MICROFLUIDIC DEVICE AND METHOD FOR RAPID HIGH THROUGHPUT IDENTIFICATION OF MICROORGANISMS

Abstract

An apparatus and method are disclosed for detecting the presence of a microorganism within sampling device. The sampling device has a plurality of reaction chambers each having a reactive reagent for reacting with the microorganism to indicate the presence of the microorganism within the reaction chamber. A grabber holding the sampling device and a motion stage connected to the grabber moves the sampling device in a plane. A detector detects each of the plurality of reaction chambers for detecting the presence of the microorganism within the reaction chamber.

Claims

1. An apparatus for detecting a microorganism within sampling device, the sampling device having a plurality of reaction chambers each having a reactive agent for reacting with the microorganism to indicate the presence of the microorganism in said reaction chamber, comprising: a grabber for holding the sampling device; a motion stage connected to said grabber for moving the sampling device in a plane; and a detector for detecting each of the plurality of reaction chambers for detecting the presence of the microorganism in said reaction chamber.

2. The apparatus for detecting the Microorganism within sampling device as set forth in claim 1, wherein the sampling device is a rotatable disk having a central hole; and said grabber engaging with the central hole for holding and rotating said rotary disk.

3. The apparatus for detecting the Microorganism within sampling device as set forth in claim 1, wherein said detector is a spectrometer for detecting the presence of the microorganism in the reaction chamber.

4. The apparatus for detecting the Microorganism within sampling device as set forth in claim 1, wherein said detector includes a light source located on one side of the sampling device and a spectrometer located on another side of the sampling device for detecting the presence of the microorganism in the reaction chamber.

5. The apparatus for detecting the Microorganism within sampling device as set forth in claim 1, wherein said detector includes a light source for irradiating the reaction chambers to indicate a reaction between the reactive agent and the microorganism; and spectrometer for detecting a florescence radiation in the reaction chamber indicative of the presence of the microorganism in the reaction chamber.

6. The apparatus for detecting the Microorganism within sampling device as set forth in claim 1, including a cartridge having a plurality of containers for introducing reactive agents into the sampling device.

7. The apparatus for detecting the microorganism within sampling device as set forth in claim 1, including a cartridge having a plurality of containers for introducing samples into the sampling device.

8. A microfluidic sampling device for identifying the presence of a microorganism from a potential patient sample, comprising: a sample body having an inlet for receiving the patient sample and reactive agents; a reservoir for receiving the patient sample and a reactive agent from said inlet; a microchannel for transferring the patient sample and said reactive agent from said reservoir to a reaction chamber; said microchannel having a path for mixing the patient sample and said reactive agent prior to entering said reaction chamber; and said reaction chamber being transparent for enabling said detection of the presence of the microorganism within said reaction chamber.

9. The microfluidic sampling device as set forth in claim 8, wherein said sample body is a rotary disk.

10. The microfluidic sampling device as set forth in claim 8, wherein said microchannel transfers the patient sample and said reactive agent from said reservoir to said reaction chamber upon rotation of said sample body.

11. The microfluidic sampling device as set forth in claim 8, wherein said microchannel has a restrictive path for creating an extra resistance for the patient sample and said reactive agent for mixing the patient sample with said reactive agent.

12. The microfluidic sampling device as set forth in claim 8, wherein said microchannel has a restrictive path including a siphon valve to create an extra resistance for the patient sample and said reactive agent for mixing the patient sample with said reactive agent.

13. The microfluidic sampling device as set forth in claim 8, wherein said channels connect to the reaction chamber by a siphon valve for backflow prevention.

14. The microfluidic sampling device as set forth in claim 8, wherein air evacuates from said reaction chamber through a vent channel.

15. A microfluidic sampling device for identifying the presence of Covid-19 from a patient sample, comprising: a rotary disk having a plurality of inlets located on and inner region of said rotary disk for receiving potential patient samples; a plurality of reservoirs for receiving the clinical sample and said reactive agent from said plurality of inlets; a plurality of reaction chambers located on the outer periphery of said rotary disk; a plurality of specific transparent patterned electrode on the bottom and cover layer for locally heating all reaction chambers at the same time; a mixer channel for transferring the patient samples and said reactive agent from each of said plurality of reservoirs to each of said plurality of reaction chambers; each of said microchannels having a restrictive path for mixing each of the patient sample with said reactive agents prior to entering each of said reaction chambers; and said reaction chamber being transparent for enabling detection of the presence of Covid-19 within said reaction chambers.

