A substrate functionalized with antioxidant for biological sample collection and preservation

Abstract

A functionalized paper product (100), including: polysaccharide fibers (102); and an antioxidizing agent (106) covalently bonded to the polysaccharide fibers.

Description

Attorney Docket No. 10457-615PC0A SUBSTRATE FUNCTIONALIZED WITH ANTIOXIDANT FOR BIOLOGICAL SAMPLE COLLECTION AND PRESERVATIONFIELD

[0001] The invention relates to a functionalized substrate for biological sample collection and preservation.BACKGROUND

[0002] From a patient's perspective, little has changed in the field of biological sample collection for disease and health testing in the past several decades. For example, for conventional blood sample collection, patients typically must travel to the collection site, present an arm, and trust that the phlebotomist hits a vein on the first try, all of which can be inconvenient and stressful. From the perspective of collecting the conventional blood samples, there are countless safety precautions, many hours of training, and a rigorous schedule of how samples must be handled and shipped to ensure the safety of everyone involved. In addition, the cost of shipping samples under cold conditions is expensive and often limited to wealthy countries.

[0003] Dried Blood Spot (DBS) sampling has gained widespread acceptance as a convenient, low stress, and minimally invasive technique for disease screening and diagnosis globally across large populations.1-3Unlike traditional venipuncture-based blood collection methods, which require large volumes of blood that are stored and shipped under stringent conditions, DBS sampling enables the collection of just a few blood drops onto filter paper, which is conducive to easy shipping and storage.2Dried blood spot samples can be easily transported and mailed without temperature control to laboratories for analysis. The versatility and simplicity of DBS sampling and analysis has made it a preferred method in various clinical and research settings.4, 5Importantly, DBS analysis facilitates the quantification of metabolites crucial for diagnosing and monitoring rare metabolic disorders across the world.6DBS sampling and resulting diagnostics helps to eliminate health disparities because DBS samples are more economical to ship, allow for home sampling, and reach rural populations where access to traditional blood collection is notAttorney Docket No. 10457-615PC0available.7, 8With enhanced adaptations, DBS samples can ameliorate downstream analytical challenges associated with the imperfect storage / shipping conditions experienced.

[0004] However, there remains room in the art for better preserving the dried blood spot samples.BRIEF DESCRIPTION OF DRAWINGS

[0005] Figure 1 shows an analysis of keto- and enol- indole-3-pyruvate in DBS paper 1) without vitamin C (top), 2) with paper functionalized with vitamin C and washed one time to remove any non-covalently bound vitamin C (middle), and 3) with paper functionalized with vitamin C and washed two times (bottom) to remove any non-covalently bound vitamin C.

[0006] Figure 2 shows an example embodiment of a functionalized paper product.

[0007] Figure 3 shows an example embodiment of a method.

[0008] Figure 4 shows an example embodiment of a blood sampling kit.

[0009] Figures 5A and 5B show example embodiments of a method.

[0010] Figure 6 pertains to a graph showing preservation of GSSG using a functionalized paper embodiment described herein.

[0011] Figure 7 provides a graph demonstrating preservation of GSH using a functionalized paper embodiment described herein.

[0012] Figure 8 provides a graph demonstration preservation of cysteine using a functionalized paper embodiment described herein.DETAILED DESCRIPTION

[0013] The present inventors have developed a unique and innovative way to help preserve biological samples.

[0014] The term “biological sample” (“biospecimen”) as used herein refers to a sample obtained from a biological source, including human or nonhuman animals, plants, or microorganisms. Biological samples may include but are not limited to, a biological fluid, tissue sample, cell sample, tissue lysate, or components thereof obtained from an animal. Biological fluids may include but are not limited to blood or blood components (e.g. serum), saliva, tears, sweat, vaginal discharge, mucous, semen, urine, gastric fluid, bile, or feces including. A biological sample may also include blood or blood component spiked with a tissue lysate from the subject.Attorney Docket No. 10457-615PC0Typically, but not necessarily, the blood or blood component sample is incubated with the tissue lysate for a period of time to allow biomolecules to interact. The disclosure herein focuses on the collection of dried blood spot (“DBS”) samples. However, the technology disclosed herein applies to the collection of other biospecimens.

[0015] An example embodiment includes modifying a substrate to prevent, not correct for, degradation of a blood sample. This is done through covalent attachment of antioxidants (antioxidizing agent) to polysaccharide fibers in the substrate.

