Method for storing and releasing protein-modified nanoparticles on a paper substrate

Abstract

The present invention relates to a method for storing protein-modified nanoparticles on a paper substrate without irreversible binding, in particular a method for storing protein-modified nanoparticles within a polymer-modified paper substrate. The present invention also relates to a reservoir containing protein-modified nanoparticles capable of establishing biological interactions obtained by this method and its use in a biosensor made of paper.

Description

[0001] The present invention relates to a method for storing protein-modified nanoparticles on a paper substrate without irreversible binding, in particular a method for storing protein-modified nanoparticles within a polymer-modified paper substrate. The present invention also relates to a reservoir containing protein-modified nanoparticles capable of establishing biological interactions obtained by this method and its use in a biosensor made of paper. Background Art

[0002] Filter paper is the preferred substrate for developing disposable biosensors because it is lightweight, inexpensive, and easy to dispose of (Reboud et al. Paper-based microfluidics for DNA diagnostics of malaria in lowresource underserved rural communities. Proc. Natl. Acad. Sci. USA 2019, 116, 4834–4842; Roig-Sanchez et al. Nanocellulose films with multiple functionalnanoparticles in confined spatial distribution Nanoscale Horiz., 2019, 4, 634--641; Tenda et al., Paper-Based Antibody Detection Devices Using BioluminescentBRET-Switching Sensor Proteins. Angew. Chemie-Int. Ed. 2018, 57, 15369–15373). It is commercially available in a variety of pore sizes and can be easily modified with biomolecules by physical adsorption or covalent linkage methods (Chuet al. Paper-based chemiluminescence immunodevice with temporal controls ofreagent transport technique. Sensors Actuators, B Chem. 2017, 250, 324–332; Wang etal. Simple and covalent fabrication of a paper device and its application insensitive chemiluminescence immunoassay. Analyst 2012 137, 3821).Its porous matrix can also be used to store reagents such as enzymes and their substrates to fabricate reservoirs integrated into paper-based analytical devices (Calabria et al. Smartphone–based enzymatic biosensor for oral fluid L-lactate detection in one minute using confined multilayer paper reflectometry. Biosens. Bioelectron. 2017, 94, 124–130). Such reservoirs must maintain the physicochemical properties of the component reagents over time while also ensuring efficient release of their contents into other paper regions upon addition of liquid. Plasmonic nanoparticles are very useful building blocks for fabricating biosensors that utilize various signal transduction mechanisms, from colorimetry (Russell and dela Rica Paper transducers to detect plasmon variations in colorimetric nanoparticle biosensors. Sensors Actuators, B Chem. 2018, 270, 327–332) and surface-enhanced Raman spectroscopy (SERS) (Catala et al. Online SERS Quantification of Staphylococcus aureus and the Application to Diagnostics in Human Fluids Adv. Mater. Technol. 2016, 1600163) to fluorescence (Ellairaja et al. Novel Pyrimidine Tagged Silver Nanoparticle Based Fluorescent Immunoassay for the Detection of Pseudomonas aeruginosa. J. Agric. Food Chem. 2017, 65, 1802–1812) and electrochemistry (Liang et al. An origami paper device for complete elimination of interferents in enzymatic electrochemical biosensors. Electrochem. Commun. 2017 82, 43–46).

[0003] However, nanoparticles tend to irreversibly adsorb onto the paper substrate after drying, making them difficult to store in reservoirs made of this material (Ruivo et al. Colorimetric detection of D-dimer in a paper-based immunodetection device. Anal. Biochem. 2017, 538, 5–12). Conventionally, this problem has been addressed by fabricating the reservoirs with glass fiber instead of conventional paper (Deraney et al., Multiplexed, Patterned-Paper Immunoassay for Detection of Malaria and Dengue Fever. Anal. Chem. 2016, 88, 6161–6165). However, this approach is not fully compatible with biosensor designs such as origami-based analytical devices. These biosensors must be made entirely of paper in order to simplify their fabrication and facilitate contact between components without the use of additional adhesives (Liu et al. Aptamer-based origami paper analytical device for electrochemical detection of adenosine. Angew. Chemie-Int. Ed. 2012, 51, 6925–6928; Liu and Crooks, Three-dimensional paper microfluidic devices assembled using the principles of origami. J. Am. Chem. Soc. 2011, 133, 17564–17566).

[0004] PSS has previously been used to avoid nanoparticle flocculation, typically in the form of alternating layers of PSS and a positively charged polymer such as poly(diallyldimethylammonium chloride) (McLintock et al., Universal surface-enhanced Raman tags: Individual nanorods for measurements from the visible to the infrared (514-1064 nm). ACS Nano 2014, 8, 8600–8609). It has also been used as a carrier for growing nanoparticles (Cai et al., Coating sulfonated polystyrene microspheres with highly dense gold nanoparticle shell for SERS application. Colloid Polym. Sci. 2013, 291, 2023–2029), and for transferring them onto a soft-lithographed receiving substrate (Basarir, Fabrication of gold patterns via multilayer transfer printing and electroless plating. ACS Appl. Mater. Interfaces 2012, 4, 1324–1329). PSS blended with PEDOT is commonly used to modify cellulose and make it conductive (Khan et al., Nano-gold assisted highly conducting and biocompatible bacterial cellulose-PEDOT:PSS films for biology-device interface applications. Int. J. Biol. Macromol. 2018, 107, 865–873).