16. The microfluidic sampling device for identifying the presence of Covid-19 as set forth in claim 15, wherein said rotary body is formed with glass or a polymeric material.

17. A method for rapid identifying the presence of Covid-19 from a potential patient sample, comprising the steps of: introducing a reagent into a reservoir as preloaded; introducing the patient sample into the reservoir; shaking the patient sample and the reagent through back and forth rotation motion; moving the mixed patient sample and reagent to a reaction chamber by a microchannel; heating the patient sample and reagent within the reaction chamber; and detecting the presence of Covid-19 within the reaction chamber.

18. A method for rapid identifying the presence of Covid-19 as set forth in claim 17, wherein the step of moving the patient sample and the reagent includes rotating the reservoir for creating a centrifugal force.

19. A method for rapid identifying the presence of a microorganism from a potential patient sample, comprising the steps of: introducing the patient sample into a reservoir; introducing the reagent into the reservoir; shaking the patient sample and the reagent by back and forth motion to mix the patient sample with the reagent; moving the patient sample and the reagent to a reaction chamber; thermal cycling the patient sample and the reagent within the reaction chamber for polymerize chain reaction (PCR); and detecting the presence of the infectious microorganisms within the reaction chamber.

20. The method for rapid identifying the presence of a microorganism as set forth in claim 19, wherein the step of moving the patient sample and the reagent includes rotating the reservoir for creating a centrifugal force.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] For a full understanding of the nature and objects of the invention, reference should be made to the following detailed description taken in connection with the accompanying drawings in which:

[0034] FIG. 1 is a side view of an apparatus for rapid identification of microorganisms in the present invention;

[0035] FIG. 2A illustrates a base layer of a microfluidic sampling device shown as a rotary disk;

[0036] FIG. 2B illustrates a main of patterned main layer of the rotary disk wherein a reaction will take place;

[0037] FIG. 2C is a magnified view of the FIG. 2B;

[0038] FIG. 2D illustrates a cover layer of the rotary disk;

[0039] FIG. 3A-B illustrate a cartridge top view and isomorphic view respectively;

[0040] FIG. 4A illustrates a first portion of a molecular biology-based methodologies; and

[0041] FIG. 4B illustrates a second portion of a molecular biology-based methodologies;

[0042] FIG. 5A presents the detected spectrum for the positive, negative controls and water;

[0043] FIG. 5B presents the detected spectrum for the positive, negative controls and water,

[0044] FIG. 5C presents the detected spectrum for the positive, negative controls and water;

[0045] FIG. 6 presents patterned transparent electrode for an accurate local heating of the disk sample; and

[0046] FIG. 7 presents the isomorphic view of the sample tube holder.

DETAILED DISCUSSION

[0047] FIG. 1 illustrates an apparatus for rapid identification of a microorganism such as a bacteria, fungi and viruses. The apparatus is suitable for use with LAMP, RT-LAMP CRISPR-CAS 12/13 diagnostics method, PCR, and RT-PCR. The apparatus receives a microfluidic sampling device 1.0. As will be described in greater detail hereinafter, the microfluidic sampling device 1.0 receives a sample for analysis. The microfluidic sampling device 1.0 is interchangeable and disposable for rapid identification of a microorganism. The microfluidic sampling device 1.0 is able to diagnose the infectious diseases in sample such as blood, mucosa, saliva, semen, urine. However, the microfluidic sampling device 1.0 can be designed for virus, bacteria or fungi detection as well. It always looks for the genetic fingerprint of the pathogen under detection.

[0048] In this example, the microfluidic sampling device 1.0 is shown as a disk 1.0 having an approximate size of a compact disk (CD). However, the specific design of the micro fluidic sampling device may take various shapes and forms depending upon the desired tests to be performed. The apparatus comprises a grabber 1.1 for holding the rotary disk 1.0. The grabber 1.1 includes a motor 1.11 mounted on the base 1.2 for rotating the rotary disk 1.0.

