WO2020030090A1

WO2020030090A1 - Systems for automated handling of fluid samples into microfluidic droplets for in vitro diagnostic

Info

Publication number: WO2020030090A1
Application number: PCT/CN2019/099942
Authority: WO
Inventors: Ho Cheung Shum; Yuk Heng TANG; Man Ho WONG; Hui Sheng ZHANG; Tong Yuan WU
Original assignee: Shenzhen University; Versitech Ltd
Current assignee: Shenzhen University; Versitech Ltd
Priority date: 2018-08-09
Filing date: 2019-08-09
Publication date: 2020-02-13
Anticipated expiration: 2021-02-09
Also published as: CN113841053B; CN113841053A

Abstract

An assembly, including hardware and software, for handling fluid samples to form emulsions. The assembly may include a robotic liquid handler to manipulate fluid from sample tubes to a cartridge, and to collect emulsions from the cartridge to vials. The assembly may apply pressure for the manipulation of fluid and emulsions within the body of the assembly. In some embodiments, the assembly may stop applying pressure to the fluid when a predefined condition is arrived. The change may indicate the endpoint of fluid manipulation is achieved.

Description

SYSTEMS FOR AUTOMATED HANDLING OF FLUID SAMPLES INTO MICROFLUIDIC DROPLETS FOR IN VITRO DIAGNOSTIC

TECHNICAL FIELD

Disclosed is an assembly which automates fluid sample handling for microfluidic droplet generation and method associated therewith.

BACKGROUND

The demand for the biochemical and biological assays increases as the society improves. For example, vast amount of patient samples is screened in the industries of in vitro diagnostic (IVD) . Thus, advancement in technology for IVD with automation and high-throughput capabilities may help to digest the demand. Recent emerging droplet microfluidic technology can accurately generate and manipulate tiny volume of droplets. The technology can help the operations in biological and chemical laboratories by precisely controlling fluids on microfluidic device of a few centimeters square in size. Basically, thousands of uniform sizes droplets can be generated in the device per minute. These droplets function as microreactors for assays, and may help to replace the bulk vials and microtiter plates in traditional in vitro diagnostic industries. This is showing important direction for biochemical analysis in the future, as this may potentially lead to breakthrough of the bottleneck of traditional robotic-based automatic biochemical analysis, which is limited by the moving speed of mechanics.

Typically, the number of clinical samples which need to be processed is tremendous in clinics and hospitals. The clinical samples are usually present in containers such as test tubes, centrifuge tubes, and multi-well plates. To transfer the clinical samples to microfluidic device, the process is mainly manually done and is not an effective approach that consumes laboring costs. The users may also require having sufficient skill and knowledge to operate the microfluidic device. Without an assembly for these containers and microfluidic devices, it may fail to persuade doctors or inspectors to comfortably use new technology to handle a large number of different samples.

SUMMARY

The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify key or critical elements of the invention nor delineate the scope of the invention. Rather, the sole purpose of this summary is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented hereinafter.

A new sampling assembly which seamlessly bridges current sampling method with microfluidic technology is provided. The subject invention provides an assembly, including hardware and software, for handling fluid samples to form emulsions. The assembly can include a robotic liquid handler to manipulate fluid from sample tubes to a cartridge, and to collect emulsions from the cartridge to vials. The assemble can apply pressure for the manipulation of fluid and emulsions within the body of the assembly. In some embodiments, the assembly can stop applying pressure to the fluid when a predefined condition is arrived. The change can indicate the endpoint of fluid manipulation is achieved.

To the accomplishment of the foregoing and related ends, the invention comprises the features hereinafter fully described and particularly pointed out in the claims. The following description and the annexed drawings set forth in detail certain illustrative aspects and implementations of the invention. These are indicative, however, of but a few of the various ways in which the principles of the invention may be employed. Other objects, advantages and novel features of the invention will become apparent from the following detailed description of the invention when considered in conjunction with the drawings.

