Multiplexed polymerase chain reaction in micropipette format

Abstract

To provide processing of samples such as biological samples.SOLUTION: This system may include a horizontal actuator to move a tray to which a microwell plate and a microfluidic chip may be coupled. The system may include a vertical actuator to move a support arm to which a plurality of pipettes or pipette tips may be coupled. The system may include a rotational actuator to move an angle bracket to which a magnet may be coupled. The system may include a heater through which the pipettes may extend. The system may include a pump to control the flow of fluids through the pipettes. The disclosed methods include performing PCR within the described system.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The technology described herein relates to the processing of samples, such as biological samples, e.g., the separation of contaminants from biological samples contained on a substrate, in conjunction with polymerase chain reaction of nucleic acids derived from complex samples, e.g., blood. [Background technology]

[0002] It is well known that nucleic acid can be collected for analytical testing.For example, the main problem in collecting nucleic acid from blood is that biological samples often contain contaminants.Therefore, nucleic acid sample purification has become an important step in experimental workflow, because the quality of sample nucleic acid can affect the performance in downstream applications, especially in applications that are sensitive to contaminants, such as polymerase chain reaction (PCR).

[0003] Many common nucleic acid purification techniques involve centrifugation, chemical separation, or solid-phase-based separation. However, these techniques are time- and labor-intensive and very often require the use of specialized equipment.

[0004] The use of microfluidics in the analysis of biological and chemical samples is well known. One such use involves a system that utilizes a microfluidic chip (sometimes referred to as a "lab-on-a-chip") to acquire one or more samples, process the samples for measurement, and then assess their composition. However, the samples must be purified to remove contaminants and ensure quality for downstream applications.

[0005] PCR is one downstream application that requires strict control of purified nucleic acid samples and reaction conditions to avoid cross-contamination. Multiplexed purification of nucleic acid samples followed by PCR of the purified samples on the same microfluidic chip is therefore cumbersome, and systems and methods for its improvement are needed.

[0006] Thus, there is a need in the art for improved processes for purifying biological samples and amplifying nucleic acids therein without the need for expensive, highly specialized equipment. Summary of the Invention [Means for solving the problem]

[0007] Embodiments of the present technology are directed to performing PCR in a multiplexed format within a cannula or tube that can also be used to transfer and / or dispense samples and reagents for sample isolation and purification in a microfluidic format. The PCR methods described herein can be used in combination with the isolation and purification techniques described herein, so that samples can be purified and the nucleic acids obtained therefrom can be amplified within a single assay using the same instrument. However, sample isolation and / or purification on the described instrument is not required, and the methods and devices described herein are equally useful for PCR of pre-purified nucleic acids obtained from various sources. PCR reagents and methods are known in the art and are described in more detail in WO 2011 / 094577 (incorporated by reference in its entirety).

[0008] The method may be summarized as including the steps of: operating a pump to draw a biological sample from a well of a well plate into the pipette through a pipette tip of the pipette; operating a first actuator to move a first valve to a closed position and seal the biological sample from the pipette tip into the pipette; operating the pump to apply a positive gauge pressure to the biological sample in the pipette; operating a second actuator to move a second valve to a closed position and seal the biological sample from the pump into the pipette; and operating a heater to heat the biological sample in the pipette, wherein a polymerase chain reaction occurs in the heated biological sample in the pipette between the first valve and the second valve.

[0009] Another method can be summarized as including the steps of: operating a pump to draw a biological sample from a well of a well plate through a first pipette tip into a first pipette conduit; operating a first actuator to move a first valve to a closed position to seal the biological sample from the first pipette tip into the first pipette conduit; operating a pump to apply a positive gauge pressure to the biological sample in the first pipette conduit; operating a second actuator to move a second valve to a closed position to seal the biological sample from the pump into the first pipette conduit; and operating a heater to heat the biological sample in the first pipette conduit, wherein a chemical reaction occurs in the heated biological sample in the first pipette conduit between the first valve and the second valve.

[0010] The system may be summarized as comprising: a first pipette including a first pipette tip and a first end of the first pipette opposite the first pipette tip along a first length of the first pipette; a second pipette including a second pipette tip and a second end of the second pipette opposite the second pipette tip along a second length of the second pipette; a three-way connector fluidly coupling the second end of the first pipette to the second end of the second pipette and fluidly coupling the second ends of the first and second pipettes to a feed conduit; and a heating element thermally coupled to at least a portion of the first and second lengths.

[0011] The isolation and purification techniques described herein, which can be performed separately from or prior to PCR, use an electric field and / or a gel, such as a polyether compound, to separate biological analytes, such as nucleic acids, from a biological sample contained on a substrate. According to a first embodiment of the present technology, a method for separating biological analytes includes providing a first well and a second well interconnected via a microchannel. A fluid, such as a buffer, is provided in the first well, into which the biological sample is placed. Magnetic beads, e.g., polyvinyl alcohol-based magnetic particles (M-PVA magnetic beads), are then introduced into the first well. The surface of the M-PVA magnetic beads can be functionalized with many different groups and modified with individually excitable loadings. In one embodiment, the M-PVA magnetic beads can be excitable to isolate nucleic acids in a biological sample, which may consist of blood.

[0012] The M-PVA magnetic beads isolate nucleic acids in the biological sample and attract the target molecules to the M-PVA magnetic beads. An electric field that interacts with negatively charged contaminants in the buffer is then applied to the first well. A magnet outside the first well is then brought close to the first well. The magnet functions to attract the M-PVA magnetic beads such that, as the magnet is moved outward toward the microchannel, the M-PVA magnetic beads, along with the target molecules attracted to them, are drawn toward and into the microchannel. However, most of the negatively charged contaminants remain in the first well due to their interaction with the electric field. The magnet continues to attract the M-PVA magnetic beads and the target molecules along their path within the microchannel toward the second well and into the second well. The microchannel also contains a buffer, and movement of the M-PVA magnetic beads through the buffer within the microchannel further functions to expel contaminants from the target molecules so that the target molecules are essentially free of contaminants by the time the M-PVA magnetic beads reach the second well.

[0013] At this point, the M-PVA magnetic beads can be withdrawn from the second well, with the target molecules still attached, and placed into a chamber where they can be demagnetized to expel the target molecules and then removed. The result is a very clean biological sample, essentially free of contaminants. There is no need for expensive or specialized equipment, such as that required for centrifugation, chemical separation, or solid-phase-based separation. Rather, the technique can be implemented in a simple microfluidic chip format with few moving parts and no need for extensive power requirements.

[0014] In some embodiments, the third well can be connected to the second well through a second microchannel. In this configuration, once the magnet has moved the M-PVA magnetic beads carrying the target molecule into the second well, the magnet can then be moved toward the second microchannel, thereby attracting the M-PVA magnetic beads from the second well into the second microchannel. The magnet can then be moved toward the third well so that the M-PVA magnetic beads are attracted into the third well. This configuration provides another cleaning step for biological samples for applications requiring it, such as PCR.

[0015] It is contemplated that, in some embodiments, additional purification of the biological sample can be achieved by generating a flow of buffer from the second well toward the first well. This can be achieved by a simple fluid volume difference between the wells. In one embodiment, the second well can be provided with a larger volume of fluid than that provided in the first well. For example, a fluid volume difference in the range of 20% to 50% can be provided, which can produce a desired flow rate. This additional volume can be added after the biological sample is inserted into the first well, such that fluid flow from the second well through the microchannel into the first well occurs during the transfer of the M-PVA magnetic beads from the first well to the second well. This fluid flow will serve to carry any contaminants that have escaped from the first well out of the microchannel and back into the first well.

[0016] In some embodiments, an electric field is applied to a first well or chamber by application of first and second conductors. A power source is connected to the first and second conductors to generate a potential energy difference between the conductors. In addition, software can be used to control the power source to develop a desired voltage between the conductors. It is contemplated that the software may actively control the magnitude of the voltage across the conductors.

[0017] In some embodiments, the system may further comprise a motor coupled to the first conductor and adapted to move the first conductor into and out of the first well. Similarly, the system may further comprise a motor coupled to the second conductor and adapted to move the second conductor into and out of the first well.

[0018] In some embodiments, the system may include a motor coupled to the delivery system for inserting the M-PVA magnetic beads into the first well and for fully automated movement of a magnet that functions to pull the M-PVA magnetic beads from the first well into the microchannel and into the second well. Similarly, the system may further include a motor coupled to the magnet for withdrawing the M-PVA magnetic beads from the second well and for insertion into the chamber.

[0019] In some embodiments, the M-PVA magnetic beads are excited to attract deoxyribonucleic acid, while in other embodiments, the M-PVA magnetic beads are excited to attract ribonucleic acid. The isolated and purified nucleic acids can then, optionally, be amplified via PCR, as described herein.

[0020] In one embodiment, a method for removing contaminants from a biological sample is provided, the method including providing a first well and a second well interconnected by a microchannel and providing a fluid in the first well, the second well, and the microchannel. The method further includes placing the biological sample in the first well, introducing magnetic beads into the first well, and attracting target molecules in the biological sample to the magnetic beads. The method still further includes applying an electric field to the first well, where the electric field interacts with the contaminants; introducing a magnet that generates a magnetic field near the first well, where the magnetic field interacts with the magnetic beads; and moving the magnet toward the microchannel, where the magnetic beads are attracted with the movement of the magnet so that the magnetic beads and the target molecules are drawn into the microchannel. The method is provided such that the electric field acts on the contaminants to maintain them in the first well as the magnetic beads and the target molecules move into the microchannel. Finally, the method includes moving a magnet toward the second well, wherein the magnetic beads and target molecules are attracted with the movement of the magnet such that the magnetic beads and target molecules are drawn into the second well. Optionally, the purified nucleic acids obtained in such a method are further amplified via PCR as described herein.

[0021] In another embodiment, a system for removing contaminants from a biological sample, e.g., for exciting the sample for PCR, is provided, comprising a first well containing a fluid and adapted to receive a biological sample, a second well adapted to contain a fluid, and a microchannel extending between the first and second wells. The system is provided such that magnetic beads are introduced into the first well, and the magnetic beads are excited to attract target molecules in the biological sample. The system further comprises a power source and two probes coupled to the power source, the two probes adapted to apply an electric field to the first well. The system is provided such that when power is applied to the two probes, the two probes generate an electric field therebetween, with the contaminants interacting with the electric field. The system still further comprises a magnet adapted to be moved near the first well, the magnet generating a magnetic field and adapted to interact with the magnetic beads. The system is further provided such that the magnet is moved toward the microchannel such that the magnetic beads are drawn into the microchannel as the magnet moves. Additionally, the electric field is adapted to interact with the contaminant so that the contaminant is maintained within the first well, and the magnet is adapted to move toward the second well so that the magnetic beads and target molecules are drawn into the second well.

[0022] The M-PVA magnetic beads isolate nucleic acids in the biological sample, attracting target molecules to the M-PVA magnetic beads. A magnet outside the first well is then brought close to the first well. The magnet functions to attract the M-PVA magnetic beads such that when the magnet is moved toward the microchannel, the M-PVA magnetic beads, along with the target molecules attracted to them, are drawn toward and into the microchannel. The microchannel is filled with a polyether compound, such as polyethylene glycol (PEG), also known as polyethylene oxide or polyoxyethylene, depending on its molecular weight. Contaminants are negatively charged particles. When negatively charged contaminants are drawn into the PEG, the PEG functions to block the progression of these negatively charged species through the microchannel.

[0023] The magnet continues to draw the M-PVA magnetic beads and target molecules along their path within the microchannel toward the second well and into the second well, and by the time the M-PVA magnetic beads reach the second well, the target molecules are essentially free of the contaminants that have been expelled within the PEG.

[0024] At this point, the M-PVA magnetic beads can be withdrawn from the second well, with the target molecules still attached, and placed into a chamber where they can be demagnetized to expel the target molecules and then removed. The result is a very clean biological sample, essentially free of contaminants. There is no need for the expense or specialized features required for centrifugation, chemical separation, or solid-phase-based separation. Rather, the technique can be implemented in a simple microfluidic chip format with few moving parts or extensive power requirements.

[0025] In some embodiments, the third well can be connected to the second well through a second microchannel. In this configuration, once the magnet has moved the M-PVA magnetic beads carrying the target molecule into the second well, the magnet can then be moved toward the second microchannel, thereby attracting the M-PVA magnetic beads from the second well into the second microchannel. The second microchannel can also contain PEG. The magnet can then be moved toward the third well, such that the M-PVA magnetic beads are attracted into the third well. This configuration provides another cleaning step for biological samples for applications requiring it.

[0026] Still further, it is envisioned that the first well and the second well may comprise a gel, such as PEG, which would function to even further clean the biological sample.

[0027] It is contemplated that, in some embodiments, additional purification of the biological sample can be achieved by generating a flow of gel from the second well toward the first well. This can be achieved by a simple fluid volume difference of gel between the wells. In one embodiment, the second well can be provided with a larger volume of gel than that provided in the first well. This additional volume can be added after the biological sample is inserted into the first well, such that the flow of gel from the second well through the microchannel into the first well occurs during the transfer of the M-PVA magnetic beads from the first well to the second well. This fluid flow will serve to carry any contaminants that have escaped from the first well out of the microchannel and back into the first well.

[0028] In some embodiments, the system may include a motor coupled to the delivery system for inserting the M-PVA magnetic beads into the first well and for fully automated movement of a magnet that functions to pull the M-PVA magnetic beads from the first well into the microchannel and into the second well. Similarly, the system may further include a motor coupled to the magnet for withdrawing the M-PVA magnetic beads from the second well and for insertion into the chamber.

[0029] In some embodiments, the M-PVA magnetic beads are energized to attract deoxyribonucleic acid, while in other embodiments, the M-PVA magnetic beads are energized to attract ribonucleic acid.

[0030] In one example, a method for removing contaminants from a biological sample, e.g., for preparing the sample for PCR as described herein, is provided, comprising: providing a first well and a second well interconnected by a microchannel; providing a fluid into the first well, the second well, and the microchannel; and placing the biological sample into the first well. The method further comprises: introducing magnetic beads into the first well; attracting target molecules in the biological sample to the magnetic beads; and introducing a magnet that generates a magnetic field near the first well, where the magnetic field interacts with the magnetic beads. The method still further comprises moving the magnet toward the microchannel, where the magnetic beads are attracted with the movement of the magnet such that the magnetic beads and the target molecules are drawn into the microchannel; and providing a gel in the microchannel, where the gel interacts with the contaminants. The method is provided such that the gel interacts with the contaminants. The method finally includes a step of moving a magnet toward the second well, and the magnetic beads and target molecules are attracted with the movement of the magnet such that the contaminants are separated from the target molecules as they are maintained within the gel, and the magnetic beads and target molecules are drawn into the second well.

[0031] In another example, a system for removing contaminants from a biological sample is provided, the system comprising: a first well adapted to contain a fluid and receive a biological sample; a second well adapted to contain a fluid; a microchannel extending between the first and second wells; and a gel positioned within the microchannel. The purified biological sample may optionally undergo PCR according to the methods described herein. The system is provided such that magnetic beads are adapted to be introduced into the first well, and the magnetic beads are excited to attract target molecules within the biological sample. The system further comprises a magnet adapted to be moved near the first well, the magnet generating a magnetic field and adapted to interact with the magnetic beads. The system is further provided such that the magnet is moved toward the microchannel, and the magnetic beads are drawn into the microchannel with the movement of the magnet; and the gel is adapted to interact with contaminants as the magnetic beads move through the gel, such that at least some of the contaminants are captured within the gel. Finally, the system is provided such that the magnet is moved towards the second well, and the magnetic beads and target molecules are drawn into the second well.

