This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in ACS Applied Materials and Interfaces, copyright © American Chemical Society after peer review. To access the final edited and published work see: https://dx.doi.org/10.1021/acsami.9b23487. Chitin Nanofiber Paper towards Optical (Bio)sensing Applications Tina Naghdi†, ‡, Hamed Golmohammadi †,*, Hossein Yousefi §, Mohammad Hosseinifard † Uliana Kostiv, ⊥ Daniel Horák, ⊥ and Arben Merkoçi ,⃰ ‡ ,‖ † Nanosensor Bioplatforms Laboratory, Chemistry and Chemical Engineering Research Center of Iran, 14335-186, Tehran, Iran, ‡ ICN2 - Nanobioelectronics & Biosensors Group, Institut Catala de Nanociencia i Nanotecnologia, Campus UAB, Bellaterra, Barcelona 08193, Spain, § Laboratory of Sustainable Nanomaterials, Department of Wood Engineering and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan 4913815739, Iran, ⊥Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského Sq. 2, Prague 6 162 06, Czech Republic, ‖ ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain. * Corresponding authors: Hamed Golmohammadi, Arben Merkoçi Email : golmohammadi@ccerci.ac.ir arben.merkoci@icn2.cat ABSTRACT Because of numerous inherent and unrivalled features of nanofibers made of chitin, the second most plentiful natural-based polymer (after cellulose), including affordability, abundant nature, biodegradability, biocompatibility, commercial availability, flexibility, transparency and extraordinary mechanical and physicochemical properties, chitin nanofibers (ChNFs) are being applied as one of the most appealing bionanomaterials in a myriad of fields. Herein, we exploited the beneficial properties offered by ChNF paper to fabricate transparent, efficient, biocompatible, flexible and miniaturized optical sensing bioplatforms via embedding/immobilizing various plasmonic nanoparticles (silver and gold 1 nanoparticles), photoluminescent nanoparticles (CdTe quantum dots, carbon dots and NaYF4:Yb3+@Er3+&SiO2 upconversion nanoparticles) along with colorimetric reagents (curcumin, dithizone and etc.,) in 3D nanonetwork scaffold of ChNF paper. Several configurations, including 2D multi-wall, 2D cuvette patterns with hydrophobic barriers/walls and hydrophilic test zones/channels were easily printed using laser printing technology or punched as spot patterns on the dried ChNF paper-based nanocomposites to fabricate the (bio)sensing platforms. A variety of (bio)chemicals as model analytes were used to confirm the efficiency and applicability of the fabricated ChNF paper-based sensing bioplatforms. The developed (bio)sensors were also coupled with smartphone technology to take the advantages of smartphone-based monitoring/sensing devices along with the Internet of Nano Things (IoNT)/the Internet of Medical Things (IoMT) concepts for easy-to-use sensing applications. Building upon the unrivaled and inherent features of ChNF as a very promising bionanomaterial, we foresee that the ChNF paper-based sensing bioplatforms will emerge new opportunities for the development of innovative strategies to fabricate cost-effective, simple, smart, transparent, biodegradable, miniaturized, flexible, portable, and easy-to-use (bio)sensing/monitoring devices. KEYWORDS: Chitin nanofibers; Optical sensor; Sensing bioplatform; Bionanocomposite, Internet of Things. 1. INTRODUCTION Despite the tremendous and profound impact of using polymeric materials in everyday life, the growing concerns about the use of these materials due to adverse effects associated with their usage on human and environmental health have 2 attracted enormous attention during the recent decades to replace them with natural- based biopolymers 1-2. The ever-increasing demand to natural-based biopolymers is attributed to their inherent non-toxicity, renewability, and biodegradability properties 1-3. Among the bio-based polymers, chitin as the second most plentiful natural-based polymer (after cellulose) and the most abundant natural amino-polysaccharide with an annual biosynthesis around 1010−1011 tons, which is made of β-(1,4)-2- acetamido-2-deoxy-D-glucose and mainly found in the exoskeletons of numerous organisms including marine shrimp and crabs, insects, fungi and yeasts, has received considerable attention in several areas on account of its inherent and beneficial features such as low cost, abundant nature, non-toxicity, renewability, biodegradability, sustainability and biocompatibility compared to conventional polymeric materials 4-16. This biopolymer is composed of chitin nanofibrils or chitin nanofibers (ChNFs) at the nanoscale with the diameter ranging from 3 to 40 nm and length longer than 5 micrometers. Interestingly, ChNFs as one of the natural-based nanomaterials, which are completely produced from chitin compounds through top- down approaches including chemical, mechanical or chemi-mechanical routes, not only have all the aforementioned beneficial properties of chitin but bring optical transparency and high reinforcing capability as well. Furthermore, because of fascinating features including high aspect ratio, strong hydrogen bonding, extended crystalline structures and other remarkably unique characteristics of nanomaterials along with the aforementioned properties of chitin, ChNFs are being exploited as one of the most desirable bionanomaterials in various fields including food, pharmaceutical, biomedical, cosmetic industries, energy, electronics, electrical and optical devices 4-18. Special examples of myriad applications of ChNFs include the low-calorie foods 19, natural biodegradable films to preserve foods from microbial 3 deterioration 20, filters for purification of water 21, medical devices and wound-healing dressings 22-23, drug delivery 8, organic light-emitting diodes (OLED) 7, thin film flexible transistors and solar cells 7, tissue engineering applications 17, energy storage devices 9, and novel aerogels 24. Nevertheless, despite the diverse uses of ChNFs, so far, they have been rarely exploited for optical (bio)sensing applications. On the other hand, although paper-based analytical devices as efficient sensing platforms have recently received great attention for (bio)sensing applications25-30 because of the advantageous properties of paper including affordability, abundant availability, versatility, biodegradability, printability, flexibility, portability, and porosity, however, there are major hurdles restricting their development. Namely, the intrinsic features of paper including large surface roughness, low stability and mechanical strength in the aqueous media, and especially its optical opaqueness can hinder the further progress of paper-based analytical devices in optical sensing applications 3, 31. Accordingly, it is urgent to develop new optical sensing bioplatforms that, while having the beneficial properties of paper-based sensors, are optically transparent and possess more chemical, mechanical, and thermal stability as well. We have already fabricated optical sensing platforms using bacterial cellulose (BC) nanopaper 31-34. In the current research, the optical sensing platforms were fabricated from ChNF paper. Unlike BC nanopaper, ChNFs are isolated from very cheap raw material of shrimp’s shells, which are available in abundant amount almost in every country. The production of BC nanopaper possesses time- consuming and costly process as well, while that of ChNF is inexpensive, easy and fast. Moreover, unlike BC nanopaper, owing to the presence of the amide and hydroxyl reactive functional groups on the surface of ChNF paper, there is no need to modify the ChNF papers through chemical reactions for immobilization of different 4 (bio)chemicals/nanomaterials in their nanoscaffold. On the other hand, the conversion of very cheap and abundant raw materials of shrimp’s shells into ChNFs and their application in the fabrication of sensing platforms can be of great importance from technical, economic and environmental points of view. Herein, we exploited the beneficial features offered by ChNF paper to fabricate transparent, flexible, biocompatible, lightweight and efficient optical sensing bioplatforms through embedding various plasmonic nanoparticles (NPs): silver and gold NPs; photoluminescent NPs (PLNPs): N-Acetyl L-Cysteine/TGA capped cadmium telluride (CdTe) quantum dots (QDs), carbon dots (CDs) and NaYF4:Yb3+@Er3+&SiO2 up-conversion