[0016] As used herein, a substrate is any material that can be used to collect a biospecimen and that is composed at least in part of polysaccharide fibers. An example substrate is paper. In an example embodiment, the polysaccharide fibers are a structural component of the paper, however, the fibers need not be structural. Other substrates include a swatch, a cotton swab, etc. Example polysaccharides include cellulose, chitin, alginate etc. The disclosure herein focuses on paper composed at least in part of cellulose. However, the technology disclosed herein applies to other polysaccharides in paper and to other substrates than paper.

[0017] The antioxidizing agent is covalently bonded to monomers of the polysaccharide. The disclosure herein focuses on covalent bonding of the antioxidizing agent to the glucose monomers in the cellulose fibers. However, the technology disclosed herein also applies to covalent bonding of the antioxidizing agent to the respective monomer (e.g., sugar or hydroxy group containing monomer) of the selected polysaccharide.

[0018] Example antioxidants include vitamin C (ascorbic acid), vitamin E, ferulic acid, vitamin A, beta-carotene, etc. Vitamin C is a potent antioxidant that has been shown to reduce reactive oxygen species (ROS) and oxidative stress.9It has been tested as an additive to prevent oxidation of red cells for blood transfusions. It has been shown that the addition of vitamin C prior to DBS collection prevents oxidative processes from occurring, thereby stabilizing metabolomes.9Importantly, the inventors discovered a key set of indole- 3 -pyruvate (IPyA) tautomers that can indicate proper preservation of the blood sample. Recently, IPyA has been reported to be an oncometabolite, raising interest in proper measurement from blood.10The disclosure herein focuses on vitamin C as the antioxidant. However, the technology disclosed herein also applies to the use of other antioxidants.

[0019] Covalently bonded antioxidants are immobilized (e.g., not released into the blood sample). The covalently bonded antioxidants maintain the same antioxidant capacity but do notAttorney Docket No. 10457-615PC0interfere with downstream analysis like unbonded antioxidants would. Paper that is functionalized with the antioxidant in this way and used for DBS sample collection can prevent oxidative damage to analytes and molecules in the blood and thereby preserve or stabilize the analytes and molecules.

[0020] The inventors now disclose attaching ascorbic acid to two-dimensional cellulose paper by adapting a process disclosed by Edwards.11The Edwards process attaches ascorbic acid to cotton fibers in three-dimensional clothing to prevent microbial activity. In the modified process, an antioxidant is covalently bonded to glucose monomers of cellulose polymers in a manner similar to how Edwards covalently bonds vitamin C to cellulose in cotton. The modified process thereby forms a mixture of functionalized cellulose (cellulose with antioxidant covalently bonded thereto) and unbonded antioxidant (plus any linking agent). Where the Edwards process stops at the formation of the mixture, the process disclosed herein removes the unbonded antioxidant (and any linking agent) from the functionalized cellulose, leaving washed functionalized cellulose. The functionalized paper is thereby composed of the washed functionalized cellulose.

[0021] To remove the unbonded antioxidant from the functionalized cellulose, the functionalized cellulose may be washed using, for example, distilled water.

[0022] The cellulose that is functionalized may already be in the form of paper that is processed as described herein into the functionalized paper product. Alternately, the cellulose may be functionalized prior to being formed into the functionalized paper product. The functionalized cellulose may be washed before or after being formed into the functionalized paper product. In each case, the functionalized paper product may be composed of washed functionalized cellulose alone or it may be composed of washed functionalized cellulose together with unfunctionalized cellulose. In an example embodiment, the washed cellulose has a concentration of unbonded antioxidant of not more than 100 nanomolar. In an example embodiment, the functionalized paper product is essentially free of the unbonded antioxidant. As used herein, essentially free means a functionalized paper product formed using washed cellulose having a concentration of unbonded antioxidant of not more than 100 nanomolar. Alternatively, it is possible for the functionalized paper product to include functionalized cellulose that has not been washed of the unbonded antioxidant or that has only been partially washed of the unbonded antioxidant.Attorney Docket No. 10457-615PC0EXPERIMENTAL

[0023] Ahlstrom 226 paper is a common substrate use for DBS sample collection, and it is used in this example. A solution that contained 9% citric acid, 5% ascorbic acid, and 3% sodium hypophosphite was prepared. Paper squares (6x6 mm) were submerged in twenty times the weight of the paper to saturate the squares with the solution. The squares were then dried for three minutes at 95° C and cured for three minutes at 100° C on a heating block. The squares were washed in some cases once and in other cases twice by submerging the squares in distilled water. The washing was followed by drying at 95° C for three minutes each time. The washing is to remove any excess linking agent (citric acid) and any antioxidant (ascorbic acid) that was not functionalized. Even with washes, it was not clear if all vitamin C had been removed because vitamin C still appeared in the analysis. Functionalization of the cellulose prior to formation of the functionalized paper containing the functionalized cellulose may reduce the amount of excess citric acid and unfunctionalized ascorbic acid. However, for now the imperfect functionalized cellulose can be assayed with the understanding residual unfunctionalized vitamin C can be interfering with preservation.

