MICROFLUIDIC DEVICE

Abstract

A microfluidic device including a serum separator, a quantum dot and antibody inlet connected to the serum separator, a quantum dot linked immunosorbent assay (QLISA) chamber connected to the serum separator, and an outlet connected to the QLISA chamber. The microfluidic device is configured to determine an amount of drug in a serum.

Claims

1. A microfluidic device, comprising: a serum separator; a quantum dot and antibody inlet connected to the serum separator; a quantum dot linked immunosorbent assay (QLISA) chamber connected to the serum separator; and an outlet connected to the QLISA chamber, wherein the microfluidic device is configured to determine an amount of drug in a serum.

2. The device of claim 1, wherein a length of the microfluidic device is 55-85 mm.

3. The device of claim 1, wherein the serum separator has a diameter of 7-17 mm.

4. The device of claim 1, wherein the QLISA chamber has a length of 20-40 mm and a width of 5-9 mm.

5. The device of claim 1, wherein the outlet has a diameter of 4-8 mm.

6. The device of claim 1, wherein the serum separator comprises a first inlet configured for sample injection.

7. The device of claim 6, wherein the quantum dot and antibody inlet comprises a second inlet configured to receive an injection of fluorescent tags and antibodies against an analytical material.

8. The device of claim 7, wherein the fluorescent tags are gold nanoparticles.

9. The device of claim 7, wherein the fluorescent tags are quantum dots.

10. The device of claim 6, wherein a width of a channel connecting the first inlet to the QLISA chamber is 1-3 mm.

11. The device of claim 1, wherein a width of a channel connecting the QLISA chamber to the outlet is 1-3 mm.

12. The device of claim 1, wherein a reaction takes place in the QLISA chamber where laser light falls and emission signal is recorded.

13. The device of claim 1, wherein unbound molecules are extracted through the outlet.

14. The device of claim 7, wherein the first and second inlets and the outlet are connected to syringes for sample injection and extraction.

15. The device of claim 1, further comprising materials of silicon and glass.

16. The device of claim 1, wherein the device is configured for serum drug level monitoring using non-competitive sandwich enzyme linked immunosorbent assay.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention, and together with the description serve to explain the principles of the invention.

[0028] FIG. 1 illustrates a microfluidic device having QLISA according to an exemplary embodiment.

[0029] FIG. 2 illustrates a thin film print of device according to an exemplary embodiment.

[0030] FIG. 3 illustrates a patterned silicon substrate according to an exemplary embodiment.

[0031] FIG. 4 illustrates an etched silicon substrate according to an exemplary embodiment.

[0032] FIG. 5 illustrates an optical microscopy images of the etched silicon according to an exemplary embodiment.

[0033] FIG. 6 illustrates an image of a microfluidic device working with inlet and outlet connections according to an exemplary embodiment.

[0034] FIG. 7A is a schematic diagram showing the injection and coating of the chamber with a biocompatible material.

[0035] FIG. 7B is a schematic diagram showing the injection and coating of the chamber coated with a primary antibody and bovine serum albumin.

[0036] FIG. 7C is a schematic diagram showing the injection of the serum sample of interest.

[0037] FIG. 7D is a schematic diagram showing the injection of a secondary antibody linked with quantum dots.

[0038] FIG. 8A is a graph showing the photoluminescence of quantum dots dispersed in chloroform.

[0039] FIG. 8B is a graph showing the photoluminescence of mercaptoacetic acid coated quantum dots in water.

[0040] FIG. 8C is a graph showing the photoluminescence of antibody coated quantum dots in water.

[0041] FIG. 8D is a graph showing a comparison of the graphs of FIGS. 8A, 8B, and 8C.

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS

[0042] In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of various exemplary embodiments. It is apparent, however, that various exemplary embodiments may be practiced without these specific details or with one or more equivalent arrangements. In other instances, well-known structures and devices are shown in block diagram form in order to avoid unnecessarily obscuring various exemplary embodiments.

