETAILED DESCRIPTION OF THE INVENTION

[0042] FIG. 1 shows two parallelly arranged dispensing devices 10 according to an embodiment of the invention, each with two pressure chambers, in perspective view. Further views of a dispensing device are shown in FIGS. 2 to 5. Each dispensing device 10 has a cuboid housing 2 with two tubular bores of 4-10 mm diameter and 120-140 mm length. Said bores form two parallel pressure chambers 5. Each bore has an open end which forms the circular supply opening 6. The opposite end is opened for system filling and emptying and is sealed tight during dispensing operation. Situated here is the leakage or vent opening 3. The side of the housings 2 on which the supply opening 6 is situated is attached to a mount 7 such that said housings protrude away from the mount 7 in a horizontal manner The mount 7 has a recess for each supply opening 6. Situated on the underside of the outwardly protruding housing 2 is a row of up to 48 conduits between each pressure chamber 5 and the underside of the housing 2, there being situated in each of the conduits a capillary tube 4 made of preferably stainless steel, the first end of which protrudes into the pressure chamber 5 and the second end of which protrudes out of the housing 2 on the underside. The capillary tubes have an inner diameter of 0.1-0.8 mm and are arranged perpendicularly. The segments of the tubes protruding out of the underside of the housing 2 are hydrophobically sheathed (preferably with Teflon).

[0043] FIG. 3 shows the dispensing device 10 in rear view with a view of the two supply openings 6.

[0044] FIG. 4 shows the dispensing device 10 in side view. The circular section A is shown enlarged in FIG. 4. In FIG. 4, it can be seen that the capillary tubes 4 protrude into the pressure chamber up to half the height thereof. This has been found to be particularly advantageous for a uniform dispensing for all 48 capillaries.

[0045] FIG. 6 shows the dispensing system according to an embodiment of the invention. The pressure chamber 5 (not visible in the housing 2) of the dispensing device 10 is connected, via a line 71, a valve 72 and a further line 70, to a liquid situated in a reservoir vessel. Via a membrane pump 66 and a pressure line 68, room air is introduced into the reservoir vessel under a pressure of 0.8 bar. It is also possible to use a different gas such as N.sub.2 instead of room air. It is also possible to use a different pressure supply system instead of the membrane pump. Solenoid valves with low dead-space volume and short switching time, as sold by Parker Hannifin Corp., Cleveland, Ohio 44124 USA, for example, have been found to be suitable valves.

[0046] Hereinafter, the operation of an imaging measurement instrument having the dispensing device according to some embodiments of the invention will be described.

[0047] The liquid 65 containing the reactant to be analyzed is pumped into the pressure chamber 5 (not visible) of the dispensing device via a valve 72 from a reservoir vessel 64 (FIG. 6). The capillary tubes 4 have an outer diameter (including the sheath) of less than 2 mm, this allowing a parallel dispensing into 48 assay wells of the microtiter plate (FIG. 6). To achieve mixing conditions of high turbulence, the orientation of the dispensing outlets of the tubes 4 is specifically adapted to microtiter plates with 384 and 1536 assay wells. According to some embodiments, best mixing results for microtiter plates with 384 assay wells were achieved by dispensing the reactant in a diagonal manner onto the microtiter plate wall. In contrast, the lower dispensing volume necessary for microtiter plates 60 with 1536 assay wells is dispensed perpendicularly, as shown in FIG. 6. Typical dispensing times for the required reactant volumes were determined by weighting of the dispensed liquid with a known time of switching of the valve 72 at a pressure of 0.8 bar.

[0048] A precise microtiter plate mount 62 can ensure the exact orientation of the dispensing device 10 in relation to the wells of the microtiter plate 60.

[0049] The progress of a reaction can be tracked by the simultaneous recording of the fluorescence intensity of all 48 assay wells in a row or of all assay wells of the microtiter plate. Dispensing from the upper side of the microtiter plate 60 situated on a microtiter plate mount 62 is combined with illumination and detection from the underside. This allows the observation of the kinetic process during the dispensing time and mixing time.

[0050] According to some embodiments, the homogeneous illumination of the base of the microtiter plate 60 can be achieved by 2 LED illumination units with in each case up to 36 UV or VIS high-output LEDs 80 which can be arranged in rows and oriented diagonally in relation to the plate (FIGS. 7 and 8). The LEDs 80 available supply light 92 within the wavelength range from 340 to 800 nm and extinction filters improve the fluorescence excitation by transmitting a selected wavelength range. The emitted fluorescence 94 is detected perpendicularly and/or at an angle of 90 by a rapid and highly sensitive back-illuminated EMCCD (electron multiplying charge coupled device) or ICCD (intensified charge coupled device) camera 82. The cameras 82 are equipped with interference filters 84 and can additionally be equipped with polarization filters 85.

