DEVICE AND METHOD FOR PRODUCING INDIVIDUALLY PROCESSED FLUID SAMPLES

Abstract

The present invention is directed to a continuous fluid sample processing device for producing individually processed fluid samples and a method for producing individually processed fluid samples. Moreover, the invention also relates to the use of the device in corresponding methods, in particular in a method for continuously mixing, incubating and analyzing a fluid sample by flow cytometry.

Claims

1. A method for producing individually processed liquid samples, the method comprising the steps: (a) providing the liquid sample processing device configured to permit continuous liquid sample processing comprising, i. a syringe plunger pump positioned vertically and including a plunger at the top and an opening at the bottom, forming a plunger displacement space between the plunger and the opening, wherein the opening is operably coupled to a multiport pump head that connects the syringe plunger pump to: 1. at least one liquid sample source, 2. at least one sample processing liquid source, 3. at least one gas inlet, and 4. at least one outlet for transferring the fluids in the syringe plunger pump, the outlet including a valve having a closed position and an open position; (b) loading at least one liquid sample through the multiport pump head into the syringe plunger pump of the continuous liquid sample processing device from one or more of the liquid sample sources; (c) loading at least one sample processing liquid through the multiport pump head into the syringe plunger pump of the device from one or more of the sample processing liquid sources; (d) loading at least one gas bubble through the multiport pump head into the syringe plunger pump of the device from one or more of the gas inlets, which floats a bubble through the liquids to the top of the syringe plunger pump, thus mixing the liquids in the syringe plunger pump and accumulating at the top; and (e) transferring, when the outlet valve is in an open position, the mixed liquids followed by the gas bubble in the syringe plunger pump through one or more of the outlets of the multiport pump head.

2. The method according to claim 1, wherein in step (e) the mixed liquids followed by the gas bubble are transferred together or sequentially from the syringe plunger pump to an incubation device that mixes the mixed liquids by floating a gas bubble through the mixed liquids.

3. The method according to claim 1, wherein in step (e), the mixed liquids followed by the gas bubble in the syringe plunger pump are transferred through one or more of the outlets of the multiport pump head to a further device.

4. The method according to claim 3, wherein the further device is a processing device, an incubating device, an analytical device, or combination thereof.

5. The method according to claim 1, further comprising step (f) assessing at least one of a physical property, a chemical property, a biological property, or a combination thereof, of the processed liquid samples.

6. The method according to claim 5, wherein the physical property, the chemical property, the biological property, or a combination thereof, of the processed one or more liquid samples are assessed either within the syringe plunger pump, in a sample taken directly from the mixed liquids in the syringe plunger pump, or after transfer and further processing of the mixed liquids from the syringe plunger pump.

7. The method according to claim 5, wherein the physical property, the chemical property, the biological property, or a combination thereof, is selected from the group consisting of photo spectrometry, mass spectrometry, microscopy, cell counting, flow cytometry, and flow cytometry for stained liquid samples.

8. The method according to claim 5, further comprising step (g) assessing at least one of a physical property, a chemical property, a biological property, or a combination thereof, of the processed samples, wherein the method comprises continuously mixing, incubating, and assessing a liquid sample by flow cytometry.

9. The method according to claim 1, wherein the multiport pump head further connects the syringe plunger pump with at least one source for: one or more further sample processing liquid, a rinse liquid, a disinfection liquid, a cleaning liquid, or a combination thereof, and the method further comprises a step (f) of loading the further sample processing liquid, the rinse liquid, the disinfection liquid, the cleaning liquid, or a combination thereof through the multiport pump head from the respective source.

10. The method according to claim 5, further comprising a step (g) assessing a physical property, a chemical property, a biological property, or a combination thereof, of the processed and incubated liquid samples.