BRIEF SUMMARY OF THE DRAWINGS

Figure 1 depicts schematics for the assembly design. The machine control unit serves as the core unit to perform different function, such as the pressure control and motion control for droplet generation and transfer. The user can control the assembly through the computer, which will be connected to the assembly by USB/RS232 to the universal asynchronous receiver/transmitter (UART) on the assembly.

Figure 2 depicts the workflow of the assembly for droplet generation (enclosed by yellow dotted line) , liquid transfer and cleaning procedures (enclosed by green and blue dotted line) .

Figure 3 depicts a schematic for waste disposal schematics.

Figure 4 shows a front side of the assembly, where the user interacts with. After the user places the sample tube, the assembly automates the process of sample transfer, droplet preparation, and droplet transfer. The user collects the droplets on the PCR tubes after the automated processing. Thus, the user does not need to concern about the whole process.

Figure 5 depicts a microfluidic adaptor is fixed by 2 clamps on the side. 2 screws are used to tighten the adaptor on the place holder.

Figure 6 depicts a transparent view of the clamp after fixing the microfluidic adaptor.

Figure 7 shows a rear side of the assembly, where the valves, sensors, and pumps rest in. The pressure module, which we developed in previous progress updates, is composed of rotary pump 1, solenoid valve 1 & 2, pressure chamber, and pressure sensor. The piston pump with valve controls the infuse-and-withdraw function of the liquid handler. The rotary pump 2 & 3, and solenoid valve 3 & 4, are involved in the washing process for the liquid handler to prevent cross-contaminations.

Figure 8 depicts a pressure detection module. The pressure sensor is embedded in between the diaphragm pump and the microfluidic outlet chamber. The sensor then collects the pressure value and send the data back to the pressure control module. With a comparison between the user defined pressure value and the value from the sensor, the control module then controls the rotary speed of the diaphragm pump thus to increase/decrease the control pressure. This feedback mechanism allows a precise control on the size of the droplet being generated.

Figure 9 depicts a pressure control module. The large electronic board supplies power and receive pressure data to and from the small electronic board, which is the pressure acquisition module.

Figure 10 shows a CAD drawing of the standard adaptor. Top view of the adaptor. The tapered corner is for correct orientation on the assembly place holder.

Figure 11 depicts an overview of the adaptor, with the blue arrow indicating the row of outlets.

Figure 12 depicts a bottom view of the adaptor, with the orange arrow indicating one of the pin structures.

Figure 13 depicts a CAD drawing of the microfluidic channels. The white parts indicate the microfluidic channels. The oil flows into the oil inlets (red) and the samples flow into the sample inlets (green) when a negative pressure is applied at the outlets (blue) . The samples meet with the oil at the cross-junction and become emulsified into droplets.

Figure 14 depicts a 3D-printed adaptor consists of 3 rows of pins corresponding to the oil inlets, samples inlets, and outlets. The pins are inserted into the ports on the PDMS chip, indicated by the direction of the yellow arrow, to finish the final assembly. The pin structure is enlarged in the dotted white square for clear representation.

Figure 15 shows that after the assembly, the oil inlets are pre-filled with oil, and the sample inlets are pre-filled with the sample before the suction starts. The white 3D-printed part seals the outlet to prevent air leak. Negative pressure is applied to the outlets to initiate the droplet generation processes. Note that in the final assembly, the white 3D-printed part belongs to the pressure sealer on the assembly.

Figure 16 graphically illustrates the polydispersity of the droplets generated from different microchannels. The droplets polydispersity are within 9%, suggesting the droplets are stable after the droplet generation using the custom-design adaptors.

Figure 17 graphically illustrates variations of the droplet generation rate of individual microfluidic channels. We apply a negative pressure (-4 psi) at the outlet and investigate the droplet generation. High speed camera is used to monitor the droplet generation. We record the generation process and analyze the droplet generation rate of the droplets in the microfluidic channels. Droplet generation process takes place in all channels, supporting the functioning of the pin-to-hole idea of the adaptor.

Figure 18 graphically illustrates pressure value against the size of the droplet generated with different oil system, silicone oil and FC40.

