Patent Application
20130337576
Embodiments of the invention relate generally to a microfluidic system, cartridge and method and more particularly to a microfluidic system, cartridge and method for preparing samples automatically.
Samples containing analytes of interest are generally required to be prepared before loading into analytical instrumentations for analysis. For example, a flow cytometer is a powerful analytical instrument to analyze individual cells or particles for a wide range of applications spanning from cellular analysis to molecular and genomic analysis. Cells or particles need to be prepared before loading into the flow cytometer with a sample preparation procedure tailored to a specific application. Many commercially available flow cytometers are expensive, large bench top instruments that require an intensive manual sample preparation before sample loading. The manual sample preparation usually requires highly skilled personnel to carry out using long hours and at high cost and it requires additional lab equipment and resources. Some automation in sample preparation and handling has been realized by a large-scale robotic system. However, those robotics increase the system size, cost and complexity, making them not suitable for point-of-care applications.
It is desirable to provide a system, cartridge and method that can be integrated into an analytical instrument like a flow cytometer to provide rapid and automated sample preparation. In addition, it is also desirable to provide a microfluidic-based system that is compact, inexpensive and suitable for use at point-of-care.
In accordance with one embodiment disclosed herein, a microfluidic system for preparing a sample containing an analyte of interest is provided. The microfluidic system includes a microfluidic cartridge and a magnetic element. The microfluidic cartridge includes a number of reservoirs, an immobilizer, a number of microfluidic flow channels and a number of microvalves. The microfluidic channels are coupled with the reservoirs and the immobilizer. The microvalves are positioned along the microfluidic flow channels. The magnetic element is positioned with respect to the immobilizer. The magnetic element is configured to generate a magnetic field to magnetically immobilize the analyte of interest in the immobilizer. The immobilizer is configured to flow one or more reagents therethrough to react with the analyte of interest.
In accordance with another embodiment disclosed herein, a microfluidic cartridge for preparing a sample containing an analyte of interest is provided. The microfluidic cartridge includes a number of reservoirs, an immobilizer, a number of microfluidic flow channels and a number of microvalves. The immobilizer is configured to immobilize an initial sample using a magnetic field. The microfluidic flow channels are coupled with the reservoirs and the immobilizer and configured to pass one or more reagents from one or more of the reservoirs through the immobilizer to forma prepared sample. The microvalves are positioned along the microfluidic flow channels for controlling the flow of the one or more reagents.
In accordance with another embodiment disclosed herein, a method for preparing a sample automatically in a microfluidic cartridge is provided. The method includes introducing an initial sample into an immobilizer. The method further includes immobilizing the initial sample in the immobilizer by a magnetic field. And the method further includes introducing one or more reagents from one or more reagent reservoirs through the immobilizer to react with the initial sample to form a prepared sample while the one or more reagents flow through the immobilizer. The method further includes releasing the prepared sample into a downstream analysis portion by removing the magnetic field from the immobilizer.
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms “first”, “second”, and the like, as used herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. Also, the terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The use of “including,” “comprising” or “having” and variations thereof herein are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Although the terms “connected” and “coupled” are often used to describe physical or mechanical connections or couplings, they are not intended to be so restricted and can include electrical connections or couplings, whether direct or indirect. Moreover, the terms “connected” and “coupled” may refer to physical or mechanical connections or couplings that together allow a carrier fluid or liquid to flow therein.
In one embodiment, magnetic particles adapted to capture the analyte of interest are used in conjunction with the initial sample as part of the sample preparation process. In one embodiment, the magnetic particles are functionalized such that the analyte of interest binds to the magnetic particles through functionalized surfaces on the magnetic particles that provide specific or non-specific binding moieties. In one embodiment, the magnetic particles comprise functionalized magnetic beads having a size on the order of 1˜10 μm, for example. The magnetic particles may be premixed with and stored with the initial sample or the magnetic particles and the initial sample may be stored separately and combined only during the sample preparation process. In an embodiment where the initial sample and the magnetic particles are stored together, they may be stored in a sample reservoir on the microfluidic cartridge or in a container separate from the microfluidic cartridge and injected into the microfluidic cartridge as part of the sample preparation process. In an alternative embodiment, the magnetic particles and the initial sample may be stored in isolation from each other and mixed only during the sample preparation process. In this case, the initial sample and/or the magnetic particles may be stored on or off of the microfluidic cartridge. In one embodiment, the magnetic particles may be loaded into and stored in a reagent reservoir prior to reacting with the analyte of interest. For the purpose of explanation, the reservoirs 11b-11d will be described as representing reagent reservoirs, while reservoir 11a will be described as representing a sample reservoir. However, in practice, the initial sample and reagents may be stored in different reservoirs and greater or fewer numbers of reservoirs may be used. The reservoirs may store one or more types of reagents such as buffer agents and various labeling agents.