[0043] Unless otherwise specified, the illustrated exemplary embodiments are to be understood as providing exemplary features of varying detail of various exemplary embodiments. Therefore, unless otherwise specified, the features, components, modules, layers, films, panels, regions, and/or aspects of the various illustrations may be otherwise combined, separated, interchanged, and/or rearranged without departing from the disclosed exemplary embodiments. Further, in the accompanying figures, the size and relative sizes of layers, films, panels, regions, etc., may be exaggerated for clarity and descriptive purposes. When an exemplary embodiment may be implemented differently, a specific process order may be performed differently from the described order. For example, two consecutively described processes may be performed substantially at the same time or performed in an order opposite to the described order. Also, like reference numerals denote like elements.

[0044] For the purposes of this disclosure, “at least one of X, Y, and Z” and “at least one selected from the group consisting of X, Y, and Z” may be construed as X only, Y only, Z only, or any combination of two or more of X, Y, and Z, such as, for instance, XYZ, XYY, YZ, and ZZ. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0045] In exemplary embodiments, modules and/or one or more components thereof, may be implemented via one or more general purpose and/or special purpose components, such as one or more discrete circuits, digital signal processing chips, integrated circuits, application specific integrated circuits, microprocessors, processors, programmable arrays, field programmable arrays, instruction set processors, and/or the like.

[0046] According to one or more exemplary embodiments, the features, functions, processes, etc., described herein may be implemented via software, hardware (e.g., general processor, digital signal processing (DSP) chip, an application specific integrated circuit (ASIC), field programmable gate arrays (FPGAs), etc.), firmware, or a combination thereof. In this manner, modules and/or one or more components thereof may include or otherwise be associated with one or more memories (not shown) including code (e.g., instructions) configured to cause modules, processors, and/or one or more components thereof to perform one or more of the features, functions, processes, etc., described herein.

[0047] FIG. 1 illustrates a microfluidic device having QLISA according to an exemplary embodiment. As shown in FIG. 1, the microfluidic device is fairly small with an elongated shape that has three inputs two inlets on the left and one outlet on the right. Channels to the upper inlet and outlet extend upward and away from the QLISA chamber (main body) of the micro fluidic device at an approximate 90° angle whereas the side inlet has a channel that extends or serum separator away from the QLISA chamber of the microfluidic device at an approximate 0° angle or a 180° angle. The microfluidic has a general overall length with a thicker portion at the QLISA chamber and a narrow portion for the channels.

[0048] In an exemplary embodiment, the microfluidic device may have the following dimensions set forth in Table 1.

TABLE-US-00001 TABLE 1 Dimensions for the Microfluidic Device Example Shown Device Portion Description Range in FIG. 1 Overall length 55-85 mm 69 ± 1 mm Overall width of the main body 1-3 mm 2 mm outside of QLISA chamber Length of the QLISA Chamber 20-40 mm 28.9 mm Width of the QLISA Chamber 5-9 mm 6.7 mm Channel length connecting the 7-17 mm 9 mm QLISA chamber to the outlet Channel width connecting the 1-3 mm 2 mm QLISA chamber to the outlet Outlet end diameter 4-8 mm 5.8 mm Channel length connecting the 10-20 mm 14.8 mm QLISA chamber to the inlet Channel width connecting the 1-3 mm 2 mm QLISA chamber to the inlet Inlet end diameter (Inlet B) 4-8 mm 5.8 mm Serum separator end diameter 7-17 mm 12.1 mm (Inlet A)

[0049] As outlined in Table 1, the overall size of the microfluidic device is quite small. It can be 55 mm in length and about 1 mm in width at its most narrow point in the main chamber. The specific example outlined in FIG. 1 illustrates many dimensions and an overall length of 69.5 mm with a narrow width of 2 mm.

[0050] Fabrication Method.

[0051] FIG. 2 illustrates a thin film print of device according to an exemplary embodiment. As shown in FIG. 2, the microfluidic device may be printed using well known thin film printing methods. FIG. 2 shows that that device may have the dimensions discussed above with respect to FIG. 1. Other methods may be used to create the microfluidic device.

[0052] FIG. 3 illustrates a patterned silicon substrate according to an exemplary embodiment. The method to obtain the illustrated image of FIG. 3 is discussed below.

