Fabrication and application of highly wicking cellulose microneedles

Abstract

The present disclosure is directed to methods for fabricating cellulose-based microneedles with exceptional wicking properties which can be used for rapid extraction of dermal interstitial fluid (ISF). Microneedle arrays are fabricated through a combination of compression molding and laser etching, utilizing microcrystalline cellulose, a biocompatible material with excellent wicking properties. These microneedles are mechanically robust and can autonomously extract fluid from the skin without the need for external instruments. As such, they can be directly integrated with paper-based lateral flow immunoassays for point-of-care disease detection.

Description

[0001] DESCRIPTION

[0002] FABRICATION AND APPLICATION OF HIGHLY WICKING CELLULOSE

[0003] MICRONEEDLES

[0004] PRIORITY CLAIM

[0005] This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 704,740, filed October 8, 2024, the entire contents of which are hereby incorporated by reference.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0007] This invention was made with government support under Grant No. 1842494 awarded by the National Science Foundation. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] 1. Field of the Disclosure

[0010] The present disclosure relates generally to the fields medicine, bioengineering and in vitro diagnostics. More particularly, the disclosure relates to methods of generating microneedles and microneedle arrays using microcrystalline cellulose (MCC) and their applications in diagnostic testing of interstitial fluid obtained through the use of said microneedles (MNs) and MN arrays.

[0011] 2. Background

[0012] Interstitial fluid (ISF), the fluid that surrounds cells and tissues in the human body, is a promising diagnostic fluid due to its nearly identical protein composition as blood [1], while being safer and less invasive to collect. Unfortunately, the collection of ISF from skin (dermal ISF) is challenging. Prior reports have demonstrated the extraction of dermal ISF using hollow [1] or solid microneedles [2, 3], however these methods are time-consuming (> 1 h) and / or require the use of external instruments (e.g., vacuum pumps). Thus, improved methods are urgently needed. SUMMARY

[0013] Thus, in accordance with the present disclosure, there is provided a method of preparing a microneedle comprising:

[0014] (a) adding microcrystalline cellulose to a rigid mold;

[0015] (b) compacting said microcrystalline cellulose;

[0016] (c) releasing compacted microcrystalline cellulose from the mold; and

[0017] (d) etching the compacted microcrystalline cellulose, thereby forming a microneedle. Step (b) may comprise compacting with a mechanical press. Step (d) may comprise etching using a CO2 laser cutter. The microneedle may be part of a microneedle array formed according to steps (a)-(d), such as a 5x5 array, and / or wherein the microneedles may have a height of 0.5-2 mm (e.g., about 1 mm), and / or wherein the microneedles may have a base diameter of 400-750 pm (e.g., about 550 pm). The microcrystalline cellulose may be prepared by mixing cellulose with about 0.5-20% glutaraldehyde. Also provided is a microneedle or an array of microneedles formed according to these methods.

[0018] Further provided is a microneedle or an array of microneedles comprising microcrystalline cellulose. The microneedle or each microneedle of the array may have a height of 0.5-2 mm (e.g., about 1 mm), and / or wherein each microneedle may have a base diameter of 400-750 pm (e.g., about 550 pm), and / or the array may be in a 5x5 configuration. The microcrystalline cellulose may comprise about 0.5-20% of a binder, such as glutaraldehyde, chitosan or genipin.

[0019] In addition, there is provided a method of obtaining interstitial fluid from a tissue, such as a tissue in a subject, comprising contact microneedle or array of microneedles as described herein. The subject may be suspected of having a disease or at risk of developing a disease. The method may further comprise the step of detecting an analyte in said interstitial fluid. The analyte may be a cell, a protein, a lipid, a metabolite, a horomone, a carbohydrate or a nucleic acid. The protein analyte may be an antigen, a cancer / tumor marker, a toxin, or a cytokine. Detecting may comprise performing an immunodetection assay on said interstitial fluid, such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), immunoblotting, or a lateral flow immunoassay, mass spectroscopy, or cell sorting, such as flow cytometry. Also provided is a kit comprising a microneedle or array of microneedles as described herein. The kit may further comprise a protein, lipid, metabolite, a horomone, or carbohydrate detection agent.

