Wick-based analytical device for multiplexed quantification of biomarkers using carbon dots integrated with molecularly imprinted polymer

Abstract

A device for assaying analytes has a wicking substrate having a sample zone, a waste zone, a plurality of channels that each support capillary flow between the sample zone and the waste zone. The channels comprise different synthetic receptors that are selected to interact with different analytes. The channels also include nanoparticles that are configured to undergo a visible state change when an interaction between an analyte and a synthetic receptor occurs nearby. An extent of the state change along a channel is indicative of concentration of the analyte that corresponds to that channel.

Description

[0001] WICK-BASED ANALYTICAL DEVICE FOR MULTIPLEXED QUANTIFICATION OF BIOMARKERS USING CARBON DOTS INTEGRATED WITH MOLECULARLY

[0002] IMPRINTED POLYMER

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to US Provisional Application No. 63 / 560,979, filed March 4, 2024, and US Provisional Application No. 63 / 662,613, filed June 21, 2024, the contents of all which are hereby incorporated by reference in their entireties.

[0005] BACKGROUND

[0006] The human immune system is a sophisticated interplay of diverse cellular components collaborating to protect our body against diseases by combatting antigens and pathogens and controlling infections and their associated symptoms.

[0007] Cytokines play a crucial role in human immunology by mediating communication between various cells to bolster the immune response. Monitoring cytokine levels within the body is of tremendous value for clinical diagnosis and prognosis. This arises in part because cytokines are associated with several diseases, including but not limited to inflammation, infection, injury, myocardial infarction, diabetes, Alzheimer’s, Parkinson’s disease, sepsis, asthma, heart disease, rheumatoid arthritis, Acquired Immune Deficiency Syndrome (AIDS), depression, and various cancers.

[0008] More recently, elevated serum cytokine levels have emerged as a crucial indicator for assessing the severity of COVID-19. Cytokine production has been found to sometimes significantly increases upon infection. This leads to a collapse of the immune system, often referred to as a “cytokine storm.” This phenomenon is not limited to COVID-19, It can occur in response to other infections and cancers, often with life-threatening consequences.

[0009] Cytokine biomarkers also play a vital role in a wide range of medical conditions, offering clinicians valuable insights for accurate diagnosis and informed treatment decisions.

[0010] The most common sensor for quantification of cytokines relies on immunological analysis, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA). While such methods provide great selectivity and sensitivity, they can only be operated by a skilled user requiring sufficient sample preparation and analysis time. Moreover, the instruments for quantitative monitoring using these methods might not be accessible to resourcelimited settings for diagnosis and prognosis.

[0011] SUMMARY

[0012] In one aspect, the invention features an analytical device for multiplexed detection and quantification of multiple biomarkers using a distance-based readout. In some embodiments, the device uses a fluorescent nanomaterial whose fluorescence is quenched when target biomolecules bind with molecularly imprinted polymers that are selective to those target biomolecules.

[0013] Among the embodiments are those that use fluorescent carbon dots integrated with molecularly imprinted polymer to enable simultaneous biomarker quantification. The resulting device represents an eco-friendly, cost-effective, and instrument-free approach that uses rulerlike visual readouts. The integration of molecularly-imprinted polymers enhances selectivity to a wide panel of biomarkers ranging from cytokines, hormones, lipids, proteins, and small molecules. It does so without relying on expensive reagents.

[0014] Further embodiments include those that are used to assay opsonin, bilirubin, creatinine, IL-2, IL-6, IL- 10, IFN-y, TNF-a, CRP, uric acid, CEA, PSA, sarcosine, cholesterol, PSA, IGg, IGA, lactoferrin, beta-amyloid and urea.

[0015] The resulting multiplexed analytical-device quantitatively monitors multiple biochemical markers using synthetic receptors and fluorescent carbon dots. This results in a user-friendly visual readout based on ruler-like distance measurements that enable a user to measure where fluorescent activity starts. This makes the device eminently suitable for at-home use without the need for trained professionals. As a result, the device enables CLIA-waived point-of-need testing that enhances telemedicine, reduces healthcare costs, and improves population health through rapid, quantitative health marker detection.