[0053] According to FIG. 3, a wafer cleaning is carried out by ultrasonification of the wafer for approximately 30 minutes in an acetone solution. After 30 minutes of ultrasonification, the mask is dried with liquid nitrogen in a clean environment. Upon drying of the mask, a spin coating of positive photoresist (AZ 40XT) is applied at up to 2000 revolutions per minute for 40 seconds on the mask. The spin coating is followed by pre-bake at 100 Celsius for 10 minutes to evaporate any residual solvent and increase adhesion of mask on the silicon wafer. The pattern from mask is transferred to the silicon wafer using photolithography with Ultraviolet exposure two cycles for 200 seconds. After transfer of pattern to the wafer, the wafer is baked at 200 Celsius for up to two minutes for complete adhesion. Development of the wafer is carried out by immersing the wafer in a developer solution where the positive resist UV exposed area is dissolved in the developer solution. Finally, a hard baking step is carried out to increase the adhesion of pattern on silicon wafer as shown in FIG. 3.

[0054] Etching

[0055] FIG. 4 illustrates an etched silicon substrate according to an exemplary embodiment. FIG. 5 illustrates an optical microscopy images of the etched silicon according to an exemplary embodiment. The method to obtain the illustrated image of FIGS. 4 and 5 are discussed below.

[0056] A reactive ion etching of the silicon wafer with patterned device design, according to FIG. 1, is carried out following the hard baking of the silicon wafer. The etching is carried out by using argon and sulfur hexafluoride (SF.sub.6) gases and is followed by molding of the QLISA device. The etching is done so that the wafer has an etched pattern with a depth of 70-120 microns.

[0057] Mold Formation

[0058] For device fabrication polydimethylsiloxane (PDMS) polymer (4 ml, 15:1 ratio of base:curing agent) was poured on clean, etched wafer and was cured at 150° for 1.5 hours. Once the mold is dried completely and hardened on the wafer; it is carefully peeled off using clean tweezers.

[0059] The PDMS device mold was bonded to clean glass slide using stamp and stick bonding approach with slight modification (spin coat 0.5 ml at 1500 rpm for 20 s).

[0060] The PDMS on glass slide was cured at 60° C.; and after 10 minutes the peeled off device mold is bonded on the glass slide and left to dry for 1 hour at 60-100° C.

[0061] Assembly of Completed and Working Device

[0062] FIG. 6 illustrates an image of a microfluidic device working with inlet and outlet connections according to an exemplary embodiment. This device is described below.

[0063] Referring to FIG. 6, the inlet and outlet syringe connections were introduced in the working microfluidic device after the PDMS direct, and perfectly bonded, to the glass slide. The microfluidic device was then assembled in proper portable optical detection setup having laser source, spectrometer, optical fiber, filters and movable stage for sample.

[0064] The microfluidic device will perform detection of any biomolecule (drug) depending on the biological technique: sandwich enzyme linked immunoassay with the help of quantum dot instead of biological fluorophores. The detection setup is explained below.

[0065] Once a sandwich configuration is completed in the detection chamber/QLIZA chamber that has the primary antibody coated on a surface of glass slide (antibody immobilization), then the primary antibody is conjugated to the biomolecule (drug) followed by conjugation of the biomolecule to a secondary antibody coated on the quantum dot.

[0066] During each step of antibody immobilization or biomolecule attachment, glass slide is coated with bovine serum albumin followed by repetitive washing with phosphate buffer saline solution. The purpose of this step is to prevent non-specific adsorption or detection of any other biomolecule except the analytical material of interest in serum sample.

[0067] FIG. 7A is a schematic diagram showing the injection and coating of the chamber with a biocompatible material. FIG. 7B is a schematic diagram showing the injection and coating of the chamber coated with a primary antibody and bovine serum albumin. FIG. 7C is a schematic diagram showing the injection of the serum sample of interest. FIG. 7D is a schematic diagram showing the injection of a secondary antibody linked with quantum dots. These figures are discussed below.

[0068] As shown in FIG. 7A, the PDMS based device is coated with any biocompatible material to prevent sticking of biomolecules to the walls of PDMS. The QLIZA chamber is first activated with carboxyl group for antibody immobilization using carbodiimide crosslinking chemistry.