nanoparticles (UCNPs); along with colorimetric reagents (curcumin, dithizone and etc.,) in 3D nanonetwork scaffold of ChNF paper. To fabricate the ChNF paper-based (bio)sensing platforms, several patterns including 2D multi-wall and 2D cuvette patterns with hydrophobic walls and hydrophilic test zones were easily printed by using an office laser printer or punched as spot patterns on the dried films of the fabricated ChNF paper-based nanocomposites. A various of (bio)chemicals were tested as model analytes to confirm the efficiency of the developed ChNF paper-based sensing bioplatforms. The fabricated ChNF paper-based sensing bioplatforms were also coupled with smartphone technology for (bio)sensing applications. Additionally, we have designed an Internet of Nano Things (IoNT)/ Internet of Medical Things (IoMT) Model to improve potential applicability and efficiency of the developed smartphone-based (bio)sensing device via IoT concept. Proposed by Ashton in 1999 35, IoT has revolutionized our world in so many different ways and offered new solutions for our 5 complex world. IoT is a combination of data from sensors and networks that provide different smart services 36. It links all kinds of connected “Things” into a comprehensive network of interrelated computing intelligence without the involvement of a human 37. It aims to add computer-based logic to plenty of things which can then be monitored or controlled by analytics or machines 38. Besides, IoT ecosystem contains web-enabled smart devices that use implanted processors, sensors and communication hardware to collect, send and use the data they acquire from their settings 36. Nowadays, IoT is known as a “megatrend” which goes beyond the limitations of all scientific and engineering disciplines and institutes an integrating technical evolution to improve our life 39. Moreover, inter-connection of Nano-sensors and Nano-devices with Internet has also led to a new evolution called “Internet of Nano Things” (IoNT) 40-41. The coupling of ChNF paper-based sensors to smartphones could significantly increase the potential applications of these sensing bioplatforms and open up new opportunities to develop affordable, portable, and easy-to-use sensing/monitoring devices, especially in the developing smart cities. 2. RESULTS AND DISCUSSION The aim of the current study is the fabrication of transparent, flexible, biocompatible, compact, portable, and efficient ChNF paper-based optical sensing bioplatforms through immobilization/embedding of various plasmonic NPs, PLNPs and colorimetric reagents in 3D nanoscaffold of ChNF paper. In fact, in this work, we are intending to exploit unrivaled and beneficial features offered by ChNF paper in combination with fascinating and unique photophysical properties of plasmonic and PLNPs and colorimetric chemicals to develop bionanocomposites that can be utilized as novel (bio)sensing platforms, which are compatible with the World Health 6 Organization’s ASSURED (affordable, sensitive, specific, user-friendly, rapid & robust, equipment free and deliverable to the end-users) criteria for the ideal diagnostic devices/assays, for easy (bio)chemical sensing applications. The ChNF paper used throughout this work, as a thin sheet fully made from ChNFs, was produced from shells of shrimp (Littopenaeus vannamei) through a top-down chemi-mechanical approach. The experimental steps to produce ChNF paper are shown in Figure S1. The fibers diameter of the produced ChNF paper was in the range of 5-55 nm (average: 26±8 nm). The crystallinity index, tensile strength, Young’s modulus and strain-at-break of the produced ChNF paper were 89%, 135 MPa, 7.7 GPa, and 8.2%, respectively (See the Experimental section and Figures S2-S4 in the Supporting Information for more details on the production and characterization of the ChNF paper). The high mechanical properties of ChNF paper are attributed to the high degree of crystallinity, high aspect ratio, and specific surface area as well as its network structure. In this section, the preparation of different ChNF paper-based nanocomposites, focusing on their fabrication, mechanism of sensing and also characterization via UV–visible spectroscopy (UV–vis), field emission scanning electron microscopy (FE- SEM), energy-dispersive X-ray spectroscopy (EDX), and Thermogravimetric analysis (TGA) techniques will be discussed. Various patterns with hydrophilic test zones/channels and hydrophobic barriers/walls were created on the fabricated ChNF paper-based sensing bioplatforms using laser printing technology. 7 Figure 1. Schematic representation for the fabrication of different chitin nanofiber (ChNF) paper-based nanocomposites. (A and B) Fabrication of plasmonic ChNF paper-based nanocomposites: (A) silver nanoparticles-ChNF paper (AgNPs-ChNF paper); (B) gold nanoparticles ChNF paper (AuNPs-ChNF paper). (C and D) Fabrication of colorimetric ChNF paper-based nanocomposites: (C) Curcumin-ChNF paper (Cur- ChNF paper); (D) Dithizon-ChNF paper (DTZ- ChNF paper). (E and F) Fabrication of photoluminescent ChNF paper-based nanocomposites: (E) CdTe@ZnS quantum dots ChNF paper (QDs- ChNF paper), and Carbon dots- ChNF paper (CDs- ChNF paper); (F) aminosilica-coated NaYF4:Yb3+@Er3+ upconversion NPs-ChNF paper (UCNPs- ChNF paper). 8 Several strategies have been employed to embed and immobilization of different reagents in ChNF paper to fabricate various ChNF paper-based nanocomposites, which are schematically illustrated in Figure 1, including in situ synthesis of metallic NPs in ChNF paper through in situ chemical reduction of noble metal ions to metallic NPs by using the hydroxyl and amide functional groups of the ChNFs as reducing agents (Pathway A; Figure 1A); by first immobilization of noble metal ions within the ChNF paper substrate and subsequently chemical reduction of noble metal ions to metallic NPs after addition of chemical reducing agents (Pathway B; Figure 1B); by in situ embedding/immobilization of hydrophobic chemical reagents into the fibril network of the ChNF paper via hydrophilic-hydrophobic interactions (Pathway C,D; Figure 1C,D); and by direct coupling the functionalized NPs with functional groups of the ChNF paper (Pathway E,F; Figure 1E,F). The plasmonic ChNF paper-based nanocomposites (AgNPs-ChNF paper and AuNPs-ChNF paper) were fabricated via Pathways A and B, respectively. The colorimetric ChNF paper-based nanocomposites (Curcumin/ChNF paper (Cur-ChNF paper) and Dithizone/ChNF paper (DTZ-ChNF paper) were fabricated via Pathways C and D; respectively. The photoluminescent ChNF paper-based nanocomposites (QDs-ChNF papers, CDs- ChNF paper and UCNP-ChNF papers) were also fabricated via Pathways E and F, respectively. To fabricate the (bio)sensing platforms, various patterns with desired designs/shapes, including 2D multi-wall and 2D cuvette patterns with hydrophobic barriers/walls and hydrophilic test zones/channels can be easily created by using an office laser printer via direct-printing the hydrophobic toner layer on the fabricated ChNF paper-based nanocomposites (see Figures 2A and 2B). The dried films of the fabricated ChNF paper-based nanocomposites can also be easily cut using a hole 9 punch to fabricate spot patterns for individual assays (See Figure 2C). The flexibility and the created hydrophobic (black parts) and hydrophilic (colorless parts) areas on the fabricated ChNF paper-based sensing platforms have also been depicted in Figure 2D, and 2E, respectively. Figure 2. Pictures of the fabricated ChNF paper-based sensing platforms with (A) 2D multi-wall, (B) 2D cuvette and (C) spot patterns. (D) A picture showing the flexibility of the fabricated ChNF paper-based sensing platform. (E) A picture of the fabricated ChNF paper-based sensing platform: comparison of hydrophobic (black parts) and hydrophilic (colorless parts) areas after applying a drop (10 µL) of colored water. To verify the applicability of the developed ChNF paper-based sensing bioplatforms, they were exploited for optical sensing of various (bio)chemicals. Interestingly, owing to optical transparency of the developed ChNF paper-based sensing bioplatforms, their color changes (e.g., fluorescence or absorbance intensity) upon the addition of analytes can be monitored spectroscopically using a spectrofluorometer/spectrophotometer or visually by a naked eye. 10 To enhance the applicability of the developed ChNF paper-based sensing bioplatforms, they were also coupled with smartphone technology as a new generation of easy-to-use, cost-effective, rapid, and portable monitoring devices. Full details on all experimental procedures and setups are presented in the Experimental section and Supporting Information. In the present work, the photoluminescence and UV-vis absorption spectra of the fabricated ChNF-based optical sensing platforms were carried out using a spectrofluorometer/spectrophotometer. The images, color intensities, and corresponding determined concentrations of the test zones of the fabricated ChNF paper-based optical sensing platforms were taken and processed using the developed smartphone-based 3D-printed platform/setup. 