[0051] The expansion of the adjustable camera structure to dual fluorescence detection (FIG. 8) allows the simultaneous detection of two emission signals, as necessary, for example, in measurements in relation to Frster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) or in relation to fluorescence polarization (FP).

[0052] According to some embodiments,it is possible to expand the measurement system shown in FIGS. 7 and 8 by further LED illumination units, which are arranged beneath the LED units shown at an altered illumination angle. If these additional LED units have a different wavelength compared to the original LED units, it is possible to effect a fluorescence excitation in multiple wavelengths.

[0053] The false-color depiction of the emission of a microtiter plate with 1536 assay wells containing fluorescent solution is depicted by way of example in FIG. 13. Data for the assay wells are collected by capturing and visualizing up to 1000 points per second per assay well and processing them by means of customized data processing software.

[0054] Performance Test

[0055] A method routinely used to test the performance of a rapid mixing device is the observance of a rapid test reaction. In the case of fluorescence studies, what is suitably tracked is the binding of the hydrophobic dye 1-anilino-8-naphthalenesulfonic acid (ANS) to bovine serum albumin (BSA), which is associated with a large increase in fluorescence yield. The fluorescence kinetics for various BSA concentrations are fitted to exponential functions and extrapolated to a common starting fluorescence. This common point provides the fluorescence of ANS in the absence of BSA at the starting time point (to) of the reaction. The time interval from this point up to the first data point which falls onto the fitted exponential curve provides an estimate of the dead time of the measurement. FIGS. 14 A-C show the corrected fluorescence kinetics at various BSA concentrations, which were measured in the imaging measurement instrument with the inventive dispensing device for rapid kinetics.

[0056] After 55 ms, 1.6 l of ANS solution were added via capillary valve switching of 9 ms to 48 assay wells of a BSA-containing microtiter plate (FIG. 10A). The capillary valve switching is indicated by the gray bar. The binding of ANS to BSA leads to an increase in ANS fluorescence, which is recorded at 460 nm (bandpass 60 nm) after excitation at 370 nm (bandpass 36 nm). The ANS and BSA solutions were prepared in 100 mM potassium phosphate (pH 7.5). Final concentration: 5 M ANS and 1.9 (open circles), 2.5 (filled circles), 3.4 (inverted triangles), 7.9 (squares) and 10.6 M (triangles) BSA (FIG. 10A).

[0057] The starting time point of the binding reaction was ascertained by double exponential fits and extrapolation of the fluorescence kinetics to the common start time to. The fluorescence kinetics (gathered from FIG. 10A) were corrected to said start time to (FIG. 10B). Solid lines show double exponential functions which were extrapolated to the common time point to.

[0058] It should be noted that to of the reaction is not equivalent to the time point of valve switching, but instead has a time delay corresponding to the entry and the mixing of the reactants in the assay wells. The dead time of the instrument, which is implemented by the period from to up to the first correctly determined point on the fitted exponential curve, is based on the dispensing time and mixing artifacts. In the case of the presence of 3 l of liquid in the assay wells of a microtiter plate and dispensing of a small volume of 1.6 l into the 1536 assay wells of said microtiter plates, it is possible to achieve a dead time of about 10 ms, which approximately corresponds to the time-resolution of commercial stopped-flow instruments of a few milliseconds.

[0059] The detected fluorescence traces (FIG. 10B) show a very low noise level, which according to some embodiments indicates the high quality of the kinetics data. FIG. 10C shows a graph of the apparent rate constants of binding, ascertained from the kinetics traces, as a function of the BSA concentration. It was possible to detect a slow (filled circles) binding phase and a rapid binding phase (open circles). The linear dependency of the binding phase with slow kinetics on the BSA concentration can confirm the accuracy and reliability of the determined traces. The observed rate constants (black) were compared with data which were obtained by means of a conventional stopped-flow apparatus (red). According to some embodiments, the apparent rate constants and the second-order rate constants, which were determined on the basis of the concentration-dependence of the slow binding phase, coincide outstandingly with the data which were obtained via addition in a stopped-flow apparatus in an individual cuvette.

REFERENCE NUMBERS

[0060] 10 Dispensing device [0061] 1 Valve [0062] 2 Housing [0063] 3 Leakage/vent opening [0064] 4 Tube [0065] 5 Pressure chamber [0066] 6 Supply opening [0067] 6a Seal [0068] 7 Mount [0069] 8 Sheath [0070] 60 Microtiter plate [0071] 62 Plate holder [0072] 64 Reservoir vessel [0073] 65 Liquid [0074] 66 Pump [0075] 68 Pressure line [0076] 70 Line [0077] 71 Line [0078] 72 Valve [0079] 80 LED module [0080] 82 Camera [0081] 84 Emission filter [0082] 85 Polarization filter [0083] 86 Excitation filter [0084] 90 Beam splitter [0085] 92 Excitation light [0086] 94 Fluorescence light