[0032] The system may be summarized as comprising a horizontal actuator, a tray coupled to the horizontal actuator, a well plate coupled to the tray, a microfluidic chip coupled to the well plate, a vertical actuator, a pipette coupled to the vertical actuator, a heater or heating element mechanically and / or thermally coupled to the pipette to control the temperature of fluid in the pipette, a pump coupled to the pipette to control movement of fluid in the pipette, and a controller communicatively coupled to the horizontal actuator to control horizontal movement of the tray, well plate, and microfluidic chip, communicatively coupled to the vertical actuator to control vertical movement of the pipette, communicatively coupled to the pump to control the pump, and communicatively coupled to the heater to control the heater.

[0033] The system may further include a rotational actuator and a magnet coupled to the rotational actuator, and the controller may be communicatively coupled to the rotational actuator to control rotation of the magnet beneath the tray. The well plate may include a plurality of conductive leads positioned beneath the microfluidic chip. The pipette may include a pipette tip held in a vertical orientation by a support arm, and an end of the pipette opposite the pipette tip may be held within the cartridge. The heater may include a stationary sidewall and a hinged sidewall rotatably coupled to the stationary sidewall by a hinge. The stationary sidewall may include a first groove, and the hinged sidewall may include a second groove, and the pipette may extend between the stationary sidewall and the hinged sidewall through the first and second grooves. The stationary sidewall may include a first bar movable outward from the stationary sidewall toward the hinged sidewall to clamp the pipette at a first location near a first side of the stationary sidewall, and a second bar movable outward from the stationary sidewall toward the hinged sidewall to clamp the pipette at a second location near a second side of the stationary sidewall opposite the first side of the stationary sidewall.

[0034] The method includes receiving a biological sample into a first well in a well plate, receiving another reagent into a second well in the well plate, operating a pump to draw the biological sample from the first well of the well plate into a pipette, operating an actuator to move the pipette from the first well of the well plate to a first well of a microfluidic chip, operating the pump to expel the biological sample from the pipette into the first well of the microfluidic chip, and operating the actuator to expel the biological sample from the first well of the microfluidic chip to a second well of the microfluidic chip. The method may be summarized as including the steps of: moving a pipette, operating a pump to draw a biological sample from a second well of the microfluidic chip into the pipette, operating an actuator to move the pipette from the second well of the microfluidic chip to a second well of the microfluidic chip, operating a pump to draw other reagents from the second well of the well plate into the pipette, operating a heater to heat the biological sample and other reagents in the pipette, and operating a pump to expel the biological sample from the pipette into a third well in the well plate. The method optionally includes performing PCR after purification of the sample as described herein.

[0035] The biological sample may include DNA, RNA, mRNA, or protein. Contaminants may be removed from the biological sample within the microfluidic chip. Polymerase chain reaction may occur within the pipette. The present invention provides, for example, the following items. (Item 1) 1. A method comprising: activating a pump to draw biological samples from the wells of the well plate through the pipette tip of the pipette and into the pipette; activating a first actuator to move a first valve to a closed position to seal the biological sample from the pipette tip into the pipette; operating the pump to apply a positive gauge pressure to the biological sample in the pipette; activating a second actuator to move a second valve to a closed position to seal the biological sample from the pump into the pipette; operating a heater to heat the biological sample in the pipette, wherein a polymerase chain reaction occurs in the heated biological sample in the pipette between the first valve and the second valve; A method comprising: (Item 2) Item 10. The method of claim 1, wherein the positive gauge pressure is at least 7 psi above atmospheric pressure. (Item 3) activating the second actuator to move the second valve to an open position; operating the pump to release the positive gauge pressure; activating the first actuator to move the first valve to an open position; operating the pump to dispense the biological sample through a pipette tip of the pipette; Item 1, the method of claim 1 further comprising: (Item 4) 4. The method of claim 3, wherein operating the second actuator to move the second valve to an open position takes at least about 5 seconds. (Item 5) 4. The method of claim 3, wherein operating the first actuator to move the first valve to an open position takes at least about 5 seconds. (Item 6) 1. A method comprising: activating a pump to draw a biological sample from a well of a well plate through a first pipette tip and into a first pipette conduit; actuating a first actuator to move a first valve to a closed position to seal the biological sample from the first pipette tip into the first pipette conduit; operating the pump to apply a positive gauge pressure to the biological sample in the first pipette conduit; activating a second actuator to move a second valve to a closed position to seal the biological sample from the pump into the first pipette conduit; operating a heater to heat the biological sample in the first pipette conduit, wherein a chemical reaction occurs in the heated biological sample in the first pipette conduit between the first valve and the second valve; A method comprising: (Item 7) activating the second actuator to move the second valve to an open position; operating the pump to release the positive gauge pressure; activating the first actuator to move the first valve to an open position; operating the pump to draw the biological sample through a three-way connector; Item 7. The method of item 6, further comprising: (Item 8) 8. The method of claim 7, wherein operating the second actuator to move the second valve to an open position takes at least about 5 seconds. (Item 9) 8. The method of claim 7, wherein operating the first actuator and moving the first valve to an open position takes at least about 5 seconds. (Item 10) activating the first actuator to move the first valve to the closed position or activating the second actuator to move the second valve to the closed position; operating the pump to push the biological sample through the three-way connector and into a second pipette conduit; 8. The method of claim 7, further comprising: (Item 11) operating the pump to force the biological sample through the second pipette conduit; operating the pump to dispense the biological sample through a second pipette tip coupled to the second pipette conduit; Item 11. The method of item 10, further comprising: (Item 12) 12. The method of claim 11, wherein the chemical reaction is a polymerase chain reaction. (Item 13) actuating a third actuator to move a third valve to a closed position to seal the biological sample into the second pipette conduit from a second pipette tip coupled to the second pipette conduit; operating the pump to apply a second positive gauge pressure to the biological sample in the second pipette conduit; activating a fourth actuator to move a fourth valve to a closed position to seal the biological sample from the pump into the second pipette conduit; operating a heater to heat the biological sample in the second pipette conduit, wherein a second chemical reaction occurs in the heated biological sample in the second pipette conduit between the third valve and the fourth valve; Item 11. The method of item 10, further comprising: (Item 14) activating the fourth actuator to move the fourth valve to an open position; operating the pump to release the second positive gauge pressure; activating the third actuator to move the third valve to an open position; operating the pump to dispense the biological sample through the second pipette tip; Item 14. The method of item 13, further comprising: (Item 15) Item 15. The method of item 14, wherein operating the fourth actuator and moving the fourth valve to an open position takes at least about 5 seconds. (Item 16) Item 15. The method of item 14, wherein operating the third actuator and moving the third valve to an open position takes at least about 5 seconds. (Item 17) Item 14. The method of item 13, wherein the second chemical reaction is a polymerase chain reaction. (Item 18) Item 14. The method of item 13, wherein the second positive gauge pressure is at least 7 psi above atmospheric pressure. (Item 19) Item 14. The method of item 13, wherein operating the fourth actuator and moving the fourth valve to a closed position seals the biological sample from the three-way connector into the second pipette conduit. (Item 20) 1. A system comprising: a first pipette including a first pipette tip and a first end of the first pipette opposite the first pipette tip along a first length of the first pipette; a second pipette including a second pipette tip and a second end of the second pipette opposite the second pipette tip along a second length of the second pipette; a three-way connector fluidly coupling a second end of the first pipette to a second end of the second pipette and fluidly coupling the second ends of the first and second pipettes to a feed conduit; a heating element thermally coupled to at least a portion of the first and second lengths; A system comprising: [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 is a diagram of one embodiment of the present technology.

[0037] [Figure 2] FIG. 2 is an enlarged view according to FIG. 1, containing the biological sample to be purified.

[0038] [Figure 3] FIG. 3 is a diagram according to FIG. 2 with magnetic beads that serve to attract target molecules.

[0039] [Figure 4] FIG. 4 is a diagram according to FIG. 3 with electrodes applying an electric field to the biological sample which serves to attract contaminants.

[0040] [Figure 4A] FIG. 4A is a view similar to that shown in FIG. 4, but in which the electrodes are placed in different wells than those shown in FIG.

[0041] [Figure 5] FIG. 5 is a diagram of the electric field interacting with the contaminant.

[0042] [Figure 6] FIG. 6 is a diagram according to FIG. 4 with a magnet introduced in the vicinity of the magnetic beads, which serves to attract the beads.

[0043] [Figure 7] FIG. 7 is a diagram according to FIG. 6, in which magnetic beads are moved into a microchannel.

[0044] [Figure 8] FIG. 8 is a diagram according to FIG. 7, in which the magnetic beads are moved through the microchannel into the second well.

[0045] [Figure 9] FIG. 9 is a diagram of the purified biological sample according to FIG.

[0046] [Figure 10] FIG. 10 is a flow diagram illustrating the sequence of operations of the technique according to FIGS. 1-8.

[0047] [Figure 11] FIG. 11 is a flow diagram illustrating additional further steps according to FIG.

[0048] [Figure 12] FIG. 12 is a diagram according to FIG.

[0049] [Figure 13] FIG. 13 is a diagram according to FIG. 3 with a controller, agitator, heater, and temperature sensor.

[0050] [Figure 14] FIG. 14 is a diagram according to FIG. 13 with a magnet introduced in the vicinity of the magnetic beads, which serves to attract the beads.

[0051] [Figure 15] FIG. 15 is a diagram according to FIG. 14, in which magnetic beads are moved into a microchannel.

[0052] [Figure 16] FIG. 16 is a diagram according to FIG. 15, in which the magnetic beads are moved through the microchannel into the second well.

[0053] [Figure 17] FIG. 17 is a diagram of the purified biological sample according to FIG.

[0054] [Figure 18] FIG. 18 is a diagram of one embodiment of the present technology according to FIG.

[0055] [Figure 19] FIG. 19 is a flow diagram illustrating the sequence of operations of the technique according to FIGS. 1-3 and 13-17.

[0056] [Figure 20] FIG. 20 is a flow diagram illustrating additional further steps according to FIG.

[0057] [Figure 21]FIG. 21 is a diagram of a chip employing the method according to FIG.

[0058] [Figure 22] FIG. 22 illustrates rear, top, and left side perspective views of a microfluidic system for processing biological samples.

[0059] [Figure 23] FIG. 23 illustrates front, bottom, and right side perspective views of the microfluidic system of FIG.

[0060] [Figure 24] FIG. 24 illustrates a left side view of the microfluidic system of FIG.

[0061] [Figure 25] FIG. 25 illustrates a right side view of the microfluidic system of FIG.

[0062] [Figure 26] FIG. 26 illustrates a top plan view of the microfluidic system of FIG.

[0063] [Figure 27] FIG. 27 illustrates a bottom plan view of the microfluidic system of FIG.

[0064] [Figure 28] FIG. 28 illustrates a rear view of the microfluidic system of FIG.

[0065] [Figure 29] FIG. 29 illustrates a front view of the microfluidic system of FIG.

[0066] [Figure 30] FIG. 30 illustrates a front, top, and left side perspective view of the microfluidic system of FIG. 22 with its housing removed.

[0067] [Figure 31] FIG. 31 illustrates front, bottom, and left side perspective views of the microfluidic system of FIG. 22 with its housing removed.

[0068] [Figure 32] FIG. 32 illustrates a front, top, and right side perspective view of the microfluidic system of FIG. 22 with its housing removed.

[0069] [Figure 33] FIG. 33 illustrates rear, top, and left side perspective views of the microfluidic system of FIG. 22 with its housing removed.

[0070] [Figure 34] FIG. 34 illustrates a rear, top, and right side perspective view of the microfluidic system of FIG. 22 with its housing removed.

[0071] [Figure 35] FIG. 35 illustrates a bottom perspective view of a microfluidic plate or chip of the microfluidic system of FIG.

[0072] [Figure 36] FIG. 36 illustrates a perspective view of a portion of the microfluidic system of FIG. 22 with the microfluidic chip of FIG. 35 removed.

[0073] [Figure 37] FIG. 37 illustrates a top perspective view of a microwell plate of the microfluidic system of FIG.

[0074] [Figure 38] FIG. 38 illustrates a perspective view of a portion of the microfluidic system of FIG. 22 with the microfluidic chip of FIG. 35 and the microwell plate of FIG. 37 removed.

[0075] [Figure 39]FIG. 39 illustrates a perspective view of a portion of the microfluidic system of FIG. 22 with its microfluidic chip, microwell plate, and conductive leads removed.

[0076] [Figure 40] FIG. 40 illustrates a perspective view of the microfluidic system of FIG. 22 with its microfluidic chip, microwell plate, conductive leads, and tray removed.

[0077] [Figure 41] FIG. 41 illustrates a perspective view of a microfluidic system as illustrated in FIG. 40 with additional components removed.

[0078] [Figure 42] FIG. 42 illustrates a perspective view of a portion of the vertical actuation system of the microfluidic system of FIG.

[0079] [Figure 43] FIG. 43 illustrates a perspective view of the micropipette system of the microfluidic system of FIG.

[0080] [Figure 44] FIG. 44 illustrates a perspective view of the cradle of the microfluidic system of FIG. 22 with its hinged door removed.

[0081] [Figure 45] FIG. 45 illustrates a perspective view of the hinged door of the cradle of the microfluidic system of FIG.

[0082] [Figure 46] FIG. 46 illustrates a syringe pump system of the microfluidic system of FIG.

[0083] [Figure 47] FIG. 47 illustrates front, left, and top perspective views of another system including a micropipette system and cradle that can be used in conjunction with the microfluidic system of FIG.

[0084] [Figure 48] FIG. 48 illustrates a rear, right, and bottom perspective view of the system of FIG.

[0085] [Figure 49] FIG. 49 illustrates a front, left, and top perspective view of the system of FIG. 47 with its fluid conduits, solenoid actuator, heat sink, and handle removed.

[0086] [Figure 50] FIG. 50 illustrates a rear, right, and bottom perspective view of the system of FIG. 47 with its fluid conduits, solenoid actuator, heat sink, and handle removed.

[0087] [Figure 51] FIG. 51 illustrates the system of FIG. 47 with the rear portion of the cradle removed to reveal additional components, as illustrated in FIG.

[0088] [Figure 52] FIG. 52 illustrates the system of FIG. 47 with the micropipette system removed to reveal additional components, as shown in FIG. 51.

[0089] [Figure 53] FIG. 53 illustrates the system of FIG. 47 with the front portion or door of the cradle removed to reveal additional components as shown in FIG.

[0090] [Figure 54] FIG. 54 illustrates the system of FIG. 47 with the micropipette system removed to reveal additional components, as shown in FIG. 53.

[0091] [Figure 55]FIG. 55 illustrates the system of FIG. 47 with the valves and heat transfer blocks removed to reveal additional components as shown in FIG. DETAILED DESCRIPTION OF THE INVENTION

[0092] Detailed Description Reference is now made to the drawings, in which like reference numerals designate corresponding structure throughout the views.