2.1. Plasmonic ChNF paper-based optical sensing platforms In this work, plasmonic sensors were made using two types of noble metals (Ag and Au), which were synthesized/loaded into the ChNF paper. Since the color changes of noble metallic NPs, which associated with their surface plasmon absorption bands, are extremely dependent on their inter-particle spacing, composition, shape, and size, as well as the dielectric properties of their surrounding medium, they have been extensively employed as versatile and efficient platforms in sensing applications 42-45. One of the beneficial ways that strongly affects the characteristics of plasmonic NPs- based sensors is their immobilization/synthesis on appropriate substrates 33. For this purpose, we firstly in situ synthesized AgNPs in the ChNF paper. For the synthesis of AuNPs in the ChNF paper, we used two methods: I) in situ synthesis of AuNPs within the ChNF paper as a reducing agent and II) synthesis of AuNPs using 11 hydrazine as a reducing agent and then embedding the synthesized AuNPs within the scaffold of ChNF paper. The images and scanning electron micrographs of bare ChNF paper, the fabricated AgNPs-ChNF paper and the fabricated AuNPs-ChNF paper (using hydrazine) are shown in Figure 3A-C, respectively. The cross-section scanning electron micrographs and phase image of bare ChNF paper, the fabricated AgNPs-ChNF paper and the fabricated AuNPs-ChNF paper (using hydrazine) are also shown in Figure S5A-C and S5D-F, respectively, which confirm the homogeneous distribution of the plasmonic NPs inside the ChNF paper. The image, UV-vis spectrum, and scanning electron micrograph of the in situ synthesized AuNPs-ChNF paper are shown in Figure S6. EDX spectra and TGA analysis were also carried out to verify the synthesis of AgNPs and AuNPs in the ChNF paper matrix (see Supporting Information Figure S7). We then used the fabricated AgNPs-ChNF paper as plasmonic sensors for optical sensing of the drug methimazole (MTZ) and the toxic compound cyanide (CN-) 32-33. We observed the color changes of the AgNPs-ChNF papers from yellow to dark yellow by increasing the MTZ concentration, along with a red-shift to longer wavelengths in the UV-vis absorption spectra (see Figure 3D) and an increase in the size of AgNPs due to aggregation of AgNPs in the AgNPs-ChNF paper (see Figure 3B-b, Supporting Information Figure S8D). A color change from yellow toward light yellow is also observed by increasing the concentration of CN-, which is accompanied with a shift to shorter wavelengths (blue-shift) in the UV-vis absorption spectra (see Figure 3E) and a decrease in the size of AgNPs owing to the etching of AgNPs in the AgNPs-ChNF paper (see Figure 3B-c). Monitoring of MTZ and CN- is based on the high affinity of the CN- ions and thiol groups of MTZ to the AgNPs- 12 ChNF paper, which consequently leads to changes in the size-dependent optical properties, surface plasmon absorption bands, and color of the AgNPs-ChNF paper. To demonstrate the potential of the fabricated AuNPs-ChNF paper as a plasmonic sensor, it was employed for optical sensing of the noxious and harmful heavy metal ion Hg2+. Figure 3F illustrates the color changes of the AuNPs-ChNF papers from red to purple, along with a red-shift in their UV-vis absorbance spectra upon addition of Hg2+. The respective FE-SEM micrograph, which shows an increase in the size of AuNPs in the presence of Hg2+ due to the aggregation of AuNPs in the AuNPs-ChNF paper, is also illustrated in Figure 3C-b. The calibration curves and analytical characteristics corresponding to these experiments (the AgNPs-ChNF papers for MTZ and CN- sensing and the AuNPs-ChNF paper for Hg2+ sensing) are presented in Supporting Information Figure S8 A-C and Table S1. The size-distribution of the fabricated plasmonic ChNF papers were roughly estimated by image processing from FE-SEM images and the results confirmed the recommended size-depending sensing mechanisms (see Supporting Information Figure S8D and E). To evaluate the selectivity of the fabricated plasmonic ChNF paper-based optical sensing platforms, the competitive experiments were independently performed at a fixed concentration of CN- (3 ppm) and MTZ (100 ppm) for AgNPs-ChNF paper, and Hg2+ (10 ppm) for AuNPs-ChNF paper in the presence of some chemicals probably existing in water samples. Figure S9-11, indicate the good selectivity of the developed plasmonic ChNF paper-based optical sensing platforms toward CN-, MTZ, and Hg2+, respectively. 13 Figure 3. The fabricated plasmonic ChNF paper-based optical sensing platforms. (A) Scanning electron micrographs and a digital photograph (the inset image up-left) of bare ChNF paper. (B) Scanning electron micrographs of the fabricated AgNPs-ChNF paper: (B-a) in situ synthesized AgNPs-ChNF; (B-b) AgNPs-ChNF in the presence of 100 ppm of MTZ; (B-c) AgNPs-ChNF paper in the presence of 3 ppm of CN-. The inset image (up-left) is a digital photograph of the fabricated AgNPs-ChNF paper. (C) Scanning electron micrographs of the fabricated AuNPs-ChNF paper: (C-a) AuNPs- ChNF paper; (C-b) AuNPs-ChNF paper in the presence of 10 ppm of Hg2+. The inset image (left) is a digital photograph of the fabricated AuNPs-ChNF paper. Changes in UV-vis spectra and corresponding colors of (D) AgNPs-ChNF paper upon addition of MTZ (0-200 ppm); (E) AgNPs-ChNF paper upon addition of CN- (0-5 ppm) and (F) AuNPs-ChNF paper upon addition of Hg2+ (0-20 ppm). 14 2.2. Colorimetric ChNF paper-based optical sensing platforms In this work, colorimetric ChNF paper-based nanocomposites were fabricated through embedding/immobilization of curcumin (Cur) and 1, 5-diphenyl thiocarbazone (dithizone) within the ChNF paper. More details on the fabrication of colorimetric ChNF paper-based sensing platforms using curcumin and DTZ (the Cur- ChNF paper and the DTZ-ChNF paper) are presented in the Experimental section. The yellow-orange color (Figure 4A (up)) and the UV-vis absorption spectrum (Figure 4B) of the fabricated Cur-ChNF paper with a characteristics absorption band (λ~440 nm), which are related to the presence of curcumin, confirm the immobilization of curcumin into the ChNF paper. The color (Figure 4A (down)) and the UV-vis absorption spectrum (Figure 4C) of the fabricated DTZ-ChNF paper also verify the immobilization of DTZ into the ChNF paper. Curcumin or diferuloylmethane, is a yellow-orange water-insoluble natural pigment and the active ingredient of turmeric “Curcuma longa”. Curcumin is broadly used as a seasoning, coloring and protective agent in a variety of foods and drugs, as well as for treatment of various infections and diseases on account of its recognized antioxidant, antibacterial, anti-carcinogenic, anti-inflammatory and wound healing properties. Curcumin has also been employed for sensing different (bio)chemicals 46- 47. Based on studies that have been previously reported, the color and keto-enol tautomerism forms of curcumin depend on pH 46-47. In the present work, as a representative example of the potential application of the fabricated Cur-ChNF paper, it was exploited as a colorimetric pH sensor. The color changing and UV-vis peaks of the Cur-ChNF paper at different pH values (from 7-13) are shown in Figure 4B. It is revealed from Figure 4B that the absorption intensities of the Cur-ChNF papers firstly decrease at λ~440 nm in the pH range of 7–10 along with a color 15 change from yellow toward red, and new red-shifted UV-vis peaks at λ~480 nm appear in the pH range of 11–13 with a color change from red toward brown. DTZ is an organic chelating chemical agent that can form colored water-insoluble complexes with some metal cations 48. We assessed the potential application of the fabricated DTZ-ChNF paper as a colorimetric sensor for determination of seven heavy metal ions, including Zn2+, Co2+, Fe3+, Ni2+, Cd2+, Hg2+, and CrO42- based on DTZ chelation. Figure 4. The fabricated colorimetric ChNF-based optical sensing bioplatforms. (A) The digital photographs of colorimetric ChNF-based nanocomposites: (up) curcumin- ChNF paper (Cur-ChNF paper) and (down) dithizone-ChNF paper (DTZ-ChNF paper). Changes in UV-vis spectra and corresponding colors of (B) Cur-ChNF paper at various pHs in the range of 7–13; (C) DTZ-ChNF paper upon the addition of different metal ions (100 ppb for Zn2+, 1 ppm for other metal ions), and (D) DTZ-ChNF paper upon addition of Zn2+ (20-200 ppb). 