[0005] The release of nanoparticles stored in unmodified paper substrates has not been fully characterized (Chen et al. Three-dimensional origami paper-based device for portable immunoassay applications. Lab Chip 2019, 19, 598–607), and has been reported to be extremely inefficient in some cases (Ruivo et al., 2017).

[0006] Accordingly, there is a desire to find a method for storing nanoparticles on a paper substrate and releasing them efficiently on demand so that they can be applied in 2D and 3D analytical devices made of paper. Summary of the Invention

[0007] A first aspect of the present invention relates to a method for obtaining a reservoir comprising protein-modified nanoparticles on a paper substrate, the method comprising the steps of:

[0008] i. treating the paper substrate with a hydrophilic negatively charged polymer on the paper substrate; and

[0009] ii. applying protein-modified nanoparticles on the PSS-modified paper substrate obtained in step (i).

[0010] In another embodiment, the present invention relates to the method as defined above, wherein the polymer is polystyrene sulfonate (PSS).

[0011] In another embodiment, the present invention relates to the method as defined above, wherein the protein is a protein capable of establishing a biological interaction, preferably wherein the protein is avidin or an antibody.

[0012] In another embodiment, the present invention relates to the method as defined above, wherein the protein is a protein capable of establishing a biological interaction and the protein is avidin.

[0013] In another embodiment, the present invention relates to the method as defined above, wherein the protein is a protein capable of establishing a biological interaction and the protein is an antibody.

[0014] In another embodiment, the present invention relates to the method as defined above, wherein the nanoparticles are PEGylated nanoparticles.

[0015] In another embodiment, the present invention relates to the method as defined above, wherein the PEGylated nanoparticles are PEGylated nanoparticles made of gold.

[0016] In another embodiment, the present invention relates to the method as defined above, wherein the treatment of step (i) is carried out by spotting the polymer, preferably PSS, on a piece of paper substrate.

[0017] In another embodiment, the present invention relates to the method as defined above, wherein the piece of paper substrate in step (i) is cut into a square form.

[0018] In another embodiment, the present invention relates to the method as defined above, wherein the volume of the added polymer, preferably the added PPS, is large enough such that the volume of the polymer-modified paper is larger than the volume to be occupied by the polyethylene glycolated nanoparticles.

[0019] In another embodiment, the present invention relates to the method as defined above, wherein the volume of the added polymer, preferably the added PSS, is 50 μL, and the volume of the polyethylene glycolated nanoparticles, preferably the polyethylene glycolated nanoparticles made of gold, is 1 μL.

[0020] In another embodiment, the present invention relates to the method as defined above, which further comprises a step (iii) of drying the paper after step (i), and preferably wherein the drying step (iii) is carried out at a temperature of 15 °C to 50 °C.

[0021] In another embodiment, the present invention relates to the method as defined above, which further comprises a step (iv) of allowing the paper to dry after step (ii), and preferably wherein the drying step (iv) is carried out at a temperature of 15 °C to 50 °C.

[0022] In another embodiment, the present invention relates to the method as defined above, which further comprises a step (v) of releasing the nanoparticles from a reservoir.

[0023] In another embodiment, the present invention relates to the method as defined above, wherein the releasing step (v) is a transferring step to a receiving substrate, preferably transferring to a wet receiving paper substrate or transferring to a polypropylene receiving substrate.

[0024] In another embodiment, the present invention relates to the method as defined above, wherein the transferring step (v) is carried out by pressing, preferably by pressing with a finger, a press or a clamp, and more preferably by pressing at the center of the spot for at least 4 minutes.

[0025] By fine-tuning the % of PSS and the concentration of the nanoparticles, the transfer of the nanoparticles to the reservoir can be adjusted to preferentially occur at the edge or to occur uniformly throughout the reservoir. For example, when the PSS % is between 1.9% and 7.5% and the concentration of the nanoparticles is 72 nM, the transfer from the edge is favored, while when the PSS % is 30% and the concentration of the nanoparticles is 210 nM or higher, a uniform transfer of the nanoparticles to the receiving substrate is obtained.

[0026] Thus, in another embodiment, the present invention relates to the method as defined above, wherein the transferring step (v) is adjusted by fine-tuning the PSS content and the concentration of the nanoparticles (preferably polyethylene glycolated nanoparticles or polyethylene glycolated nanoparticles made of gold).

[0027] In another embodiment, the present invention relates to the method as defined above, wherein the transfer step (v) is adjusted by fine-tuning the PSS content between 1.9% and 7.5% and setting the concentration of the gold-made polyethylene glycolated nanoparticles at 72 nM.

[0028] In another embodiment, the present invention relates to the method as defined above, wherein the transfer step (v) is adjusted by fine-tuning the PSS content to 30% and setting the concentration of the gold-made polyethylene glycolated nanoparticles at least at 210 nM.