[0069] As shown in FIG. 7B, the chamber is then coated with primary antibody (50 μl, 0.001 dilution in PBS buffer) by injecting the primary antibody through a second inlet.

[0070] The chamber is then washed with phosphate buffer saline (PBS) solution to remove any unbound antibody from the chamber.

[0071] Referring to FIG. 7B, Bovine serum albumin (BSA) (50 μl, 0.1% solution) is then injected to coat the unbound glass surfaces in order to prevent any non-specific adsorption or detection of irrelevant analytical material in serum.

[0072] As shown in FIG. 7C, serum sample containing analytical material of interest (drug) (50 μl, 0.8 ng/ml) is injected from the first inlet. The serum sample then binds to available primary antibody sites in the QLIZA chamber.

[0073] The device is again washed with PBS buffer solution to remove any unbound analytical material form the chamber, to prevent non-specific detection.

[0074] As shown in FIG. 7D, the secondary antibody (50 μl, 0.001 dilution in PBS) linked with quantum dots (1 mg, 500 μl) is injected in the second inlet. The secondary antibody will bind to the available antigen binding sites.

[0075] PBS washing step is preformed again to remove unbound antibodies from the chamber.

[0076] Now the device with the sample is ready for detection.

[0077] The sample may be placed in a proper sample holder in the optical setup and excited with a laser light of wavelength 404 nm. The emitted light from the sample is collected by a collimating lens which aligns the emitted light rays. The lens is connected to optical fiber, which transmits light collected from the collimating lens to spectrometer.

[0078] The spectrometer, connected to computer, displays the results in from of an emission spectra. The changes in the intensity of emitted light are helpful in detection or assessing the level of any biomolecule in serum.

[0079] FIG. 8A is a graph showing the photoluminescence of quantum dots dispersed in chloroform. FIG. 8B is a graph showing the photoluminescence of mercaptoacetic acid coated quantum dots in water. FIG. 8C is a graph showing the photoluminescence of antibody coated quantum dots in water. FIG. 8D is a graph showing a comparison of the graphs of FIGS. 8A, 8B, and 8C. These figures are discussed below.

[0080] To monitor the change in emission intensity of dots before and after biofunctionalization of the quantum dots (QDs) photoluminescence (PL) spectra were acquired using the detection system described earlier.

[0081] FIG. 8A shows the PL spectra of QDs dispersed in chloroform with emission wavelength of 665 nm.

[0082] In order to make QDs water soluble (as they are soluble in chloroform) and for antibody conjugation, the surface of QDs were coated with mercaptoacetic acid (MAA). The MAA acts as a linker with thiol at a first end and carboxyl group at a second end. At the first end, sulfur will bind with the QDs sulfur (CdSe/ZnS) through S—S bond. The carboxyl end (second end) of MAA renders the QDs water soluble.

[0083] FIG. 8B shows a decrease in emission intensity of dots with a blue shift of 650 nm after coating with MAA or in water when compared to chloroform.

[0084] After making the dots water soluble, next step is conjugation of an antibody. Using carbodiimide chemistry, a carboxyl group of QDs will bind with the antibody amine group through C—N bond as shown in FIG. 8C. The PL spectra of MAA QDs with an antibody dispersed in water is 645 nm.

[0085] The comparison of change in emission of dots after coating with MAA and finally with an antibody is shown in FIG. 8D.

[0086] Proposed detection scheme:

[0087] Conventional therapeutic level of detection of digoxin drug is 0.8 ng-2.0 ng per ml.

[0088] With said device the detection methodology would involve detection of different concentrations of drug spiked in PBS and then in serum from 0.1 ng to 1 μg including the therapeutic range of 0.8 ng-2.0 ng per ml.

[0089] Another set of experiment would run in parallel as confirmation of results. Acquiring the spectra of chamber/glass surface after coating with primary antibody, then with drug immobilized on the primary antibody (blank spectra) followed by final spectra with secondary antibody immobilized.

[0090] The limit of detection (LOD) achievable with the said device is to be about 0.1 ng per ml. Experiments of quantum dot conjugation with antibody and detection using the said optical setup has been completed FIG. 8A-8C.

[0091] The said device is portable, reusable and produce results within 30 minutes as compared to time consuming and laborious procedures in conventional machine.