Figure 19 depicts droplet stability after the droplet generation process, with different oil and surfactant as the outer phase: Silicon oil and DC749, FC40 and commercial surfactant, FC40 and self-synthesized surfactant. Under the same pressure setting, the FC40 groups show better stability performance than the silicone oil group, suggesting FC40 is the suitable choice for the emulsion system.

Figure 20 graphically illustrates droplets generated with the improved pressure modules, under the pressure value of -4 psi, -5 psi, -6 psi, -7 psi, -8 psi, and -10 psi. Qualitatively, the droplets are of similar sizes under the pressure values of -5 psi, -6 psi, and -7 psi, regardless of the surfactant used. Scale bar: 200μm.

Figure 21 graphically illustrates quantitative analysis of the droplet size uniformity based on the cross-sectional area of droplets generated using the commercial surfactant. Box plot showing the size distribution of droplets against pressure values of -4 psi, -5 psi, -6 psi, -7 psi, -8 psi, and -10 psi.

Figure 22 graphically illustrates the polydispersity of the droplets are within 10%under the pressure value of -5 psi, -6 psi, which provides a narrower operating pressure range than that generated using the custom-synthesized surfactant.

Figure 23 graphically illustrates beads with different diameter and concentrations investigated in the experiments.

Figure 24 depicts selected bright field images of the droplets with beads. Three sizes of beads, 1μm, 9.51μm, and 20μm were encapsulated in the droplets. The beads are indicated by the yellow arrows, suggesting that the beads can be encapsulated inside the droplets with the current setup. With a high concentration of beads, most of the droplets encapsulated the beads. By further diluting the bead concentration, the number of beads in the droplet decreased, and the probability of the encapsulation followed Poisson statistics. Scale bar: 200μm.

Figure 25 depicts bright field and fluorescence images of the droplets with 50nm blue fluorescent polystyrene beads. (a, b) With a high concentration of beads, their fluorescence could be easily detected by their uniform blue fluorescence in the droplets. (c, d) The decrease in the bead concentration results in a decrease in fluorescence in the droplets. Scale bar: 200μm.

Figure 26 depicts fluorescence images of the droplets with 1μm green fluorescent polystyrene beads. The droplets were observed under a fluorescence microscope at the outlet of the microfluidic chip. A 1,000 times higher concentration of fluorescence beads (left) gives a brighter fluorescence than at a low bead concentration in the droplets (right) . Scale bar: 200μm.

Figure 27 depicts the CAD drawing of the microfluidic channels for mixing. The white parts indicate the microfluidic channels. The oil flows into the oil inlets (red) and the samples flow into the sample inlets (green) when a negative pressure is applied at the outlets (blue) . The samples meet with the oil at the cross-junction and become emulsified into droplets. The droplets flow downstream to a serpentine channel, which create vortices within droplet to enhance mixing.

Figure 28 graphically illustrates calibration of droplet fluorescence. The droplets are collected after generation, and their fluorescence are monitored under a fluorescence microscope. The fluorescence intensities are analyzed using ImageJ. More than 30 droplets are analyzed in each data point.

Figure 29 graphically illustrates calibration curve of the droplet fluorescence intensities. Fluorescence dye solution of 46.875 nM, 93.75 nM, 187.5 nM, 375 nM, and 750 nM are monitored with a custom-built platform. The droplets are scanned by a laser line and the corresponding fluorescence intensities are recorded. More than 1,000 droplets are analyzed in each data point.

Figure 30 graphically illustrates characterization of the fluorescence of mixture after 0, 1, 3, 5, and 10 pipette up-and-down motions. After different degree of mixing, the samples are dispersed into droplets. The droplet fluorescence are monitored by a laser line scan. The solution is homogenous when its fluorescence intensities fall in the + and –10%range of the expected mean signal. More than 1,000 droplets are analyzed in each data point.