[0029] In another embodiment, the present invention relates to the method as defined above, wherein the receiving paper substrate of step (v) has been modified with a bioligand (preferably biotinylated bovine serum albumin (BSA)) and saturated with phosphate buffered saline with Tween-20 (PBST), and the bioligand specifically interacts with the receptor protein (preferably avidin) bound to the nanoparticles.

[0030] In another embodiment, the present invention relates to the method as defined above, wherein in step (v), the protein (preferably avidin or antibody) bound to the nanoparticles establishes a biological interaction with the target (preferably biotin or antigen) bound to the receiving substrate (preferably a wet paper substrate or a polypropylene substrate).

[0031] In another embodiment, the present invention relates to the method as defined above, which further comprises a step (vi) of removing the reservoir and removing the excess reagent after performing step (v), and preferably wherein step (vi) is performed by washing 3 to 5 times with PBST.

[0032] The second aspect of the present invention relates to a reservoir comprising protein-modified nanoparticles on a paper substrate, which reservoir is obtained by the method as defined above.

[0033] The third aspect of the present invention relates to the use of the protein-modified nanoparticle reservoir as defined above in the preparation of a biosensor made of paper.

[0034] The present invention introduces a new method for fabricating a nanoparticle reservoir on filter paper. It includes modifying the paper substrate with the negatively charged polymer poly(sodium 4-styrenesulfonate) (PSS) to avoid the irreversible binding of the polyethylene glycolated nanoparticles to the cellulose matrix ( Figure 1 A). The reservoir prepared in this way can efficiently release nanoparticles.

[0035] According to the present invention, PSS can be used to avoid the irreversible binding of nanoparticles to the cellulose matrix. In addition to being able to release nanoparticles from the cellulose matrix on demand, PSS can also transfer nanoparticles from a dry reservoir to a receiving wet paper substrate by simply pressing the dry reservoir onto the receiving wet paper substrate ( Figure 1 B). This enables the proposed reservoir to be used for developing biosensors with simplified liquid handling protocols, as there is no need to add volumetrically metered buffer to the reservoir to transfer its contents to the detection area. Additionally, nanoparticles modified with proteins (avidin, antibodies) retain their ability to specifically recognize their targets (biotin, antigen) immobilized on the receiving paper sheet in a dose-dependent manner, demonstrating the applicability of this method for developing biosensors ( Figure 1 C).

[0036] A method for storing protein-modified nanoparticles on a paper substrate is introduced here, which also allows for their release on demand. The method involves spotting the nanoparticles onto filter paper pre-modified with poly(sodium 4-styrenesulfonate) (PSS). By simply pressing with a finger, gold nanoparticles modified with avidin can be easily transferred from a dry reservoir to a receiving wet paper sheet. Additionally, the nanoprobes can establish biospecific avidin-biotin interactions with substrate-binding molecules, making the reservoir suitable for fabricating nanoparticle-based biosensors. Adjusting the PSS% and nanoparticle concentration in the reservoir enables uniform transfer between paper layers without forming a "coffee ring". These features make the method of the present invention ideal for developing origami biosensors made of filter paper and incorporating a nanoprobe-based signal generation mechanism.

[0037] The reservoir proposed by the present invention is easy to fabricate, has a long shelf life, and avoids common defects associated with paper biosensors, such as the generation of unevenly distributed colorimetric signals. These characteristics make them ideal for fabricating biosensors made of paper.

[0038] In summary, the present invention describes a method for storing nanoparticles modified with PEG and proteins in filter paper when the filter paper is pre-treated with PSS. This treatment with PSS avoids irreversible interactions with the paper, enabling complete release of the nanoparticles upon addition of an aqueous solution. It also enables the transfer of nanoparticles from the reservoir to the receiving substrate by pressing one paper sheet against another with a finger. Fine-tuning the PSS% avoids the formation of a "coffee ring" in the reservoir and the receiving substrate. This results in a uniformly distributed colorimetric signal generated by biomolecular interactions between the nanoprobes and substrate-binding molecules. The presence of PSS in the reservoir does not interfere with the generation of avidin-biotin or antibody-antigen interactions and preserves the nanoprobes for at least one month. These features make the proposed reservoir ideal for fabricating pure paper biosensors incorporating biomolecule-modified nanoprobes.

[0039] Throughout the present invention, the term "hydrophilic negatively charged polymer" refers to a polymer that establishes a thermodynamically favorable interaction with water and contains groups that can be deprotonated to produce a negatively charged moiety. Examples include polystyrenesulfonate (PSS), polyglutamic acid, and nucleic acids, among others.

[0040] A "protein capable of establishing a biological interaction" refers to a protein that interacts with another molecule through specific biomolecular interactions. Examples include avidin-biotin interactions, streptavidin-biotin interactions, antibody-antigen interactions, agonist-receptor interactions, enzyme-substrate interactions, and enzyme-inhibitor interactions, among others.