[0020] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0021] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024] FIGS. 1A-D. Overview of the cellulose MN array fabrication process. The mold is filled with MCC (FIG. 1 A), compressed using a mechanical press (FIG. 1 B), and etched using a CO2 laser cutter (FIG. 1C). (FIG. ID) Optical micrograph of the cellulose MN array. Scale bar, 1000 pm.

[0025] FIGS. 2A-D. Characterization of MNs. (FIG. 2A) Influence of the laser etching parameters on the MN height. Key = top to bottom same as left to right. (FIG. 2B) Optical micrograph of porcine skin with dyed MN penetration sites. Optical micrographs of the dyed MNs before (FIG. 2C) and after (FIG. 2D) insertion into porcine skin. Scale bars, 1000 pm.

[0026] FIG. 3. Fluid wicking characteristics of the cellulose MNs. Sequential still frame images showing blue dye being wicked by the MN array. Arrows indicate the liquid front. Timestamps (min:s) are located in the upper right corners. Scale bars, 100 pm.

[0027] FIGS. 4A-D. (FIG. 4A) Photograph of the MN-LFIA device. (FIG. 4B-C) Sequential still frame images showing fluid extraction and transport in the LFIA. Scale bars, 5 mm. (FIG. 4D) Enlarged view of the test and control dots, indicating a positive result. Scale bar, 1 mm. Timestamps (min:s) are located in the upper right corners.

[0028] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0029] As discussed above, ISF is a promising diagnostic fluid, but there are challenges in in obtaining ISF samples that limit its used. Here, the inventors describe highly wicking cellulose microneedles (MNs), enabling rapid, instrument-free fluid extraction, transport, and analysis. The methods for fabricating cellulose-based MNs with exceptional wicking properties provide materials that can be used for rapid extraction of dermal ISF. MN arrays are fabricated through a combination of compression molding and laser etching, utilizing MCC, a biocompatible material with excellent wicking properties. Cellulose can be mixed with a concentration (0.5- 20%) of glutaraldehyde, a cross-linking polymer, or other binders such as chitosan or genipin, and packed into a rigid mold then compressed using a mechanical press. The resulting compressed cellulose block may then be etched with a CO2 laser cutter, producing sharp MNs with a controlled and consistent structure. Alternatively, a MN or MN array can be dip-coated with glutaraldehyde or other binders, such as chitosan or genipin. These MNs are mechanically robust and can autonomously extract fluid from the skin without the need for external instruments, a significant advancement over previous methods that relied on hollow or solid MNs, which were either time-consuming or required additional equipment such as vacuum pumps. Additionally, the MNs can be directly integrated with paper-based lateral flow immunoassays and biosensors for point-of-care disease detection.

[0030] These and other aspects of the disclosure are described in detail below.

[0031] I. Interstitial Fluid

[0032] Interstitial fluid, or ISF, is the body fluid between blood vessels and cells, containing nutrients from capillaries by diffusion and holding waste products discharged by cells due to metabolism. It is the main component of extracellular fluid. Eleven liters of the ECF are ISF and the remaining three liters are plasma. Plasma and ISF are very similar because water, ions, and small solutes are continuously exchanged between them across the walls of capillaries, through pores and capillary clefts. ISF is essentially comparable to plasma. The ISF and plasma make up about 97% of the ECF, and a small percentage of this is lymph.

[0033] ISF consists of a water solvent containing sugars, salts, fatty acids, amino acids, coenzymes, hormones, neurotransmitters, white blood cells and cell waste-products. This solution accounts for 26% of the water in the human body. The composition of ISF depends upon the exchanges between the cells in the biological tissue and the blood. This means that tissue fluid has a different composition in different tissues and in different areas of the body. The plasma that filters through the blood capillaries into the interstitial fluid does not contain red blood cells or platelets as they are too large to pass through but can contain some white blood cells to help the immune system.

[0034] Once the extracellular fluid collects into small vessels (lymph capillaries) it is considered to be lymph, and the vessels that carry it back to the blood are called the lymphatic vessels. The lymphatic system returns protein and excess interstitial fluid to the circulation.

[0035] The ionic composition of the interstitial fluid and blood plasma vary due to the Gibbs- Donnan effect. This causes a slight difference in the concentration of cations and anions between the two fluid compartments.