11. The method according to claim 10, wherein the physical property, the chemical property, the biological property, or a combination thereof, of the processed and incubated liquid samples are assessed either within the syringe plunger pump, in a sample taken directly from the mixed liquids in the syringe plunger pump, or after transfer and incubation of the mixed liquids from the syringe plunger pump.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In the following the device and method of the present invention are illustrated by a representative embodiment, none of which are to be construed as limiting the invention beyond the scope of the appended claims.

[0060] FIG. 1 depicts a preferred embodiment of the fluid sample processing device (20) of the present invention.

DETAILED DESCRIPTION

[0061]

TABLE-US-00001 Reference signs used in FIG. 1 (1)-(4) Outlet ports (5)-(12) Inlet ports (13) Syringe plunger pump (14) Multiport pump head (15) Temperature controlled chamber (16) Further device, such as a flow cytometer (17) Incubation device (18) Tubing (19) Sample, processing and/or gas sources, waste container (20) Fluid sample processing device

[0062] FIG. 1 depicts a preferred embodiment of the fluid sample processing device (20) of the present invention. The syringe plunger pump (13) is positioned at the top of the multiport pump head (14) in a vertical manner allowing for a gas bubble to float to its top. Syringe plunger pump (13) has an opening at its bottom and is in fluid communication with the multiport pump head (14). The plunger of syringe plunger pump (13) operates in a vertical direction and moves away from the multiport pump head (14) when loading fluids or gases. In this figure, multiport pump head (14) features 8 inlet ports (5) to (12) and 4 outlet ports (1) to (4). The number of ports as depicted in FIG. 1 is not limiting for practicing the present invention and any number of inlet and outlet ports is within the scope of the present invention, as long as there are at least one fluid sample source, at least one sample processing fluid source, at least one gas inlet, and at least one outlet for transferring the fluids, e.g. to a further device (16). The ports of the multiport pump head (14) are preferably connected via connecting tubing (18) to incubation devices (17), syringe plunger pump (13) and to the sources, containers, interfaces or exits (19). Upon activation, the syringe plunger pump (13) creates a negative pressure within the plunger displacement space and draws at least one fluid sample and at least one sample processing fluid through the respective ports into the syringe plunger pump (13) followed by a gas bubble. The gas enters the syringe plunger pump (13) at its bottom and preferably spans the whole width of the bottom before it floats through the fluid components to the top of the syringe plunger pump (13), thus mixing the fluids in the syringe plunger pump (13) and accumulating at its top. The displacement of the fluids by the gas bubble(s) forces the fluids to the sidewalls of the plunger displacement space, preferably forming a narrow film when the bubble floats by. Subsequently, the plunger of the syringe plunger pump (13) is moved towards the multiport pump head (14) creating a positive pressure and the mixed fluids followed by the gas bubble are transferred through, e.g., connecting tubing (18), preferably to the bottom of an incubation device (17), more preferably an incubation device (17) configured to allow the floating of the gas bubble through the mixed fluid components. Most preferably, the incubation device (17) is configured so that in use at least one gas bubble spans the whole width of the bottom of the incubation device (17) before the bubble floats through the fluid components to the top of the incubation device (17). In the incubation device (17) the mixed sample fluid components arrive first and the gas bubble follows. In the incubation device (17) the gas bubble then floats through the fluid components from bottom to top, thus mixing the fluids a second time. The incubation devices (17) are either configured to be used for direct, e.g. photometric, analysis or are open at their tops to allow for the sample to be drawn, e.g. by a needle of a flow cytometer (16) interface or any other analytical device (16) such as, for example, described above. Also, the incubation devices (17) can alternatively comprise a pressure relief valve at their tops. Preferably, the incubation devices (17) are located in a temperature controlled chamber (15) that provides for different incubation temperatures depending on the processing requirements of the individual samples.