[0016] A device as described herein is thus able to simultaneously quantify a broad panel of host biochemical markers for rapid and cost-effective assessment of host inflammatory response and of cardiac, liver, and kidney function. This addresses major health security concerns by facilitating early detection and at-home monitoring of infectious diseases, autoimmune disorders, and inflammatory conditions for timely intervention and public health management.

[0017] A device as described herein therefore enhances health security by providing a user- friendly, at-home testing solution for multiplexed monitoring of wide variety of host biochemical markers. This enables early detection of infections, cardiac distress, sepsis, and other inflammatory conditions. This decentralized approach supports timely interventions, reduces healthcare burdens, helps predict pandemic outbreaks, and contributes to overall public health resilience.

[0018] In one aspect, the invention features an apparatus for assaying analytes from a set of analytes that includes at least first and second analytes. The apparatus includes a substrate that is able to wick a liquid. The substrate has a sample zone, waste zones, and a plurality of channels, each of which is a detection channel that is configured to support capillary flow between the sample zone and a corresponding waste zone, the plurality of channels comprising a first channel and a second channel. The first channel comprises a first synthetic receptor and nanoparticles that are configured to undergo a state change in response to interaction of the first synthetic receptor with the first analyte. Similarly, the second channel comprises a second synthetic receptor and nanoparticles that are configured to undergo a state change in response to interaction of the second synthetic receptor with the second analyte. An extent of the state change along the first channel is indicative of concentration of the first analyte, and wherein an extent of the state change along the second channel is indicative of a concentration of the second analyte.

[0019] Embodiments include those in which the wick comprises paper and those in which it comprises a fabric or textile, including a textile formed into a thread.

[0020] Embodiments include those in which the state change is a change between being fluorescent and not being fluorescent. Among these are embodiments in which the state change is a transition from a fluorescent state of the nanoparticles to a non-fluorescent state of the nanoparticles, the transition being caused by exposure of the synthetic receptor to an analyte from the set of analytes. Still other embodiments include those in which the nanoparticles comprise carbon nanodots, those in which the nanoparticlcs comprise cellulose nanocrystals, those in which the nanoparticles comprise silicon dots, and those in which the nanoparticles comprise quantum dots.

[0021] In some embodiments, the first synthetic receptor comprises a first molecularly-imprinted polymers and the second synthetic receptor comprises a second molecularly-imprinted polymer. Other embodiments include those in which the first and second synthetic receptors comprise imprinted polymers and those in which the first and second synthetic receptors comprise ion- imprinted polymers.

[0022] Embodiments further include those in which the analytes comprise cytokines. In such embodiments, the synthetic receptors comprise receptors that are configured to bind to or otherwise interact with cytokines.

[0023] In still other embodiments, the first channel comprises a first molecularly-imprinted polymer that has been configured to interact with the first analyte, as a result of which fluorescence of nanoparticles proximate to a site of the interaction is quenched and wherein the second channel comprises a second molecularly-imprinted polymer that has been configured to interact with the second analyte, as a result of which fluorescence of nanoparticles proximate to a site of the interaction is quenched.

[0024] Also among the embodiments are those in which the plurality of analytes further comprises a third analyte. In such embodiments, the plurality of channels further comprises a third channel that is configured to support capillary flow between the sample zone and the waste zone. Preferably, the third channel has been prepared with a third synthetic receptor that has been configured to bind to or otherwise interact with the third analyte.

[0025] Still other embodiments include those in which the first channel comprises a wicking- material via that divides the channel into a proximal section and a distal section. In such embodiments, analyte in the proximal section and analyte in the distal section flow in opposite directions on opposite faces of the wicking-material substrate. Also among the embodiments are those in which the waste zone and / or the sample zone is one of a plurality of waste zones and / or sample zones, each of which is connected to a corresponding one of the channels.

[0026] Further embodiments include those having either an absorbent pad or a semi semi- permeable membrane disposed on the sample zone. An example of such a membrane is a blood-filtration membrane that separated plasma from blood.