[0049] As will be described in greater detail hereinafter, a light source 1.4 is located under the rotary disk 1.0 and aligned to reaction chambers 2.7 in the rotary disk 1.0. A spectrometer detector 1.6 measures the light passing through and/or emitted from selective reaction chambers 2.9 of the rotary disk 1.0. Cartridge 1.7 is located on top of the disk 1.0 to inject biomaterials and clinical samples to the disk 1.0 through needle 1.75. The cartridge 1.7 is inserted on a linear rail 1.8 as such cartridge can move towards the center of the disk or reverse. The combination of this motion and rotation of the disk 1.0 make sures that all six needles in the cartridge can access the inlet 2.4 (FIG. 2C). In another embodiment the disk is preloaded with biomaterials and only patient samples are inserted into the disk. Also sample tube 1.9 is located on a rotation stage 1.85 (cross section is shown) and needle 1.95 well aligned with the inlet 2.4 of the disk 1.0. By rotation of the stage 1.85, 30 sample tubes inject in 60 inlets. Each patient sample injects in two inlets for the internal control and the gene of interest in the pathogen (N and E genes for COVID-19). There is a heating system 1.3 that can heat the disk up to 65° C. This heater can be set at a constant temperature or perform like a thermal cycler.

[0050] FIGS. 2A-D are enlarged views of a microfluidic sampling device 1.0 incorporated into a rotary disk 1.0. The rotary disk 1.0 comprises a base layer 2.1 formed from transparent material such as glass or any other material with preferred thickness of 100-1100 m as a structural base of the rotatable disk 1.0. The base layer 2.1 stabilizes the remaining thinner layers of the rotary disk 1.0. A hole 2.2 is defined in rotary disk 1.0 for enabling the grabber 1.1 to hold and to rotate the rotary disk 1.0.

[0051] FIG. 2B is the main layer of the rotary disk 1.0. It has a concentric central hole forming a part of the hole 2.02 of the rotary disk 1.0. The main layer 2.2 is formed from transparent material such as glass or any other materials. All the rotary disks 1.0 have specific assigned identification number such as a barcode 2.25 that is printed on the main layer 2.2.

[0052] FIG. 2C is a magnified view of the disk 1.0 presenting three levels of microfluidic which is called leaf. All these three leaf design perform same function. An inlet 2.4 is defined in the main layer 2.2 to inject the reactive agent and sample. The reaction agent will be injected to the reservoir 2.5. The volume of this reservoir is intentionally designed to be few micrometers more than the volume of total injected material.

[0053] When the sample is injected through inlet 2.4, the sample is pushed to the middle of reactive reagent in the reservoir 2.5. The mixing of the sample and reagents start here due to intrinsic liquid diffusion and shaking. After loading the materials (reactive reagents and the samples) the rotary disk 1.0 begins to shake for few seconds then rotate at 2000 rpm for 10 seconds. The intrinsic centrifugal force accurately regulates the motion of the mixture during rotation. The microchannels 2.6 define a resistive path to provide extra resistance to the flow of liquid therethrough. Microchannel 2.6 carries mixed liquids outward towards reaction chamber 2.7. This extra resistance promotes mixing of the sample and reactive agents. There is no full access from microchannel 2.6 to the reaction chamber 2.7. A siphon valve 2.65 is installed in between. This valve ensures the liquid will trap in the reaction chamber 2.7 and will not come back to the microchannel 2.6 when disk stops the rotation. An outlet microchannel 2.8 through exit hole of 2.9 is designed to vent all possible trapped air during rotation or biochemical reaction. Similarly another siphon valve 2.85 is installed in the path of reaction chamber 2.7 and vent channel 2.8 to prevent skipping of the liquid from reaction chamber.

[0054] The excess of the air in the microchannels 2.6 and 2.8 as well as the chambers 2.5, and 2.7 push the liquid back towards the inlet 2.4 when rotary disk 1.0 stops the rotation. The outlet microchannel 2.8 evacuates any trapped air through siphon channel 2.85. However, the location of the exit of the outlet microchannel 2.8 is very important. Due to Coriolis force that happens in the rotary disk 1.0, air tends to move towards 5 O'clock position. If the outlet microchannel 2.8 is located at 12 O'clock position, the liquid goes through the outlet microchannel 2.8 and the air will be trapped inside the reaction chamber 2.7 creating air bubbles. This means losing some portion of the liquid resulting in a lower signal. The outlet microchannel 2.8 is intentionally made towards the center of the disk as such liquid itself cannot be discharged from the rotary disk 1.0 while rotating. When the rotary disk 1.0 stops, then sample and reactive agents are mixed and placed in the reaction chamber 2.7.

[0055] The existence of a siphon valve structure 2.65 at the inlet of the reaction chamber 2.7 and a siphon valve 2.85 at the outlet of the reaction chamber 2.7 creates resistance against capillary effect and traps the liquid in the reaction chamber 2.7. This type of valving is referred to as siphon valving. Siphon valving is very effective and simple and performs with no additional valving cost.