[0093] Embodiments of the present technology involve systems and methods for separating biological material from a sample via magnetic beads and electric fields. Embodiments of the present technology are generally well suited for use with complex samples, such as blood, that consist of nucleic acids in the form of deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), which can be amplified via PCR, as described herein below.

[0094] 1 illustrates a system 100 for magneto-electrophoretic separation to purify nucleic acids. This example utilizes a microfluidic chip 102 that includes a first well or reservoir 104, a microchannel 106, and a second well or reservoir 108. While the first and second wells 104, 108 are illustrated as square, it will be understood by those skilled in the art that they may comprise virtually any desired shape, such as rounded or oval.

[0095] In one embodiment, a first well 104 having a diameter of approximately 2 mm may be provided. Additionally, in one embodiment, the microchannel 106 may be approximately 2-3 cm in length, approximately 100 μm deep, and approximately 50-200 μm in width.

[0096] 2 and 3, a first well 104 is illustrated with a biological sample therein, consisting of both target molecules 110 as well as contaminants 112. Note that the biological sample may consist of blood, and the first well 104 is provided with a fluid or buffer into which the biological sample is placed.

[0097] Magnetic beads 114, such as M-PVA magnetic beads (FIG. 3), are then introduced into the first well 104. The magnetic beads 114 are conjugated to collect target molecules, such as nucleic acids, from a complex sample, such as blood. As shown in FIG. 3, the target molecules 110 are attracted to the magnetic beads 114, while the contaminants 112 are not. The result is that the target molecules 110 cluster around individual magnetic beads 114, as shown.

[0098] In some embodiments, the biological sample can be gently magnetically stirred, for example, when magnetic beads are placed into the biological sample to ensure mixing of the sample for proper attachment of target molecules. It is also contemplated that the biological sample can be locally heated.

[0099] 4 and 5 illustrate one embodiment of a system 100a that includes the use of an electrical energy source 116a controlled by a controller 118a. The controller 118a may be any type of computer that is programmed to control the voltage source 116a as desired.

[0100] It is also contemplated that controller 118a may also control magnetic stirrer 117a and heater 119a. The heater may also comprise, for example, a resistive metal coating. The resistive metal coating may be indium tin oxide (ITO) lining the well or reservoir. While heater 119a is shown on the outside of the well, it is understood that the coating may line the inside or outside of the well. Similarly, controller 118a may provide direct control over the metal coating, or an intermediate controller adapted to apply a 0-12 volt signal to the heater may be provided. Additionally, it is understood that temperature sensor 121a may be provided to provide feedback information and maintain the temperature at a set point. While various parts and components are illustrated with connecting lines to indicate connections, it is understood that these are merely schematic and that the connections may comprise wired or wireless connections.

[0101] 4 is a first probe 120a configured to have a positive charge and a second probe 122a configured to have a negative charge. The magnitude of the voltage difference developed between the first and second probes 120a, 122a depends on the power source.

[0102] 5, an electric field 124a is generated between the first and second probes 120a, 122a. The electric field is illustrated with field lines (shown as dashed lines) that interact with the negatively charged contaminants 112a. The electric field 124a is generated such that it interacts relatively strongly with the negatively charged contaminants 112a, but does not essentially interact with the magnetic beads 114a or the target molecules 110a.

[0103] 6 illustrates the introduction of a magnet 126a introduced near the first well 104a. The magnet 126a is designed to attract the magnetic beads 114a. In one example, the magnet 126a may be positioned at one end of the first well 104a and moved across the first well 104a toward the microchannel 106a such that the magnetic beads 114a and associated target molecules 110a are moved toward the microchannel 106a. However, it will be understood by those skilled in the art that the electric field 124a will function to hold the negatively charged contaminants 112a in place due to their interaction with the electric field 124a.

[0104] 6, the magnet 126a and magnetic beads 114a are moved toward the microchannel 106a, while most of the contaminants 112a are left behind in the first well 104a. Note, however, that a small number of the contaminants 112a may be attracted toward the microchannel along with the target molecules 110a.

[0105] Furthermore, it should be noted that the movement of the magnet 126a may be controlled by the controller 118a and may include fully automatic movement by software programming. The movement may be simply linear or any combination of complex movements that may be programmed.

[0106] 7 illustrates a magnet 126a moving into a microchannel 106a, along which magnetic beads 114a and target molecules 110a are being drawn. The microchannel 106a will be filled with a fluid (e.g., a buffer) such that the relatively high speed movement of the magnetic beads 114a through the fluid (e.g., on the millimeters per second scale) will cause any contaminants 112a drawn along with the target molecules 110a to be expelled within the microchannel. This is illustrated in FIG. 8, which shows a magnet 126a moving into a second well 108a through the opposite end of the microchannel.

[0107] At this point, any contaminants 112a that may have been attracted along with the target molecules 110a are left behind in the fluid within the microchannel 106a.

[0108] The magnet 126a may then be moved into the second well 108a in any motion or sequence of motions as desired and programmed into the controller 118a. The magnetic beads 114a may then be removed from the fluid in the second well 108a and placed into a separate container 128a (FIG. 9). The magnetic beads 114a may then be demagnetized so that the target molecules 110a are no longer attracted toward the magnetic beads 114a, which may then be removed from the container 128a.

[0109] The result is a container 128a containing a buffer solution and target molecules 110a essentially free of contaminants 112a. The process is simple and easy to perform, without requiring expensive or highly sophisticated equipment.

[0110] 4A, the system shown therein is substantially similar to and operates in a similar manner to the system shown in FIG. 4, except that rather than the first and second probes 120a, 122a being disposed within the first and second wells 104a, 108a, the first and second probes 120a, 122a are disposed within separate third and fourth wells 105a, 109a that also communicate with the microchannel 106a. With this configuration, the magnet 126a attracts material from the first well 104a to the second well 108a (as described above), and the presence of the third and fourth wells 105a, 109a with the probes 120a, 122a disposed therein generates an electric field 124a across the microchannel 106a traversed by the magnetic beads 114a.

[0111] Thus, in a manner similar to that discussed above, the microchannel 106a would be filled with a fluid (e.g., a buffer) such that the relatively high speed movement of the magnetic beads 114a through the fluid (e.g., on the millimeter / second scale) would cause any contaminants 112a attracted along with the target molecules 110a to be expelled within the microchannel 106a. Thus, similar to the previous example, the microchannel 106a provides relative motion of the liquid via bead motion and power via the applied electric field to purify the sample.

[0112] Turning now to FIG. 10, a flow diagram of process 200a is provided. First, a biological sample to be purified is deposited into a well (201a), which may comprise a lab-on-a-chip. Next, magnetic beads are introduced into the biological sample contained in the first well (202a). As described above, in one example, the magnetic beads can be M-PVA magnetic beads that are excited to attract target molecules. Still further, the target molecules can be nucleic acids. The magnetic beads are then maintained within the biological sample for a period of time (an "incubation" period) to allow for attraction of the target molecules. In one example, the period of time can be, for example, less than one minute.

[0113] The incubation cycle may be further supplemented with periodic mixing or stirring of the biological sample, which will further aid in the binding of target molecules. In one example, a magnetic stirrer may be used inside the well / reservoir for gentle mixing, for example, to increase the bead / molecule interaction rate and therefore reduce the overall time for the same binding efficiency.

[0114] One benefit of these process steps is that capture / binding of target molecules occurs within the wells / reservoirs, making the method uniquely suited for processing large volumes of sample for rapid separation. While previously known methods must employ multiple washes per sample, embodiments of the present method can remove bead-bound molecules from all excess molecules in a single step on a one-minute timescale. This allows for increased throughput capacity.

[0115] Once the selected time period has elapsed, an electric field can be applied to the biological sample (204a). This can be accomplished by application of leads coupled to a power source. It will be understood by those skilled in the art that application of the electric field will also generate a magnetic field. Contaminants within the biological sample will have a negative charge and will interact with the electric field, which acts to "hold" or maintain these contaminants within the electric field.

[0116] Once the contaminants are held within the electric field, a magnet is then brought into proximity with the magnetic beads (206a). The magnet will function to attract the magnetic beads such that as the magnet is moved into proximity with the first well, the magnetic beads will be drawn along with the movement of the magnet. The movement of the magnet can be fully automatic and can be moved in a pre-programmed manner.

[0117] The magnet may then be moved (208a) to attract the magnetic beads toward the microchannel connected to the first well. It should be understood that an electric field will now be applied such that the contaminants interact with the electric field to be held in place within the fluid in the first well while the magnetic beads are moving toward the microchannel. This effectively allows the magnetic beads with the attracted target molecules to move away from the majority of the contaminants.

[0118] The magnet is then moved such that the magnetic beads are drawn into and move through the microchannel (210a). This can be done at a relatively fast rate, such as on the millimeter / second scale. Because there is fluid (buffer) within the microchannel, movement of the magnetic beads through the fluid will act to flush out any unwanted contaminants that were unintentionally drawn along with the target molecules from the first well.

[0119] The magnet then continues to draw the magnetic beads into a second well located at the opposite end of the microchannel (212a). The result is a purified biological sample in which only the target molecule has been transferred into the second well on the lab-on-a-chip.

[0120] Another benefit of the process described above is that it allows for highly efficient separation without requiring the use of any membranes or pumps, which is advantageous for resource-limited settings.

[0121] From this point, the purified biological sample (e.g., target nucleic acid) can then be removed from the second well and placed into a container (214a), after which the magnetic beads can be demagnetized (216a) so that the target molecules become unbound from the magnetic beads. Finally, the magnetic beads can be removed from the container and discarded.

[0122] At this point, the purified target molecule is located in a separate container and is ready for downstream processing. Note that the step of removing the purified biological sample from the second well is optional. For example, the first well can be drained, and the purified sample in the second well can be ready for on-chip amplification / detection. Alternatively, the purified biological sample can be in a separate container and ready for off-chip amplification / detection.

[0123] 11 illustrates an alternative embodiment that includes several additional process steps if improved purification is desired. For example, rather than removing magnetic beads with associated target molecules in step 212a, it is contemplated that the system may include a second microchannel and a third well. In this example, a magnet is moved toward the second microchannel (218a) and through the second microchannel (220a). This movement can be performed relatively quickly, as discussed in connection with movement through the first microchannel. Similarly, movement of the magnet can be fully automatic, with the magnet being moved according to a preprogrammed software program.

[0124] The magnetic beads can then be moved into a third well located at the opposite end of the second microchannel (222a), and the magnetic beads can then be removed and placed into a new container (224a). As previously described, the magnetic beads can then be demagnetized and removed (226a).

[0125] In other examples, the fluid provided in the second well can be such that the volume provides a flow of fluid from the second well to the first well, which acts to carry any contaminants or unbound molecules into the first well. In examples utilizing a second microchannel and a third well, the fluid can be provided such that the volume provided in the third well provides a flow from the third well to the second well, as well as from the second well to the first well. This flow of fluid, combined with the relatively high speed movement of the magnetic beads through the microchannel, acts to remove even more unbound molecules that may be unintentionally drawn from the first well.

[0126] In other embodiments, the method may further provide localized heating of a biological sample to enable thermally driven processes such as PCR (polymerase chain reaction). This may be provided by applying 0-12 volts to an indium tin oxide (ITO) chip with a resistive metal coating. The heating may maintain a temperature localized to the sample without overheating other areas of the chip.

[0127] While various functions and methods are described and presented in a sequence of steps, it should be noted that the sequence is provided merely as an illustration of one advantageous embodiment and that it is not necessary to perform these functions in the specific order illustrated. Furthermore, it is contemplated that any of these steps may be moved relative to any of the other steps and / or combined. Additionally and still further, it is contemplated that, depending on the application, it may be advantageous to utilize all or any portion of the functions described herein.

[0128] 12, an alternative embodiment is illustrated for a lab-on-a-chip for magnetic electrophoretic separation to purify nucleic acids. This embodiment utilizes a microfluidic chip 302a that includes a first well or reservoir 304a, a first microchannel 306a, and a second well or reservoir 308a. The first microchannel 306a extends from the first well 304a to the second well 308a.

[0129] Additionally, this embodiment utilizes a second microchannel 310a and a third well or reservoir 312a. The second microchannel 310a extends from the second well 308a to the third well 312a. As previously described, the first, second, and third wells 304a, 308a, 312a are illustrated here as square-shaped, but it will be understood by those skilled in the art that they may comprise virtually any desired shape, such as rounded or oval.

[0130] Polyethylene glycol (PEG), polyethylene oxide (PEO), or polyoxyethylene (POE) refers to an oligomer or polymer of ethylene oxide. The structure of PEG is commonly represented as H-(0-CH2-CH2)n-OH. PEG is a liquid, referred to herein as a gel. Different forms of PEG are also available depending on the initiator used in the polymerization process. One common initiator is monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), abbreviated as mPEG. Low molecular weight PEG is also available as purer oligomers, referred to as monodisperse.

[0131] FIG. 13 illustrates one embodiment of system 100b, including controller 118b. Controller 118b may be any type of computer programmed to control equipment used in connection with microchips. It is contemplated that controller 118b may control magnetic stirrer 117b and heater 119b. The heater may comprise, for example, a resistive metal coating. The resistive metal coating may be indium tin oxide (ITO) lining the well or reservoir. While heater 119b is shown on the outside of the well, it should be understood that the coating may line the inside or outside of the well. Similarly, controller 118b may provide direct control over the metal coating, or an intermediate controller adapted to apply a 0-12 volt signal to the heater may be provided. Additionally, it should be understood that temperature sensor 121b may be provided to provide feedback information and maintain the temperature at a set point. While various parts and components are illustrated with connecting lines to indicate connections, it will be understood that these are schematic only and the connections may comprise wired or wireless connections.

[0132] 14 illustrates the introduction of a magnet 126b introduced near a first well 104b. The magnet 126b is designed to attract the magnetic beads 114b. In one example, the magnet 126b may be positioned at one end of the first well 104b and moved across the first well 104b toward the microchannel 106b such that the magnetic beads 114b and associated target molecules 110b are moved toward the microchannel 106b.

[0133] The magnet 126b and magnetic beads 114b are moved toward the microchannel 106b, leaving some of the contaminants 112b in the first well 104b. Note, however, that some of the contaminants 112b may be attracted toward the microchannel along with the target molecules 110b.

[0134] Furthermore, it should be noted that the movement of magnet 126b may be controlled by controller 118b and may include fully automatic movement by software programming. The movement may be simply linear or any combination of complex movements that may be programmed.

[0135] 15 illustrates a magnet 126b moving into a microchannel 106b, along which magnetic beads 114b and target molecules 110b are drawn. The microchannel 106b will be filled with a gel 107b (e.g., PEG), shown as a dashed line within the microchannel 106b. The negatively charged contaminants 112b interact with the gel 107b such that the contaminants 112b are held in place within the gel 107b as the magnetic beads 114b and target molecules 110b move forward through the microchannel 106b.

[0136] In one example, the movement of the magnetic beads 114b through the gel 107b is relatively fast (e.g., on the order of millimeters per second), which causes any contaminants 112b that are attracted along with the target molecules 110b to be expelled into the gel 107b within the microchannel 106b. This is illustrated in Figure 16, which shows a magnet 126b moving through the opposite end of the microchannel into a second well 108b.