[0027] In another aspect, the invention features a method that comprises introducing a sample comprising first and second analytes onto a sampling zone on an assay device that comprises a plurality of channels that support capillary flow between the sampling zone and waste zones, the plurality of channels comprising first and second channels, each of which comprises nanoparticles, the first channel comprising a first synthetic receptor that is configured to interact with the first analyte and the second channel comprising a second synthetic receptor that is configured to interact with the second analyte, measuring a first distance along the first channel, measuring a second distance along the second channel, determining a concentration of the first analyte in the sample based on the first distance, and determining a concentration of the second analyte in the second sample based on the second distance. In such a method, the first distance is a distance between a first end of the first channel and a boundary on the first channel and the second distance is a distance between a first end of the second channel and a boundary on the second channel. The boundary on the first channel separates the first channel into a fluorescing segment and a non-fluorescing segment and the boundary on the second channel separates the second channel into a fluorescing segment and a non-fluorescing segment.

[0028] Among the practices of the method are those that include exposing the device to electromagnetic radiation having energy that is selected to initiate fluorescence of the first and second channels.

[0029] These and other features of the invention will be apparent from the following detailed description and the accompanying figures, in which:

[0030] BRIEF DESCRIPTION OF THE FIGURES

[0031] FIG. 1 shows an isometric view of a wicking-material-based analytic device in which the wicking material comprises paper; FIG. 2 shows the analytic device of FIG. 1 after analytes have interrupted fluorescence below a particular boundary;

[0032] FIG. 3 shows a calibration curve for inferring analyte concentrations;

[0033] FIG. 4 shows a process for manufacturing the device of FIG. 1;

[0034] FIG. 5 shows a dual-layer device that uses a paper via;

[0035] FIG. 6 shows several dual-layer devices stacked to form a multilayer device;

[0036] FIG. 7 shows the multilayer device of FIG. 6 spread apart like a fan;

[0037] FIG. 8 shows an isometric view of a wicking-material-based analytic device in which the wicking material comprises thread;

[0038] FIG. 9 shows a multiplexed wicking-material based analytic devices having plural threads of the type shown in FIG. 8;

[0039] FIG. 10 shows the sample zone in FIG. 5 having an absorbent pad disposed thereon; and

[0040] FIG. 11 shows the sample zone in FIG. 5 having a semi-permeable membrane disposed thereon.

[0041] DETAILED DESCRIPTION

[0042] Referring to FIG. 1, a wick-based analytic device 10 comprises a wicking substrate 26 that is made of material that wicks liquid. Examples of such materials include porous or fibrous materials, such as paper and textile. The wicking substrate 26 includes a sample zone 12, a pretreatment zone 14, at least one waste zone 16, and a detection channel 18 that extends between the pre-treatment zone 14 and the waste zone 16. Because of the wicking substrate’s ability to wick, a liquid sample dropped on the sample zone 12 will tend to flow towards the waste zone 16 by capillary action.

[0043] Each detection channel 18 has been provided with a synthetic receptor 32. This synthetic receptor 32 interacts with a particular analyte from a set of analytes, for example by binding to that analyte. Examples of a suitable synthetic receptor 32 include an imprinted polymer, examples of which include a molecularly-imprinted polymer and an ion-imprinted polymer. Each detection channel 18 is also loaded with nanoparticlcs 30 (sec FIG. 4). These nanoparticlcs 30 change state in response to a nearby interaction of the synthetic receptor 32 with that analyte. One way to load the detection channel 18 is to provide a layer of a polymer that has been molecularly imprinted to form cavities and to then add nanoparticles 30. These nanoparticles 30 then collect in the cavities, as shown in FIG. 4.

[0044] A particularly useful state change for the nanoparticles 30 to undergo is one in which the nanoparticles 30 transition between fluorescing and not fluorescing states. In one embodiment, the nanoparticles 30 are carbon nanodots that fluoresce until an interaction with an analyte quenches that fluorescence. Examples of nanoparticles 30 that transition between fluorescent and non-fluorescent states are graphene dots, cellulose nanocrystals, silicon dots, and quantum dots.

