Bioinkjet Printing and Protein Tagging of Camouflaged Biosafe Quick Response Codes for Medicine Authentication

Online pharmacies and social media platforms are responsible for the growing presence of counterfeit medicines, and the verification and authentication of dosage levels are imperative for protecting individual medicines. However, the existing anticounterfeit methods for medicines and exterior box‐level protection are often limited, and they focus on pharmaceutical supply chains instead of empowering patients. Here, bioprinting and taggant construction of camouflaged biosafe quick response (QR) codes are introduced for on‐dose (or in‐dose) medicine security, integrated with the dosage form. Machine‐readable color QR codes contain concealed invisible patterns with biologically safe near‐infrared absorption properties, which help enhance the security of conventional QR codes. The reported bioinkjet printing and protein taggant construction guarantee printability, imperceptibility, stability, biocompatibility, digestibility, and tamper resistance to be an inherent part of each unit of medicine in solid dosage formats. Camouflaged biosafe QR code taggants can offer various medicine security applications including anticounterfeit measures, authentication features, track‐and‐trace, and serialization at the dosage level. This approach is expected to empower patients to play an active role in fighting illicit medicines and pharmaceutical products.


Introduction
Fake pharmaceutical drugs are not a new problem; however, they pose a considerable threat to public and patient safety DOI: 10.1002/admt.202301438worldwide.Fake and counterfeit medicines include a wide array of substandard, falsified, and diverted pharmaceutical products. [1,2]The World Health Organization estimates that over one million people die annually because of counterfeit pharmaceutical products and that several disasters can be attributed to poor medicine security. [3]or example, counterfeit malaria and pneumonia medications have been estimated to result in 250 000 child deaths annually in Africa. [4]Cambodia witnessed the loss of 30 lives because of counterfeit antimalarial drugs that included outdated components. [1]In Nigeria, over 50 000 individuals were administered fraudulent meningitis vaccines, leading to a death toll of 2500. [1]he consumption of cough syrups laced with paracetamol-containing diethylene glycol resulted in 89 deaths in Haiti, and similar cough syrups were linked to the tragic demise of 30 infants in India. [1]Recently, the sale of counterfeit pharmaceuticals containing illicit substances in Mexican pharmacies has made its way into the United States. [5]he proliferation of online (or internet) pharmacies has intensified the illicit trade of medications across the global, national, and local levels.Globally, ≈ 40 000 online pharmacies exist; however, a strikingly low 3% of them operate legally. [6,7]vidently, there has been a surge in counterfeit medical treatments and supplies amid the COVID-19 pandemic driven by heightened demand. [8]Social media and online platforms allow even high school students to acquire controlled substances (e.g., OxyContin, Vicodin, Xanax, and Adderall) along with counterfeit renditions. [5,9]An estimated 18% of high school students have access to controlled substances without a prescription. [10]Drug dealers target teenagers by distributing counterfeit medicines in the form of legitimate prescriptions.Recently, an increase in accidental youth deaths has been closely associated with the proliferation of illicit fentanyl distributed on Snapchat and Instagram. [11,12]Unfortunately, there is a lack of public awareness regarding the perils associated with counterfeit medicines, and therefore, it is imperative to implement advanced medicine security technologies.
Pharmaceutical companies conventionally apply anticounterfeit measures that rely on the exterior box (e.g., secondary packaging) used to pack multiple medicine products together.However, exterior/secondary box-level protection is fundamentally limited because medicines must be removed from the initial packaging and separated into individual doses for sale and at-home use, often rendering exterior box-level protection useless.As a result, each medicine cannot be individually verified or authenticated when separated from the secondary package.[15][16] In the United States, the Drug Supply Chain Security Act has mandated the establishment of unit-level traceability by 2023.Therefore, many US pharmaceutical manufacturers, retailers, and distributors have agreed to create blockchain technology for better managing complex pharmaceutical supply chains (also known as the MediLedger Network). [17]deal medicine security should be integrated with the dosage form itself (i.e., on-dose or in-dose authentication) because it offers medicine verification and traceability as part of each unit of medicine, instead of on the secondary package.In this case, each individual pill, tablet, or capsule can be verified as genuine in the absence of packaging.Therefore, even if the original packaging is not retained by pharmacists or patients, the risk of ingesting counterfeit medicines can be minimized.Analytical chemistry and spectroscopy methods (e.g., Raman, fluorescence, and near-infrared spectroscopy) can be used to analyze the exact biochemical compositions of drugs [13,18] for detecting counterfeit medicines at the dosage level; however, these approaches require expensive devices, specialized readers, and highly trained personnel, and patients and healthcare professionals often have limited access to such advanced tools.