[0137] At this point, at least a significant portion of the contaminants 112b that were attracted along with the target molecules 110b remain within the gel 107b in the microchannel 106b.

[0138] The magnet 126b may then be moved into the second well 108b in any motion or sequence of motions as desired and programmed into the controller 118b. The magnetic beads 114b may then be removed from the fluid in the second well 108b and placed into a separate container 128b (FIG. 17). The magnetic beads 114b may then be demagnetized so that the target molecules 110b are no longer attracted toward the magnetic beads 114b, which may then be removed from the container 128b.

[0139] The result is a container 128b containing a buffer solution and target molecules 110b essentially free of contaminants 112b. The process is simple and easy to perform, without requiring expensive or highly sophisticated equipment.

[0140] 18 illustrates a first well 104b, a microchannel 106b, and a second well 108b, all containing a gel 107b. Note that the gel 107b can be contained only in the microchannel. Alternatively, the gel 107b can be contained in both the microchannel 106b and the first well 104b. The idea is that the negatively charged contaminants 112b will interact with the gel 107b such that the contaminants are retained by the gel 107b, allowing the magnetic beads 114b, along with the target molecules 110b, to be moved away from and separated from the contaminants 112b.

[0141] Turning now to Figure 19, a flow diagram of process 200b is provided. First, a biological sample to be purified is deposited into a well (201b), which may comprise a lab-on-a-chip. Next, magnetic beads are introduced into the biological sample contained in the first well (202b). As described above, in one example, the magnetic beads can be M-PVA magnetic beads that are excited to attract target molecules. Still further, the target molecules can be nucleic acids. The magnetic beads are then maintained within the biological sample for a period of time (an "incubation" period) to allow for attraction of the target molecules. In one example, the period of time can be, for example, less than one minute.

[0142] The incubation period may be further supplemented with periodic mixing or stirring of the biological sample, which will further aid in the binding of target molecules. In one example, a magnetic stirrer may be used inside the well / reservoir for gentle mixing.

[0143] One of the benefits of these process steps is that the capture / binding of target molecules occurs within the wells / reservoirs so that the method uniquely processes large volumes of sample for rapid separation.

[0144] The next step is to place a magnet in proximity to the magnetic beads (206b). The magnet will function to attract the magnetic beads such that as the magnet is moved near the first well, the magnetic beads will be attracted along with the movement of the magnet. The movement of the magnet can be fully automatic and can be moved in a pre-programmed manner.

[0145] The magnet may then be moved (208b) to attract the magnetic beads toward the microchannel connected to the first well. The magnet is moved (210b) so that the magnetic beads are drawn into the microchannel. It should be understood that the microchannel is filled with a gel, such as PEG, which will function to interact (211b) with negatively charged contaminants. This interaction means that the magnet attracts the magnetic beads along the microchannel and thus through the gel, and the negatively charged contaminants are expelled into the gel within the microchannel. This serves to purify the sample as it moves through the microchannel.

[0146] It is further contemplated that movement of the magnetic beads through the microchannels can occur at relatively fast rates, e.g., on the millimeter / second scale. Movement of the magnetic beads through the gel will function to expel unwanted contaminants, not only due to PEG interacting with negatively charged contaminants, but also due to fluidic resistance that serves to expel contaminants attracted along with the target molecules from the first well.

[0147] The magnet then continues to draw the magnetic beads into a second well located at the opposite end of the microchannel (212b). The result is a purified biological sample in which only the target molecule has been transferred into the second well on the lab-on-a-chip.

[0148] Another benefit of the process described above is that it allows for highly efficient separation without requiring the use of any membranes or pumps, which is advantageous for resource-limited settings.

[0149] From this point, the purified biological sample (e.g., targeted nucleic acid) can then be removed from the second well and placed into container 214b, after which the magnetic beads can be demagnetized so that the target molecules become unbound from magnetic beads 216b. Finally, the magnetic beads can be removed from the container and discarded.

[0150] At this point, the purified target molecule is located in a separate container and is ready for downstream processing. Note that the step of removing the purified biological sample from the second well is optional. For example, the first well can be drained, and the purified sample in the second well can be ready for on-chip amplification / detection. Alternatively, the purified biological sample can be in a separate container and ready for off-chip amplification / detection.

[0151] 20 illustrates an optional additional process step when improved purification is desired. For example, rather than removing magnetic beads with associated target molecules in step 212b, it is contemplated that the system may include a second microchannel and a third well. In this example, a magnet is moved toward the second microchannel (218b) and through the second microchannel (220b). This movement can be performed relatively quickly, as discussed in connection with movement through the first microchannel. Similarly, movement of the magnet can be fully automatic, with the magnet being moved according to a pre-programmed software program.

[0152] The magnetic beads can then be moved into a third well located at the opposite end of the second microchannel (222b), and the magnetic beads can then be removed and placed into a new container (224b). As previously described, the magnetic beads can then be demagnetized and removed (226b).

[0153] In other examples, the fluid provided in the second well may comprise a gel, as illustrated in connection with FIG. 18. In one example, a gel can be provided such that a volume provides a flow of gel from the second well to the first well, which further functions to transport any contaminants or unbound molecules into the first well. In an example utilizing a second microchannel and a third well (FIG. 21), a gel can be provided such that a volume provided in the third well provides a flow of fluid from the third well to the second well, and from the second well to the first well. This flow of gel, combined with the direct interaction of the contaminants with the gel and the relatively high speed movement of the magnetic beads through the microchannel, functions to remove even more unbound molecules that may be drawn from the first well.

[0154] In other embodiments, the method may further provide localized heating of the biological sample. This may be provided, for example, by applying 0-12 volts to an indium tin oxide (ITO) with a resistive metal coating. The heating may maintain a temperature localized to the sample without overheating other areas of the chip.

[0155] While various functions and methods are described and presented in a sequence of steps, it should be noted that the sequence is provided merely as an illustration of one advantageous embodiment, and that it is not necessary to perform these functions in the specific order shown. Furthermore, it is contemplated that any of these steps may be moved relative to any of the other steps and / or combined. Additionally, and still further, it is contemplated that, depending on the application, it may be advantageous to utilize all or any portion of the functions described herein.

[0156] Referring now to Figure 21, an embodiment is illustrated for a lab-on-a-chip for the purification of nucleic acids employing a further optional enhanced purification process according to Figure 20. This embodiment utilizes a microfluidic chip 302b that includes a first well or reservoir 304b, a first microchannel 306b, and a second well or reservoir 308b. The first microchannel 306b extends from the first well 304b to the second well 308b.

[0157] Additionally, this embodiment utilizes a second microchannel 310b and a third well or reservoir 312b. The second microchannel 310b extends from the second well 308b to the third well 312b. As previously described, the first, second, and third wells 304b, 308b, 312b are illustrated here as square-shaped, but it will be understood by those skilled in the art that they may comprise virtually any desired shape, such as rounded or oval.

[0158] Gel 307b is minimally provided within first microchannel 306b. However, gel 307b is also illustrated as optionally provided within second microchannel 310b. Furthermore, it should be understood that gel may be provided within any of first, second, or third wells 304b, 308b, 312b, as desired. Similarly, the volume and placement of gel 307b can be selected to generate a flow rate toward first well 304b, as desired.

[0159] Figure 22 illustrates rear, top, and left side perspective views of a microfluidic system 400 including its housing 402 for processing biological material. Figure 23 illustrates front, bottom, and right side perspective views of the housing 402. Figure 24 illustrates a left side view of the housing 402. Figure 25 illustrates a right side view of the housing 402. Figure 26 illustrates a top plan view of the housing 402. Figure 27 illustrates a bottom plan view of the housing 402. Figure 28 illustrates a rear view of the housing 402. Figure 29 illustrates a front view of the housing 402.

[0160] As used herein, terms such as "front," "forward," "rear," "back," "behind," and other similar terminology, when used in the context of microfluidic system 400, are used with respect to a viewer positioned to the side of system 400, where the viewer is typically expected to interact with and operate system 400. Thus, in some cases, "front," "forward," and other similar terms refer to features positioned in the direction of such a viewer, while terms such as "rear," "rear," "behind," and other similar terms refer to features positioned in the opposite direction. As used herein, relative height terms such as "top," "bottom," "upper," "lower," "above," "below," "upper," and "below," when used in the context of microfluidic system 400, are used in their ordinary sense, i.e., relative to the direction of gravity, as gravity pulls objects downward. As used herein, terms such as "right" and "left," when used in the context of microfluidic system 400, refer to locations as viewed toward the front of microfluidic system 400.

[0161] 22-29 illustrate that the housing 402 of the microfluidic system 400 includes a bottom portion or plate 404 that spans across the entire bottom surface of the housing 402 and provides a base or foundation to which various other components of the system 400 and the housing 402 can be coupled. The housing 402 also includes a rear portion 406 that is rigidly coupled to the bottom plate 404 and may be integrally formed therewith. The rear portion 406 spans across the entire rear surface of the housing 402 except for openings formed therein, and across rear portions of the top, left, and right sides of the housing 402. The housing 402 also includes a panel 408 that is removably disposed and coupled to the rear portion 406 to cover the openings formed therein. The panel 408 includes slots 410 formed therein to allow air to flow in and out of the housing 402 and ports 412 formed therein to allow wires or cables, such as for carrying communication and / or power, to extend in and out of the housing 402. In some cases, panel 408 may also be removed from the remainder of housing 402 to allow an operator or technician to access components inside housing 402, such as for inspection or repair, through an opening formed in rear portion 406.

[0162] The housing 402 also includes a front portion 414 that is hingedly or otherwise rotatably coupled to the rear portion 406 such that the front portion 414 can be rotated away from the rear portion 406 to open the housing 402 and allow an operator or technician to interact with the internal components of the microfluidic system 400. The front portion 414 spans across the entire front of the housing 402 and across front portions of the top, left, and right sides of the housing 402. A bottom end or edge of the front portion 414 can abut against the outer edge of the bottom plate 404, and a rear edge of the front portion 414 can abut against a front edge of the rear portion 406 when the front portion 414 is in its closed position. In some implementations, the front portion 414 is rotatably coupled to the top of the rear portion 406, such as by one or more hinges, such that the front portion 414 can rotate about a horizontal axis that extends upward along the top surface of the housing 402 and away from the rest of the system 400 to provide access to the remaining components inside the housing 402. In other implementations, the front portion 414 is rotatably coupled to the left or right side of the rear portion 406, such as by one or more hinges, such that the front portion 414 can rotate about a vertical axis that extends laterally outward along the left or right surface of the housing 402 and away from the rest of the system 400 to provide access to the remaining components inside the housing 402.

[0163] The housing 402 also includes a single external physical button 416 that may allow an operator or technician to manually interact with the microfluidic system 400. In some implementations, the operator may open the housing 402 by moving its front portion 414, dispense a biological sample and / or other substance into a well or set of microwells inside the housing 402, close the housing 402 by moving its front portion 414, and then press or push the button 416 to begin operation of the microfluidic system 400 and processing of the biological sample or other substance therein. In some implementations, the operator or technician may also press the button 416 to stop or interrupt operation of the microfluidic system 400 prior to completion of processing, for example, in the event of an emergency or other unforeseen situation or event.

[0164] 30-34 illustrate various perspective views of a microfluidic system 400 with its housing 402 removed to show its internal components. As illustrated in FIGS. 30-34, the microfluidic system 400 includes a microfluidic chip or plate 418 within which biological samples or other materials can be processed. The microfluidic system 400 also includes a microwell plate 420 having a plurality of microwells and a recess, slot, or cavity for receiving the microfluidic chip 418 such that the microwell plate 420 can carry the microfluidic chip 418. The microfluidic system 400 also includes a carriage or tray 422 to which the microwell plate 420 can be secured or coupled. The microfluidic system 400 also includes a horizontal actuation system 424 to which the tray 422 can be coupled. In operation, the horizontal actuation system 424 can move the tray 422, and therewith the microwell plate 420 and microfluidic chip 418, back and forth and side to side in a horizontal direction.

[0165] As illustrated in FIGS. 30-34 , the microfluidic chip 418 includes a plurality of wells 418 a extending from its top surface vertically through the thickness of the chip 418 to its bottom surface. FIG. 35 illustrates a perspective view of the back or bottom surface of the microfluidic chip 418. As illustrated in FIG. 35 , the microfluidic chip 418 includes a plurality of microchannels 418 b and other features formed on its bottom surface. The microchannels 418 b and other features are interconnected with each other and with the wells 418 a to form a plurality of chambers and pathways extending between the wells 418 a. When the system 400 is in use, the microchannels 418 b and other features formed on the bottom surface of the chip 418 can direct a fluid, biological sample, or other substance along a pathway, such as from a first one of the wells 418 a to a second one of the wells 418 a, such as in accordance with any of the embodiments of the microfluidic system described herein.

[0166] FIG. 36 illustrates a portion of the microfluidic system 400 with the microfluidic chip 418 removed to illustrate additional features of the microwell plate 420. For example, FIG. 36 illustrates that the microwell plate 420 includes a cavity 426 on its right side and a set of conductive tracks or leads 428 that extend horizontally across the bottom edge of the cavity 426 from its front end to its rear end. FIG. 37 illustrates a rear perspective view of the microwell plate 420 isolated from the rest of the microfluidic system 400. As illustrated in FIG. 37, the microwell plate 420 includes the cavity 426 on its right side and an array of multiple microwells 430 on its left side.

[0167] Cavity 426 has a geometric shape comprising a regular rectangular prism and has dimensions, including a vertical depth, horizontal length, and horizontal width, that match, are identical to, or are the same as corresponding dimensions of microfluidic chip 418. As shown in FIG. 37 , the front end of cavity 426 is set rearward relative to the front end of plate 420, the right end of cavity 426 is set inward relative to the right end of plate 420, the rear end of cavity 426 extends to and coincides with the rear end of plate 420, and the left end of cavity 426 is located between one-third and one-half of the distance across the width of plate 420 from the right end of the plate toward the left end of the plate. Thus, the cavity forms a slot or enlarged groove that extends into plate 420 from its rear end toward its front end. In use, the microfluidic chip 418 can be positioned within the cavity 426 so that the bottom surface of the cavity 426 faces the microfluidic channel 418b within the bottom surface of the chip 418, defining or forming the bottom or lower boundary of the chamber and the pathway formed by the microfluidic channel 418b.

[0168] FIG. 37 also illustrates that the plate 420 includes a plurality of channels 432 formed in the bottom end or surface of the cavity 426. In the illustrated implementation, the plate 420 includes four such channels 432. Each channel 432 includes a relatively narrow top portion that extends downward into the plate 420 from the floor or bottom surface of the cavity 426 to a larger, wider bottom portion. In the illustrated implementation, the top portion of each of the channels 432 is rectangular in cross section and the bottom portion of each of the channels 432 is square in cross section, although in alternative implementations, the features may have different shapes. Each of the channels 432 extends horizontally from front to back through the plate 420 from a location adjacent and set rearward from the front end of the plate 420 to the rear end of the plate 420.