16 Figure 4C illustrates the variations in the UV-vis absorption spectra along with corresponding colors of the fabricated DTZ-ChNF paper as a result of the addition of different metal ions (100 ppb for Zn2+, 1 ppm for other metal ions). In this case, the reaction between transition metal ions with DTZ ligand in the fabricated DTZ-ChNF paper results in formation of highly colored-complexes owing to cation−π interactions, electronic transitions, and also formation of coordination bonds between thiol group of DTZ and metal cations 48. Figure 4D also shows the changes in color and the UV-vis absorption spectra of the DTZ-ChNF paper upon addition of different doses of Zn2+ ions (20-200 ppm). 2.3. Photoluminescent ChNF paper-based optical sensing platforms Among different nanomaterials, photoluminescent nanoparticles (PLNPs) particularly colloidal semiconductor nanocrystals or quantum dots (QDs), carbon dots (CDs) and more recently upconversion nanoparticles (UCNPs) have given rise to enormous interest in various (bio)sensing applications on account of their unique, fascinating and tunable physicochemical features 45, 49-51. QDs recognize as zero-dimensional (0-D) semiconductor nanocrystals with sizes in the range of 1-10 nm. The outstanding photophysical features of QDs such as high emission quantum yields, stable and size-tunable photoluminescence emissions, broad absorption spectra and long term photostability, which originate from the quantum confinement effect, as well as wide chemical-modification capabilities, make them among the most interesting PLNPs with significant potential in (bio)sensing applications 50, 52-54. Recently, CDs as a new class of 0-D carbon nanomaterials have been widely investigated and applied in different (bio)sensing applications because of their low 17 cost, low toxicity, biocompatibility, good aqueous solubility, easy fabrication, and functionalization, as well as other common photophysical properties with conventional semiconductor QDs 49, 55. Unlike QDs and CDs, which are commonly excited through UV-vis light to produce Stokes-shifted visible photoluminescence emissions, more recently a novel class of PLNPs known as upconversion nanoparticles (UCNPs) have emerged that can be excited in the near infrared (NIR) region to trigger anti-Stokes-shifted photoluminescence emission in the visible region 51, 56-58. Because of unique and extraordinary optical and chemical features of UCNPs including high quantum yield, high resistance to photo-bleaching, low toxicity, long- term lifetime, narrow emission bandwidths, as well as very low optical background noise, they can be potentially considered as appealing alternatives to traditional fluorophores in design and fabrication of novel (bio)chemical sensors 51, 56-58. Along with the beneficial properties of PLNPs, a pivotal step that can significantly boost their potential applications in sensors technology is the immobilization/embedding of PLNPs in appropriate sensing substrates/platforms. In the current work, various photoluminescent NPs-ChNF paper-based nanocomposites were fabricated through embedding/immobilization of different PLNPs (including N-Acetyl L-Cysteine (NAC)-capped cadmium telluride (CdTe) quantum dots (QDs) with different emission: green-, yellow, and red-emitting QDs (GQDs, YQDs, and RQDs); thioglycolic acid (TGA) CdTe QDs with greenish-yellow emission (GyQDs); carbon dots (CDs) and amine-functionalized NaYF4:Yb3+@Er3+&SiO2 upconversion nanoparticles (UCNPs)) within the ChNF paper. Interestingly, to use ChNF paper as a biotemplate to embed the PLNPs, there is no need to modify the ChNF papers via chemical reactions owing to the presence 18 of the amide and hydroxyl functional groups on the surface of ChNF paper, which can facilitate the direct coupling of amine/carboxyl-functionalized PLNPs with the ChNF paper. The fabricated QDs-ChNF based sensing platforms and the corresponding photoluminescence emissions are shown in Figure 5A. The fabricated CDs-ChNF based sensing platform and the corresponding photoluminescence emission are shown in Figure 5B. The fabricated UCNPs-ChNF-based sensing platform is also shown in Figure 5F. More details on the fabrication of different types of PL-ChNF paper-based sensing platforms are presented in the Experimental section. As representative examples of the potential of the fabricated PLNPs-ChNF paper platforms for photoluminescent sensing applications, we assessed the CDs-ChNF paper for sensing of quercetin and the YQDs-ChNF paper for sensing of sulfide ions (S2-). Figures 5B and 5C illustrate the changes in photoluminescence intensity of the CDs-ChNF and YQDs-ChNF papers upon addition of different doses of quercetin (from 40 to 300 ppm) and sulfide ions (from 20 to 200 ppm), respectively. The calibration curves and LOD values corresponding to these experiments are presented in Supporting Information Figure S12. Photoluminescence spectra from the CDs-ChNF paper and YQDs-ChNF paper spots were obtained using a spectrofluoremeter (excitation wavelength (λEx), 370 nm; emission wavelengths (λEm) for the CDs-ChNF and YQDs-ChNF papers were 445 nm and 560 nm, respectively). The photoluminescence of all the fabricated PLNPs-ChNF paper nanocomposites was also modulated by graphene oxide (GO) as a well-known, highly efficient photoluminescence quenchers. 19 Figures 5D-5F show vividly how the photoluminescence intensity of the fabricated CDs/YQDs/UCNPs ChNF paper-based sensing platforms is decreased upon addition of various concentrations of GO. Figures 5G-5L also demonstrate the confocal microscopy images of the ChNF paper and the fabricated PLNPs-ChNF papers before and after addition of GO (320 ppm), which clearly show the photoluminescence quenching caused by GO due to fluorescence resonance energy transfer (FRET) mechanism, where GO and PLNPs- ChNF papers operate as FRET acceptor and donor, respectively. 20 Figure 5. The photoluminescent nanoparticles-ChNF paper-based optical sensing platforms. The photoluminescence spectra and corresponding colors of the fabricated (A) N-Acetyl L-Cysteine (NAC)-functionalized cadmium telluride (CdTe) quantum dots (QDs)-ChNF paper based sensing platforms with different emission: green-emitting QDs (GQDs), yellow-emitting QDs (YQDs), red-emitting QDs (RQDs); thio-glycolic acid (TGA) CdTe QDs with greenish-yellow emission (GyQDs); (B) carbon dots (CDs)-ChNF paper upon addition of different concentrations of quercetin; (C) YQDs-ChNF paper upon addition of different concentrations of S2-. Modulation of the photoluminescence intensity of the fabricated (D) CD-ChNF paper; (E) YQD-ChNF paper and (F) amine-functionalized NaYF4:Yb3+@Er3+&SiO2 upconversion nanoparticles (UCNPs)-ChNF paper based sensing platforms by addition of graphene oxide (GO) at different concentrations. Confocal microscopy images of the fabricated (G) ChNF paper; (H) CD-ChNF paper (λEx=370 nm, maximum λEm~445 nm); (I) YQD- ChNF paper (λEx=370 nm, maximum λEm~560 nm); (J) GyQD-ChNF paper (λEx=370 nm, maximum λEm~555 nm).; (K) GQD-ChNF paper (λEx=370 nm, maximum λEm~450 nm); and (L) RQD-ChNF paper (λEx=370 nm, maximum λEm~610 nm) based sensing platforms before (left) and after (right) addition of GO. 2.4. ChNF paper-based assay kits To demonstrate the general applicability of the ChNF paper in the fabrication of (bio)chemical assay kits, as a representative example of the potential of the ChNF paper-based sensing platforms for a wide range of sensing applications, it was exploited in the fabrication of a colorimetric assay kit for simultaneous