Given this background, several dose-level authentication methods for medicines and pharmaceutical products have been developed (Table S1, Supporting Information, for comprehensive comparisons); for example, silica microtaggants, [19] DNA taggants, [19,20] plasmonic nanotags, [21] multicolor nonpareil coatings, [22] all-protein-based physical unclonable functions, [23] watermark bioprinting, [24] QR code drug labels, [25,26] and matrix codes. [27][30][31][32] However, the existing dosage-level identification methods have several limitations: First, the constituent materials are not easily accessible, procured, or fabricated on a large scale.Second, some materials used in on-dose technologies may not be safe for oral intake because of the potential hazard and cytotoxicity associated with artificial and foreign materials.Further, there are concerns regarding certain medicine-printing materials (e.g., phthalates) that possess endocrine-disrupting properties. [33]Third, the existing ondose technologies often require skilled and trained personnel, as well as high-cost sophisticated analyzers and specialized readers equipped with optical components. [34]eanwhile, the QR code-based authentication offers not only simple and effective data storage for speedy retrieval, but is also robust against variations in illumination, scale, coverage, and camera angles for reading.This method enables end users (e.g., patients and healthcare professionals) to extract data via online or offline modes on Android and iOS smartphones.Furthermore, smartphone ownership is widespread in developing countries with rates of 43% in sub-Saharan Africa and 62% in the Asia-Pacific region. [35,36]37] However, a major drawback of this approach is the reduced security: QR codes can be duplicated and copied easily because of their visibility.
In this study, we introduce camouflaged biosafe QR code bioprinting and taggant construction as medicine security technologies at the dosage level.To this end, we modify an inkjet printing method, which is potentially scalable for printing edible materials and additive pharmaceutical manufacturing.Further, we fabricate inkjet printing inks using biologically safe and edible materials, such as Food and Drug Administration (FDA)-approved edible coloring and near-infrared (NIR) dyes, for camouflaged effects with increased QR code security.We also construct edible and digestible natural protein-based print sheets, on which the QR code is printed, and we generate QR codes with color and invisible (hidden) patterns by determining the optimal printing parameters.We evaluate the biocompatibility, enzymatic digestibility, and reliability of the proposed QR codes.In addition, we test the resistance to copy attack when a scanner or copier is used to duplicate QR codes.Finally, we demonstrate the reading of QR codes affixed to individual medicines in a solid dosage format (e.g., tablet, pill, or capsule) using a built-in QR code scanner in a smartphone.

Concept of Camouflaged Biosafe QR Code Taggants
Figure 1 illustrates the concept of a camouflaged QR code taggant using edible and biologically safe inks and substrates via inkjet printing for protecting individual medicines at the dosage level.The proposed QR code taggant is constructed using bioinkjet printing on a protein print sheet (Figure 1a).A QR code is designed using a QR code generator, which allows us to generate a URL hyperlink. [38]The generated digital QR code is printed on a protein (silk fibroin) print sheet using a commercially available inkjet printer with color and invisible NIR inks.FDA-approved food coloring is the main ingredient in the color inks.[41] Furthermore, ICG combines well with the silk fibroin. [42]We exploit IR820 (also known as the new ICG) as an ICG alternative because it has advantages over conventional ICG, including lower costs and enhanced stability. [39,43]IR820 is invisible to the naked eye because of its low absorption in the visible wavelength range of 400−650 nm. [43,44]he proposed QR code taggant exhibits three features for ondose authentication.First, this QR code contains an additional invisible hidden pattern as part of the QR code to overcome the security limitations of conventional QR codes.It is unlikely that a counterfeiter will be able to successfully duplicate the entire QR code using a simple scan-print method (i.e., a copy attack).Second, all constituent materials, including silk, are biologically safe and edible.This proposed QR code taggant can be affixed to a solid medicine (e.g., tablet or capsule) using an edible adhesive by the pharmaceutical manufacturer or pharmacist (Figure 1b).Third, an end user (e.g., a patient) can easily scan the QR code immediately before oral intake using an onboard QR code scanner on a smartphone (Figure 1c).As part of the authentication, the end user can access additional information on the dose and track/trace, including product data (e.g., dosage strength, dose frequency, cautions, and expiration date), manufacturing details (e.g., location, date, batch, and lot number), and distribution paths (e.g., country, distributor, and wholesaler).