[0045] The process of using the device 10 begins by exposing the wick-based analytic device 10 to light having an energy that is appropriate for causing the nanoparticles 30 to fluoresce. With the nanoparticles 30 now fluorescing, the next step is to drop a sample with three analytes into the sample zone 12. One then waits for a pre-determined period to allow the sample to be wicked through the device 10. As sample moves through the detection channel 18, it quenches fluorescence. Therefore, in those portions of the detection channel 18 that have yet to be exposed to the analyte, the nanoparticles 30 fluoresce. In contrast, in those portions of the detection channel 18 that have been exposed to the sample, the nanoparticles 30 will no longer fluoresce.

[0046] As a result, each detection channel 18 develops a boundary 20 between a fluorescing region 22 and a quenched region 24, as shown in FIG. 2. The location of this boundary 20 depends on the initial concentration of the relevant analyte. Therefore, the location of this boundary 20 provides a basis for estimating the concentration of each analyte. To establish a relationship between the initial concentration and the location of the boundary 20, one uses calibration curves, as shown in FIG. 3.

[0047] Referring now to FIG. 4, a process for manufacturing the wick-based analytic device 10 begins with using a wax printer to print a design on the wicking substrate 26. The process continues with sealing the back side of the substrate 26 to prevent solution from leaking through the wicking substrate 26. The next steps are to add phosphate buffer solution and carboxymethylcellulose 28 to the pre-treatment zones 14 and the detection channels 18, respectively.

[0048] At this point, the device 10 is ready to receive the nanoparticles 30. In the preferred embodiment the nanoparticles 30 are carbon nanodots. A drop of a solution containing a solution of nanoparticles 30 is placed on the detection channels 18. The solution is allowed to evaporate, thus leaving behind only the nanoparticles 30.

[0049] This is followed by a step of adding drops of prepared MIP solution to form the synthetic receptor 32, which in this case is a layer of the imprinted polymer. Each layer of the imprinted polymer has been tuned to a different analyte. In the preferred embodiment, the analytes are CRP, TNF-a, and IL-6.

[0050] Finally, the resulting template is removed by using a weak acid, such as acetic acid.

[0051] Another process for manufacturing the wick-based analytic device 10 includes designing a paper pattern having a sample zone 12, pretreatment zones 14, detection channels 18, and waste zones 16. A suitable sample zone 12 is a circle having a twelve-millimeter diameter. Suitable pre-treatment zones 14 are circles having diameters of six millimeters. Suitable detection channels 18 are three millimeters wide, forty millimeters long, have a one-millimeter- thick line disposed inside, and arc spaced apart by one millimeter. Each waste zone 16 is a circle having a six-millimeter diameter. The foregoing structures are on a wicking substrate 26 that is 31 millimeters wide and 118 millimeters long.

[0052] The foregoing structures are printed on a wicking substrate 26, such as filter paper, using a wax printer. A suitable filter paper is Whatman No.l filter paper. A suitable wax printer is the “XEROX COLORQUBE” ™. The wax is melted at 120 °C for two minutes before being cooled at room temperature. This is followed by sealing the paper’s back side with adhesive tape (3M) to prevent the solution from leaking through the wick-based analytic device 10.

[0053] A coating of 3.5% CMC solution is applied to each detection channel 18 and allowed to dry at 25°C for thirty minutes. This is followed by deposition, onto the detection channels 18, of eight microliters of CD solution at a concentration of four grams per liter. After that, 8.0 pL of CD solution at 4.0 g / L was deposited onto these detection channels 18 and then stood at room temperature (25 °C) for 30 min. Subsequently, 10.0 u L of each MIP solution for each analyte was immobilized onto each detection channel 18 and left them dry at room temperature (25 °C) for 30 min for bilirubin, cortisol, CRP, and IL-6, and for 2 hrs. for only creatinine. Next, the templates were removed by pipetting 3 x 8.0 p L of 1.0% acetic acid and then 3 x 8.0 pL of DI water. Later, a single drop at 8.0 pL of the mixture solution of EDC (5.0 mg / mL) and NHS (5.0 mg / mL) was introduced onto each detection channel 18 to block amino groups of polydopamine and then let it stand at room temperature (25 °C) for 30 min.