determination of glucose, total bilirubin and albumin in human blood samples. On the other hand, given the weak microfluidic property of the ChNF paper as well as to separate plasma from whole blood samples without centrifugation, which is an essential step 21 in the development of plasma-based assays, the ChNF paper was combined with plasma separation membrane and filter paper in a specially designed pattern. Full details on design and fabrication of the ChNF paper-based assay kit and its application in blood samples analysis are provided in the Experimental section and Figures 6A and 6B. The images and calibration curves corresponding to these experiments for simultaneous colorimetric determination of glucose, total bilirubin and albumin in human blood samples using the developed smartphone-based ChNF paper assay kit are presented in Figure 6C-6E, respectively. The clinical efficiency of the developed smartphone-based ChNF paper assay kit was also validated by analyzing the blood samples of 5 volunteers and comparing their results with glucose, total bilirubin, and albumin Assay Kits as clinical reference methods. The results of both methods for glucose, total bilirubin, and albumin are plotted against each other for comparison in Figure S14A-C, respectively. The obtained regression coefficients for each analyte indicate the results of the developed smartphone-based ChNF assay kit are comparable to reference methods, which are routinely exploited in clinical laboratories for glucose, total bilirubin, and albumin detection level in blood samples. The obtained results show that the developed smartphone-based ChNF paper assay kits as user-friendly, cost-effective and portable sensors can be potentially utilized for fast and easy detection of biological analytes at the point-of- care (POC) as well as in the clinical laboratories. 22 Figure 6. (A) Schematic representation for the fabrication of ChNF paper-based assay kit. (B) The digital photographs of the fabricated ChNF paper-based assay kit, which was used for colorimetric determination of glucose, total bilirubin and albumin in blood samples, before (left) and after (right) applying a drop of blood. The images and corresponding calibration curves for colorimetric determination of (C) glucose; (D) total bilirubin; and (E) albumin using the developed smartphone-based ChNF paper assay kit. (Colorimetric reagents for glucose: phosphate buffer (pH= 7.5, 250 mM) phenol (5 mM), 4-aminoantipyrine (0.5 mM), glucose oxidase>10 kU/; for total bilirubin: EDTA-Na2 (0.07 mM), NaCl (6.6 gL-1), sulfamic acid (70 mM), 2,4- dichlorophenyl-diazonium salt (0.09 mM), HCl (130 mM), EDTA-Na2 (0.02 mM); for albumin: Citrate buffer (pH =4.2, 30 mM), bromocresol green (0.26 mM)). 23 2.5. Integration of IoT in the developed smartphone-based ChNF paper assay kits Since its arrival, nanotechnology has been offered effective and valuable solutions in so many diverse fields such as biomedical, industry, agriculture and military. Moreover, inter-connection of Nano-devices and Nano-sensors with Internet has led to a new revolution in this field called “Internet of Nano Things” (IoNT) 59-60. Developing and establishing such technical revolution into the value chain of multifaceted areas such as healthcare requires the implementation and connection of hi-tech analytical innovations 39. On the other hand, successful implementation and integration of IoT technology in healthcare and especially in POC diagnostic tests include several challenges in different layers such as data storage, data management, exchange of data between devices, security and privacy, and unified and ubiquitous access. The most possible solution to aforementioned challenges is cloud computing technology and specially fog-assisted computing. Cloud computing is backbone of IoT healthcare systems. It has scalability, mobility and security benefits by providing on-demand computing resources (e.g. storage, services, networks, servers, applications, and hardware) to users 61. Herein, we have designed our model of IoNT/IoMT to improve potential applicability and efficiency of our developed smartphone-based assay kits via IoT concept. To have a fully-integrated smartphone-based ChNF paper assay kits, we need to design different but inter-related layers based on the concept of IoT healthcare systems. As a result, we added network and application layers to our model based on the pre- supposition of data security for all layers (Figure 7). Besides, to optimize results from our self-developed mobile’s app, a machine learning layer has also been designed. 24 Machine learning is closely connected to computational statistics which concentrates on making predictions using computers 62. It has been witnessed huge progression in mathematical optimization and computing hardware since its coinage in 1959 by Artur Samuel 63. In our proposed architecture, we have used such notion through a machine learning algorithm in Computational Analysis & Machine Learning layer. As illustrated in Figure 7, our IoNT - IoMT based model has four but inter-related layers with some intermediating layers in and out of each layer constructed on the pre-supposition of storage (MD5: Message-digest algorithm & RSA: Rivest–Shamir– Adleman one of the first public-key cryptosystems) and communication (WPA: Wi-Fi Protected Access, WAP: Wireless Application Protocol, TLS: Transport Layer Security) security using different encryption and safety methods for different type of data usage. It also takes advantage of our self-developed application (Android & iOS) for data processing and results obtained from fabricated ChNF paper-based sensing bioplatforms which coupled with smartphone technology for (bio)sensing applications. The first layer is Device & Perception layer using an access layer (2G/3G/4G/LTE/LTE-A/Broadband/Wi-Fi/WiBro/GPRS) for connectivity. Real-time processing, data visualization, embedded systems, gateways, micro-data storage, access layer are some of first layer characteristics. Besides, both storage and communication encryption and safety methods have been applied for security in this layer. Data mined from smartphone will be transferred into Computational Analysis & Machine Learning layer before further development in order to make predictions or decisions without being explicitly programmed to accomplish the tasks 62. It would assist us to optimize our services and have better results. Decision tree–based 25 machine learning algorithm has been applied to create smart data for further usages63. Smart data created in this layer will be filtered and transcoded in Data Pre- processing layer. Computational & big data analysis, machine learning algorithm, prediction and analysis, smart data are parts of this layer. Besides, both storage and communication encryption and safety methods have been applied for security in this layer. Cloud, Fog and Service layer have been integrated in layer three. After pre- processing of data, it will be transferred to cloud and fog servers. Cloud servers will store patients’ medical records. But their speeds are not sufficient compared to fog servers for real-time processing and giving service to end-users. Thus, fog servers will be used to solve such problems. Moreover, flow of information and data from layer one to three has designed in such a way to have a customer-oriented value chain based on their needs. Therefore, Data has been aggregated in this layer for all clinical usages. Doctors, health professionals and caregivers can take advantages of all kinds of embedded services. Besides, both storage and communication encryption and safety methods have been applied for security in this layer. Primary focus of the present IoT architecture is on medical and diagnostic tests e.g. POC diagnostic tests. Consequently, Business layer has been designed to fit into aforementioned aim. Our user-friendly self-developed mobile application is an added-value in our model and helped us to reach such a goal. Therefore, Business layer has been proposed a hybrid solution to have a better service not only for private but also for public sector as well. It has followed the same approaches in IoT healthcare and IoMT. As a result, we have targeted not only home-users but also experts and medical centers as our potential customers. Business ethics, Customer Relationship Management (CRM), service