Synthesis of Inkjet Bioinks for Color and Invisible QR Code Printing
For bioinkjet printing, we use biologically safe coloring and NIR absorbing dyes (Figure 2a).Specifically, the main ingredients of color inks include FDA-approved food colorings: FD&C Blue No. 1 (brilliant blue FCF) for cyan, FD&C Red No. 3 (erythrosine), and FD&C Red No. 40 (Allura Red AC) for magenta, and FD&C Yellow No. 5 (tartrazine) for yellow.IR820 is the main ingredient for the NIR-absorbing invisible ink; IR820 (1 μM) dissolved in deionized water is transparent due to its low absorption in the visible range, while it absorbs NIR light in a wavelength range of 650−1000 nm (Figure 2b,c).The viscosity of all inks is maintained in a range of 3−5 mPa•s (cP) for ensuring that it is comparable to water-based inkjet inks. [45,46]Colorimetric analyses show that cyan, magenta, and yellow inks have colors corresponding to the absorption spectra of the food colorings (Figure 2d,e).However, all color inks are clear upon NIR light illumination at a center wavelength of 850 nm.The IR820 ink at a concentration of 1 mm is dark green, while highly concentrated IR820 inks at >5 mm appear black under white light illumination because of strong absorption in the visible wavelength region of 400-650 nm (Figure S1, Supporting Information).As expected, all IR820 inks are black under 850-nm NIR light illumination because of the high absorption peak at 850 nm.Therefore, the invisibility of the IR820 ink is achieved by determining the optimal concentration.Finally, the prepared ink solutions are injected into refillable ink cartridges of a commercially available inkjet printer (Figures S2,S3, Supporting Information).

Optimization of Camouflaged Biosafe QR Code Printing on Protein Print Sheets
We implement a QR code as a taggant because the solid dosage formats of medicines cannot be fed directly into a commercially available inkjet printer to print a QR code onto the surface of medicines.We use a silk fibroin print sheet easily fed into an inkjet printer.][50][51][52][53] From an optical perspective, silk fibroin exhibits negligible optical absorption in the visible and NIR wavelength range of 400−1000 nm (Figure S4, Supporting Information).From a manufacturing perspective, the easy processability of the silk fibroin offers a scalable production option (Figures S5,S6, Supporting Information); however, printing fine patterns using water-based inkjet inks on a silk fibroin print sheet is not straightforward because the printing ability is affected by the surface properties of the print sheets.Unlike paper, silk fibroin print sheets with a highly dense surface do not adsorb inkjet droplets because of their water-repellent surfaces (Figure S7, Supporting Information).The inkjet droplets often spread because of wetting, which results in droplet aggregation. [24,54,55]he density of the droplet-jetted inks on silk fibroin print sheets in bioinkjet printing can be controlled by varying the opacity level in digital printing.QR codes with color and invisible NIR dual patterns are printed on silk fibroin print sheets at different opacity levels of red (R), green (G), and blue (B) colors in a range of 20−100% [Figure 3a; Figure S8 (Supporting Information), and Experimental Section].Photographs of the QR codes are captured using two smartphones either equipped with a standard visible camera or an NIR-imaging-enabled camera under visible (white) light or/and NIR ( = 850 nm) light illumination (Figures S9,S10, Supporting Information).Conventional smartphone cameras already have decent sensitivity to NIR light; typical complementary metal-oxide-semiconductor (CMOS) image sensors are sensitive to light in the ranges of visible (400−700 nm) and NIR (700−1000 nm) wavelengths.58] On the other hand, recent smartphone cameras often include a short-pass filter (i.e., ≤ 700 nm) to eliminate the NIR light and enhance the quality of photos.We use smartphones without a short-pass filter to enhance the NIR sensitivity without using additional NIR sensors.In QR codes, the RGB color pattern is captured by both the visible camera and NIR-enabled camera under visible light illumination, while the invisible NIR pattern printed with