[0054] In the foregoing embodiments, the flow along the detection channel 18 between the sample zone 12 and the waste zone 16 is generally constrained to be predominantly in one dimension that lies along the plane of the paper. However, this is not a requirement.

[0055] FIG. 5 shows an alternative embodiment in which the detection channel 18 includes a wicking via 34. This wicking via 34 redirects the flow along a direction perpendicular to the plane of the wicking substrate 26 and in a direction towards the sample zone 12. In a preferred embodiment, the wicking via 34 comprises paper. In this embodiment, the sample zone 12 and waste zone 16 are stacked onto each other with an intervening barrier to prevent flow therebetween.

[0056] In the configuration shown in FIG. 5, the wicking via 34 divides the detection channel 18 into a proximal section 36 and a distal section 38.

[0057] In the detection channel’s proximal section 36, the slope of a line that relates concentration to distance to the boundary 20 is steep. This proximal section 36 is used for measurements of concentrations in the nanomolar range of concentrations. Because of the steep slope, this proximal section 36 has picomolar sensitivity.

[0058] The detection channel’s distal section 38 is used for measurements of concentrations in the micromolar range. Because the rate of change of the distance to the boundary as a function of concentration is less steep, this distal section 38 has only nanomolar sensitivity.

[0059] The principle illustrated in FIG. 5 is extensible to further increase the dynamic range of measurement. For example, in FIG. 6, the two-sided device shown in FIG. 5 is coupled to further two-sided devices by additional wicking vias 40 to form extremely long detection channels 18 between the sample zone 12 and the waste zone 16. In this embodiment, it is useful to have a shaft 42 to align the various wicking vias 40. This permits the individual layers to be spread out like a fan as shown in FIG. 7. In some embodiments, the wicking via 40 comprises paper.

[0060] FIG. 8 shows an alternative embodiment of the wick-based analytic device 10 in which the wicking substrate 26 comprises a thread 44 that includes a sample zone 12, a waste zone 16, and a detection channel 18 extending therebetween. The thread is shown disposed on a chassis 46. Suitable threads 44 are those that comprise cotton, polyester, or combinations thereof. In this embodiment, the nanoparticles 30 are integrated into the thread 44.

[0061] A wick-based analytic device 10 that uses a thread 44 as a wicking substrate 26 arises avoids the need for a wax printer. This is useful because wax printers are increasingly difficult to acquire and maintain.

[0062] FIG. 9 shows a multiplexed version of the embodiment shown in FIG. 8 in which each thread 44 in a plurality of threads has been functionalized for a different molecule. The operation of the embodiments shown in FIGS. 8 and 9 is similar to that described in connection with FIGS. 1 and 2.

[0063] In some cases, it is useful to use a fixed volume of liquid as a sample. For small quantities, the relevant volume can be placed directly on the sample zone 12. However, the sample zone 12 is only able to hold a small amount of liquid without becoming saturated. To address this difficulty, it is useful to provide an absorbent pad 48 on the sample zone 12. When saturated, the absorbent pad 48 acts as a reservoir of the liquid. As liquid wicks away from the sample zone 12, the absorbent pad replaces what has been wicked way until its own supply has been depleted.

[0064] In other cases, an analyte of interest is found in a mixture that contains substances that would interfere with the operation of the wick-based analytic device 10. For example, there exist cases in which the analyte of interest is found in blood plasma. If one were to drop whole blood onto the sample zone 12, the various solid constituents of whole blood can interfere with the wicking process. One solution to this difficulty is to separate plasma from whole blood and dropping just the plasma onto the sample zone 12. However, this can be inconvenient.

[0065] An alternative solution, shown in FIG. 12, is to introduce a semi-permeable membrane 50 above the sample zone 12. A useful type of semi-permeable membrane 50 is one that allows plasma to pass through and that retains those components of whole blood that would otherwise interfere with the wicking process.