management, marketing & sales are 26 important parts of this layer. Besides, both storage and communication encryption and safety methods have been applied for security in this layer. Figure 7. The proposed IoT architecture the developed smartphone-based ChNF paper assay kits. 3. CONCLUSIONS The present work deals with the fabrication of efficient, transparent, flexible, and biocompatible ChNF paper-based bionanocomposites. Various optical (plasmonic, photoluminescent and colorimetric) sensing bioplatforms were fabricated by taking 27 the beneficial properties of ChNF paper including low cost, commercial availability, biocompatibility, biodegradability, flexibility, printability, optical transparency and other extraordinary mechanical and physicochemical properties, in combination with the unique and tunable photophysical and physicochemical properties of plasmonic NPs (AgNPs and AuNPs), PLNPs (different color emitting QDs, CDs, and UCNPs) and colorimetric reagents. A fast and simple patterning method by using an office laser printer, through printing the hydrophobic ink on the ChNF paper, was used to fabricate the ChNF paper-based optical (bio)sensing platforms with different desired patterns including 2D multi-wall, 2D cuvette patterns with hydrophilic test zones and hydrophobic walls/barriers. The applicability and efficiency of the developed ChNF paper-based platforms for analytical tasks were confirmed by optical sensing a wide range of (bio)chemicals as model analytes. Owing to the optical transparency of the developed ChNF paper-based (bio)sensors, their color/fluorescence changes upon the addition of tested analytes can be monitored visually by the naked eye and also spectroscopically using a spectrophotometer/spectrofluorometer. To digitize the images of the obtained colorimetric/fluorometric measurements using the developed ChNF paper-based sensing bioplatforms, they were coupled with mobile phones. Indeed, the coupling of ChNF paper-based sensors to smartphone technology and the designed IoNT model can enhance the potential applications of these novel (bio)sensing platforms as affordable, user-friendly, and portable monitoring/sensing devices, especially in the developing smart cities. On the other hand, the reasons why ChNF-paper can be considered as a novel, attractive and high potential substrate alongside paper and BC nanopaper for making optical sensing platforms have been presented as a comparative table (Table 1). As seen in this table, many properties of ChNF paper, from technical, 28 economic and environmental points of view, are comparable and in some cases better compared to paper and BC nanopaper for making optical sensing platforms. Building upon the unrivalled features of ChNFs as one of very promising bionanomaterial, we believe that the ChNF paper-based sensing bioplatforms have the promising potential to be exploited in the development of new strategies to fabricate simple, cost-effective, easy-to-use, green, transparent, flexible, miniaturized, and smart (bio)sensing/monitoring devices. Table 1. Comparison of normal cellulose paper, BC nanopaper and ChNF paper properties for making optical sensing platforms from technical, economic and environmental points of view. Normal Cellulose Paper BC nanopaper ChNF paper Production cost Very low Medium Low Simplicity of production Very simple Medium Simple method Raw material availability Very high Low High 2 Very short Very long (~two weeks) Short (less than 2 hr) Production time (For 1 m ) (Commercially available) (Commercially available) (Commercially available) Transparency Non-transparent ~ 40% ~50% Functionalizing for sensing Usually needed Usually needed Usually not needed applications Stability in aqueous media Not stable Stable Stable Fabrication of sensing Require expensive Very cheap, easy (one Very cheap, easy (one platforms/Creation of test equipment and multistep step) and fast using step) and fast using zones processing normal printers normal printers Reflectance-based Scanometric, Scanometric, Optical detection methods measurements spectrophotometric, and spectrophotometric, and (scanometric) spectrofluorometric spectrofluorometric Compatibility with bio)chemical High High High for sensing applications Mechanical properties Very low Very high Very high 29 4. Experimental Section 4.1. Reagents and Equipment. Milli-Q ultrapure water was used in all experiments. Curcumin (CAS Number: 458- 37-7, ≥94%), dithizone (CAS Number: 60-10-6, ≥98%), methimazole (CAS Number: 60-56-0, ≥99%), ethylenediamine (CAS Number: 107-15-3, ≥99.5%), ethanol (CAS Number: 64-17-5, ≥99.8%), tellurium powder (CAS Number: 13494-80-9, 99.997%), CdCl2.2H2O (CAS Number: 654054-66-7, 99.995%), N-Acetyl L-Cysteine (NAC, CAS Number: 616-91-1, ≥99%), thioglycolic acid (TGA, CAS Number: 68-11-1, ≥99%) and quercetin (CAS Number: 117-39-5, ≥95%) were obtained from Sigma Aldrich (Taufkirchen, Germany). KCN (CAS Number: 151-50-8, ≥96%), NaOH (CAS Number: 1310-73-2, ≥97%), AgNO3 (CAS Number: 7761-88-8, 99.0-100.5%), HAuCl4. 3H2O (CAS Number: 16961-25-4, 99-100%), acetone (CAS Number: 67-64-1, ≥99.8%), FeCl3.6H2O (CAS Number: 10025-77-1, 97%), K2CrO4 (CAS Number: 7789-00-6, ≥99.5%), ZnCl2 (CAS Number: 7646-85-7, 98.0-100.5%), Na2S.9H2O (CAS Number: 1313-84-4, ≥98.0%), Ni(NO3)2.6H2O (CAS Number: 13478-00-7, ≥99.0%), Co(NO3)2.6H2O (CAS Number: 10026-22-9, ≥98%), Hg(NO3)2.H2O (CAS Number: 7783-34-8, ≥99.99%), 3CdSO4.8H2O (CAS Number: 7790-84-3, ≥98%), and NaBH4 (CAS Number: 16940- 66-2, ≥96.0%) were purchased from Merck (Germany). Britton–Robinson buffers (pH range of 7–12) were prepared through mixing phosphoric acid (Merck, Germany, CAS Number: 7664-38-2, 99%), citric acid (Merck, Germany, CAS Number: 77-92-9, 99.5-100.5%), and boric acid (Merck, Germany, CAS Number: 10043-35-3, 99.5- 100.5%) (0.04 M each). KCl (Merck, Germany, CAS Number: 7447-40-7, ≥99.5%) (0.2 M) was used for preparation of buffer solutions in the pH range of 12–13. Hydrazine hydrate (CAS Number: 7803-57-8, 100%) was purchased from Royalex 30 (India). Graphene oxide suspension was acquired from Angstron Materials, Inc. (Dayton, OH). Water-soluble N-Acetyl L-Cysteine (NAC)-capped CdTe QDs with different emission capability and also highly efficient photoluminescent CDs were produced according to the process described in the Experimental section. Chitin nanofiber paper (ChNF- paper) was supplied from Nano Novin Polymer Co., Iran. Albumin, total bilirubin, and glucose Assay Kits were supplied from Man Co., Iran. MF1, blood separation membrane, was purchased from GE Healthcare; the United States, to separate plasma from blood samples without centrifugation. A spectrophotometer model DR5000TM (Hach, Canada) was used to obtain the UV- vis absorption spectra and absorbance measurements through placing the fabricated ChNF paper-based sensing platforms in a hand-made paperboard sample holder. The photoluminescence spectra and measurements were obtained with a Jasco Model FP-6500 Spectrofluorometer. The images were taken by Samsung Galaxy A7 (16 megapixels). The FE-SEM images and EDX analysis were obtained through a Magellan 400L High-Resolution SEM and a Quanta 650F Environmental SEM(FEI, Hillsboro, OR), respectively. High-resolution transmission electron microscopy (HRTEM) images and dynamic light scattering (DLS) analysis of synthesized CDs were performed by a transmission electron microscope (FEI, TEC9G20, USA) and a particle size analyzer (Scatteroscope I; Qudix, Inc., South Korea), respectively. Confocal microscopy was performed through a Leica TCS SPE. Thermogravimetric analysis (TGA) was performed using the thermogravimetric analyzers (Netzsch-TGA 209 F1, Germany) at a scan rate of 10 °C /min under N 2 atmosphere. A HP LaserJet P1102 official printer was utilized to print the hydrophobic patterns on the dried ChNF papers. 