the IR820 ink is revealed only under NIR light illumination ( = 850 nm).We determine the optimal opacity of RGB color printing to be 80% with a scan success rate of 96 ± 4.9% (mean ± standard deviation).At lower opacity levels, the absorption spectra intensity of RGB colors is too weak, although the droplets of the color inks are individually well distributed on the silk fibroin print sheet (Figures S11-S13, Supporting Information).In the cases (≤ 50%), the average scan success rate is low ≤ 18% (Figure 3b and Table S2, Supporting Information).At high opacity levels ≥ 90%, the readability of color QR codes decreases because the high density of droplets forms significant aggregations of droplets, and the optimal opacity level for RGB color printing limits the minimum printable size.The minimum size of the reported QR codes can be determined based on a tradeoff between inkjet printing and camera image resolutions, although smartphone cameras have high-performance (optical, digital, or hybrid) zoom options.When QR codes are printed in different sizes and imaged under visible and NIR light illumination (Figure S14, Supporting Information), QR codes with sizes larger than 7 × 7 mm 2 show scan success rates ≥ 90% without using the zoom option of the NIR-enabled camera (Table S3, Supporting Information).
For the IR820 ink, we consider the opacity level of IR820 printing and the concentration of the IR820 ink.An opacity level of 100% is selected to ensure a sufficient printing resolution (Figure S15, Supporting Information): Unfortunately, unlike the available control of opacity levels in color printing, the variable opacity level of the black color in digital printing is not implemented on the print sheets using only IR820 ink.At an opacity level of 100%, only the IR820 ink is present on the silk fibroin print sheet, whereas color droplets are printed at opacity levels ≤ 90%, which reveals the absorption in the visible wavelength range.Highly concentrated IR820 ink has a dark greenish color (Figure 2d), and therefore, its concentration must be optimized to minimize visibility under visible light and maximize visibility under NIR illumination.The optimal concentration of IR820 is determined to be 10 mm for ensuring that the NIR QR code pattern is not visible to the naked eye (Figure 3c,d; Figure S16, Supporting Information).Under NIR light illumination, the print pattern becomes clearer with an increase in the concentration of IR820 ink, and this results in the enhanced readability of QR codes with scan success rates of 96−100% at ≥10 mm (Table S4, Supporting Information).Below 10 mm, the pattern printed in the QR codes using IR820 ink is invisible to the naked eye under visible light illumination; however, it shows light greenish colors at 20 and 30 mm.The QR code printed with 30 mm IR820 ink could be read using a QR code scanner under visible light without NIR light (Movie S1, Supporting Information).

Biocompatibility and Enzymatic Digestibility of Camouflaged Biosafe QR Code Taggants
The biocompatibility of the main constituent materials used in a QR code taggant is investigated.Using sheep erythrocytes, we conduct hemocompatibility tests for IR820, food colorings (Blue No. 1, Yellow No. 5, Red No. 3, and Red No. 40), and silk fibroin (Figure 4a and Experimental Section).Phosphate-buffered saline (PBS) and Triton X-100 (0.1%) solutions are used as negative and positive controls, respectively.The positive control (0.1% Triton X-100) clearly exhibits a dark red color resulting from the uniform breakdown of red blood cells.In contrast, all test samples and negative control (i.e., PBS) exhibit pale yellow colors because of red blood cell membrane preservation (Figure S17, Supporting Information).Further, we calculate the hemolysis efficiency: (AS-AN)/(AP-AN) × 100 (%), where AS, AP, and AN represent the optical absorption values at  = 580 nm for the testing samples, positive control, and negative control, respectively.The hemolysis efficiency values of test samples are not statistically significantly different from those of the negative control (pvalue of ANOVA test = 0.10).This result supports the hypothesis that the constituent materials are hemocompatible.