[0024] Next, a blood sample was added and was evaluated for metabolite response with and without the antioxidant vitamin C in the paper product. The initial markers of successful preservation (as disclosed in U.S. patent application number 18 / 409,738 by Garrett et al.) were used to evaluate performance, (i.e., the keto- and enol- tautomers of indole-3-pyruvate).

[0025] Figure 1 shows an analysis of keto- and enol- indole-3-pyruvate 1) in the paper without vitamin C (top), 2) with the paper functionalized with vitamin C and washed one time to remove any non-covalently bound vitamin C (middle), and 3) with the paper functionalized with vitamin C and washed two times to remove any non-covalently bound vitamin C (bottom). The peaks at 6.8 and 7.0 min are the tautomers of indole-3-pyruvate. The DBS samples on the functionalized paper clearly show increased signals for both keto- and enol indole-3-pyruvate.

[0026] In addition, stabilization of 2-hydroxyglutarate (2-HG), a well-known oncometabolite related to isocitrate dehydrogenase (IDH), was similarly observed. The preliminary data withAttorney Docket No. 10457-615PC0vitamin C functionalized to cellulose indicates a dramatic increase in the signal for 2-HG and thereby shows the potential to stabilize the oncometabolome.

[0027] EMBODIMENTS

[0028] As shown in FIG. 2, in an example embodiment, a functionalized paper product 100 includes cellulose fibers 102 composed of glucose monomers 104, and an antioxidizing agent 106 covalently bonded to the glucose monomers 104 of the cellulose fibers 102 via an intervening linking agent 108. Cellulose fibers 102 and antioxidizing agent 106 covalently bonded thereto collectively form functionalized cellulose. Functionalized cellulose may or may not be washed to reduce or eliminate unbonded antioxidizing agent 106 (and any linking agent used in the formation of the functionalized cellulose). Functionalized cellulose that has been washed is deemed to be washed functionalized cellulose 102WF.

[0029] The functionalized paper product 100 may be composed of functionalized cellulose and optionally unfunctionalized cellulose. The functionalized cellulose may be all (unwashed) functionalized cellulose, may be all washed functionalized cellulose 102WF, or may be a combination of (unwashed) functionalized cellulose and washed functionalized cellulose 102WF. The functionalized cellulose of the functionalized paper product 100 of FIG. 2 is all washed functionalized cellulose 102WF.

[0030] Although this example embodiment discloses cellulose fibers, other polysaccharide fibers may be used. Similarly, while this example embodiment discloses paper as the substrate, other substrates may be used.

[0031] As shown in FIG. 3, in an example embodiment a method includes the following:

[0032] 200: applying a liquid blood sample to a functionalized paper product 100;

[0033] 202: drying the liquid blood sample into a dried blood spot sample;

[0034] 204: preserving the dried blood spot sample via an antioxidizing agent 106;

[0035] wherein the functionalized paper product comprises cellulose fibers 102; and

[0036] wherein the antioxidizing agent is covalently bonded to the cellulose fibers 102.

[0037] The method may include the additional following steps:Attorney Docket No. 10457-615PC0

[0038] 206: evaluating the dried blood sample to determine if a select metabolite is present; and

[0039] 208: determining if a metabolite is present by determining if a tautomer of the metabolite is present.

[0040] As detailed above, the functionalized paper product 100 may be composed of functionalized cellulose and optionally unfunctionalized cellulose. The functionalized cellulose may be all (unwashed) functionalized cellulose, may be all washed functionalized cellulose 102WF, or may be a combination of (unwashed) functionalized cellulose and washed functionalized cellulose 102WF.

[0041] Although this example embodiment discloses blood samples, other biological samples may be used. Similarly, while this example embodiment discloses cellulose fibers, other polysaccharide fibers may be used. Further, while this example embodiment discloses paper as the substrate, other substrates may be used.