[0041] "PEGylated nanoparticles" refer to nanoparticles modified with polyethylene glycol (PEG). Examples include metal nanoparticles or metal oxide nanoparticles (such as gold, silver, platinum, iron oxide, or zinc oxide) modified with PEG, semiconductor nanoparticles (e.g., quantum dots and upconversion nanoparticles) modified with PEG, liposomes and protein nanoparticles modified with a PEG shell, and polymer nanoparticles containing PEG in their outer layer, among others.

[0042] The term "receiving substrate" refers to any solid onto which the nanoparticles can be transferred. This term includes "wet receiving paper substrates" and "polypropylene receiving substrates", etc. A "wet receiving paper substrate" refers to a paper sheet wetted with an aqueous solution. Examples include filter papers with different pore sizes, bacterial cellulose, nitrocellulose, polypropylene masks, textiles such as cotton or polyester, skin, animal tissues, metals, silicon wafers, elastomers, and hydrogels, among others.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used to practice the present invention. Throughout the specification and claims, the word "comprising" and its variants should not be construed as excluding other technical features, additives, ingredients, or steps. Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the specification, or can be learned by practicing the present invention. The following examples and drawings are provided by way of illustration only and are not intended to limit the present invention. Description of the Drawings

[0044] Figure 1. Schematic of the nanoparticle reservoir and the process of transferring its contents to the receiving paper substrate: (A) The paper reservoir (i) contains poly(sodium 4-styrenesulfonate) (PSS) and protein-modified gold nanoparticles; the receiving substrate (ii) contains physically adsorbed ligands; (B) The dry reservoir (i) is placed on the wet receiving substrate (ii) and pressed, for example, with a finger, to transfer the nanoparticles from the dry reservoir (i) to the wet receiving substrate (ii); (C) After washing away the excess reagents, the nanoparticles remain bound to the receiving substrate (ii) through biorecognition ligand-receptor interactions (v);

[0045] Figure 2 . Fabrication of nanoparticle reservoirs with different PSS%: (A) Scanning images of the reservoir before (top row) and after (bottom row) three washes with 1 mL of PBST; (B) Pixel intensity (PI) distribution of the reservoir; (C) Colorimetric signal (S) in the reservoir before (circles) and after (squares) three washes with PBST. Error bars are standard deviations (n = 3). The trend lines are a guide to the eye;

[0046] Figure 3 . Fabrication of reservoirs with 30% PSS and different concentrations of avidin-modified nanoparticles: (A) Scanning images of the reservoir before (top row) and after (bottom row) three washes with 1 mL of PBST; (B) Pixel intensity (PI) distribution of the reservoir; (C) Colorimetric signal (S) in the reservoir before (circles) and after (squares) three washes with PBST. Error bars are standard deviations (n = 3). The trend lines are a guide to the eye;

[0047] Figure 4 . Transfer of avidin-modified nanoparticles from reservoirs with different PSS% to biotinylated protein-modified receiving substrates; (A) Scanning images of the receiving substrate after pressing the reservoir for 4 minutes, removing the receiving substrate, and washing three times with 1 mL of PBST; (B) Pixel intensity (PI) distribution on the receiving substrate; (C) Colorimetric signal (S) in the receiving paper substrate. Error bars are standard deviations (n = 3). The trend lines are a guide to the eye;

[0048] Figure 5 . Transfer of avidin-modified nanoparticles from a reservoir containing 30% PSS to a biotinylated protein-modified receiving substrate in a time-dependent manner: (A) Scanning images of the receiving substrate after pressing the reservoir for different times; (B) Pixel intensity (PI) distribution on the receiving substrate; (C) Colorimetric signal (S) in the receiving paper substrate. The trend lines are a guide to the eye;

[0049] Figure 6. The stored avidin-modified nanoparticles at different concentrations were transferred onto receiving paper substrates with different pore sizes modified with biotinylated proteins (Whatman paper #41, #6, and #1 have pore sizes of 22 μm, 11 μm, and 3 μm, respectively); (A) Scanning images of the receiving substrates modified with biotinylated BSA (biotin) or unmodified BSA (control); (B) Colorimetric signals in the receiving substrates made of Whatman paper #41 (triangle), #6 (circle), and #1 (square); The control experiment with non-biotinylated BSA is represented by a dashed line; (C) Pixel intensity (PI) distribution of the receiving substrate made of Whatman paper #41. Error bars are standard deviations (n = 3). The trend line is for visual guidance;

[0050] Figure 7 . When measurements were performed using dry nanoparticle reservoirs stored for different times, the changes in the colorimetric signals (pixel intensity PI) caused by the biospecific avidin-biotin interaction. The percentage signals were calculated relative to the reservoir stored for 1 hour. Error bars are standard deviations (n = 3);

[0051] Figure 8 . Antibody-modified nanoparticles stored in paper reservoirs detected Escherichia coli (E. coli) (A) and glycoprotein B from cytomegalovirus (B) (semi-logarithmic scale). Error bars are standard deviations (n = 3);

[0052] Figure 9 . Antibody-modified nanoparticles stored in paper reservoirs detected Pseudomonas aeruginosa (semi-logarithmic scale). Error bars are standard deviations. The colorimetric signal S is an integer obtained by subtracting the pixel intensity of the background from the pixel intensity of the target area;