Figure 31 graphically illustrates characterization of the mixing efficiency after 0, 1, 3, 5, and 10 pipette up-and-down motions. After different degree of mixing, the samples are dispersed into droplets. The droplet fluorescence is monitored by a laser line scan. From the experimental result, at least 5 pipette up-and-down motions is required to obtain a >95%homogenous solution.

Figure 32 depicts a physical model of the assembly.

Figure 33 charts droplet sizes and standard deviations.

Figure 34 graphically illustrates the variation of pressures inside the chamber during a 32-second interval experiment. The percentage of pressure variations is within 2%.

Figure 35 depicts an exemplary user interface.

DETAILED DESCRIPTION

Several embodiments in the following description with reference to the Figures are described.

The present disclosure provides a micro-fluidic platform to generate droplets for analysis by application of robotic arm. First, a physical model was developed to describe the model. Second, the system was applied to generation of droplets. Finally, the system was tested with different parameters.

There are 4 major parts of the assembly which the user can interact with: (1) Place holder for the sample tube, (2) Reservoirs for waste buffer, (3) Place holder for microfluidic chip, and (4) Rack for PCR tubes. We optimise the assembly design by aligning all parts on the same axis. Thus, a liquid handler with movement along only 2 axes (Z and Y) is enough to fulfil our requirement. Three components are required on hand to use the assembly: (1) Sample tube, (2) Microfluidic chip, already pre-filled with the oil, and (3) Empty PCR tubes. The sample tube has to be placed on the place holder, the microfluidic chip on the place holder, and the PCR tubes on the rack. The positions of the place holders and rack are indicated in Figure 4.

Characterised in that the adaptor was designed to standardise the dimension of microfluidic chips. In the assembly structure, a pressure sealer will be pressed onto the outlets of the adaptor along the Z-axis. Therefore, there will be a bending moment on the adaptor, leading to potential air leakage of the sealer. To resist the bending moment after the pressure sealer presses on the adaptor, the microfluidic adaptor is fixed by 2 clamps on the side. After inserting the microfluidic adaptor, the microfluidic adaptor is tightened by the clamps with 2 screws on the place holder, as shown in Figure 5. After the microfluidic adaptor is fixed, the adaptor slides along the X-axis inside the assembly.

After inserting the components and initiating the process on the control software, the assembly runs and the process is automated. The liquid handler will first go to the sample tube to withdraw the sample. The samples are then transferred into the 8 sample wells on the microfluidic adaptor. The fluid transfer function of the liquid handler is achieved by the piston pump with valve module at the back of the assembly. After the liquid handler transfers the sample, the pressure sealer covers all outlets on the microfluidic adaptor and the assembly starts the negative pressure. The negative pressure is applied by the pressure module, which have been developed previously. Figure 34 shows the pressure variation of pressure module during 32 seconds interval. The module is placed at the back side of the assembly, and is composed of rotary pump1, solenoid valve 1 & 2, pressure chamber, and pressure sensor (Figure 7) . After the droplet generation process, the pressure sealer is released from the microfluidic adaptor. The liquid handler moves to the outlets of the adaptor, and transfers the droplets into PCR tubes. PCR tubes filled with droplets can now be collected. To prevent cross-contaminations, the nozzle of the liquid handler is washed between each operation. The nozzle of the liquid handler will dip into the reservoirs for washing. This washing step is done with the washing modules, which are composed of rotary 2 & 3, and solenoid valve 3 & 4. The components and modules are protected from the back side of the assembly, as shown in Figure 7. By setting up the solenoid values, pumps, and motors, the sample preparation steps are then automated. The physical model of the assembly is shown in Figure 32.

On the microfluidic adapter, there are 8 sets of inlets for the oil, 8 sets of inlets for the sample, and 8 sets of outlets for the negative pressure. Since the smallest dimension of the channel is ～35 μm, any dust in the environment can clog the channels, leading to operation failure. This problem was tackled by designing passive filter structures at the inlets. The passive filters consist of patterns with gaps of only ～15 μm. Thus, dust or any objects that is larger than 15 μm are filtered and the flow downstream is preserved. Flow resistor is also designed at the droplet generation junction to stabilize the flow against fluctuations introduced during the droplet generation process. Moreover, 6 ‘+’ signs on the pattern are included to aid our fabrication process. After the PDMS is cured on the mould, the edge was trimmed closely of the 6 ‘+’ signs to minimize the size variation of the PDMS replica from batch to batch. The pattern of the microfluidic channel is shown in Figure 13.