[0169] An array of multiple microwells 430 may include any suitable number of individual microwells 430. In the illustrated implementation, the array of microwells 430 includes 112 microwells 430 arranged in 14 equally spaced rows extending from front to back and 8 equally spaced columns extending laterally across the plate 420. As illustrated in FIG. 37 , the front end of the array of microwells 430 is positioned rearward relative to the front edge of the plate 420, the left end of the array of microwells 430 is positioned inward relative to the left edge of the plate 420, and the rear end of the array of microwells 430 is positioned forward relative to the rear edge of the plate 420, with the left end of the array of microwells 430 located between two-thirds and one-half of the distance across the width of the plate 420 from the left edge of the plate 420 toward the right edge of the plate 420.

[0170] 37 also illustrates that plate 420 includes multiple arms or knobs or protrusions 434 that extend horizontally forward and rearward from the front and rear ends, respectively, of plate 420. In the illustrated implementation, plate 420 includes two protrusions 434 that extend rearward from the rear end of plate 420 and two protrusions 434 that extend forward from the front end of plate 420.

[0171] FIG. 38 illustrates a portion of a microfluidic system 400 with the microfluidic chip 418 and microwell plate 420 removed to illustrate the additional features of a tray 422 and conductive leads 428. For example, FIG. 38 illustrates that the tray 422 includes an angled bracket 440 having a first vertical leg portion 440a extending laterally and side-to-side and coupled to the horizontal actuation system 424, and a second horizontal leg portion 440b aligned at a right angle to the first vertical leg portion 440a. The tray 422 also includes a front rail 436 coupled to the top surface of the front end of the horizontal leg portion 440b of the bracket 440, and a rear rail 438 coupled to the top surface of the rear end of the horizontal leg portion 440b of the bracket 440. The front and rear rails 436, 438 are parallel and extend laterally and side-to-side along the top surface of the bracket 440.

[0172] 38 , the front and rear rails 436, 438 each include a plurality of recesses or grooves 442 configured to mate with protrusions on the plate 420 and lock or secure the plate 420 to the top surface of the bracket 440 between the front and rear rails 436 and 438. For example, the rear rail 438 includes two grooves 442 that extend rearward from its front surface partially into the rail 438. In the illustrated implementation, the grooves 442 in the rear rail 438 also each extend from its top end to its bottom end, then from left to right along the bottom end of the rail 438, into and down through the rail 438. As another example, the front rail 436 includes two grooves 442 that extend forward from its rear surface partially into the rail 436. In the illustrated implementation, the grooves 442 in the front rail 436 also extend downward into and through the rail 436, from its top end to its bottom end, and then from left to right along the bottom end of the rail 436, respectively.

[0173] To secure the plate 420 to the tray 422, the plate 420 is positioned over the tray 422 so that its protrusions 434 are aligned with the grooves 442. The plate 420 can then be lowered into position on the tray 422 between the front and rear rails 436, 438 as the protrusions 434 travel down through the grooves 442 until the bottom surface of the plate 420 rests on the upper surface of the horizontal legs 440b of the brackets 440 of the tray 422. The plate 420 is then moved to the right so that the protrusions 434 travel right through the grooves 442, thereby securing the plate 420 to the tray 422.

[0174] FIG. 38 also illustrates the location of the conductive tracks or leads 428 when the plate 420 and chip 418 are secured to the tray 422. Each of the conductive leads 428 includes a relatively narrow top portion that extends downward from the top of the lead 428 to its larger, wider bottom portion. The top portion of each of the conductive leads 428 is rectangular in cross section and has a size and shape corresponding to the size and shape of the top portion of the channel 432, while the bottom portion of each of the conductive leads 428 is square in cross section and has a size and shape corresponding to the size and shape of the bottom portion of the channel 432. Thus, each conductive lead 428 can be positioned closely within a respective one of the channels 432. Each of the conductive leads 428 extends horizontally from front to back between the front and rear rails 436 and 438, from a location adjacent to and set rearward from the rear end of the front rail 436 to the front end of the rear rail 438. In use, the conductive lead 428 will not typically be in the position illustrated in FIG. 38 without the plate 420 present, but the conductive lead 428 is illustrated in such position in FIG. 38 with the plate 420 removed for purposes of clarity and illustration.

[0175] 39 illustrates a portion of the microfluidic system 400 with the microfluidic chip 418, the microwell plate 420, and the conductive leads 428 removed to illustrate additional features of the tray 422. For example, FIG. 39 illustrates that the tray 422 includes a set of conductive terminals 444, which may include conductive balls, each mounted within a rear rail 438 and biased forward from the rear rail 438, such as by a spring within the rear rail 438. The terminals 444 can be electrically coupled to a control system configured to operate the microfluidic system 400 such that the control system can control the voltage and / or current supplied to each of the conductive leads 428.

[0176] 36 and 37 , conductive leads 428 extend through plate 420 and are exposed to cavities 426 within plate 420. Accordingly, conductive leads 428 may also be exposed to one or more of wells 418 a and / or one or more of microchannels 418 b of microfluidic chip 418. A control system can therefore be configured to control the voltage and / or current supplied to conductive leads 428, such as to control the processing of fluids, biological samples, and / or other substances within microfluidic chip 418, such as in accordance with any of the embodiments of microfluidic systems described herein.

[0177] 39 also illustrates that the microfluidic system 400 includes a rotational actuation system including a first actuator 446, which may include an electric motor, a servo motor, or any other suitable actuator capable of generating torque, such as from electrical power, as well as an angle bracket 448 and a magnet 450. The first actuator 446 may be firmly affixed to the top surface of the bottom plate 404 of the housing 402, for example, below the tray 422, below the plate 420 when the plate 420 is affixed to the tray 422, and at a location where the chip 418 is positioned within the cavity 426 in the plate 420 and below the chip 418 when the plate 420 is affixed to the tray 422.

[0178] The output or drive rod of a first actuator 446 is rigidly coupled to an angle bracket 448 that includes a first arm 448a and a second arm 448b perpendicular to the first arm 448a, such as the first arm 448a. The first actuator 446 can generate a torque that rotates the first arm 448a about a horizontal axis from a first position shown in FIG. 39 , where the first arm 448a extends to the right of the output of the actuator 446, to a second position, where the first arm 448a extends upward from the output of the actuator 446, where the first arm 448a extends to the left of the output of the actuator 446, to a third position, where the first arm 448a extends from front to back, and back again, or through some lesser portion of such range of progression.

[0179] The second arm 448b of the angle bracket 448 can be secured to the first arm 448a at a right angle so as to extend rearward. Thus, when the first arm 448 extends upward, the second arm 448b extends rearward directly above the actuator 446 and horizontally from front to back below the tray 422, below the plate 420 when the plate 420 is secured to the tray 422, with the chip 418 positioned within the cavity 426 in the plate 420, and below the chip 418 when the plate 420 is secured to the tray 422. The magnet 450 is secured to the second arm 448b of the angle bracket 448 so that when the angle bracket 448 extends upward from the actuator 446, the magnet 450 is above the second arm 448b and adjacent to the tray 422.

[0180] The rotational actuation system and its first actuator 446 can be electrically or otherwise communicatively coupled to a control system, which can be configured to operate the first actuator 446 such that the control system can control the rotation of the magnet 450 relative to the actuator 446, tray 422, plate 420, and chip 418 when the plate 420 and chip 418 are secured to the tray 422. Thus, the control system can be configured to control the movement of the magnet 450 as well as the magnetic field generated by the magnet 450, such as to control the processing of fluids, biological samples, and / or other substances within the microfluidic chip 418, such as according to any of the embodiments of the microfluidic system described herein.

[0181] FIG. 40 illustrates a perspective view of the microfluidic system 400 with the microfluidic chip 418, microwell plate 420, conductive leads 428, and tray 422 removed to show the other components, including the horizontal actuation system 424, in more detail. As illustrated in FIG. 40, the horizontal actuation system 424 includes a second actuator 452 that can generate torque, such as from an electric motor, a servo motor, or any other suitable second actuator 452. The horizontal actuation system 424 also includes an elongated guide rail 454 that is securely affixed and coupled to the second actuator 452 and extends laterally and to the right from the actuator 452. The second actuator 452 and guide rail 454 may be securely affixed to the top surface of the bottom plate 404 of the housing 402, for example, at a location below the tray 422.

[0182] The horizontal actuation system 424 also includes a threaded rod 456 coupled to the output or drive rod of the second actuator 452. The second actuator 452 can generate a torque that rotates the threaded rod 456 about its own central longitudinal axis, which is a horizontal axis that extends laterally from the second actuator 452 to the right and below the tray 422. The horizontal actuation system 424 also includes a travel block 458 that is secured to and mounted on the guide rail 454 so that the travel block 458 can travel linearly from side to side along the length of the guide rail 454. For example, the travel block 458 can include one or more grooves, such as with an undercut portion thereof, and the guide rail 454 can include one or more ridges having a shape corresponding to that of the grooves, such that the ridges can be positioned within the grooves to secure the travel block 458 to the guide rail 454. As another example, the guide rail 454 may include one or more grooves, such as with an undercut portion thereof, and the travel block 458 may include one or more ridges having a shape corresponding to that of the grooves, such that the ridges can be positioned within the grooves to secure the travel block 458 to the guide rail 454.

[0183] 40 , the threaded rod 456 extends through a conduit that extends through the progression block 458. In some implementations, the conduit extending through the progression block 458 is threaded, with threads on the conduit corresponding to the threads on the threaded rod 456, and the threads on the conduit may engage and interlock with the threads on the threaded rod 456. Thus, due to the engagement of these threads and the engagement of the progression block 458 with the guide rail 454, when the actuator 452 generates a torque and induces rotation of the threaded rod 456, the rotation of the threaded rod 456 induces linear movement of the progression block 458 laterally along the length of the guide rail 454. By pivoting the threaded rod 456 in a first direction, such as clockwise or counterclockwise, the threaded rod 456 can advance the progression block 458 in a first direction, such as right or left. By rotating the threaded rod 456 in a second direction opposite the first direction, such as clockwise or counterclockwise, the threaded rod 456 can advance the advancement block 458 in the second direction opposite the first direction, such as right or left.

[0184] 39 and 40 , the tray 422 can be securely coupled and affixed to the advancement block 458, such as by adhesive or a plurality of mechanical fasteners, such as screws or bolts 460. Thus, movement of the advancement block 458 can induce corresponding or matching movement of the tray 422, and thus the microwell plate 420 and microfluidic chip 418. Accordingly, the actuator 452 can be used as described herein to move the microwell plate 420 and microfluidic chip 418 laterally and side-to-side within the system 400. The horizontal actuation system 424 and its second actuator 452 can be electrically or otherwise communicatively coupled to a control system, which can be configured to operate the second actuator 452 such that the control system can control the horizontal and lateral movement of the microwell plate 420 and microfluidic chip 418 when the plate 420 and chip 418 are affixed to the tray 422.

[0185] FIG. 41 illustrates a perspective view of the microfluidic system 400 as illustrated in FIG. 40 with additional components removed to more clearly illustrate the other components, including the rotational actuation system and horizontal actuation system 424. As illustrated in FIG. 41, the microfluidic system 400 includes a vertical actuation system 462, a micropipette system 464, a cradle 466 enclosing a portion of the micropipette system 464, and a pump system 468 configured to control at least a portion of the operation of the micropipette system 464. As illustrated in FIG. 41, the vertical actuation system 462 includes a third actuator 470 that can generate torque, such as an electric motor, a servo motor, or any other suitable third actuator 470. The vertical actuation system 462 also includes an elongated guide rail 472 that is securely attached and coupled to the second actuator 470 and extends above and below the actuator 470, as well as upwardly from the actuator 470. The second actuator 470 may be, for example, firmly affixed to the top surface of the bottom plate 404 of the housing 402 at a location behind the horizontal actuation system 424 .

[0186] Vertical actuation system 462 also includes a threaded rod 474 coupled to the output or driving rod of a third actuator 470. Third actuator 470 can generate a torque that rotates threaded rod 474 about its own central longitudinal axis, which is a vertical axis extending up and down from third actuator 470. Vertical actuation system 462 also includes a travel block 476 that is secured to and mounted on guide rail 472 so that travel block 476 can travel linearly up and down along the length of guide rail 472. For example, travel block 476 may include one or more grooves, such as with an undercut portion thereof, and guide rail 472 may include one or more ridges having a shape corresponding to that of the grooves, such that the ridges can be positioned within the grooves to secure travel block 476 to guide rail 472. As another example, guide rail 472 may include one or more grooves, such as with an undercut portion thereof, and progression block 476 may include one or more ridges having a shape corresponding to that of the grooves, such that the ridges can be positioned within the grooves to secure progression block 476 to guide rail 472.

[0187] 41 , the threaded rod 474 extends through a conduit that extends through the progression block 476. In some implementations, the conduit extending through the progression block 476 is threaded, with threads on the conduit corresponding to the threads on the threaded rod 474, and the threads on the conduit may engage and interlock with the threads on the threaded rod 474. Thus, due to the engagement of these threads and the engagement of the progression block 476 with the guide rail 472, when the actuator 470 generates a torque and induces rotation of the threaded rod 474, the rotation of the threaded rod 474 induces linear movement of the progression block 476 along the length of the guide rail 472, up or down. By pivoting the threaded rod 474 in a first direction, such as clockwise or counterclockwise, the threaded rod 474 can advance the progression block 476 in a first direction, such as up or down. By rotating the threaded rod 474 in a second direction opposite the first direction, such as clockwise or counterclockwise, the threaded rod 474 can advance the advancement block 476 in a second direction opposite the first direction, such as up or down.

[0188] FIG. 42 illustrates components of vertical actuation system 462 with other components of system 400 removed for greater clarity. As shown in FIG. 42, travel block 476 includes a body coupled to guide rail 472 and threaded rod 474, and a bar or arm 478 extending forward from the body. As shown in FIG. 42, travel block 476 also includes a latch 480 rotatably coupled to an upper surface of arm 478. Latch 480 includes an enlarged paddle 480 a at its first, rear end and a tooth 480 b at its second, front end. Latch 480 can be biased, such as by a spring, to rotate paddle 480 a away from the upper surface of arm 478 and tooth 480 b toward the upper surface of arm 478. In operation, an operator or technician can press down on paddle 480a, overcoming the bias and rotating paddle 480a towards the top surface of arm 478 and rotating tooth 480ba away from the top surface of arm 478.

[0189] FIG. 43 illustrates a micropipette system 464, with other components of the system 400 removed for greater clarity. As illustrated in FIG. 43, the micropipette system 464 includes a plurality of individual micropipettes 482, each including an individual micropipette tip 482a coupled to an individual pipette or micropipette (referred to herein as a (micro)pipette) conduit 482b. As illustrated in FIG. 43, the micropipette system 464 also includes a horizontally extending support bar or arm 484 through which each micropipette 482, such as its micropipette tip 482a, extends. The support arm 484 can maintain the micropipette tips 482a in a vertical orientation and can hold the micropipette tips 482a in an array or grid having eight equally spaced rows extending laterally and two spaced columns extending front to back across the support arm 484.

[0190] 43, the support arm 484 is hollow and has an opening 486 at its rear end. The opening 486 is sized and dimensioned such that the arm 478 can be inserted into and through the opening 486 into the hollow support arm 484. The support arm 484 also includes a recess, indentation, or small groove 488 at its rear end above the opening 486 that extends downwardly into the top surface of the support arm 484. Thus, to position the micropipette system 464 on the vertical actuation system 462, an operator or technician depresses the paddle 480a of the latch 480, inserts the arm 478 into the opening 486 in the hollow support arm 484, and then releases the paddle 480a, allowing the tooth 480b of the latch 480 to move into and seat within the groove 488, thereby securing and locking the support arm 484 of the micropipette system 464 to the arm 478 of the vertical actuation system 462.