[0036] IL Microneedles (MNs) and Microneedle Arrays

[0037] Microneedles (MNs). MNs are micron-scaled medical devices used to administer vaccines, drugs, and other therapeutic agents, or to sample biofluids such as blood or ISF. MNs are usually applied through a single needle or an array of needles, called a MN patch or microarray patch. MN arrays can range from only a few microneedles to several hundred, attached to an applicator, sometimes a patch or other solid stamping device. The height of each needle can range from 25 .m to 2000pm. The arrays are applied to the skin and are given time to allow for the effective administration of drugs or sampling of biofluids.

[0038] MNs can be constructed through various methods, usually involving photolithographic processes, micromolding or stereolithography. These methods involve etching microscopic structure into resin or silicon in order to cast microneedles. MNs are made from a variety of materials ranging from silicon, titanium, stainless steel, and polymers. A variety of MNs (solid, hollow, coated, hydrogel) have been developed to possess different functions. Some MNs are made of a drug to be delivered to the body but are shaped into a needle so they will penetrate the skin. The MNs range in size, shape, and function but are all used as an alternative to other delivery methods like the conventional hypodermic needle or other injection apparatus. Stimuli-responsive MNs are advanced devices that respond to environmental triggers such as temperature, pH, or light to release therapeutic agents.

[0039] Microcrystalline cellulose. In the present disclosure, MCC is used with significant advantage. A naturally occurring polymer, it is composed of glucose units connected by a 1-4 beta glycosidic bond. These linear cellulose chains are bundled together as microfibril spiraled together in plant cell walls.

[0040] Each microfibril exhibits a high degree of three-dimensional internal bonding resulting in a crystalline structure that is insoluble in water and resistant to reagents. There are, however, relatively weak segments of the microfibril with weaker internal bonding. These are called amorphous regions; some argue that they are more accurately called dislocations, because of the single-phase structure of microfibrils. The crystalline region is isolated to produce MCC.

[0041] MN arrays. In some embodiments, a single MN is provided on the support base. In other cases, a plurality of MNs may be disposed closely together on the support base. When a plurality of MNs are disposed, the MNs may be arranged in an array. As used herein, the term “array” means that microneedles are arranged in a specific pattern, for example in a matrix arrangement, concentric circle arrangement, or random arrangement.

[0042] As described herein, MNs can be hollow or solid. MNs can be produced by any method that yields MNs and MN arrays that are smooth and sharp enough to penetrate skin.

[0043] The MN array described herein may comprise needles with different lengths, e.g., as measured from a base or support. In some embodiments, the needles are about 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm, 1250 pm, 1500 pm, 1750 pm, 2000 pm in, e.g., in length, e.g., as measured from a base or support. In some embodiments, the needles are greater than about 100 pirn, 150 pirn, 200 pirn, 250 pirn, 300 pirn, 350 pirn, 400 pirn, 450 pirn, 500 pm, 550 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm, 1250 pirn, 1500 pm, 1750 pirn, 2000 pm in, e.g., in length, e.g., as measured from a base or support. In some embodiments, the needles are less than about 100 pirn, 150 pirn, 200 pirn, 250 pirn, 300 pm, 350 pirn, 400 pirn, 450 pm, 500 pirn, 550 pirn, 600 pirn, 650 pun, 700 pun, 750 pun, 800 pm, 900 pm, 950 pm, 1000 pm, 1250 pm, 1500 pm, 1750 pm, 2000 pm in, e.g., in length, e.g., as measured from a base or support.

[0044] The MN array described herein may comprise needle tips with different radii. In some embodiments, the needle tip radii are about 10 pun, 25 pun, 50 pm, 60 pm, 70 pun, 80 pun, 90 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm in, e.g., diameter or longest linear dimension. In some embodiments, the needle tip radii are greater than about 10 pm, 25 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm in, e.g., diameter or longest linear dimension. In some embodiments, the needle tip radii are less than about 10 pm, 25 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, 600 m, 650 pm, 700 pm, 750 pm, 800 pm, 900 pun, 950 pirn, 1000 pun in, e.g., diameter or longest linear dimension.

[0045] In certain embodiments, MCC is formed into a MN using laser cutting / etching. A particular form of laser etching uses a carbon-dioxide laser (CO2 laser).