The PDMS replica and the glass slide were treated under air plasma; subsequently, the PDMS replica and the glass slide are bonded together to form an irreversible seal. Following the plasma treatment, Aquapel was infused into the channel. This can enhance the wettability of the fluorocarbon oil on the PDMS channel wall. Excess Aquapel are flushed out carefully with pressurized air, and the PDMS chip is heated at 90℃ to evaporate the residual Aquapel. To assemble the final device for the experiment, the PDMS chip is connected with the 3D-print adaptor. The pins on the adaptor are aligned on top of the PDMS chip and compressed to form one piece. This “pin to hole, and go” design facilitates a quick preparation of the final device, as shown in Figure 14.

To test the chip, another 3D-printed part (white) was glued to the outlet of the adaptor. The white 3D-printed part connects to the transparent tubing to the negative pressure source. Thus, all the microfluidic channels are exposed to equal negative pressure. Samples are then dispersed into droplets when the pressure pump operates. The experimental setup is illustrated in Figure 15.

The robustness of the adaptor was examined during the droplet generation process. An important parameter was the droplet generation rate. If there is any air leakage between the pin and the ports on the PDMS chip, the fluid flow will be affected and stop the droplet generation. The droplet generation process was therefore monitored with a high speed camera. Video of the droplet generation was recorded, and the number of droplets generated per second was counted. The droplet generation rate of the channels shows a variation of 5%to 20%from mean, as shown in Figure 17. The experimental result is better than the reported result in literature, which shows a 4-25%variations [1] . The remaining variations may originate from error in measuring the droplet generation rate in the video, and the fabrication defects on the adaptor. Other means, such as injection molding, can enhance the quality of the adaptor. In addition, the droplet size was investigated under an inverted microscope. The size of the droplets was analyzed using ImageJ (NIH) . The size of the droplets and their standard deviations are shown in Figure 33. The polydispersity was plotted against each outlet of the adaptors, as shown in Figure 16. The droplets polydispersity are within 9%, suggesting the droplets are stable after the droplet generation using the custom-design adaptors.

The mixing steps are done by liquid handler in the assembly. To mimic the mixing step, water was first pipetted into the wells on the adaptor. Another water sample was then transferred with fluorescence dye to the adaptor. The sample was mixed with the pipette-up-and-down motion. To determine how well the mixing is, a calibration curve was set up with different concentration of fluorescence dye in the water. Dye solution was prepared with concentrations of 46.875 nM, 93.75 nM, 187.5 nM, 375 nM, and 750 nM and their corresponding fluorescence intensities was monitored. The result is plotted in figure 33. More than 1,000 droplets were analyzed in each data point.

The minimal rounds of pipetting motion required was further investigated to achieve a thorough mixture. The mixture was pipetted with 0, 1, 3, 5, and 10 up-and-down motions, and the sample was dispersed into droplets. The fluorescence intensities of the droplets was monitored with laser line scan on a custom-built platform. When the droplets flow across the laser line, the fluorescence signal is acquired by a photomultiplier tube, and recorded in the computer. If the mixing is complete, the mixture is homogenous and the fluorescence signal should fit the values predicted in the calibration curve. 10 μL of water was mixed with 10 μL of 750 nM fluorescence dye solution, and the fluorescence of the droplets was monitored. A fluorescence intensity of around 2 a.u. was expected. The observed droplet fluorescence is indeed around 2 a. u. after 5 pipette up-and-down motions, as shown in Figure 34. Based on the assumption that the droplets are monodisperse, the mixing efficiency is defined as:

The plot of the mixing efficiency is shown in Figure 35. From the experimental result, it is concluded that at least 5 up-and-down motions is required to achieve a throughout mixture in the well before the droplet generation starts.