[0191] Thus, movement of the advance block 476 can induce a corresponding or matching movement of the support arm 484, and thus the micropipette tip 482a. Accordingly, the actuator 470 can be used as described herein to move the micropipette tip 482a up and down within the system 400. As illustrated in FIGS. 30-34 , the micropipette system 464 can be disposed within the system 400 such that the micropipette tip 482a is positioned directly above the tray 422 and / or directly above the array of wells 418a in the microfluidic chip 418 or the wells 430 in the microwell plate 430. The vertical actuation system 462 and its actuator 470 can be electrically or otherwise communicatively coupled to a control system, which can be configured to operate the actuator 470 such that the control system can control the vertical movement of the micropipette tip 482a when the micropipette system 464 is disposed within the system 400 and the support arm 484 is coupled to the arm 478.

[0192] 43 also illustrates that the micropipette system 464 includes a cartridge 490 that includes an outer frame 492, which may have an overall square or rectangular shape. As illustrated in FIG. 43, the outer frame 492 includes a male portion of a fluid connector 494, as well as multiple openings 496 through which protrusions or knobs of another component may be inserted to secure the cartridge 490 thereto. Each end of the (micro)pipette conduit 482b opposite the individual micropipette tip 482a extends into and through the cartridge 490 and terminates in a respective port in the male portion of the fluid connector 494.

[0193] 41 and 44, the pedestal 466, which may also be referred to as a "heating assembly," includes an arm or post or stand 498 upon which various other components of the pedestal 466 may be supported. The stand 498 of the pedestal 466 may be securely affixed to the rear end surface of the guide rails 454 of the horizontal actuation system 424, such as at locations behind the horizontal actuation system 424, including, as examples, behind the tray 422, behind the plate 420, and / or behind the tip 418. Also, as shown in FIGS. 41 and 44, the stand 498 can be securely coupled and secured to the guide rails 454, such as by adhesive or a number of mechanical fasteners, such as screws or bolts 500.

[0194] 41 and 44, the pedestal 466 includes a stationary plate or sidewall 502 extending upward and from front to back, and a hinge component 504 located at the bottom end of the sidewall 502 and extending toward and facing the right of the sidewall 502. The pedestal 466 also includes a heat transfer block 506 including 16 individual grooves or channels 508 extending horizontally and from front to back therethrough, each of the channels 508 having a semicircular cross-sectional shape. The pedestal 466 also includes a pair of bars 510 extending to the right from the right-facing surface of the sidewall 502 and extending vertically up and down through the right-facing surface of the sidewall 502 along their own respective central longitudinal axes.

[0195] As also shown in FIGS. 41 and 44 , the pedestal 466 includes a plurality of solenoid actuators 512 coupled to the bar 510 and configured to actuate the bar 510 to move laterally and left and right relative to the sidewall 502, and in and out of the right-facing surface of the sidewall 502. As also shown in FIGS. 41 and 44 , the pedestal 466 includes a heat sink 514 with tines or teeth facing leftward, away from the heat transfer block 506. The pedestal 466 may also include one or more heating systems, which may be integrated with the heat transfer block 506, integrated with the heat sink 514, or positioned between the heat transfer block 506 and the heat sink 514. In some implementations, the heating system may include a heat pump, such as a solid-state heat pump or a thermoelectric heat pump, a Peltier device, a Peltier heater, or a Peltier heat pump. The heat transfer block 506 may be made of copper or other highly thermally conductive material.

[0196] 41 and 45 illustrate perspective views of the hinged door or hinged sidewall 516 of the pedestal 466. As shown in FIGS. 41 and 45, the hinged sidewall 516 extends upwardly and from front to back and includes a hinge component 518 located at the bottom end of the sidewall 502 and extending toward and facing the left of the sidewall 502. The component 518 can be coupled with the component 504 to form a complete hinge such that the hinged sidewall 516 can rotate to the left and inward toward the stationary sidewall 502 or to the right and outward away from the stationary sidewall 502 about a horizontal axis extending front to back through the hinge components 504 and 518. The hinged sidewall 516 also includes 16 individual grooves or channels 520 extending horizontally and from front to back therethrough, each of the channels 520 having a semicircular cross-sectional shape.

[0197] 45, hinged sidewall 516 includes a plurality of protrusions or knobs or PEGs 522 having a shape corresponding to that of openings 496 such that knobs 522 can be inserted into openings 496 to secure cartridge 490 to hinged sidewall 516. Also, as shown in FIG. 45, hinged sidewall 516 includes a female portion of fluid connector 524 that provides a plurality of ports, each of which can be fluidly coupled to a respective one of the ports of the male portion of fluid connector 494. As shown in FIG. 41, micropipette system 464 can be disposed within system 400 such that cartridge 490 is positioned within cradle 466 and between stationary sidewall 502 and hinged sidewall 516.

[0198] FIG. 46 illustrates pump system 468 with other components of system 400 removed for greater clarity. Pump system 468 may be a hydraulic or pneumatic pump system and may also be referred to as an “air handling system.” As shown in FIG. 46 , pump system 468 includes a stationary frame 526 to which the other components of pump system 468 may be coupled. Frame 526 may be firmly affixed to the top surface of bottom plate 404 of housing 402, for example, at a location behind horizontal actuation system 424. As shown in FIG. 46 , pump system 468 also includes an electric motor, servo motor, or any other suitable fourth actuator 528 capable of generating torque, such as from electrical power. Pump system 468 also includes a threaded rod 530 coupled to the output or drive rod of fourth actuator 528. The fourth actuator 528 can generate a torque that rotates the threaded rod 530 about its own central longitudinal axis, which is a vertical axis that extends up, down, and upward from the fourth actuator 528.

[0199] Pump system 468 also includes a travel block or plate 532 secured to and mounted on frame 526 such that travel block 532 can travel linearly up and down along a portion of frame 526. For example, travel plate 532 may include one or more holes or openings therein, and frame 526 may include one or more posts or columns having a cross-sectional shape corresponding to that of the openings, such that the columns can be positioned within the openings to secure travel plate 532 to frame 526. Threaded rod 530 extends through a conduit or opening that extends through travel plate 532. In some implementations, the conduit extending through travel plate 532 is threaded, with conduit threads corresponding to the threads of threaded rod 530, and the conduit threads may engage and interlock with the threads of threaded rod 530.

[0200] Thus, due to the engagement of these threads and the engagement of advancement plate 532 with frame 526, when actuator 528 generates torque and induces rotation of threaded rod 530, the rotation of threaded rod 530 induces linear movement of advancement plate 532 up or down along the height of the column of frame 526. By turning threaded rod 530 in a first direction, such as clockwise or counterclockwise, threaded rod 530 can advance advancement plate 532 in the first direction, such as up or down. By turning threaded rod 530 in a second direction opposite the first direction, such as clockwise or counterclockwise, threaded rod 530 can advance advancement plate 532 in the second direction opposite the first direction, such as up or down.

[0201] 46 , pump system 468 also includes multiple (e.g., eight) syringe pumps 534, each including a respective pump barrel 534 a and a respective pump plunger 534 b extending into the respective pump barrel 534 a. Thus, pump system 468 can also be referred to as an “eight-channel syringe pump.” Each pump plunger 534 b is coupled to an advancement plate 532 such that movement of advancement plate 532 upward pulls the pump plunger upward, retracting pump plunger 534 b out of the respective pump barrel 534 a, and such that movement of advancement plate 532 downward pushes the pump plunger downward, extending pump plunger 534 b into the respective pump barrel 534 a. FIG. 46 also illustrates that pump system 468 includes a separate pair of conduits 536 for each syringe pump 534, with the inlet of each pair of conduits 536 fluidly coupled to the outlet of a respective one of syringe pumps 534, such as by a two-way selector valve, and the outlet of each of conduits 536 fluidly coupled to a respective one of the ports on the female portion of fluid connector 524.

[0202] To operate the microfluidic system 400, an operator or technician can approach the system 400 and open the housing 402 by rotating the front portion 414 of the housing 402 away from the rest of the housing 402. The technician can then arrange the microfluidic chip 418 and microwell plate 420b by positioning the microfluidic chip 418 into the cavity 426 in the microwell plate 420 and then securing the microwell plate 420 to the tray 422 by sliding the protrusions 434 downward and horizontally along and through the grooves 442. The technician can then load biological samples and PCR reagents and other substances into the array of microwells 430 of the microwell plate 420 and / or into the wells 418a of the microfluidic chip 418, depending on the processing to be performed. In some implementations, these substances may include substances to facilitate qPCR, gel electrophoresis, or any of the other processing techniques described herein. In some specific implementations, these substances may include RNA polymerase or DNA polymerase, and may include any of the various DNA polymerases from thermophilic organisms used in PCR, which may also be referred to as "TAQ polymerase."

[0203] The technician can then install the micropipette system 464. Installing the micropipette system 464 can include rotating the hinged sidewall 516 of the cradle 466 outwardly away from the stationary sidewall 502, then securing the cartridge 490 to the hinged sidewall 516 by inserting the knob 522 of the sidewall 502 into the opening 496 in the cartridge 490 and securing the male portion of the fluid connector 494, including its fluid port, into the female portion of the fluid connector 524, including its fluid port, and then rotating the hinged sidewall 516 of the cradle 466 inwardly toward the stationary sidewall 502, and securing the cartridge between the stationary sidewall 502 and the hinged sidewall 516 such that the (micro)pipette conduit 482b is received between the groove 508 in the heat transfer block 506 and the groove 520 in the hinged sidewall 516. The step of disposing the micropipette system 464 may also include the steps of depressing the paddle 480a of the latch 480 downward, inserting the arm 478 into the opening 486 in the hollow support arm 484, and then releasing the paddle 480a, allowing the tooth 480b of the latch 480 to move into and seat in the groove 488, thereby securing and locking the support arm 484 of the micropipette system 464 to the arm 478 of the vertical actuation system 462.

[0204] Once these actions are completed, the technician can close the system 400 by rotating the front portion 414 of the housing 402 toward the rest of the housing 402. The technician can then precisely and once, etc., press or push the button 416 to begin operation of the microfluidic system 400 and processing of the biological sample or other material therein. In some implementations, the operator or technician may also press the button 416 to stop or suspend operation of the microfluidic system 400 prior to completion of processing, for example, in the event of an emergency or other unforeseen situation or event.

[0205] Once a technician presses button 416 to initiate operation of microfluidic system 400, microfluidic system 400 can automatically control the movement and operation of components therein to process materials in a prescribed manner. For example, in some implementations, system 400 can use horizontal actuation system 424 to move tray 422 horizontally until micropipette tip 482a is positioned directly above a microwell 430 in microwell plate 420 containing a desired material. System 400 can then use vertical actuation system 462 to move micropipette tip 482a downward until it is positioned within the desired material in microwell 430 of microwell plate 420. System 400 can then use fourth actuator 528 to drive syringe pump 534 to draw the desired material into (micro)pipette conduit 482b. Generally, such operation of fourth actuator 528 drives each of eight syringe pumps 534 in unison.

[0206] The system 400 can then use the vertical actuation system 462 to move the micropipette tip 482a upward until it is positioned above the microwell plate 420. The system 400 can then use the horizontal actuation system 424 to move the tray 422 horizontally until the micropipette tip 482a is positioned directly above the well 418a in the microfluidic chip 418 where material processing will begin. The system 400 can then use the vertical actuation system 462 to move the micropipette tip 482a downward until it is positioned within the desired well 418a in the microfluidic chip 418. The system 400 can then use the fourth actuator 528 to drive the syringe pump 534 to expel the material from the (micro)pipette conduit 482b into the well 418a. This process can be repeated to transfer as many materials as desired from the microwells 430 of the microwell plate 420 into the wells 418a in the microfluidic chip 418.

[0207] Once materials are thus delivered to wells 418a, the materials may undergo various processing steps within microfluidic chip 418, such as to separate or remove contaminants from such materials, such as in accordance with descriptions of such processing elsewhere herein. During such processing, conductive leads 428 may be energized to generate an electric field, and / or magnet 450 may be moved to provide a magnetic field that interacts with the materials being processed, thereby affecting their behavior within microfluidic chip 418 and assisting in the processing of these materials within microfluidic chip 418. Once such processing is complete, quality control checks may be performed to ensure that sufficient material is available for further processing, such as for use in PCR processing. Such quality control checks may be performed inside or outside of system 400.

[0208] The system 400 can then use the horizontal actuation system 424 to move the tray 422 horizontally until the micropipette tip 482a is positioned directly above the well 418a in the microfluidic chip 418 containing the desired substance. The system 400 can then use the vertical actuation system 462 to move the micropipette tip 482a downward until it is positioned within the desired substance in the well 418a of the microfluidic chip 418. The system 400 can then use the fourth actuator 528 to drive the syringe pump 534 to draw up the desired substance into the (micro)pipette conduit 482b.

[0209] System 400 can then use vertical actuation system 462 to move micropipette tip 482a upward until it is positioned above microfluidic chip 418. System 400 can then use horizontal actuation system 424 to move tray 422 horizontally until micropipette tip 482a is positioned directly above a microwell 430 in microwell plate 420 where additional desired substances, such as PCR reagents, will be located. System 400 can then use vertical actuation system 462 to move micropipette tip 482a downward until it is positioned within the desired microwell 430 in microwell plate 420. The system 400 can then use the fourth actuator 528 to drive the syringe pump 534 to draw desired substances, such as PCR reagents, up into the (micro)pipette conduit 482b, etc., until the desired substances are mixed with each other inside the (micro)pipette conduit 482b and are located within the portion of the (micro)pipette conduit 482b inside the cartridge 490.

[0210] Once the desired substance is located within the portion of the (micro)pipette conduit 482b inside the cartridge 490, a solenoid actuator can be used to move the bar 510 to the right, out of the right-facing surface of the side wall 502, and toward the hinged side wall 516, until the bar 510 pinches each of the (micro)pipette conduits 482b in two places, preventing any substance from escaping from the portion of the (micro)pipette conduit 482b located inside the cartridge 490. A heater in the cradle 466 can then be used to heat the substance held within the portion of the (micro)pipette conduit 482b inside the cartridge 490 to promote a chemical reaction or other processing step therein. In some cases, this involves using a heater to generate a constant heat flow to the (micro)pipette conduit 482b, while in other cases, it involves cycling the heater to provide a cyclical heat flow to the (micro)pipette conduit 482b. In some implementations, the (micro)pipette conduit 482b is made from a thermally conductive plastic to more effectively conduct heat to the material.

[0211] As an example, heat can promote a PCR reaction occurring within the portion of the (micro)pipette conduit 482b inside the cartridge 490. Once this process is complete, the heater in the cradle 466 can be turned off to stop heating the material held within the portion of the (micro)pipette conduit 482b inside the cartridge 490, and a solenoid actuator can be used to move the bar 510 left into the right-facing surface of the side wall 502 and away from the hinged side wall 516 until the bar 510 no longer pinches the (micro)pipette conduit 482b. Once such a process is complete, a quality control check may be performed to confirm that the process, such as a PCR process, was successful or met certain performance standards. Such a quality control check may be performed inside or outside the system 400.