[0042] As shown in FIG. 4, in an example embodiment, a blood sampling kit 300 includes: a functionalized paper product 100, including the cellulose fibers 102 and the antioxidizing agent 106 covalently bonded to the cellulose fibers 102; and a sampling and storage apparatus 302 configured to hold the functionalized paper product 100 ready to receive a liquid blood sample. The sampling and storage apparatus 302 is further configured to store a dried blood sample composed of blood from the liquid blood sample that has dried on the functionalized paper product 100.

[0043] As detailed above, the functionalized paper product 100 may be composed of functionalized cellulose and optionally unfunctionalized cellulose. The functionalized cellulose may be all (unwashed) functionalized cellulose, may be all washed functionalized cellulose 102WF, or may be a combination of (unwashed) functionalized cellulose and washed functionalized cellulose 102WF.

[0044] The blood sampling kit 300 optionally further includes a tool 304 (e.g., a lancet or the like) configured to pierce the skin to obtain the liquid blood sample. The sampling and storage apparatus 302 is optionally further configured to provide a predetermined volume of the dried blood sample for analysis. Examples of such a sampling and storage apparatus 302 include products of the Capitainer® product line manufactured by Capitainer Inc. of East Providence, RI.Attorney Docket No. 10457-615PC0

[0045] Although this example embodiment discloses a blood sampling kit, the sampling kit may be configured for other biological samples. Similarly, while this example embodiment discloses cellulose fibers, other polysaccharide fibers may be used. Further, while this example embodiment discloses paper as the substrate, other substrates may be used.

[0046] As shown in FIG. 5A, in an example embodiment, a method of making a functionalized paper product includes the following:

[0047] 400: exposing cellulose fibers 102 to an antioxidant 106;

[0048] 402: forming covalent bonds between the cellulose fibers 102 and the antioxidant 106 to form a mixture of functionalized cellulose fibers and unbonded antioxidant;

[0049] 404: washing the unbonded antioxidant from the mixture to form washed functionalized cellulose fibers 102WF; and

[0050] 406: forming the washed functionalized cellulose fibers 102WF into a functionalized paper product 100.

[0051] In an example embodiment, washing the unbonded antioxidant from the mixture results in a remaining concentration of the unbonded antioxidant in the washed functionalized polysaccharide fibers of not more than 100 nanomolar.

[0052] In an example embodiment, any linking agent used when forming the functionalized cellulose is also washed away when the unbonded antioxidant is washed away.

[0053] Although this example embodiment discloses cellulose fibers, other polysaccharide fibers may be used. Similarly, while this example embodiment discloses paper as the substrate, other substrates may be used.

[0054] As shown in FIG. 5B, in a variation of the example embodiment of Fig. 5 A, a method of making the functionalized paper product includes the following:

[0055] 500: exposing cellulose fibers 102 within paper to an antioxidant 106;

[0056] 502: forming covalent bonds between the cellulose fibers 102 and the antioxidant 106 to form a mixture of functionalized cellulose fibers and unbonded antioxidant; and

[0057] 504: washing the unbonded antioxidant from the paper and thereby from the mixture to form the functionalized paper product 100 having washed functionalized cellulose fibers 102WF therein.Attorney Docket No. 10457-615PC0

[0058] In an example embodiment, washing the unbonded antioxidant from the mixture results in a remaining concentration of the unbonded antioxidant in the washed functionalized polysaccharide fibers of not more than 100 nanomolar.

[0059] In an example embodiment, any linking agent used when forming the functionalized cellulose is also be washed away when the unbonded antioxidant is washed away.

[0060] Although this example embodiment discloses cellulose fibers, other polysaccharide fibers may be used. Similarly, while this example embodiment discloses paper as the substrate, other substrates may be used.

[0061] Figures 6-8 show that glutathione disulfide (GSSG), glutathione (GSH) and cysteine are stabilized using the techniques described herein.

[0062] The protocol for the experiments represented in the graphs for Figures 6-8 for testing Cysteine, GSSG and GSH. Cotton paper was functionalized with ascorbic acid through a chemical linking process such as is described above. Capillary blood was collected from a finger using a lancet. The blood was dropped on to the paper containing ascorbic acid and to a separate paper sheet that did not have ascorbic acid added. The samples were allowed to dry over night and out of direct sunlight. Metabolites were extracted from the paper by adding 100% methanol followed by sonication. The methanol was removed, transferred to a separate tube and dried under nitrogen. The dried residue was reconstituted in LC-MS grade water. The samples were analyzed on a ThermoScientific Q-Exactive mass spectrometry with Dionex 3000 UHPLC. Chromatographic separation was achieved with a C18 column (100x2.1mm, 2um) under gradient elution. Peaks for GSH, GSSG and cysteine were evaluated using exact mass of the collected data. Extracted ion chromatograms were created to evaluate changes in metabolites levels based on the presence of ascorbic acid.