31 4.2. Production of ChNF paper Figure S1 shows the experimental steps to produce ChNF paper. In brief, the shells of shrimp (Littopenaeus vannamei) was first treated with HCl to remove minerals and then refluxed with NaOH two times, as shown in Figure S1 to remove proteins. In the second step of alkaline treatment, 0.1 wt % H2O2 was used to remove pigments. After completion of these treatments, the white shells were ground using a chicken grinder to obtain chitin powder. For mechanical nanofibrillation, the powder was converted to chitin suspension 1 wt% followed by passing through a super disk grinder (MKCA6-3; Masuko Sangyo Co., Ltd.) available at Nano Novin Polymer Co. (Iran). After grinding, chitin suspension was converted to the highly viscous wet gel of ChNF. The well-dispersed ChNF suspension (0.2 wt%) was filtered by vacuuming, followed by hot pressing at 100 °C and 2MPa for 1 h. This process resulted in transparent ChNF paper with a grammage of 60±3 g/m2 and a density of 1.1 g/cm3. The thickness of the produced ChNF paper was ~45 µm as shown in Figure S5A. The characterization of the produced ChNF paper is presented in the Supporting Information. 4.3. Fabrication of plasmonic ChNF paper 4.3.1. Fabrication of AgNPs-ChNF paper The method for in situ synthesis of AgNPs within ChNF papers was represented as follows: Five pieces of dried ChNF papers (6 cm2) were soaked in 50 mL water. The pH of this mixture was then adjusted to 12 by addition of NaOH solution (2 M). After that, the reaction mixture was heated at 60 °C for 15 min, followed by stirring. Following that, 5 mL of 0.1% (w/v) AgNO3 solution was added dropwise (about 1 mL/min) into the reaction mixture under the same temperature conditions and 32 continuous stirring. Then, the contents were kept under stirring and heating at 60 °C for a further 45 min. The color change of ChNF papers from colorless to light yellow and then to amber during the synthesis procedure confirms the successful in situ synthesis of AgNPs through the gradual reduction of Ag+ using ChNF paper. The contents were then cooled, and the fabricated AgNPs-ChNF papers were separated and washed thoroughly with water to ensure that any unreacted Ag+ and free/weakly attached AgNPs have been completely removed from the ChNF papers. The fabricated AgNPs-ChNF papers were finally stored in the refrigerator before being used. The UV-vis absorption spectra, FE-SEM, EDX, and TGA analysis confirm the successful synthesis of AgNPs in the ChNF paper matrix. The fabricated AgNPs- ChNF papers were stable for more than nine months no change in their color or UV- vis spectra. 4.3.2. Fabrication of AuNPs-ChNF paper 4.3.2.1. ChNF paper as reducing agent for in situ synthesis of AuNPs The method for in-situ synthesis of AuNPs within ChNF paper was represented as follows: Five pieces of dried ChNF paper (6 cm2) were soaked in 100 mL of the boiling solution of 0.5 mM HAuCl4 in a covered beaker under stirring. The reaction mixture was kept under boiling and stirring for 2 h. During this procedure, the adsorbed AuCl4- ions in the nanonetwork scaffold of ChNF papers are gently reduced to AuNPs using the hydroxyl and amide functional groups of ChNFs, which act as reducing agents for in-situ synthesis of AuNPs within ChNF papers. The color change of ChNF papers from colorless to pale purple endorses the in situ synthesis of AuNPs within ChNF paper. The fabricated AuNPs-ChNF papers were then separated from the mixture and rinsed thoroughly with water to ensure that any 33 unreacted gold ions or free/loosely attached AuNPs have been completely removed from the ChNF papers. The fabricated AuNPs-ChNF papers were finally stored in the refrigerator before being used. The UV-vis absorption spectra, FE-SEM, EDX, and TGA analysis confirm the in situ synthesis of AuNPs in the ChNF paper matrix. 4.3.2.2. ChNF paper as nanonetwork embedding AuNPs Here, AuNPs are synthesized using hydrazine as reducing agent and then the synthesized AuNPs are embedded within the nanonetwork scaffold of ChNF paper. In brief, ten pieces of the ChNF paper spots (circle shape with a diameter of 5 mm), which have been cut before with a hole punch, were immersed in 2 mL of 0.5 mM HAuCl4 solution for 30 min. After separation and rinsing the ChNF paper spots from the reaction mixture, they were soaked in 25 mL water followed by stirring. 500 μL of 500 μM hydrazine solution was then rapidly added into the reaction mixture. After 10 sec, the fabricated AuNPs-ChNF papers were rapidly separated and washed thoroughly with water to remove any free/loosely attached AuNPs. During this procedure, the color of ChNF papers changes from colorless to wine red, which confirms the presence of the synthesized AuNPs in the nanonetwork scaffold of ChNF papers. The UV-vis absorption spectra, FE-SEM, EDX, and TGA analysis also verify the fabrication of AuNPs-ChNF papers. 4.4. Fabrication of colorimetric ChNF paper 4.4.1. Fabrication of Cur-ChNF paper To fabricate the Cur-ChNF paper, five pieces (6 cm2) of dried ChNF papers were immersed in 25 mL of curcumin solution (0.5 %w/v in water/acetone 6:4(V/V)) followed by stirring at room temperature for 20 min. In this procedure, dissolved 34 curcumin molecules immobilize within the nanonetwork scaffold of the ChNF papers, which is subsequently verified by the color change of the colorless ChNF papers to yellow-orange (color of curcumin). After that, the ChNF paper pieces containing embedded curcumin molecules (Cur-ChNF paper) were separated and washed several times with water to remove any unimmobilized curcumin from the ChNF papers. The fabricated Cur-ChNF papers were kept in the refrigerator before being used. The fabrication of Cur-ChNF paper was also verified by UV-visible absorption spectra. The fabricated Cur-ChNF papers were stable for more than nine months no change in their color or UV-vis spectra. 4.4.2. Fabrication of DTZ-ChNF paper To fabricate the DTZ-ChNF paper, five pieces (6 cm2) of dried ChNF papers were immersed in 20 mL of ethanolic solution of 0.1% w/v dithizone (DTZ) followed by gentle stirring for 30 min. Then, the ChNF paper pieces containing embedded DTZ (DTZ-ChNF paper) were separated and completely washed with water several times to eliminate any unimmobilized DTZ from the ChNF papers. The UV-vis peak and color of the fabricated DTZ-ChNF paper confirm the immobilization of DTZ into the ChNF paper. The fabricated DTZ-ChNF paper was stable for more than nine months no change in its color or UV-vis spectrum. 4.5. Fabrication of photoluminescent (QDs/CDs/ UCNPs) ChNF papers In a typical procedure to embed CDs in the ChNF papers, two pieces (6 cm2) of dried ChNF papers were soaked in 5 mL of the 10-times diluted solution of the as- synthesized CDs followed by gentle stirring for 60 min. The fabricated CDs-ChNF 35 papers were separated and then rinsed with water several times to remove any unattached/unimmobilized CDs from the CDs-ChNF papers. The fabricated CDs- ChNF papers were stored in the refrigerator before being used. The procedures for fabrication of other PLNPs-ChNF papers (QDs/UCNPs-ChNF papers) through embedding/immobilization of different functionalized QDs (including N-Acetyl L-Cysteine (NAC) functionalized CdTe QDs with different emission: green-, yellow, and red-emitting QDs (GQDs, YQDs, and RQDs; Thioglycolic acid (TGA) CdTe QDs with greenish-yellow emission (GyQDs)) and amine-functionalized NaYF4:Yb3+@Er3+&SiO2 upconversion nanoparticles (UCNPs) within the ChNF papers are similar to the aforementioned procedure for fabrication of CDs-ChNF papers, except the as-synthesized PLNPs solutions are used instead of CDs solution. 4.6. Design and fabrication of ChNF paper-based sensing platforms To use the fabricated ChNF paper-based nanocomposites as templates for (bio)sensing, it is necessary to create the test zones on their surface. To meet this end, various patterns with specific patterned layouts (2D multi-wall and 2D cuvette patterns) with hydrophobic walls/barriers and hydrophilic test zones/channels were created using an office laser printer via direct-printing the hydrophobic ink on the fabricated ChNF paper-based nanocomposites. Initially, the desired patterns were created in Microsoft Office Word (2015) and then printed by an office printer on the dried films of the fabricated ChNF paper-based nanocomposites, which have already been attached on normal paper by an adhesive tape. The printed ChNF paper-based sensing platforms were then separated from the paper. Figure 3E depicts the hydrophilic (colorless parts) and hydrophobic (black parts) areas of the printed ChNF 36 paper-based sensing platforms. The percolation of the colored water drops into the hydrophilic area (yellow part) and their lack of percolation into the hydrophobic area because of the ink/toner coating, confirm the creation of hydrophilic test zones (with hydrophobic barriers) on the surface of fabricated ChNF paper-based sensing platforms. The printed ChNF paper-based sensing platforms were finally stored in dark conditions before being used. The ChNF paper-based nanocomposites dried films can also be cut by using a hole punch to fabricate spot patterns for individual assays (see Figure 3C). 