[0056] FIG. 2D illustrates a cover layer 2.3. The cover layer 2.3 has a concentric central hole of 2.03 for grabber 1.1. It has inlet holes 2.45 in alignment with the inlets 2.4 in the main layer 2.2 to inject the liquids as well as outlet holes 2.95 in alignment with the air outlet holes 2.9 in the main layer 2.2 to evacuate air.

[0057] FIG. 3A illustrates top view of the cartridge 3.0 for the present invention. In this example, the content of the cartridge 1.7 as shown in FIG. 1 or 3.0 as shown in FIG. 3A will be explained with specific reference to Covid-19, but it should be understood that the content of the cartridge 3.0 may be adapted for other types of testing.

[0058] Preferably, cartridge 3.0 is a molded plastic that has six different containers. Container 3.1 holds the ddH.sub.2O. Container 3.2 holds the common biomaterial such as enzymes. Container 3.3 carries primers for housekeeping gene, for example, rActin primer is used as the internal control. Container 3.4 contains primer for N gene and E gene of Covid-19 and container 3.5 contains primer for O gene of Covid-19. Container 3.6 holds the synthesized Covid-19 RNA.

[0059] The cartridge 3.0 can vary in size. For example, a 100 disks load to run 3000 tests requires 45 mL of common biomaterial, 4.25 mL of N gene primer, 4.25 mL of E gene primer, 4.25 mL of primers for housekeeping gene, and 300 μL of synthesized Covid-19 RNA and finally 32.5 mL of ddH.sub.2O. FIG. 3B is the isomorphic view of the cartridge. It presents the six needles 3.01 to 3.06 for the injection. In another embodiment of present invention all the material explained for cartridge 3.0 are preloaded in the disk and use only prepare and loads the patient sample.

[0060] Loop-mediated isothermal amplification (LAMP) method relies on the auto cycling strand displacement of DNA molecules [1-4]. The assay is based on using 2-3 primer pairs (4-6 primers) which specifically recognize 6-8 different areas of target DNA. A strand-displacing DNA polymerase initiates synthesis at a constant temperature with greater efficiency. To improve the amplification process, there are also 2 specially designed primers to create loop structures. The DNA products of LAMP assay include several repeats of the short target sequences which is linked together through single-stranded loop sequences. Although LAMP products are not applicable for further manipulations, LAMP products are very suitable for the detection of pathogens due to the extensive amplification.

[0061] The following publications investigate the process of amplifying nucleic acids. K Nagamine, T. H., T Notomi, Method of synthesizing single-stranded nucleic acid. 2000, Eiken Chemical Co Ltd. Notomi, T., et al., Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 2000. 28(12): p. E63. TSUGUNORI, H. T. N., Method of synthesizing nucleic acid. EIKEN CHEMICAL. Tsugunori Notomi, K. N., Method of amplifying nucleic acid by using double-stranded nucleic acid as template. 2003, Eiken Chemical Co Ltd.

Primers

[0062] FIGS. 4A and 4B show the FIP primer having two regions of F2 which is complementary to F2c region of the template, and F1 c which is identical to the F1c sequence of the target. Similarly, BIP primer is consisted of B2 and B1c regions which are complementary to B2c and identical to Blc regions of the target sequence in order. FOP (also known as F3) and BOP (also known as B3) are complementary to the F3c and B3c regions of the template DNA.

Stages of LAMP

[0063] Briefly, the F2 sequence of FIP primer is hybridized to F2c sequence of the template and initiates the amplification process. The F3 primer is then hybridized to F3c sequence of the template and starts the extension process. At the same time, F3 displaces the linked FIP strand and creates a single strand loop at the 5′ end of the extending. This looped-end single stranded DNA molecule operates as a target sequence for the BIP primer. At this point, B2 region is linked to B2c sequence of the target DNA and initiates the extension of the DNA molecule. The loop at the end of this template molecule is then opened at the end. The B3 primer is then hybridized to the B3c region of the molecule and displaces the linked BIP molecule and make a dumbbell shaped DNA molecule with two single stranded loops at each end. At this point, the DNA polymerase starts extending the DNA at the 3′ end of F1, opening the 5′ end-loop and forming a stem loop structure of the DNA. This structure performs as another template for LAMP. The FIP primer again hybridizes to the loop of the stem-loop DNA structure, initiates the extension of the DNA, displacing the F1 which leads to the formation of a new loop at the at the 3′ end. DNA polymerase then adds nucleotides to the 3′ end of B1, and displaces FIP strand, which leads to the formation of another dumbbell shaped DNA molecule. At this point, there will be a stem loop DNA and a gap repaired stem loop DNA, both of which serve as template other rounds of strand displacement reaction and elongation in the following cycles, which produce a mixture of stem looped-DNA with different stem lengths and several loops [2].