[0053] Figure 10 . Anti-rabbit IgG-modified nanoparticles stored in paper reservoirs detected rabbit IgG (dots) and BSA (triangles) in polypropylene masks. Error bars are standard deviations. The colorimetric signal S is an integer obtained by subtracting the pixel intensity of the background from the pixel intensity of the target area. Examples

[0054] Synthesis and modification of gold nanoparticles with polyethylene glycol and avidin: Citrate-capped gold nanoparticles with a diameter of approximately 40 nm were synthesized using the Turkevich method as described previously (Russell et al., Augmented Reality for Real-Time Detection and Interpretation of Colorimetric Signals Generated by Paper-Based Biosensors. ACS Sensors 2017, 2, 848–853). The nanoparticles were then modified overnight with 0.1 mM thiolated polyethylene glycol (PEG) molecules terminated with carboxylate moieties (poly(ethylene glycol) 2-mercaptoethyl ether acetate, Mn 2100, Sigma). The resulting PEGylated nanoparticles were concentrated and washed 5 times with water by centrifugation at 8000 rpm for 6 minutes. Finally, the nanoparticles were suspended in 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES, Sigma) with the pH adjusted to 5.5. The carboxylate moieties around the nanoparticles were then converted to sulfo-NHS esters by adding 1 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, Sigma) and 2 mg of N-hydroxysulfosuccinimide sodium salt (sulfo-NHS, Sigma) over 20 minutes. The nanoparticles were then precipitated by centrifugation and the supernatant was replaced with a solution of 0.1 M phosphate buffer (pH 7.4) containing 1 mg·mL -1 avidin. After 1 hour, the unreacted sulfo-NHS esters were blocked with 0.1 M glycine and 10 mg·mL -1 bovine serum albumin (BSA) for 30 minutes. The nanoparticles were then washed 5 times with phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST). The resulting avidin-coated nanoparticles were kept at 4 °C until use. Antibody-modified nanoparticles were obtained by incubating biotinylated antibodies with avidin-coated nanoparticles for 1 hour and then washing away the excess reagents by 3 centrifugation steps.

[0055] Optical density measurement: Gold nanoparticles on paper substrates generate a concentration-dependent colorimetric signal that can be evaluated by optical density measurement as follows. First, the paper substrate was scanned using an MFC-1910W scanning printer (Brother). The pixel intensity (PI) distribution was obtained using ImageJ. In grayscale mode, pure white generates a pixel intensity of 255, while pure black generates a pixel intensity of 0. The colorimetric signal S was obtained as follows. First, the grayscale pixel intensity was measured in a circular region within the target area using the histogram function of ImageJ. The colorimetric signal S is the integer value after subtracting the background signal. Note that subtracting the background pixel intensity produces an inverse signal compared to the original data.

[0056] Fabrication of nanoparticle reservoirs: Whatman filter papers No. 41, No. 1, and No. 6 with pore sizes of 20 μm to 25 μm, 11 μm, and 3 μm, respectively, were used. The papers were cut into squares and modified with 50 μL of poly(sodium 4-styrenesulfonate) (PSS, 30%, Sigma) diluted with water to different %(v / v) when needed. After drying, 1 μL of avidin-modified PEGylated gold nanoparticles was added and dried at room temperature. To study the release of nanoparticles from the reservoirs, the nanoparticle-modified dry paper substrates were placed on folded filter paper pieces, and 1 mL of PBST was added three times. The presence of nanoparticles after this step was evaluated by allowing the paper to dry and measuring any change in the color of the nanoparticle reservoirs by densitometry.

[0057] Nanoparticle transfer and biorecognition: The transfer of nanoparticles from the reservoirs to the receiving paper substrates was studied by the following procedure. As Figure 8 shown, the receiving paper substrates were modified with 2 μL of biotinylated BSA (100 μg·mL -1 ) in PBS or different concentrations of antigens. Biotinylated BSA was obtained using the EZ-Link TM Sulfo-NHS-LC-Biotinylation Kit (Thermo Scientific). To evaluate the contribution of non-specific interactions to the colorimetric signal, a control experiment using non-biotinylated BSA was performed. After drying, 1 mL of PBS supplemented with 5 mg·mL -1 BSA (PBS-BSA) was added. Then the reservoir was placed on top of the wet receiving paper, and the transfer of nanoparticles was facilitated by pressing on the center of the spot for 4 minutes. Subsequently, the reservoir was removed, and the receiving substrate was washed three times with PBST to remove non-specific interactions. After drying, the colorimetric signal was evaluated by densitometry as detailed above.