We assess the digestibility of a QR code taggant by examining its enzymatic degradation in vitro under physiologically relevant conditions (Figure 4b).The two major proteolytic enzymes produced in the gastrointestinal tract, pepsin, and trypsin, are responsible for denaturation (unfolding of proteins) and degradation (breaking down primary amino acid chains).In the stomach, pepsin breaks down food proteins and acts as a nonspecific protease in extremely acidic environments. [59]In the small intestine, trypsin produced by the pancreas continues to break down proteins under neutral pH conditions. [60]For in vitro testing, QR code taggants with a size of 10 × 10 mm 2 are immersed in pepsin (0.1%, pH 2.2) and trypsin (0.25%, pH 7.2) solutions.The samples are monitored to observe the denaturation and degradation processes (Figure S18, Supporting Information).Gastric enzyme exposure for just 8 min significantly degrades the QR code taggants.For comparison, QR code taggants are immersed in control solutions (pH 2.2 PBS and pH 7.2 PBS) without these enzymes.Although cloudy swelling and shape distortion of the QR code taggants occur over 30 min, they remain intact in the solution.This supports the idea that constituent materials are easily digestible.

Reliability Tests of Camouflaged Biosafe QR Code Taggants
We evaluate the printing reproducibility, photostability, and longterm stability of the QR code taggants.First, the print reproducibility, defined as the ability to produce a large number of identical QR codes on silk fibroin print sheets, is tested by producing 200 different taggants with the same digital QR code (Figure 5a).A total of 199 QR code taggants are successfully read, with an average scan success rate of 99.5%.The reading failure of only one QR code taggant is attributed to the distortion of the printed QR code patterns caused by the uneven surface condition of the print sheet.The photostability of the QR code taggants is tested, and the taggants are illuminated with white light (D65 LEDs) for 600 h at an intensity of 500 lx, which is the recommended office workspace light intensity (Figure 5b; Figures S19,S20, Supporting Information).Clearly, the QR code scan success rate decreases with longer durations of light exposure.At a low concentration of 10 mm, the readability is maintained for up to 180 h, because of the photodegradation of IR820, whereas the three RGB colors maintain their vibrancy for 600 h without undergoing bleaching.The use of commonly available pharmaceutical packaging designed for light protection (e.g., dark or opaque packaging covers) substantially prolongs shelf life.In addition, the QR code taggants show good long-term reliability for 91 d, even without the use of protective packaging (Figure 5c; Figure S21, Supporting Information).

Copy (Scan-Print) Attack Simulation of Camouflaged Biosafe QR Code Taggants
We simulate a possible copy (scan print) attack scenario, which assumes that an illicit counterfeiter possesses unrestricted access to the technologies and materials employed to produce the proposed QR code taggants, including IR820 and coloring dyes, identical inkjet printers and cartridges, and silk fibroin print sheets (Figure 5d).We assume that a counterfeiter uses a highresolution digital scanner (600 dots per inch) to acquire a digital (input) image of a QR code taggant.Subsequently, the counterfeiter reprints the scanned digital QR code image onto a silk fibroin print sheet.The colors and patterns captured by both the standard and NIR-enabled cameras appear similar when comparing the original and copied QR codes under visible illumination.However, under NIR light illumination, the invisible pattern in the scan-printed QR code is not detected because the IR820 ink pattern is not scanned or printed at all, which results in QR code reading failure.Bioinkjet printing technology using invisible IR820 ink can be applied to print completely hidden QR codes on the surface of silk fibroin print sheets included with various colors (Figure S22, Supporting Information), and this can potentially be used for other cryptography and security applications.