Method of Operation

[0064] In this example, the rotary disk 1.0 contains 63 reaction chambers enabling 63 different tests to be simultaneously run from a single rotary disk 1.0. In the matter of 30 minutes, samples will be examined for 30 individual samples. However, it should be understood by those skilled in the art that the rotary disk 1.0 may be modified in design to accommodate higher or lower number of testing chambers.

[0065] An example of the method of operation of the apparatus is set forth below for a disk design with channels numbered 1 to 63 for each chamber. Chambers 1 to 3 will be used for negative and positive control tests. Chambers 5 and 6 will be used for the first patient and chambers 7 and 8 for the next patient and so on. Two chambers will be used for each sample, one for the detection of Covid-19 RNA, and one for the detection of the housekeeping gene as a proof of the accuracy of the DNA/RNA extraction prior to the test in the absence of Covid-19 genetic material.

[0066] Here is a non-limiting example of the injection protocol: First 1 to 9 μL of ddH.sub.2O from Cartridge 3.1 will be injected in all 63 inlets. Then 12.5 μL of common biomaterial will be injected from cartridge 3.2 in all 63 inlets. Then from cartridge 3.3, 2.5 μL of housekeeping primer mix will be injected in inlets of chamber 2, 5, 7, 9, . . . , 63. Then from cartridge 3.4, 2.5 μL of N-gene and E-gene primer mix of Covid-19 will be injected in the inlet of chamber 3, 6, 8, 10, . . . , 62. The last step is to insert 1 μL of Covid-19 RNA from cartridge 3.6 into chamber 1, and 2. This step is called initialization and now disk is ready to accept clinical samples.

[0067] Sample 1 is injected into both chambers of 4 and 5. As explained above, chamber 4 has the human housekeeping gene primer, ddH.sub.2O, common material whereas chamber 5 has common material, ddH.sub.2O, N gene primer and E gene primer. If the device is tuned to do CRISPER or PCR, then the florescence emitting material can be engineered to have shift in the spectrum for both E and N genes.

[0068] The PCR process is conducted via thermal cycler, which is a laboratory apparatus capable of heating and cooling the samples in a holding block in multiple cycles to create the conditions necessary for the in vitro replication of the DNA molecule by DNA polymerase. Thermal cycling for PCR involves three main phases: 1) Denaturation (94° C. to 98° C.), in which double-stranded DNA templates are heated to separate the DNA strands; 2) Annealing (48° C. to 72° C.), in which primers bind to specific regions of the target DNA: and 3) Extension (68° C. to 72° C.), in which DNA polymerase extends the 3′ end of each primer based on the template strands. These steps are repeated in cycles to exponentially replicate the copies of the target DNA.

[0069] FIGS. 5A-C illustrate the absorption spectrum of the radiated light from chamber. The sample is irradiated with a light source and spectrometer detects the presence of the amplified DNA through absorption spectrum, indicating the presence of Covid-19 in the original sample. FIG. 5A presents the absorption spectrum of the sample, indicating yellow color. This is the positive control and sample contains Covid-19 genes. Before reaction starts the original color in the reaction chamber was violet-purple.

[0070] FIG. 5B presents the absorption spectrum of negative control. It was originally violet-purple and it stays violet-purple.

[0071] FIG. 5C presents the absorption spectrum from water only as reference signal.

[0072] FIG. 6 presents another method of heating the sampling disk 1.0. In this method the base glass layer 2.1 is printed with transparent electrode such as indium Tin Oxide (ITO). The coating 2.05 is done only in the reaction chamber locations. By applying voltage at the electrodes 2.06 and 2.07 all the reaction chambers reach to 65° C. in 30 minutes. This type of local heating can be well controlled specially in the case that thermal cycling is needed.

[0073] FIG. 7 is a schematic view of sample cartridge holder. In the FIG. 1 only the cross section is shown. The samples will be loaded in tubes 7.1. Each tube has barcode to assign for each individual sample. It has one needle 7.2. Entire holder 7.4 after filled up will be pushed in the machine to analyze.

[0074] The present disclosure includes that contained in the appended claims as well as that of the foregoing description. Although this invention has been described in its preferred form with a certain degree of particularity, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangement of parts may be resorted to without departing from the spirit and scope of the invention.