The microfluidic flow channels 15a-15e include a main channel 15e and a number of branching channels 15a-15d. The main channel 15e is coupled with the immobilizer 18 and each of the branching channels 15a-15d respectively couple each of the reservoirs 11a-11d to the main channel 15e, so the reservoirs 11a-11d are in fluid communication with the immobilizer 18. As used herein, the term “channel” is used to refer to a fluid path. In some embodiments a channel may represent a continuous fluid path while in other embodiments a channel may represent a discontinuous fluid path. A discontinuous fluid path may include one or more features or protrusions which in coordination with the microvalves function to block or enable fluid flow. The microvalves 17a-17d are respectively positioned within or adjacent to each of the branching channels 15a-15d between each respective reservoir 11a-11d and the main channel 15e to control the flow of reagents and samples from the reservoirs 11a-11d into the main channel 15e. Further, the microvalve 17e is positioned at an end of the main channel 15e near the immobilizer 18 to control the flow of material into the immobilizer 18. In one embodiment, the microvalves 17a-17e represent microfluidic components that may each be independently controlled by a fluidic controller (not shown). In one embodiment, the fluidic controller includes one or more computer-controlled pumps to pneumatically control the microvalves 17a-17e The pumps are connected to each of the microvalves 17a-17e via small tubes (not shown) which are located off of the microfluidic cartridge. Pressure and vacuum are applied by the pumps to the microvalves 17a-17e through the tubes so as to control the microvalves 17a-17e. In one embodiment, the microvalves 17a-17e are initially closed and are subsequently actuated one at a time, so that the initial sample and the reagents can be injected sequentially into the immobilizer 18.
In step 54, the reagents are sequentially introduced from the reagent reservoirs 11b-11d into the trapping area 184. In one embodiment, the microvalve 17f is controlled to open and the microvalves 17b-17d are actuated sequentially to open such that the stored reagents are sequentially introduced from the reagent reservoirs 11b-11d into the trapping area 184. As previously noted, the reagents could be initially stored in either a liquid form or a dried form and then reconstituted into fluid form. The liquefied reagents are sequentially flowed through and react with the magnetic sample immobilized in the trapping area 184 to generate the prepared sample, as the excess reagents are flowed out of the discharge channel 187. The microvalve 17f is controlled to open before the first reagent is flowed out of the discharge channel 187 and is controlled to close after the last reagent is flowed out of the discharge channel 187. In a specific embodiment, the microvalves 17b and 17e are controlled to open and a first reagent such as a buffer agent is flowed from the reagent reservoir 11b and through the trapping area 184 and then flowed out of the discharge channel 187. Next, the microvalve 17c is controlled to open and a second reagent, such as tracer-antibodies, is flowed through the trapping area 184 to react with the sample, and flowed out of the discharge channel 187. Finally, the microvalve 17d is controlled to open and a third reagent, such as a fluorescent label, is flowed through the trapping area 184 to react with the sample and then flowed out of the discharge channel 187 resulting in a prepared sample. In certain embodiments, other types of reagents may be introduced to react with the initial sample, for example, one type of labeling agent may be introduced to quantify the analyte of interest. In one embodiment, some amount of liquid from the initial sample and the reagents is left in the reservoirs 11a-11d, so as to prevent air from flowing into the sample preparation portion 10. In step 55, once the initial sample has been processed into a prepared sample in the trapping area 184, the microvalve 17f is controlled to close, the microvalve 17g is controlled to open, and the prepared sample is then released and flowed into the analysis portion 22 through the outlet 183. In one embodiment, the prepared sample is released by removing the magnetic field generated by the magnetic element 19. In one embodiment, the magnetic field is removed by physically moving the magnetic element 19 away from the immobilizer 18. However, in other embodiments, a mechanical actuator or an electromagnet could be used.
Such a dynamic sample preparation process using flow through reactions allows continuous and constant concentration of the flowing reagents to interact with the immobilized sample. This results in a more uniform and efficient reagents-sample interaction in a certain time duration as compared to the conventional, manual static incubation in a chamber, leading to a shorter reaction time. In addition, in a typical manual sample preparation process involving one or more reagents to react or be incubated with the sample, washing steps in between incubations are essential to remove any non-specifically bounded agents to reduce the background noise. A flow through reaction such as that described herein, however, enables a wash-free process since the continuously flowing reagents sweep away the non-specifically bound agents resulting from the previous reaction step. Furthermore, in such a flow through reaction, a previous reagent is more likely to be completely removed by the incoming reagents as compared to the manual static incubation where the addition or removal of reagents occurs in a chamber or other container rendering complete removal of previous reagents difficult.
While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.
This invention was made with Government support under contract number N00173-08-2-0003 that was awarded by the Naval Research Laboratory. The Government has certain rights in the invention.