[0212] The system 400 can then use the horizontal actuation system 424 to move the tray 422 horizontally until the micropipette tip 482a is positioned directly above a well 430 in the microfluidic plate 420. The system 400 can then use the vertical actuation system 462 to move the micropipette tip 482a downward until it is positioned within the desired well 430 in the microwell plate 430. The system 400 can then use the fourth actuator 528 to drive the syringe pump 534 to expel the substance from the (micro)pipette conduit 482b into the well 430.

[0213] Once such processing is complete, the technician can open the housing 402 by rotating the front portion 414 away from the remainder of the housing 402. The technician can then remove the micropipette system 464. Removing the micropipette system 464 can include rotating the hinged sidewall 516 of the cradle 466 outwardly away from the stationary sidewall 502, and then removing the cartridge 490 from the hinged sidewall 516 by moving the knob 522 of the sidewall 502 out of the opening 496 in the cartridge 490 and by removing the male portion of the fluid connector 494, including its fluid ports, from the female portion of the fluid connector 524, including its fluid ports. Removing the micropipette system 464 can also include depressing the paddle 480a of the latch 480, removing the arm 478 from the opening 486 and the hollow support arm 484, and then releasing the paddle 480a.

[0214] The technician can also remove the microfluidic chip 418 and microwell plate 420 from the tray 422 by sliding the protrusions 434 horizontally and then upward along and through the grooves 442. The technician can then remove the processed materials from the wells 430 in the microwell plate 420. Once these materials have been removed and stored elsewhere, the micropipette system 464, microwell plate 420, and microfluidic chip 418 can be discarded as waste. Subsequent processing can use a new micropipette system 464, a new microwell plate 420, and a new microfluidic chip 418. In some implementations, the microfluidic chip 418, the microwell plate 420, and / or any other components of the system 400 described herein may include RFID chips or tags to help identify specific components and track their location within a larger collection of such components.

[0215] The processes described herein proceed by transferring material from the microwell plate 430 to the microfluidic chip 418, then from the microfluidic chip 418 to the cradle 466, and then from the cradle 466 back to the microwell plate 430. Such processes may serve to remove contaminants from a biological sample and its desired components, such as DNA, RNA, mRNA, or various proteins, including various amino acid-based proteins, and then perform PCR on the biological sample. However, in alternative implementations, the processes may proceed by transferring material from any component to any other component any number of times, depending on the actions required by the desired process. In one example of an alternative implementation, the processes described herein may proceed by transferring material from the microwell plate 430 to the cradle 466, then from the cradle 466 to the microfluidic chip 418, and then from the microfluidic chip 418 back to the microwell plate 420. Such treatments can serve to perform PCR on a biological sample and its desired components, such as DNA, RNA, mRNA, or various proteins, including various amino acid-based proteins, and then remove contaminants from the biological sample.

[0216] 47-55 illustrate components of another system, including another pipette or micropipette (herein referred to as (micro)pipette) system 600 and another cradle 700, that may be used with the microfluidic system 400 of FIG. 22 or in combination with any of the other features, components, systems, and / or methods described herein. In particular, FIG. 47 illustrates front, left, and top perspective views of the (micro)pipette system 600 and cradle 700, and FIG. 48 illustrates rear, right, and bottom perspective views of the (micro)pipette system 600 and cradle 700. As illustrated in FIGS. 47 and 48, the (micro)pipette system 600 includes a plurality of individual pipettes or micropipettes (herein referred to as (micro)pipettes) 602, each of which includes an individual (micro)pipette tip 602 a coupled to an individual (micro)pipette conduit 602 b. 47 and 48, the cradle 700 includes a plurality of fluid conduits 702. The (micro)pipette system 600 includes a cartridge 604 located inside the cradle 700 that includes fluidic components that fluidically couple the (micro)pipettes 602 to the fluid conduits 702.

[0217] In some implementations, (micro)pipette system 600 can include or be used in conjunction with any of the components or features described herein with respect to micropipette system 464 and can be used in the techniques, actions, or methods described herein with respect to micropipette system 464. In some implementations, (micro)pipette 602 can include or be used in conjunction with any of the components or features described herein with respect to micropipette 482 and can be used in the techniques, actions, or methods described herein with respect to micropipette 482. In some implementations, fluid conduit 702 can include or be used in conjunction with any of the components or features described herein with respect to conduit 536 and can be used in the techniques, actions, or methods described herein with respect to conduit 536. In some implementations, cartridge 604 can include or be used in conjunction with any of the components or features described herein with respect to cartridge 490 and can be used in the techniques, actions, or methods described herein with respect to cartridge 490.

[0218] 47 and 48, the exemplary (micro)pipette system 600 includes exactly 16 (micro)pipettes 602, and the cradle 700 includes exactly 8 fluid conduits 702. However, in different implementations, the (micro)pipette system 600 may include any suitable number of (micro)pipettes 602, and the cradle 700 may include any suitable number of fluid conduits 702. In some specific implementations, the number of (micro)pipettes 602 is twice the number of fluid conduits 702.

[0219] 47 and 48 also illustrate that cradle 700 includes a fixed or stationary rear portion, plate, or wall 704 and a movable and removable front portion, plate, wall, or door 706. As illustrated in FIGS. 47 and 48, rear portion 704 and front portion 706 extend generally parallel to one another and are adjacent to one another but spaced apart from one another so as to define a gap therebetween. In use, cartridge 604 is positioned and secured in the gap between rear portion 704 and front portion 706. Also, as shown in Figures 47 and 48, rear portion 704 includes a pair of guide rods or guide pins 708 extending therethrough forward from rear portion 704 toward front portion 706, which has a complementary pair of openings 710 configured to receive, such as to snugly receive, guide pins 708 to guide movement of front portion 706 toward and away from rear portion 704 (Figure 49).

[0220] In some implementations, cradle 700 can include or be used in conjunction with any of the components or features described herein with respect to cradle 466 and can be used in the techniques, actions, or methods described herein with respect to cradle 466. In some implementations, rear portion 704 can include or be used in conjunction with any of the components or features described herein with respect to stationary sidewall 502 and can be used in the techniques, actions, or methods described herein with respect to stationary sidewall 502. In some implementations, front portion 706 can include or be used in conjunction with any of the components or features described herein with respect to hinged sidewall 516 and can be used in the techniques, actions, or methods described herein with respect to hinged sidewall 516.

[0221] 47 and 48 , the rear portion 704 includes a top flange 712a extending forward from the top end of the rear portion 704, forward toward, above, and beyond the front portion 706, and a bottom flange 712b extending forward from the bottom end of the rear portion 704, forward toward, below, and beyond the front portion 706. The top flange 712a is connected to a vertically extending rod 714a extending downward from the bottom surface of the top flange 712a, and the bottom flange 712b is connected to a vertically extending rod 714b extending upward from the top surface of the bottom flange 712b. The front portion 706 includes a handle 716 supported thereon and hinged thereto for rotation about a vertical axis aligned with the vertically extending rods 714a, 714b. As shown in Figures 47 and 48, handle 716 includes an upper semicircular or semicylindrical coupling element 718a that engages with vertically extending rod 714a and a lower semicircular or semicylindrical coupling element 718b that engages with vertically extending rod 714b.

[0222] To assemble the cradle 700, the opening 710 (FIG. 49) in the front portion 706 can be moved along the guide rod 708 of the rear portion 704 toward the rear portion 704, and the handle 716 can then be rotated in a first direction, such as counterclockwise when viewed from above, to engage the coupling elements 718a, 718b with the vertically extending rods 714a, 714b and secure the front portion 706 to the rear portion 704. To disassemble the cradle 700, the handle 716 can be rotated in a second direction opposite the first, such as clockwise when viewed from above, to disengage the coupling elements 718a, 718b from the vertically extending rods 714a, 714b and release the front portion 706 from the rear portion 704, and the opening 710 (Figure 49) in the front portion 706 can then be moved away from the rear portion 704 along the guide rod 708 in the rear portion 704.

[0223] 47 and 48, pedestal 700 includes a first upper-left solenoid actuator 720a, a second lower-left solenoid actuator 720b, a third upper-right solenoid actuator 720c, and a fourth lower-right solenoid actuator 720d. Solenoid actuators 720a, 720b, 720c, and 720d are configured to operate valves of pedestal 700, as described in more detail elsewhere herein. As also shown in FIGS. 47 and 48, pedestal 700 includes a heat sink 722 with teeth or tines facing rearward, away from a rear portion 704 of pedestal 700.

[0224] FIG. 49 illustrates front, left, and top perspective views of the (micro)pipette system 600 and cradle 700 with the fluid conduit 702, solenoid actuator 720, heat sink 722, and handle 716 removed, and FIG. 50 illustrates rear, right, and bottom perspective views of the (micro)pipette system 600 and cradle 700 with the fluid conduit 702, solenoid actuator 720, heat sink 722, and handle 716 removed. FIG. 50 illustrates that the rear portion 704 includes a plurality of (e.g., eight) fluid ports 724 to which the fluid conduits 702 are connected and through which the fluid conduits 702 supply fluid to the cartridge 604 within the cradle 700. FIG. 50 also illustrates that the rear portion 704 includes a plurality of (e.g., four) openings or apertures through which a corresponding plurality of (e.g., four) valves 726 extend. Each of the valves 726 may include a bar that extends forward out from the front surface of the rear portion 704 and extends vertically and up and down through the rear portion 704 along its own respective longitudinal axis.

[0225] In use, the solenoid actuators 720 are each coupled to a respective one of the valves 726 and are configured to actuate the respective valve 726 to move forward or rearward through a respective opening in the rear portion 704 relative to the rear portion 704, the front portion 706, and the cartridge 604. In particular, the first upper-left solenoid actuator 720a is configured to actuate the first upper-left valve 726a, the second lower-left solenoid actuator 720b is configured to actuate the second lower-left valve 726b, the third upper-right solenoid actuator 720c is configured to actuate the third upper-right valve 726c, and the fourth lower-right solenoid actuator 720d is configured to actuate the fourth lower-right valve 726d.

[0226] FIG. 51 illustrates the (micro)pipette system 600 and cradle 700 as illustrated in FIG. 50 with the rear portion 704 of the cradle 700 removed. FIG. 51 illustrates a cartridge 604 including an outer frame 606 that holds multiple fluidic components together in place within the cradle 700. For example, (micro)pipette conduits 602b extend into the cartridge 604 and are retained and supported therein by the outer frame 606. As illustrated in FIG. 51 , the (micro)pipette conduits 602b are grouped into two groups 602b′ and 602b″ of equal numbers of (micro)pipette conduits, with the first of the two sets positioned above the second of the two sets. In particular, the 16 (micro)pipette conduits 602b are grouped into two groups of eight adjacent (micro)pipette conduits 602b.

[0227] As further illustrated in FIG. 51 , each of the (micro)pipette conduits 602b is exposed at two different locations, thereby providing four distinct sealing areas: a first upper left sealing area 608a, where the first of the two sets of (micro)pipette conduits 602b′ can be sealed by a valve 726a ( FIG. 50 ) adjacent to the (micro)pipette tip 602a; The seal areas 608 form a second, lower-left seal area 608b in which a first of two sets of (micro)pipette conduits 602b' can be sealed by a valve 726b (FIG. 50) adjacent to the fluid conduit 702, a third, upper-right seal area 608c in which a first of two sets of (micro)pipette conduits 602b' can be sealed by a valve 726c (FIG. 50) adjacent to the fluid conduit 702, and a fourth, lower-right seal area 608d in which a second of two sets of (micro)pipette conduits 602b'' can be sealed by a valve 726d (FIG. 50) adjacent to the fluid conduit 702. In use, the valves 726 can each engage with a (micro)pipette conduit 602b in a respective one of the seal areas 608 to seal the (micro)pipette conduit 602b therein.

[0228] 51 , the cartridge 604 includes a plurality (e.g., eight) three-way fluid valves or connectors 610. The three-way fluid connectors 610 are each coupled to a respective one of the first set of (micro)pipette conduits 602 b′, a respective one of the second set of (micro)pipette conduits 602 b″, and a respective feed conduit 612, such that a seal area 608 is located between the (micro)pipette tip 602 a and the fluid connector 610, such as mechanically and / or fluidically. In use, the feed conduits 612 are each fluidly coupled to one of the fluid ports 724. Thus, in use, fluid pressure, such as air pressure, provided by a pump system, such as pump system 468, can be directly and simultaneously supplied to one of the first set of (micro)pipette conduits 602 b′ and one of the second set of (micro)pipette conduits 602 b″. As further illustrated in FIG. 51, the (micro)pipette conduit 602b is exposed from the frame 606 within the heating zone 614, which is mechanically and / or fluidically located between the first and third sealing zones 608a, 608c, and mechanically and / or fluidically located between the second and fourth sealing zones 608b, 608d, respectively.

[0229] Figure 52 illustrates the components of Figure 51 with the (micro)pipette system 600 removed. As shown in Figure 52, the front portion 706 of the cradle 700 includes a first vertically extending protrusion, ridge, or bar 728 positioned and sized to engage the (micro)pipette conduit 602b in the first and second seal areas 608a, 608b, and a second vertically extending protrusion, ridge, or bar 730 positioned and sized to engage the (micro)pipette conduit 602b in the third and fourth seal areas 608c, 608d. In some implementations, when a solenoid actuator 720 is used to actuate movement of the valve 726 toward the front portion 706, the valve can be actuated to move directly toward the bars 728 and 730 to pinch the (micro)pipette conduit 602b between the valve 726 and the bars 728 and 730 in the respective seal areas 608a, 608b, 608c, and 608d. As also shown in FIG. 52 , the front portion 706 of the cradle 700 includes a first heater or heating element 732 that is thermally coupled to and / or positioned and sized to engage the (micro)pipette conduit 602b′ in the heated area 614, and a second heater 734 that is positioned and sized to engage the (micro)pipette conduit 602b″ in the heated area 614. In use, the heaters 732, 734 can be operated independently of each other to heat the (micro)pipette conduits 602b' and / or 602b'' within the heating zone 614 and any fluid therein.

[0230] Figure 53 illustrates the (micro)pipette system 600 and cradle 700 as illustrated in Figure 49, with the front portion 706 of the cradle 700 removed. Figure 53 illustrates the (micro)pipette 602, cartridge 604, outer frame 606, seal area 608, three-way fluid connector 610, feed conduit 612, and heating area 614. As illustrated in Figure 54, the first and third valves 726a, 726c are retracted and positioned rearward relative to the (micro)pipette conduits 602b' such that the first set of (micro)pipette conduits 602b' are not sealed within the first or third seal areas 608a, 608c. 54, the second and fourth valves 726b, 726d are extended forward and positioned relative to the (micro)pipette conduits 602b" such that the second set of (micro)pipette conduits 602b" are sealed within the second and fourth seal areas 608b, 608d. In use, the solenoid actuator 720 may be used to actuate the valves 726 to seal the (micro)pipette conduits 602b in any one, any two, any three, or all four of the seal areas 608.