[0046] III. Diagnostic Applications

[0047] In still further embodiments, the present disclosure concerns immunodetection methods for quantifying and otherwise generally detecting protein biomarkers in ISF, such as protein antigens, antibodies, cytokines, tumor markers and infectious disease markers. A wide variety of assay formats are contemplated. The assays may be advantageously formatted for nonhealthcare (home) use, including lateral flow immunoassays (see below) analogous to home pregnancy and rapid COVID-19 antigen tests. These assays may be packaged in the form of a kit with appropriate reagents and instructions to permit use by the subject of a family member.

[0048] Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation is contemplated. In general, the immunodetection methods include obtaining a sample suspected of containing the target protein, and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[0049] These methods include methods for detecting and quantifying other analytes from a sample. The bioreceptor, such as an antibody, will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the target analyte will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the target analyte immunocomplexed to the immobilized antibody.

[0050] The immunobinding methods also include methods for detecting and quantifying the amount of target analyte in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing the target analyte and contact the sample with a bioreceptor, i.e., antibody that binds components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of target analyte detection, the biological sample analyzed may be any biofluid sample, including those from individuals suspect of having a disease or condition. Contacting the chosen biofluid sample with the bioreceptor under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to target protein(s). After this time, the protein-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0051] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art.

[0052] The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0053] Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[0054] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, for example, with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0055] Lateral flow immunoasays, also known as lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in sample (matrix) without the need for specialized and costly equipment, though many laboratory-based applications exist that are supported by reading equipment. Typically, these tests are used for home testing, point of care testing, or clinical testing. A widely spread and well-known application is the home pregnancy test or rapid COVID- 19 antigen test.

[0056] The technology is based on a series of capillary beds, such as pieces of porous paper or sintered polymer. Each of these elements has the capacity to transport fluid spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) which has stored the so-called conjugate, a dried format of bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner e.g., antibody) that has been immobilized on the particle's surface. While the sample fluid dissolves the salt-sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third 'capture' molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripearea changes color. Typically, there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked fine, the second contains a specific capture molecule and only captures those particles onto which an analyte molecule has been immobilized. After passing these reaction zones, the fluid enters the final porous material - the wick - that simply acts as a waste container, lateral flow immunoassays can operate as either competitive or sandwich assays. Lateral flow assays are disclosed in U.S. Patent 6,485,982.

[0057] Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.

[0058] IV. Kits

[0059] In still further embodiments, the present disclosure concerns kits for use with the MNs and MN arrays described herein. Analyte detection molecules, such as antibodies, may be included in the kit. The kits will thus comprise, in suitable container means, the aforementioned components. Solid support, such as a column matrix and / or well of a microtiter plate, may be included along with agents for performing control reactions.

[0060] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the various reagents may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the reagents in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. V. Examples

[0061] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0062] Example 1 - Materials and Methods

[0063] MN arrays were fabricated via a compression molding and laser etching technique (FIGS. 1 A-C). Briefly, MCC was packed into a rigid mold and compressed using a mechanical press. The compressed cellulose block was released from the mold and etched using a CO2 laser cutter, resulting in the formation of sharp MNs (FIG. ID). MN arrays were applied to cadaver porcine skin and colored dye to evaluate their skin penetration performance and wicking characteristics. For proof-of-concept demonstration, a MN array was integrated with a LFIA, which was prepared as previously described [4]. 1,600 pg / mL of anti-PfHRP2 IgM and 400 pg / mL of mouse IgG were spotted on the LFIA strip and used as test and control dots, respectively. The integrated MN-LFIA device was tested on an artificial skin model (2% agar gel covered with Parafilm) spiked with 1,000 ng / mL of PfHRP2, a malaria parasite biomarker.

[0064] Example 2 - Results

[0065] The inventors briefly characterized the laser etching process, which revealed that the MN dimensions could be accurately controlled by adjusting the laser power and rastering speed (FIG. 2A). Using the optimized parameters, they fabricated 5 x 5 MN arrays with a needle height of -1 mm and a base diameter of ~55O pm. The penetration capability of the MNs was verified by inserting the MN array into porcine skin (FIGS. 2B-D), resulting in confined penetration sites. The wicking performance of the MN array revealed its ability to extract liquid and become fully wetted within 5 min (FIG. 3). Finally, using the integrated MN-LFIA device, the inventors demonstrate capillary -based fluid extraction from the skin model in 18 min and in situ PfHRP2 detection in 30 min (FIGS. 4A-D). Example 3 - Conclusions

[0066] The inventors report a novel cellulose MN array, which exhibits exceptional wicking properties, while being mechanically robust, low cost and highly biocompatible. By integrating the MN array with a LFIA, they demonstrate rapid, instrument-free fluid extraction and in situ protein biomarker detection, validating the functionality of this technology for rapid diagnostic testing.