Reference: [1] Bardin, D., Kendall, M.R., Dayton, P.A., & Lee, A.P. (2013) , Parallel generation of uniform fine droplets at hundreds of kilohertz in a flow-focusing module, Biomicrofluidics, 7 (3) , 034112, http: //doi. org/10.1063/1.4811276, incorporated herein by reference.

Advantageously, the techniques described herein can be applied to any device and/or network including a multiprocessor system. It is to be understood, therefore, that handheld, portable and other computing devices and computing objects of all kinds are contemplated for use in connection with the various non-limiting embodiments, i.e., anywhere that a device may wish to implement automated handling systems. Accordingly, a computer for implementing the automated handling systems is but one example, and the disclosed subject matter can be implemented with any client having network/bus interoperability and interaction. Thus, the disclosed subject matter can be implemented in an environment of networked hosted services in which very little or minimal client resources are implicated, e.g., a networked environment in which the client device serves merely as an interface to the network/bus, such as an object placed in an appliance.

Although not required, some aspects of the disclosed subject matter can partly be implemented via an operating system, for use by a developer of services for a device or object, and/or included within application software that operates in connection with the component (s) of the disclosed subject matter. Software may be described in the general context of computer executable instructions, such as program modules or components, being executed by one or more computer (s) , such as projection display devices, viewing devices, or other devices. Those skilled in the art will appreciate that the disclosed subject matter may be practiced with other computer system configurations and protocols.

Unless otherwise indicated in the following examples and elsewhere in the specification and claims, all parts and percentages are by weight, all temperatures are in degrees Centigrade, and pressure is at or near atmospheric pressure.

With respect to any figure or numerical range for a given characteristic, a figure or a parameter from one range may be combined with another figure or a parameter from a different range for the same characteristic to generate a numerical range.

Other than in the operating examples, if any, or where otherwise indicated, all numbers, values and/or expressions referring to parameters, measurements, conditions, etc., used in the specification and claims are to be understood as modified in all instances by the term "about. "

While the invention is explained in relation to certain embodiments, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. Therefore, it is to be understood that the invention disclosed herein is intended to cover such modifications as fall within the scope of the appended claims.

Claims

An assembly, comprising:

a cartridge comprising an adaptor comprising multiple sets of wells, a channel extending from the multiple sets of wells to units of pins that insert into openings of a microfluidic device; and the microfluidic device comprising microchannels in a horizontal plane and openings upright to the microchannels, with at least one droplet generator comprising channel prefilters, sample inlet (s) , an immiscible fluid inlet, a serpentine channel, an outlet, and a channel junction, wherein the channel junction and serpentine channel is positioned between sample inlet (s) and the outlet, the at least one droplet generator is configured to generate droplets at the channel junction surrounded by an immiscible fluid, the fluid inside droplets are mixed when passing through the serpentine channel;

a robot for handling fluid samples, comprising a liquid handler with fluidic setup being configured to drive fluid samples into the multiple sets of wells, to transfer emulsions from the multiple sets of wells, to mix the fluid within the multiple sets of wells, and to wash the inner wall of liquid handler; a sensor that monitors a position of a nozzle of the liquid handler on the fluid surface; and a positioning apparatus for moving the liquid handler within the assembly;

a setup component for receiving the cartridge, comprising mechanical frames to secure the cartridge; a mechanical slider to translate the cartridge; and a fluidics control and pressure sensor to apply pressure to emulsion wells on the cartridge for droplet generation; and

software, comprising a user interface to communicate with and to control the assembly, and to provide feedback of a status of the assembly.
The assembly of claim 1, wherein the adaptor comprises sample wells, immiscible fluid wells, and emulsion wells.
The assembly of claim 1, further comprising a processor and memory to implement the software.
The assembly of claim 1, wherein at least one droplet generator has a variation of 5%to 20%droplet generation rate from mean.