[0231] FIG. 54 further illustrates the components of FIG. 53 with the (micro)pipette system 600 removed. As illustrated in FIG. 54, the rear portion 704 of the cradle 700 includes a support plate, which may also be a heat transfer block 736, including 16 individual grooves or channels extending horizontally and laterally therethrough, each having a semicircular cross-sectional shape configured to receive a respective one of the (micro)pipette conduits 602b. FIG. 55 illustrates the components of FIG. 54 with the valve 726 and heat transfer block 736 removed. As illustrated in FIG. 55, the rear portion 704 of the cradle 700 includes a first heater 738 positioned and sized to engage the heat transfer block 736 within the heating zone 614, and a second heater 740 positioned and sized to engage the heat transfer block 736 within the heating zone 614. In use, heaters 738, 740 are operated independently of each other to heat heat transfer block 736, thereby heating (micro)pipette conduits 602b' and / or 602b'' within heating zone 614 and any fluid therein.

[0232] A microfluidic system including the (micro)pipette system 600 and the cradle 700 may be used in combination with any of the other components, features, systems, methods, actions, or steps described herein with respect to the microfluidic system 400, etc. For example, in some implementations, such a system can use a solenoid actuator 720 to move the lower left valve 726b and the lower right valve 726d forward to clamp the (micro)pipette conduit 602b″ within the second and fourth seal areas 608b, 608d. The horizontal actuation system 424 can then move the tray 422 horizontally until the (micro)pipette tip 602a is positioned directly above a microwell containing a desired substance, such as nucleic acid and / or reagents for PCR. The system can then use the vertical actuation system 462 to move the (micro)pipette tip 602a downward until it is positioned within the desired substance in the microwell. The system can then use the fourth actuator 528 to drive the syringe pumps 534 to draw desired substances into the (micro)pipette conduits 602b′, such as until the desired substances mix with each other inside the (micro)pipette conduits 602b′ and are located within the portions 602b′ of the (micro)pipette conduits inside the cartridge 604 and within the heated zone 614. Generally, such operation of the fourth actuator 528 drives each of the eight syringe pumps 534 in unison, thereby drawing desired substances into the first upper set of (micro)pipette conduits 602b′ but not into the second lower set of (micro)pipette conduits 602b″ because they are sealed. The system can then use the vertical actuation system 462 to move the (micro)pipette tips 602a upward until they are located above the microwells.

[0233] Once the desired substance is located within the portion 602b' of the (micro)pipette conduit inside the cartridge 604 and within the heated zone 614, the solenoid actuator 720a can be used to move the first valve 726a outward until it pinches each of the (micro)pipette conduits 602b' within the first sealed zone 608a, preventing any substance from escaping therethrough. Once the (micro)pipette conduits 602b' are thus sealed within the first sealed zone 608a, the system can then use the fourth actuator 528 to drive the syringe pump 534 to apply a relatively high pressure to the desired substance within the (micro)pipette conduits 602b', such as to prevent such substance from evaporating or boiling. Such pressure may be 7 psi or 10 psi above atmospheric pressure (positive gauge pressure), or greater than 7 psi above atmospheric pressure, greater than 10 psi above atmospheric pressure, or between 7 psi and 10 psi above atmospheric pressure. Once such pressure is applied to the desired substances in the (micro)pipette conduits 602b', solenoid actuator 720c can be used to move third valve 726c outward until it pinches each of the (micro)pipette conduits 602b' within third seal area 608c and prevents any substance from escaping therethrough.

[0234] Heaters 732 and 738 (or heaters 732, 734, 738, and 740) in cradle 700 can then be used to heat the substance held in portion 602b' of conduit inside cartridge 604 to facilitate a chemical reaction (e.g., ligation) or other processing step therein, such as PCR. In some cases, this involves using a heater to generate a constant heat flow to conduit 602b', while in other cases, it involves cycling the heater to provide a cyclical heat flow to conduit 602b'. In some implementations, conduit 602b' is made from a thermally conductive plastic to more effectively conduct heat to the substance. In some implementations, clamping the (micro)pipette conduits 602b' and sealing them within the seal area 608 as described herein can improve the rate of heat transfer to the material within the (micro)pipette conduits 602b' by increasing the contact surface area between the (micro)pipette conduits 602b' and a heater or other heat transfer component. In some implementations, once the material is delivered as described herein, the material undergoes various processing steps, such as "pre-PCR" processing steps such as ligation within the (micro)pipette conduits 602b', and heat can facilitate such processing steps.

[0235] Once such a processing step has occurred within the (micro)pipette conduit 602b', the solenoid actuator 720c can be used to move the third valve 726c away from the (micro)pipette conduit 602b' until it no longer pinches each of the (micro)pipette conduits 602b' within the third seal area 608c to prevent material from escaping therethrough. This opening of the valve, and in a similar manner any of the other valves described herein, can be performed slowly, particularly when material is pressurized behind the valve, to prevent any undesirable events as the valve opens and pressure equalizes. For example, the valve 726 can be moved from a fully closed position to a fully open position over the course of about or at least 10 seconds, although such a time period may depend on the size of the valve 726 and / or the size of the (micro)pipette conduit 602b'. Thus, in some embodiments, the time period may be as short as about 5 seconds, and in some implementations, the rate at which the valve moves may increase over the course of such a time period, such that the initial opening rate is particularly slow.

[0236] Once the third valve 726c has been moved to the open position in this manner, the system can then use the fourth actuator 528 to drive the syringe pump 534 to release the pressure in the (micro)pipette conduits 602b', and the solenoid actuator 720a can then be used to move the first valve 726a away from the (micro)pipette conduits 602b' until it no longer pinches each of the (micro)pipette conduits 602b' in the first seal area 608a to prevent material from escaping therethrough. The material held in the (micro)pipette conduits 602b' can then be dispensed into the micro-wells, as described elsewhere herein, to allow further processing to occur as desired. In some alternative implementations, once the first valve 726a has been moved to the open position in this manner, the system can then use the fourth actuator 528 to drive the syringe pump 534 to draw material from within the first upper set of (micro)pipette conduits 602b' and within the heated zone 614 through the three-way connector 610 and into the feed conduit 612.

[0237] Once material has been drawn into the feed conduit 612 in this manner, solenoid actuators 720a and 720c can be used to move the first and third valves 726a and 726c outward until they pinch the (micro)pipette conduit 602b′ in the first and third seal areas 608a, 608c, respectively, preventing any material from escaping therethrough. Solenoid actuators 720b and 720d can then be used to move the second and fourth valves 726b and 726d away from the (micro)pipette conduit 602b″ until they no longer pinch the (micro)pipette conduit 602b″ in the second and fourth seal areas 608b, 608d, respectively, preventing any material from escaping therethrough. The system can then use the fourth actuator 528 to drive the syringe pump 534 to push material from within the feed conduit 612 through the three-way connector 610 into the second lower set of (micro)pipette conduits 602b'', through the second lower set of (micro)pipette conduits 602b'', through the heating zone 614 and into the microwells, as described elsewhere herein, to allow further processing to occur as desired.

[0238] In some alternative implementations, the system can then use the fourth actuator 528 to drive the syringe pump 534 to push the material from within the feed conduit 612, through the three-way connector 610, into the second lower set of (micro)pipette conduits 602b" and into the heated zone 614 for further processing therein. Once the desired material is thus located within the portions 602b" of the (micro)pipette conduits inside the cartridge 604 and within the heated zone 614, the solenoid actuator 720b can be used to move the second valve 726b outward until it pinches each of the (micro)pipette conduits 602b" within the second seal zone 608b, preventing any material from escaping therethrough. Once the (micro)pipette conduits 602b" are sealed in this manner within the first seal area 608a, the system can then use the fourth actuator 528 to drive the syringe pump 534 to apply a relatively high pressure to the desired substance within the (micro)pipette conduits 602b", such as to prevent such substance from evaporating or boiling. Such a pressure may be 7 psi or 10 psi above atmospheric pressure (positive gauge pressure), or greater than 7 psi above atmospheric pressure, greater than 10 psi above atmospheric pressure, or between 7 psi and 10 psi above atmospheric pressure. Once such a pressure has been applied to the desired substance within the (micro)pipette conduits 602b", the solenoid actuator 720d can be used to move the fourth valve 726d outward until it pinches each of the (micro)pipette conduits 602b" within the fourth seal area 608d and prevents any substance from escaping therethrough.

[0239] Heaters 734 and 740 (or heaters 732, 734, 738, and 740) in cradle 700 can then be used to heat the substance held in portion 602b″ of the conduit inside cartridge 604 to facilitate a chemical reaction such as PCR or other processing step therein. In some cases, this involves using a heater to generate a constant heat flow to conduit 602b″, while in other cases, it involves cycling the heater to provide a cyclical heat flow to conduit 602b″. In some implementations, conduit 602b″ is made from a thermally conductive plastic to more effectively conduct heat to the substance. In some implementations, pinching (micro)pipette conduits 602b″ and sealing them in seal area 608, as described herein, can improve the rate of heat transfer to the substance in (micro)pipette conduit 602b″ by increasing the contact surface area between (micro)pipette conduit 602b″ and the heater or other heat transfer component. In some implementations, once the material is delivered as described herein, the material undergoes various processing steps, such as PCR processing steps, within the (micro)pipette conduit 602b'', and heat can facilitate such processing steps.

[0240] Once such a processing step has occurred within (micro)pipette conduit 602b″, fourth valve 726d can be moved away from (micro)pipette conduit 602b″ until solenoid actuator 720d is used to no longer pinch each of (micro)pipette conduits 602b″ within fourth seal area 608d to prevent substance from escaping therethrough. This opening of this valve, and in a similar manner any of the other valves described herein, can be performed slowly, particularly when substance is pressurized behind the valve, to prevent any undesirable events as the valve opens and pressure equalizes, etc. For example, valve 726 can be moved from a fully closed position to a fully open position over the course of about or at least 10 seconds, although such a time period may depend on the size of valve 726 and / or the size of (micro)pipette conduit 602b″. Thus, in some embodiments, the time period may be as short as about 5 seconds, and in some implementations, the rate at which the valve moves may increase over the course of such a time period, such that the initial opening rate is particularly slow.

[0241] Once the fourth valve 726d has been moved to the open position in this manner, the system can then use the fourth actuator 528 to drive the syringe pump 534 to release the pressure in the (micro)pipette conduits 602b'', and the solenoid actuator 720b can then be used to move the second valve 726b away from the (micro)pipette conduits 602b'' until it no longer pinches each of the (micro)pipette conduits 602b'' in the second seal area 608b to prevent substance from escaping therethrough. The substance held in the (micro)pipette conduits 602b'' can then be dispensed into the micro-wells, as described elsewhere herein, to allow further processing to occur as desired.

[0242] As described herein, substances may be drawn into the first upper set 602b' of (micro)pipette conduits for processing and then dispensed into the microwells therefrom. However, in an alternative implementation, substances may be drawn into the second lower set 602b'' of (micro)pipette conduits for processing and then dispensed into the microwells therefrom. As described herein, substances may be drawn into the first upper set 602b' of (micro)pipette conduits for processing and then dispensed into the microwells through the second lower set 602b'' of (micro)pipette conduits. However, in an alternative implementation, substances may be drawn into the second lower set 602b'' of (micro)pipette conduits for processing and then dispensed into the microwells through the first upper set 602b' of (micro)pipette conduits. As described herein, materials may be drawn into the first upper set of (micro)pipette conduits 602b' for processing, then moved to the second lower set of (micro)pipette conduits 602b'' for additional processing, and then dispensed into the microwells from there. However, in an alternative implementation, materials may be drawn into the second lower set of (micro)pipette conduits 602b'' for processing, then moved to the first upper set of (micro)pipette conduits 602b' for additional processing, and then dispensed into the microwells from there.

[0243] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in Application Data Sheets, including U.S. Patent Application No. 62 / 983,479, filed February 28, 2020, are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified where necessary to employ concepts from various patents, applications, and publications to provide still further embodiments.

[0244] The various embodiments described above can be combined to provide further embodiments. These and other changes can be made to the embodiments in light of the description detailed above. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full range of equivalents to which such claims are entitled.

Claims

1. A system comprising: a first pipette including a first pipette tip at a first end of the first pipette and a second end of the first pipette opposite the first pipette tip along a first length of the first pipette; a second pipette including a second pipette tip at a first end of the second pipette and a second end of the second pipette opposite the second pipette tip along a second length of the second pipette; a three-way connector fluidly coupling a second end of the first pipette to a second end of the second pipette and fluidly coupling the second end of the first pipette and the second end of the second pipette to a feed conduit; a heating element thermally coupled to at least a portion of the first length and the second length; A system comprising:

2. The system described in claim 1, wherein the heating elements include a first heating element thermally coupled to at least a portion of the first length of the first pipette and a second heating element thermally coupled to at least a portion of the second length of the second pipette.

3. The system described in claim 2, wherein the first heating element and the second heating element are capable of operating independently.

4. The system of claim 1, further comprising a first valve configured to seal the first length of the first pipette, a second valve configured to seal the first length of the first pipette, a third valve configured to seal the second length of the second pipette, and a fourth valve configured to seal the second length of the second pipette.

5. The system described in claim 4, wherein the heating elements include a first heating element thermally coupled to at least a portion of the first length of the first pipette and a second heating element thermally coupled to at least a portion of the second length of the second pipette.

6. The system described in claim 5, wherein the first heating element and the second heating element are capable of operating independently.

7. The system described in claim 5, wherein the first heating element is thermally coupled to at least a portion of the first length of the first pipette between the first valve and the second valve, and the second heating element is thermally coupled to at least a portion of the second length of the second pipette between the third valve and the fourth valve.

8. A system comprising: a first pipette including a first pipette tip at a first end of the first pipette and a second end of the first pipette opposite the first pipette tip along a first length of the first pipette; a second pipette including a second pipette tip at a first end of the second pipette and a second end of the second pipette opposite the second pipette tip along a second length of the second pipette; a first three-way connector fluidly coupling a second end of the first pipette to a second end of the second pipette and fluidly coupling the second end of the first pipette and the second end of the second pipette to a first feed conduit; a third pipette including a third pipette tip at a first end of the third pipette and a second end of the third pipette opposite the third pipette tip along a third length of the third pipette; a fourth pipette including a fourth pipette tip at a first end of the fourth pipette and a second end of the fourth pipette opposite the fourth pipette tip along a fourth length of the fourth pipette; a second three-way connector fluidly coupling a second end of the third pipette to a second end of the fourth pipette and fluidly coupling the second end of the third pipette and the second end of the fourth pipette to a second feed conduit; a first heating element thermally coupled to at least a portion of the first length and the third length; a second heating element thermally coupled to the second length and at least a portion of the fourth length; A system comprising:

9. The system described in claim 8, wherein the first heating element and the second heating element are capable of operating independently.

10. The system of claim 8, further comprising a first valve configured to seal the first length of the first pipette and the third length of the third pipette, a second valve configured to seal the first length of the first pipette and the third length of the third pipette, a third valve configured to seal the second length of the second pipette and the fourth length of the fourth pipette, and a fourth valve configured to seal the second length of the second pipette and the fourth length of the fourth pipette.

11. The system described in claim 10, wherein the first heating element and the second heating element are capable of operating independently.

12. The system described in claim 10, wherein the first heating element is thermally coupled to at least a portion of the first length of the first pipette and the third length of the third pipette between the first valve and the second valve, and the second heating element is thermally coupled to at least a portion of the second length of the second pipette and the fourth length of the fourth pipette between the third valve and the fourth valve.

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