[0063] As is shown in FIG. 6, GSSG was dramatically preserved on the ascorbic acid linked paper relative to paper with no ascorbic acid. Similar preservation effect for GSH is shown in FIG. 7. FIG. 8 shows that cysteine is clearly detectable on the ascorbic acid linked paper while there is no detection on paper without ascorbic acid.

[0064] U.S. patent application number 18 / 409,738 (US20240230657) by Garrett et al. provides supporting background information and is incorporated by reference in its entirety herein.Attorney Docket No. 10457-615PC0

[0065] As has been disclosed above, the present inventor has devised an apparatus with features that are improvements in the art. All features disclosed in the specification, including the claims, abstract, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise.

[0066] While various embodiments of the present invention have been shown and described herein, it will be obvious that such embodiments are provided by way of example only.Numerous variations, changes and substitutions may be made without departing from the invention herein. Accordingly, it is intended that the invention be limited only by the spirit and scope of the appended claims.Attorney Docket No. 10457-615PC0REFERENCES1. Chace DH, Millington DS, Terada N, Kahler SG. Roe CR. Hofman LF. Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry. Clin Chem. 1993;39(l):66-71. PubMed PMID: 8419060.2. Chace DH, Hannon WH. Filter Paper as a Blood Sample Collection Device for Newborn Screening. Clin Chem. 2016;62(3):423-5. Epub 20160121. doi: 10.1373 / clinchem.2015.252007. PubMed PMID: 26797689.3. Wagner M, Tonoli D, Varesio E, Hopfgartner G. The use of mass spectrometry to analyze dried blood spots. Mass Spectrom Rev. 2016;35(3):361-438. Epub 20140922. doi:10.1002 / mas.21441. PubMed PMID: 25252132.4. Chace DH, De Jesus VR, Spitzer AR. Clinical chemistry and dried blood spots: increasing laboratory utilization by improved understanding of quantitative challenges.Bioanalysis. 2014;6(21):2791-4. doi: 10.4155 / bio,14.237. PubMed PMID: 25486226; PMCID: PMC4528187.5. Spooner N, Stove C. DBS and beyond. Bioanalysis. 2015;7(16): 1971 -6. doi:10.4155 / bio.15.138. PubMed PMID: 26327178.6. Gelb MH, Basheeruddin K, Burlina A, Chen HJ, Chien YH, Dizikes G, Dorley C, Giugliani R, Hietala A, Hong X, Kao SM, Khaledi H, Klug T, Kubaski F, Liao HC, Martin M, Manning A, Orsini J, Peng Y Matem D. Liquid Chromatography-Tandem Mass Spectrometry in Newborn Screening Laboratories. Int J Neonatal Screen. 2022;8(4). Epub 20221128. doi: 10.3390 / ijns8040062. PubMed PMID: 36547379; PMCID: PMC9781967.7. Riches NO, Johnson EP, Frost CJ, Goldenberg AJ, Rothwell E. The limited use of US residual newborn screening dried bloodspots for health disparity research. Genet Med.2020;22(10): 1723-6. Epub 20200612. doi: 10.1038 / s41436-020-0858-6. PubMed PMID:32533133; PMCID: PMC7529877.Attorney Docket No. 10457-615PC08. Williams SR, McDade TW. The use of dried blood spot sampling in the national social life, health, and aging project. J Gerontol B Psychol Sci Soc Sci. 2009;64 Suppl l(Suppl l):il31-6. Epub 20090225. doi: 10.1093 / geronb / gbn022. PubMed PMID: 19244547; PMCID:PMC2763524.9. Christopher MW, Klug AC, Lee JH, Ericson AC. Feizbakhsh Bazargani S. Dinglasan RR, Prentice BM, Garrett TJ. Indole- 3 -pyruvate: Analysis and Control of Tautomerism and Reactivity. Analytical chemistry. 2024. doi: 10.1021 / acs.analchem.4c01584.10. Venkateswaran N, Garcia R, Lafita-Navarro MC, Hao Y-H, Perez-Castro L, Nogueira PAS, Solmonson A, Mender I, Kilgore JA, Fang S, Brown IN. Li L, Parks E. Lopes dos Santos I, Bhaskar M, Kim I, lia Y, Lemoff A, Grishin NV, ..., Conacci-Sorrell M. Tryptophan fuels MYC-dependent liver tumorigenesis through indole 3-pyruvate synthesis. Nature Communications. 2024; 15(1):4266. doi: 10.1038 / s41467-024-47868-3.11. Edwards JV, Prevost NT, Yager D, Mackin R, Santiago M, Chang S, Condon B, Dacorta J. Ascorbic Acid as an Adjuvant to Unbleached Cotton Promotes Antimicrobial Activity in Spunlace Nonwovens. International Journal of Molecular Sciences. 2022;23(7):3598. PubMed PMID: doi:10.3390 / ijms23073598.