4.7. Recommended procedure for detection of (bio)chemicals utilizing the fabricated ChNF paper-based sensing platforms 5 µL (for 2D multi-wall and spot designs) and 20 µL (for 2D cuvette design) of analytes solutions were dropped to each of the test zones of the fabricated ChNF paper-based sensing platforms and then allowed to dry for 15 min at room temperature. For spectrophotometric or spectrofluorometric determination of analytes, the photoluminescence and UV-vis absorption spectra/intensities of the test zones were measured, after inserting them in hand-made paperboard sample holders. For visual or scanometric determination, the images, color intensities and corresponding determined concentrations of the test zones were taken and processed by using the 3D-printed smartphone-based sensing setup. 4.8. Fabrication of 3D-printed smartphone-based sensing setup A dark chamber, as seen in Figure S13, with well-defined places for smartphone camera and UV/white lamps was fabricated using a 3D printer 34. Two UV LED lamps with λ=365 nm (for excitation of the fabricated QDs/CDs–ChNF papers), and 37 two white LED lamps from Epileds, Taiwan with 3.3 V, which placed in defined places of the fabricated smartphone platform, were used for PL and colorimetric sensing, respectively. The photoluminescence of the fabricated UCNPs-ChNF paper was appeared using a 980 nm Infrared Line Laser Module with output power 150 mW and power supply 3mA from SHHO, China, which placed in the defined place of the fabricated smartphone platform. To take the photoluminescence of the fabricated UCNPs-ChNF paper, the 3D-printed smartphone-based platform was equipped with a UV/IR cut filter to block the NIR light. The assembled smatphone-based setup is depicted in Figure S13. The fabricated ChNF paper-based sensing platforms were first placed within the dark chamber via a strip hole. Lamps were powered using a smartphone via a USB port. A self-developed mobile application was also used to quantitate the changes in the color intensities of the fabricated ChNF paper-based sensing platforms, which were taken by integrated smartphone’s camera, related to the concentration of the tested analytes (See Figure S13). 4.9. Fabrication and application of ChNF paper-based assay kit for blood sample analysis For fabrication of ChNF paper-based assay kit, as seen in Figure 6A, the blood separation membrane MF1 (circle shaped with 5 mm diameter) and filter paper Whatman No.1 with the designed pattern were first attached on a waterproof sticker paper. The printed ChNF paper with a specific pattern (three circle-shaped test zones (with 3 mm diameter) for glucose, total bilirubin and albumin; and a punched part (with 5 mm diameter) as sample inlet zone, which designed for the blood separation membrane) was then placed on the attached set. After fabrication of the assay kit substrate, 5 µL of the pre-mixed colorimetric reagents for glucose 38 (phosphate buffer (pH= 7.5, 250 mM), phenol (5 mM), 4-aminoantipyrine (0.5 mM), glucose oxidase>10 kU/), total bilirubin (EDTA-Na2 (0.07 mM), NaCl (6.6 gL-1), sulfamic acid (70 mM), 2,4-dichlorophenyl-diazonium salt (0.09 mM), HCl (130 mM), EDTA-Na2 (0.02 mM)) and albumin (citrate buffer (pH=4.2, 30 mM), bromocresol green (0.26 mM)) were separately drop-dried on each corresponding test zones of the fabricated ChNF paper-based assay kit. To evaluate the clinical applicability of the developed smartphone-based ChNF paper assay kit, as seen in Figure 6B one drop of blood was applied directly to the sample inlet zone of the fabricated assay kit and then subjected for colorimetric determination of glucose, total bilirubin, and albumin. 4.10. Synthesis of PLNPs 4.10.1. Synthesis of N-Acetyl L-Cysteine (NAC)-capped CdTe QDs (GQDs, YQDs and RQDs) NAC-capped CdTe QDs with different emission colors (green, yellow and red) were synthesized according to our previously reported method after slight modifications of the method described by Liu et al. 64-65. In brief, 2.87 mmol of NAC was first added to 10 mL of CdCl2 solution (0.290 M), and its pH was adjusted to 9 by NaOH solution. Following that, the reaction mixture was stirred for 5 min under argon flow. Tellurium powder (0.76 mmol) was then added to 75 mL of 64 mM NaBH4 solution at 50 °C in a three-neck round bottom flask under argon atmosphere. After 20 min, 10 mL of CdCl2 solution (pH=9) was added, followed by temperature increasing to 150 °C. NAC-capped CdTe QDs with different emission colors were obtained at different reaction times, followed by cooling in an ice bath. Photoluminescence spectra of as-prepared CdTe QDs with peaks placed at 450, 560 and 610 nm were recorded for GQDs, YQDs, and RQDs, respectively. 39 4.10.2. Synthesis of thio - glycolic acid (TGA) capped CdTe QDs (GyQDs) TGA capped CdTe QDs were synthesized according to the procedure described above for the synthesis of NAC-capped CdTe QDs by using 2.87 mmol of TGA instead of NAC 66. 4.10.3. Synthesis of CDs Highly PL CDs were synthesized by mixing 500 mg citric acid and 500 µL ethylenediamine in 10 mL water. Afterward, the reaction mixture was transferred to a 30 mL autoclave (Teflon-lined stainless-steel) and then heated at 200 °C for 5 hours 34, 66. The HRTEM images, DLS analysis, and photoluminescence spectrum of the synthesized CDs are depicted in Figure S15. 4.10.4. Synthesis of upconversion NPs The synthesis and characterization of amine-functionalized NaYF4:Yb3+@Er3+&SiO2 upconversion NPs is provided in Supporting Information. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at ….  Additional details on characterization of ChNF paper (experimental steps, AFM images, XRD analysis, EDX spectra, TGA analysis, and tensile stress- strain curve), plasmonic ChNF paper (cross-section/FE-SEM and AFM images, EDX spectra, TGA analysis, estimation of the size distribution, and UV-vis spectrum of in situ synthesized AuNPs-ChNF paper), synthesized CDs (HRTEM images, photoluminescence spectrum and DLS analysis); synthesis 40 and characterization of upconversion NaYF4:Yb3+/Er3+@SiO2-NH2 nanoparticles (TEM images and histograms, ATR FT-IR and Photoluminescent spectra). Also, details about analytical figures of merit and the influence of coexisting substances for determination of MTZ, CN -, and Hg2+ using the plasmonic ChNF paper; calibration plots of determination of MTZ, CN-, Hg2+, quercetin and S2-; the illustration of the 3D-printed smartphone-based sensing setup, the results related to correlation between obtained results of glucose, total bilirubin, and albumin analysis using the fabricated ChNF paper-based assay kit and reference methods and (Table S1 and Figures S1–S18). ACKNOWLEDGMENT Financial support from Iran's National Elites Foundation (INEF), Chemistry & Chemical Engineering Research Centre of Iran (Tehran, Iran), Nano Novin Polymer Co. (Iran), and the Nano Match program of Iran Nanotechnology Initiative Council (INIC) are gratefully acknowledged. The ICN2 is funded by the CERCA Programme / Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa program of the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, grant No. SEV-2017-0706 and MAT2017-87202-P). The financial support of “the Czech Science Foundation (No. 19-00676S)" is also greatly appreciated. NOTES The authors declare no competing financial interest. 41 AUTHOR CONTRIBUTIONS T.N. and H.G. conceived of the original idea and planned the experiments. T.N., H.G., and A.M. analyzed the data. T.N. carried out the experiment. T.N. and H.G. wrote the manuscript. H.Y. produced and characterized the ChNF paper samples and wrote the related part of the manuscript. U.K. and D.H. synthesized and characterized the upconversion NPs and wrote the related part of the manuscript. M.H. designed the IoT model and wrote the related part of the manuscript. H.G. and A.M. supervised the overall project. All authors discussed the results and contributed to the final manuscript. REFERENCES (1) Babu, R. P.; O'connor, K.; Seeram, R. 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