[0058] Modifying paper substrates with PSS helps store nanoparticles in the cellulose matrix while enabling their efficient release. To study this, filter paper was cut into squares, and then 50 μL of PSS at different dilutions was added and allowed to dry. The paper substrate with a pore size of 22 μm (Whatman #41) remained mostly flat at all PSS concentrations and was thus subsequently used to fabricate nanoparticle reservoirs. These were obtained by pipetting 1 μL of gold nanoparticles at the center of the PSS-modified paper and allowing it to dry at room temperature. The most widely available gold nanoparticle suspension was synthesized according to the Turkevich method, which renders them capped with negatively charged citrate molecules. To avoid nanoparticle aggregation in solutions containing high concentrations of cations or proteins, the citrate molecules are typically replaced with thiolated polyethylene glycol ligands (de la Rica et al., Enzyme-responsive nanoparticles for drug release and diagnostics. Adv. Drug Deliv. Rev. 2012, 64, 967–978). This not only prevents nanoparticle aggregation but also enables the introduction of reactive groups, such as carboxylate moieties, for further covalent linkage to biomolecules. With this in mind, the fabrication of reservoirs containing PEGylated nanoparticle probes modified with avidin via amide bond formation has been investigated. Figure 2 A (top row) shows pictures of nanoparticle reservoirs prepared with 72 nM gold nanoparticles modified with different PSS% and avidin. In these images, as the PSS% in the paper increases, the spot diameter tends to decrease. At the same time, the color intensity increases with increasing PSS%. This can also be observed from Figure 2 the pixel intensity distribution obtained from these images in B. In grayscale mode, the pixel intensity is highest when the color is white (255) and lowest when the color is black (0). In Figure 2In B, the pixel intensity of the entire reservoir decreases with an increase in PSS%. The spot diameter also follows the same trend. This indicates that with an increase in PSS%, a higher concentration of nanoparticles is found and in a smaller volume of the paper matrix. Thus, the formation of these smaller and more concentrated nanoparticle reservoirs is related to the slower diffusion of nanoparticles within the cellulose matrix. It is well known that the viscosity of a solution increases with an increase in PSS concentration (Boris and Colby, Rheology of sulfonated polystyrene solutions. Macromolecules 1998, 31, 5746–5755). Since, according to the Stokes-Einstein equation, the diffusion coefficient is inversely proportional to the viscosity, a highly concentrated PSS reduces the radial diffusion and results in smaller spots containing a higher concentration of nanoparticles. Note that although PSS makes the paper substrate slightly yellowish, its contribution to the pixel intensity cannot fully account for the color change observed within the reservoir. For example, in Figure 2 In B, due to modification with 30% PSS, the pixel intensity outside the nanoparticle region decreased from 254 to 234, but decreased more at the center of the reservoir where nanoparticles were found (PI ≈ 62). This means that the main contribution to the observed color change is the higher concentration of nanoparticles in the reservoir rather than the added PSS. In Figure 2 In B, it is also worth noting that the pixel intensity at the edge of the reservoir is lower than that at the center. This is attributed to a higher concentration of nanoparticles at the edge of the spot due to the uneven distribution of solutes during the drying process (the so-called "coffee-ring effect") (Zhang et al. Converting color to length based on the coffee-ring effect for quantitative immunoassays using a ruler as readout. Lab Chip 2018, 18, 271–275). Finally, it was determined whether the nanoparticles could be effectively released from the reservoir, which is an important requirement for integrating the reservoir into a biosensor. For this purpose, 1 mL of PBST was added to the reservoir three times. In Figure 2 In A (bottom row), there was no color change in the absence of PSS, indicating that without the polymer, the nanoparticles irreversibly bind to the cellulose matrix. However, with an increase in PSS%, the color within the spot gradually disappeared, indicating that when the PSS% in the reservoir is higher, the nanoparticles leave the reservoir more effectively. These experiments show that PSS prevents the formation of irreversible interactions between the nanoparticles and the paper matrix. In Figure 2In C, signal quantification using densitometry showed that when the PSS% used for fabricating the reservoir was 15% or higher, there was no color residue in the reservoir, indicating that in this case, the avidin-modified nanoparticles were completely released. In summary, Figure 2 The results shown in indicate that PEGylated nanoparticles modified with proteins can be stored in dry paper sheets previously modified with PSS, and the nanoparticles can be released on demand simply by adding an aqueous solution.

[0059] Next, it was demonstrated that the nanoparticle concentration has an impact on the fabrication of paper-based reservoirs containing avidin-modified gold nanoparticles. To this end, 1 μL of nanoparticles with different concentrations was pipetted onto a 30% PSS-modified paper substrate ( Figure 3 A). Distribution analysis revealed that when the nanoparticles were dispensed at a concentration of 140 nM or higher, the formation of a "coffee ring" in the Figure 2 reservoir observed in could be prevented, as the pixel intensity was the same at the center and the edge of the spot ( Figure 3 B). To study the effect of nanoparticle concentration on the release of the content from the PSS-modified cellulose matrix, the reservoir was washed with PBST as described above, and the remaining colorimetric signal was measured using densitometry. In the Figure 3 bottom row of A and Figure 3 C, the remaining colorimetric signal was very low at all measured concentrations. Only a slight increase in color could be detected at the highest measured concentration of the nanoparticles. This indicates that even when the reservoir contains a high concentration of gold nanoparticles, 30% PSS effectively prevents irreversible interaction with the paper. In summary, Figure 3 The experiments in indicate that the optimal fabrication parameters for obtaining the reservoir using the proposed method are 30% PSS and gold nanoparticles with a concentration of 140 nM to 210 nM. Under these conditions, the nanoparticles in the reservoir are evenly distributed (i.e., no coffee ring), and the colloid is completely released from the reservoir (no color residue in the paper reservoir).