[0066] Having described the invention and a preferred embodiment thereof, what we claim as new and secured by letters patent is:

Claims

CLAIMS1. An apparatus for assaying analytes from a set of analytes, wherein said analytes comprise first and second analytes, said apparatus comprising a wick-based analytic device that comprises a wicking substrate having a sample zone, a waste zone, and a plurality of channels, each of which is a detection channel that is configured to support capillary flow between said sample zone and said waste zone, said plurality of channels comprising a first channel and a second channel, wherein said first channel comprises a first synthetic receptor and nanoparticles that are configured to undergo a state change in response to interaction of said first synthetic receptor with said first analyte, wherein said second channel comprises a second synthetic receptor and nanoparticles that are configured to undergo a state change in response to interaction of said second synthetic receptor with said second analyte, wherein an extent of said state change along said first channel is indicative of concentration of said first analyte, and wherein an extent of said state change along said second channel is indicative of a concentration of said second analyte.

2. The apparatus of claim 1, wherein said state change is a transition between a fluorescent state of said nanoparticles and a non-fluorescent state of said nanoparticles.

3. The apparatus of claim 1, wherein said state change is a transition from a fluorescent state of said nanoparticles to a non-fluorescent state of said nanoparticles, said transition being caused by exposure of said first synthetic receptor to said first analyte.

4. The apparatus of claim 1, wherein said nanoparticles comprise carbon nanodots.

5. The apparatus of claim 1, wherein said nanoparticles comprise cellulose nanocrystals.

6. The apparatus of claim 1, wherein said nanoparticles comprise silicon dots.

7. The apparatus of claim 1, wherein said nanoparticles comprise quantum dots.

8. The apparatus of claim 1, wherein said first synthetic receptor comprises a first molecularly-imprinted polymers and said second synthetic receptor comprises a second molecularly-imprinted polymer.

9. The apparatus of claim 1, wherein said analytes comprise cytokines.

10. The apparatus of claim 1, wherein said first channel comprises a first molecularly- imprinted polymer that has been configured to interact with said first analyte, as a result of which fluorescence of nanoparticles proximate to a site of said interaction is quenched and wherein said second channel comprises a second molecularly-imprinted polymer that has been configured to interact with said second analyte, as a result of which fluorescence of nanoparticles proximate to a site of said interaction is quenched.

11. The apparatus of claim 1, wherein said plurality of analytes further comprises a third analyte and wherein said plurality of channels further comprises a third channel that is configured to support capillary flow between said sample zone and said waste zone.

12. The apparatus of claim 1, wherein said first channel comprises a wicking via that divides said channel into a proximal section and a distal section, wherein analyte in said proximal section and analyte in said distal section flow in opposite directions on opposite faces of said wicking substrate.

13. The apparatus of claim 1, wherein said waste zone is one of a plurality of waste zones, each of which is in fluid communication with a corresponding one of said channels.

14. The apparatus of claim 1, wherein said sample zone is one of a plurality of sample zones, each of which is in fluid communication with a corresponding one of said channels.

15. The apparatus of claim 1, wherein said wicking substrate comprises paper.

16. The apparatus of claim 1, wherein said wicking substrate comprises a thread.

17. The apparatus of claim 1, further comprising an absorbent pad that is disposed on said sample zone.

18. The apparatus of claim 1, further comprising a semi-permeable membrane disposed on said sample zone.

19. A method comprising introducing a sample comprising first and second analytes onto a sampling zone on a wick-based analytic device that comprises a plurality of channels that support capillary flow between said sampling zone and waste zones, said plurality ofchannels comprising first and second channels, each of which comprises nanoparticles, said first channel comprising a first synthetic receptor that is configured to interact with said first analyte and said second channel comprising a second synthetic receptor that is configured to interact with said second analyte, measuring a first distance along said first channel, measuring a second distance along said second channel, determining a concentration of said first analyte in said sample based on said first distance, and determining a concentration of said second analyte in said second sample based on said second distance, wherein said first distance is a distance between a first end of said first channel and a boundary on said first channel, wherein said second distance is a distance between a first end of said second channel and a boundary on said second channel, wherein said boundary on said first channel separates said first channel into a fluorescing segment and a non-fluorescing segment, and wherein said boundary on said second channel separates said second channel into a fluorescing segment and a non-fluorescing segment.

20. The method of claim 19, further comprising, prior to introducing said sample, exposing said device to electromagnetic radiation having energy that is selected to initiate fluorescence of said first and second channels.

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