Example 1

[0063] In the following, a representative experimental method for producing individually processed fluid samples according to the present invention is provided. A complete setup for a fully automated, discrete online (i.e. in-situ) flow cytometry experiment included (i) a flow cytometer (e.g. Accuri C6, BD, USA; CytoFLEX, Beckman-Coulter, USA; NovoCyte, ACEA Biosciences, USA) including respective software, (ii) an automated sampling, pipetting, mixing, and temperature controlled incubation unit connected (a) both physically (via the sample acquisition needle) and via software to the flow cytometer, (b) to the system/sample (e.g., flowing water line, batch reactor) to be measured, and (c) to the relevant substance/chemical sources used for sample treatment and system maintenance, (iii) a software suite for operating the different operations of the automated unit (via commands) and triggering start and stop as well as the saving of measured data by the flow cytometer, and (iv) an optional software suite for batch and/or real-time data analysis.

[0064] The automation unit comprised as depicted in FIG. 1 (i) a syringe pump (Tecan, Switzerland), which was installed vertically upside-down (13) with a multi-port pump head (14) offering 12 connections (1-12) for thin tubing (18), (ii) a temperature controlled chamber (15), (iii) incubation devices (open or with a pressure release valve on the top) (17), and (iv) a connecting interface chamber to the flow cytometer (16) needle. The syringe pump was connected via the thin tubes fixed to the multi-port head to (a) the incubation chambers, (b) the connecting interface chamber, (c) the system/sample to be measured, and (d) a number of containers comprising relevant substances/chemicals used for sample treatment and system maintenance.

[0065] A typical experimental sequence for water analysis consisted of the following steps: (i) 200 l of sample water from a stream to be monitored for cellular load were introduced from container (19) through the respective thin tube via the multi-port pump head (14) into the syringe pump (13). (ii) The sample was discarded (pre-rinse sample) through the respective thin tube via the multi-port pump head (14) into a waste container (19) and then the sampling step was repeated (measurement sample). (iii) 200 l of staining solution (SYBR Green, Life Technologies, USA) were drawn from the respective container (19) through the respective thin tube via the multi-port pump head (14) into the syringe pump (13). (iv) Air was drawn through the respective thin tube via the multi-port pump head (14) into the syringe pump (13), resulting in an air bubble forming at and completely spanning the bottom of the upside-down syringe pump (13), where the two previously drawn liquids (water sample and staining solution) were located. As the head of the syringe pump was moving upwards and more air was drawn into the syringe, the air bubble spontaneously floated through the liquids, resulting in mixing of these. (v) The mixed liquid was transferred from the syringe to one of the incubation devices (17) located in the temperature-controlled chamber (15) through the respective thin tube via the multi-port pump head (14) followed by the air previously drawn through the liquids and still captured in the syringe. This resulted in another floating of the air bubble through the liquid in the incubation chamber (17) and thus a second mixing. The air escaped through the opening or pressure release valve of the incubation chamber (17). (vi) The mixed sample was incubated at a suitable temperature (37 C.) for a suitable time period (10 min). (vii) 500 l of rinsing solution (nanopure water) were drawn from the respective container (19) through the respective thin tube via the multi-port pump head (14) into the syringe pump (13). (viii) The rinsing solution was discarded through the respective thin tube via the multi-port pump head (14) into a waste container (19). (iv) Upon completion of incubation, the mixed liquid from the incubation chamber was transferred through the respective thin tube via the multi-port head (14) to the interface to the flow cytometer (16) needle. (x) The operating software then triggered the measurement of the flow cytometer and stopped it a after the desired time/volume (100 sec/50 l). (xi) The measured and incubated mixed liquid was transferred through the respective thin tube via the multi-port pump head (14) into a waste container (19). (xii) 500 l of rinsing solution (nanopure water) were drawn from the respective container (19) through the respective thin tube via the multi-port head (14) into the syringe pump and further into the incubation chamber (17). (xiii) The rinsing solution was discarded through the respective thin tube via the multi-port pump head (14) and the syringe pump (13) into a waste container (19). (xiv) The rinsing step was repeated for the interface to the flow cytometer(16) needle.