[0060] After studying the optimal conditions for storing and releasing nanoparticles in cellulose, the ability to transfer the colloid in the dry reservoir to a receiving wet paper by pressing the dry reservoir onto the receiving wet paper was tested ( Figure 1B). Meanwhile, it was tested whether the avidin around the nanoparticles was still able to bind to the biotinylated molecules in the receiving substrate. In other words, it was investigated whether the presence of PSS interfered with the biological interactions of the model of the present invention. To conduct this study, the dry reservoir was pressed onto a receiving paper substrate modified with biotinylated BSA and saturated with PBST. This transferred the liquid from the wet receiving paper to the dry paper reservoir. The resulting rehydration of the reservoir allowed the nanoparticles to transfer from the reservoir to the receiving substrate. After peeling off the reservoir, the presence of the nanoparticles bound to the receiving paper substrate through the avidin-biotin interaction was evaluated after washing three times with PBST. Figure 4 A shows images of the receiving substrate when reservoirs were made using the same concentration of nanoparticles (72 nM) and different PSS%. In these images, as the PSS% increased, the color distribution in the spots became more uniform and intense. In fact, at low PSS%, only a ring of nanoparticles was generated, and the ring of nanoparticles gradually filled at high PSS% to produce colored spots. Figure 4 B also shows that when the concentration of PSS was low, the avidin-modified nanoparticles tended to accumulate at the periphery of the receiving spots. As the PSS% increased, the diameter of the nanoparticle ring decreased, and more nanoparticles were observed at the center of the spots. In Figure 4 C, the colorimetric signal increased as the PSS% increased because more nanoparticles were transferred into the target area. It was proposed that there might be two nanoparticle transfer mechanisms that could lead to ring formation. The first mechanism means that at low PSS%, the nanoparticles radially diffused and aggregated at the edge during transfer, while at high PSS%, the degree of radial diffusion of the nanoparticles was smaller, and they were transferred uniformly onto the receiving paper. As the PSS% increased, this would result in a more concentrated distribution of nanoparticles at the center of the spots, which was consistent with the Figure 4 observations in. In the second hypothesis, the nanoparticles preferentially transferred from the edge of the reservoir to the receiving paper substrate. That is to say, a ring was generated first, which was then filled with more nanoparticles. To distinguish which mechanism controlled the transfer of nanoparticles from the reservoir to the receiving paper, the same experiment was repeated with reservoirs made with the highest concentration of PSS (30%), but with different contact times between the paper layers. In Figure 5 A and Figure 5 B, even when the PSS% was very high, when a shorter transfer time was applied, the nanoparticles were transferred in the form of a ring. In Figure 5In C, the colorimetric signal increases with time. These results refute the first mechanism because in the first stage of the nanoparticle process, no nanoparticles were observed at the center of the reservoir. Therefore, it was proposed that the nanoparticles and PSS preferentially transfer from the edges of the reservoir. This creates a diffusion barrier that causes the subsequent transfer of the polymer and nanomaterial to the central region of the receiving paper, which is consistent with the second hypothesis proposed above. The following shows that the nanoparticle concentration and paper type have an impact on the generation of specific and non-specific signals in biosensing applications.

[0061] Figure 6 Shows the effect of the receiving paper pore size on the generation of colorimetric signals when the reservoir is loaded with 30% PSS and different concentrations of avidin-grafted gold nanoparticles. Non-specific interactions were evaluated with non-biotinylated (BSA) Figure 6 A bottom row). At Figure 6 B, spectrophotometry showed that although the three paper types produced similar colorimetric signals in the biotinylated substrate, Whatman paper #6 and #1 produced higher non-specific interactions compared to Whatman paper #41. This is attributed to the higher specific surface area of paper types #6 and #1 due to their smaller pore sizes (11 and 3 μm, respectively), which facilitates non-specific interactions between the nanoparticles and the receiving substrate. At Figure 6 C, when the concentration of nanoparticles in the reservoir in Whatman paper #41 was 140 nM or higher, the pixel intensity within the colorimetric signal was uniformly distributed (i.e., no ring formation). In addition to Figure 4 the results obtained in, these results indicate that the paper-based reservoir containing 30% PSS and 140 nM nanoparticle probe is the best candidate for generating uniform and high-intensity plasmon signals in biosensors.