[0067] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0068] VI. References

[0069] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0070] 1. P. R. Miller et al., Commun. Biol., vol. l , no. l , 2018, doi: 10.1038 / s42003-018-0170- z.

[0071] 2. X. Jiang, E.C. Wilkirson, et al., Cell Reports Phys. Sci. 101975, 2024, doi: 10.1016 / j.xcrp.2024.101975.

[0072] 3. P. P. Samant et al., Sci. Transl. Med., vol. 12, no. 571, pp. 1-16, 2020, doi: 10.1126 / scitranslmed.aaw0285.

[0073] 4. X. Jiang et al., Microsystems Nanoeng., vol. 6, no.l, pp. 1-11, 2020, doi: 10.1038 / s41378-020-00206-l.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a microneedle comprising:(a) adding microcrystalline cellulose to a rigid mold;(b) compacting said microcrystalline cellulose;(c) releasing compacted microcrystalline cellulose from the mold; and(d) etching the compacted microcrystalline cellulose, thereby forming a microneedle.

2. The method of claim 1 , wherein step (b) comprises compacting with a mechanical press and / or wherein step (d) comprises etching using a CO2 laser cutter.

3. The method of claim 1 or claim 2, wherein the microneedle is part of a microneedle array formed according to steps (a)-(d), such as a 5x5 array, and / or wherein the microneedles have a height of 0.5-2 mm (e.g., about 1 mm), and / or wherein the microneedles have a base diameter of 400-750 pm (e.g., about 550 pm).

4. The method of any one of claims 1-3, wherein the microcrystalline cellulose is prepared by mixing cellulose with about 0.5-20% of a binder, such as glutaraldehyde, chitosan or genipin.

5. The method of any one of claims 1-4, wherein the microneedle or microneedle array is coated with a chemical, such as glutaraldehyde, chitosan or genipin.

6. A microneedle or an array of microneedles formed according to the method of claims 1-5.

7. A microneedle or an array of microneedles comprising microcrystalline cellulose.

8. The microneedle or array of microneedles of claim 7, wherein microneedle(s) has / have a height of 0.5-2 mm (e.g., about 1 mm), and / or wherein each microneedle has / have a base diameter of 400-750 pm (e.g., about 550 pm) and / or the array is in a 5x5 configuration.

9. The microneedle or array of microneedles of claim 7 or claim 8, wherein the microcrystalline cellulose comprises about 0.5-20% of a binder, such as glutaraldehyde, chitosan or genipin.

10. A method of obtaining interstitial fluid from a tissue, such as a tissue in a subject, comprising contact microneedle or array of microneedles according to claims 6-9 with said tissue.

11. The method of claim 10, wherein the subject is suspected of having a disease or at risk of developing a disease.

12. The method of claim 10 or 11, further comprising the step of detecting an analyte in said interstitial fluid.

13. The method of claim 12, wherein said analyte is cell, a protein, a lipid, a carbohydrate, a metabolite, a hormone or a nucleic acid.

14. The method of claim 13, wherein said protein is an antigen, a cancer / tumor marker, a toxin, or a cytokine.

15. The method of any one of claims 11-14, wherein detecting comprises performing an immunodetection assay on said interstitial fluid.

16. The method of claim 15, wherein said immunodetection assay is an ELISA, an RIA, immunoblotting, or a lateral flow assay.

17. The method of any one of claims 11-14, wherein detecting comprises mass spectroscopy.

18. The method of any one of claims 11-14, wherein detecting comprises cell sorting, such as flow cytometry.

19. A kit comprising a microneedle or array of microneedles according to claims 6-9.

20. The kit of claim 19, further comprising a protein, lipid, carbohydrate, metabolite, or hormone detection agent.

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