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 continuous fluid sample processing device (20)for producing individually processed fluid samples, the device comprising: (i) a syringe plunger pump positioned vertically with a plunger at the top and an opening at the bottom, wherein said opening is operably coupled to (ii) a multiport pump head that connects the syringe plunger pump with (a) at least one fluid sample source, (b) at least one sample processing fluid source, (c) at least one gas inlet, and (d) at least one outlet for transferring the fluids in the syringe plunger pump, wherein the plunger displacement space in the syringe plunger pump forms a mixing chamber, and wherein the syringe plunger pump receives fluids followed by gas, so that the gas forms at least one bubble that floats through the fluids to the top of the syringe plunger pump, thus mixing the fluids in the syringe plunger pump and accumulating at the top.

2. The continuous fluid sample processing device according to claim 1, wherein the device uses at least one gas bubble that spans the whole width of the bottom of the syringe plunger pump before the bubble floats through the fluid components to the top of the syringe plunger pump.

3. The continuous fluid sample processing device according to claim 1, wherein the at least one outlet (d) in the multiport pump head transfers the mixed fluids and the at least one gas bubble sequentially.

4. The continuous fluid sample processing device according to claim 1, further comprising: (i) at least one incubation device that mixes the mixed fluids with gas bubbles that float to the top of the incubation device, wherein the incubation device is connected at its bottom to outlet (d) of the multiport pump head so that the mixed fluids followed by the gas bubble can be transferred from the syringe plunger pump to the incubation device; (ii) at least one analytical device for assessing at least one of a physical property, a chemical property, a biological property, or a combination thereof, of the processed fluid samples, or (iii) a combination thereof.

5. (canceled)

6. The continuous fluid sample processing device according to claim 4, wherein the analytical device is selected from the group consisting of a photo spectrometer, a mass spectrometer, a microscope, a cell counter, a flow cytometry device, and a flow cytometry device for stained fluid samples.

7. The continuous fluid sample processing device according to claim 4, wherein the continuous fluid sample processing device comprises one or more of the incubation devices, and the at least one analytical device includes at least one flow cytometry analytical device for analyzing continuously mixed and incubated fluid samples.

8. A method for producing individually processed fluid samples, the method comprising the steps: (a) providing the continuous fluid sample processing device according to claim 1, (b) loading at least one fluid sample through the multiport pump head into the syringe plunger pump of the continuous fluid sample processing device from one or more of the fluid sample sources, (c) loading at least one sample processing fluid through the multiport pump head 4-44 into the syringe plunger pump of the device from one or more of the sample processing fluid 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 fluids to the top of the syringe plunger pump, thus mixing the fluids in the syringe plunger pump and accumulating at the top, and (e) transferring the mixed fluids followed by the gas bubble in the syringe plunger pump through one or more of the outlets of the multiport pump head.

9. The method according to claim 8, wherein in step (e) the mixed fluids followed by the gas bubble are transferred together from the syringe plunger pump to an incubation device that mixes the mixed fluids by floating a gas bubble through the mixed fluids.

10. The method according to claim 8, further comprising a step (g) of assessing at least one of a physical property, a chemical property, a biological property, or a combination thereof, of the processed fluid 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 one or more fluid samples are assessed either within the syringe plunger pump, in a sample taken directly from the mixed fluids in the syringe plunger pump, or after transfer and further processing of the mixed fluids from the syringe plunger pump.

12. The method according to claim 10, wherein the assessment of the physical, 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 fluid samples.

13. The method according to claim 8, further comprising a step (g) of 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 fluid sample by flow cytometry.

14. (canceled)

15. (canceled)

16. The continuous fluid sample processing device according to claim 1, wherein one or more of the outlets for transferring the fluids in the syringe plunger pump is for transferring the fluids to a further device.

17. The continuous fluid sample processing device according to claim 16, wherein the further device is a processing device, analytical device, or both.

18. The continuous fluid sample processing device according to claim 1, wherein the multiport pump head further connects the syringe plunger pump with at least one of: (e) at least one waste fluid exit; and (f) at least one source for: at least one further sample processing fluid, a rinse fluid, a disinfection fluid, a cleaning fluid, or a combination thereof.

19. The continuous fluid sample processing device according to claim 4, wherein the gas bubble exits the incubation device.

20. The method according to claim 8, wherein in step (e), the mixed fluids 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.

21. The method according to claim 20, wherein the further device is a processing device, an analytical device, or both.

22. The method according to claim 8, wherein the multiport pump head further connects the syringe plunger pump with at least one source for: one or more further sample processing fluid, a rinse fluid, a disinfection fluid, a cleaning fluid, or a combination thereof, and the method further comprises a step (f) of loading the further sample processing fluid, the rinse fluid, the disinfection fluid, the cleaning fluid, or a combination thereof through the multiport pump head from the respective source.

23. The method according to claim 9, further comprising a step (g) of assessing a physical property, a chemical property, a biological property, or a combination thereof, of the processed and incubated fluid samples.

24. The method according to claim 23, wherein the physical property, the chemical property, the biological property, or a combination thereof, of the processed and incubated fluid samples are assessed either within the syringe plunger pump, in a sample taken directly from the mixed fluids in the syringe plunger pump, or after transfer and incubation of the mixed fluids from the syringe plunger pump.

Description

EXAMPLE 1

[0062] 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.

[0063] 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 needle (16). 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.

[0064] 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 needle (16). (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 multiport 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 needle (16).