[0062] In addition to being able to transfer biomolecule-modified nanoparticles, the reservoir containing biomolecule nanoprobes should also be able to store them for a long time under dry conditions. This is particularly important for incorporating the reservoir into biosensors for field applications because they are not always stored under ideal conditions (i.e., low temperature, dry conditions). To test the shelf life of the proposed reservoir, paper substrates modified with 30% PSS and 140 nM avidin-modified gold nanoparticles were stored for up to one month. The reservoirs were kept in envelopes at room temperature. No preservatives were added, and no additional measures (such as lyophilization or co-storage with silica gel) were taken to further preserve the probes. Then experiments were conducted with biotinylated and non-biotinylated BSA to evaluate the ability of the nanoparticles to establish biospecific interactions after being stored in the paper reservoir for different times. Figure 7Shows the remaining percentage of the colorimetric signal compared to the reservoir used within 1 hour after fabrication. In this figure, the signal never drops below 85% of the original signal, indicating that the proposed method can be used to fabricate biosensors with an extended shelf life without imposing strict temperature or humidity storage conditions.

[0063] Finally, it was demonstrated that reservoirs containing nanoparticles modified with useful antibodies can be used for the specific detection of antigens immobilized on a paper substrate at different concentrations. For this purpose, a receiving paper substrate was modified with a drop of Escherichia coli or glycoprotein B and allowed to dry. After blocking and adding PBST, the reservoir was placed on top and pressed onto the receiving substrate for 5 minutes. After peeling off the reservoir and washing, the increase in pixel intensity was calculated. In Figure 8 the calibration graph shows that the colorimetric signal increases with increasing antigen concentration, indicating that the proposed reservoir can be used for storing and releasing antibody-modified nanoparticles and that the antibody retains its biorecognition function after being stored in the reservoir. Experiments were also conducted to detect Pseudomonas aeruginosa, which were carried out using the same procedure but with nanoparticles modified with antibodies against this pathogen. The results are as shown in Figure 9 Figure []. Additional experiments were also conducted to demonstrate that antibody-modified nanoparticles can be transferred from a paper reservoir to a substrate not made of paper. For this purpose, a model analyte (rabbit IgG) was sprayed onto a clinical-grade mask. Then, the polypropylene inner layer was removed and the presence of rabbit IgG was detected, which was carried out using the same method as for detecting Escherichia coli, Pseudomonas aeruginosa, and glycoprotein B but with a reservoir containing nanoparticles modified with anti-rabbit IgG. In Figure 10 Figure []. The mask sprayed with rabbit IgG produced a dose-dependent signal that was higher than the signal obtained in a control experiment sprayed with BSA. These experiments show that antibody-modified nanoparticles can be transferred to a polypropylene face mask, where they are able to establish specific antibody-antigen interactions.

Claims

1. A method for obtaining a reservoir comprising protein-modified nanoparticles on a paper substrate, the method comprising the steps of: i. treating the paper substrate with poly(sulfonated styrene) on the paper substrate; and ii. applying protein-modified nanoparticles on the poly(sulfonated styrene)-modified paper substrate obtained in step i; wherein the reservoir is a dry reservoir.

2. The method according to claim 1, wherein, The protein is a protein capable of establishing a biological interaction.

3. The method according to claim 2, wherein, The protein is avidin or an antibody.

4. The method according to claim 3, wherein, The protein is an antibody.

5. The method according to claim 1, wherein The nanoparticles are polyethylene glycolylated nanoparticles.

6. The method according to claim 5, wherein, The polyethylene glycolylated nanoparticles are polyethylene glycolylated nanoparticles made of gold.

7. The method according to claim 1, wherein The treatment of step i is carried out by spotting poly(sulfonated styrene) on the paper substrate sheet.

8. The method according to claim 1, wherein The paper substrate sheet of step i is cut into a square form.

9. The method according to claim 1, wherein The volume of poly(sulfonated styrene) added is large enough such that the volume of the poly(sulfonated styrene)-modified paper is larger than the volume to be occupied by the nanoparticles.

10. The method according to claim 9, wherein, The volume of poly(sulfonated styrene) added is 50 μL, and the volume of the polyethylene glycolylated nanoparticles is 1 μL.

11. The method according to claim 1, further comprising step iii of drying the paper after step i.

12. The method according to claim 1, further comprising step iv of allowing the paper to dry after step ii.

13. The method according to claim 12, wherein, Step iv of allowing the paper to dry is carried out at a temperature of 15 °C to 25 °C.

14. The method according to claim 1, further comprising step v of releasing the nanoparticles from the reservoir.

15. The method according to claim 14, wherein Step v is a transfer step to a receiving substrate.

16. The method according to claim 15, wherein The transfer step is carried out by pressing.

17. The method according to claim 16, wherein The transfer step is carried out by pressing with a finger, a press or a clamp.

18. The method according to claim 15, wherein, The transfer step is adjusted by fine-tuning the poly(sulfonated styrene) content and the nanoparticle concentration.

19. The method according to claim 15, wherein, The protein bound to the nanoparticles establishes a biological interaction with the target bound to the receiving substrate in step v.

20. The method according to claim 19, wherein, The target is biotin or an antigen.

21. A reservoir comprising protein-modified nanoparticles on a paper substrate, the reservoir being obtained by the method according to any one of claims 1 to 20.

22. Use of the protein-modified nanoparticle reservoir according to claim 21 in the preparation of a biosensor made of paper, wherein the protein is a protein capable of establishing a biological interaction, the nanoparticles are polyethylene glycolylated nanoparticles.

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