Claims

Attorney Docket No. 10457-615PC0Claims1. A functionalized paper product, comprising:polysaccharide fibers; andan antioxidizing agent covalently bonded to the polysaccharide fibers.

2. The functionalized paper product of claim 1,wherein the polysaccharide fibers comprise cellulose fibers;wherein the cellulose fibers comprise glucose monomers; andwherein the antioxidizing agent is covalently bonded to the glucose monomers.

3. The functionalized paper product of claim 2,wherein the antioxidizing agent is cross-linked to the glucose monomers via a linking agent.

4. The functionalized paper product of claim 3,wherein the antioxidizing agent comprises ascorbic acid and the linking agent comprises citric acid.

5. The functionalized paper product of claim 1,wherein the antioxidizing agent comprises at least one of vitamin E, Ferulic acid, betacarotene, and vitamin A.

6. The functionalized paper product of claim 1,wherein the functionalized paper product is essentially free of antioxidizing agent that has not covalently bonded to the polysaccharide fibers.Attorney Docket No. 10457-615PC07. A method, comprising:applying a liquid biological sample to a functionalized paper product;drying the liquid biological sample into a dried biological sample;preserving the dried biological sample via an antioxidizing agent;wherein the functionalized paper product comprises polysaccharide fibers; and wherein the antioxidizing agent is covalently bonded to the polysaccharide fibers.

8. The method of claim 7,wherein the functionalized paper product is essentially free of antioxidizing agent that has not covalently bonded to the polysaccharide fibers.

9. The method of claim 7,wherein the dried biological sample comprises a dried blood spot sample; and wherein the method further comprises evaluating the dried blood spot sample to determine if a select metabolite is present.

10. The method of claim 9,further comprising determining if a metabolite is present by determining if a tautomer of the metabolite is present.

11. A biological sampling kit, comprising:a functionalized paper product, comprising polysaccharide fibers and an antioxidizing agent covalently bonded to the polysaccharide fibers; anda sampling and storage apparatus configured to hold the functionalized paper product ready to receive a liquid biological sample.

12. The biological sampling kit of claim 11,wherein the liquid biological sample comprises a liquid blood sample; andwherein the sampling and storage apparatus is further configured to store a dried blood sample composed of blood from the liquid blood sample that has dried on the functionalized paper product.Attorney Docket No. 10457-615PC013. The biological sampling kit of claim 12,wherein the sampling and storage apparatus is further configured to provide a predetermined volume of the dried blood sample for analysis.

14. The biological sampling kit of claim 11,further comprising a lancet.

15. A method of forming a functionalized paper product, comprising:exposing polysaccharide fibers to an antioxidant;forming covalent bonds between the polysaccharide fibers and the antioxidant to form a mixture of functionalized polysaccharide fibers and unbonded antioxidant;washing the unbonded antioxidant from the mixture to form washed functionalized polysaccharide fibers; andwherein the functionalized paper product comprises the washed functionalized polysaccharide fibers.

16. The method of claim 15,wherein the functionalized paper product consists of:the washed functionalized polysaccharide fibers, and optionally unfunctionalized polysaccharide fibers.

17. The method of claim 15,wherein the polysaccharide fibers are present in paper of the functionalized paper product when the polysaccharide fibers are exposed to the antioxidant.

18. The method of claim 15,wherein washing the unbonded antioxidant from the mixture results in a remaining concentration of the unbonded antioxidant in the washed functionalized polysaccharide fibers of not more than 100 nanomolar.Attorney Docket No. 10457-615PC019. The method of claim 18,wherein the washed functionalized polysaccharide fibers are subsequently formed into the functionalized paper product.

20. The method of claim 15,wherein the polysaccharide fibers comprise cellulose fibers.

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