On-Dose Authentication Using Camouflaged Biosafe QR Code Taggants
We demonstrate on-dose authentication using a camouflaged biosafe QR code taggant affixed to an oral dosage tablet (Figure 6).A smartphone equipped with a portable NIR LED flashlight and an onboard QR code scanner is used in the simulated setting (Movie S2, Supporting Information).The QR code taggant is attached to a solid-type medicine (Alka Seltzer) using a glucose syrup adhesive, comprising food-safe or food-grade additives of sugar, pectin, citric acid, potassium sorbate, and water (FONDX America Corp.).After launching the QR code scanner app, the end user (e.g., a patient or pharmacist) views and focuses the QR code affixed to the solid medicine through the smartphone screen, and then, the user turns on the NIR LED flashlight.The scanner application automatically recognizes the color and invisible QR code patterns, immediately opens the embedded hyperlink to confirm authentication, and provides important dose information.QR codes can only be scanned under both visible and NIR light illumination, which can potentially be used to overcome the limited security of conventional QR code technologies.

Conclusion
We developed camouflaged biosafe QR code taggants that can be affixed to individual medicines in solid dosage formats, enabling serialization, track-and-trace solutions, anticounterfeit measures, and authentication features at the dosage level, instead of the package level.The reported bioinkjet printing and taggant construction methods enable the printing of a static QR code with RGB color and invisible NIR dual patterns on silk fibroinprinted substrates.Camouflaged biosafe QR codes overcome the low-security limitations of conventional QR codes by combining color and invisible hidden patterns through the bioinkjet printing of color and NIR light-absorbing inks.All constituent materials of QR code taggants, including color/NIR inks and protein print substrates, are hemocompatible, edible, and digestible.The proposed inkjet printing method is an attractive option for scalable QR code production and can serve as an additive in the pharmaceutical manufacturing process.Camouflaged biosafe QR code taggants can facilitate the development and implementation of single-unit or unit-dose packages in hospital pharmacy settings, reducing the risk of dispensing errors, improving inventory tracking, enhancing security, and minimizing labor costs.In addition to these benefits, the reported QR code taggants can empower patients to counteract illicit pharmaceutical products and maintain a sustainable healthcare system.Further, we predict their potential for deployment in other security and cryptographic applications.
Silk Cocoon Degumming: The outer protein layer (sericin) of the silk cocoons was removed, leaving only the inner fibroin.The silk cocoons were cut into thin strips with a size of 3−5 mm and treated two times with a NaHCO 3 (0.3%) solution at a boiling temperature of 100 °C for 30 min, subsequently washed with deionized water several times.The degummed cocoons were dried naturally in the dark under ambient conditions.
Preparation of Silk Fibroin Print Sheets: The degummed silk (i.e., fibroin) was dissolved in a LiBr solution (9.5 m) at a temperature of 65 °C for 6 h to produce silk fibroin print sheets.The solution was filtered using Miracloth and subsequently dialyzed in deionized water several times for at least 48 h to remove the salt (LiBr).Afterward, the solution was filtered and centrifuged at 12 000 rpm at 4 °C for 10 min, resulting in the final concentration of 5% w v −1 .For the colored silk fibroin print sheets, brilliant blue FCF (Blue No. 1), fast green FCF (Green No. 3), tartrazine (Yellow No. 5), and Allura red AC (Red No. 40) color dyes were dissolved in deionized water to obtain concentrations of 50, 50, 100, and 100 mm, respectively.Brilliant blue FCF, fast green FCF, and Allura red AC color-dye-dissolved solutions were mixed at a ratio of 1:1:1 to produce a black solution.A color silk fibroin solution (3% w v −1 ) was prepared for each color.The resulting supernatant was poured into a petri dish and allowed to solidify.Finally, the film (printed sheet) was carefully removed from the dish.[63] Preparation of IR820 Ink and Color Inks: Food-grade polysorbate 80, food-grade propylene glycol, and ethyl alcohol (200 proof) were formulated to create inkjet inks using IR820 and FDA-approved coloring dyes.The IR820 is dissolved in deionized water at 37 °C for 30 min to prepare the IR820 ink, resulting in concentrations of 1, 5, 10, 20, and 30 mm.The composition of the IR820 ink consists of IR820/water (85.8%), ethyl alcohol (9%), propylene glycol (5%), and polysorbate 80 (0.2%).Brilliant blue FCF (Blue No. 1, 50 mm), tartrazine (Yellow No. 5, 100 mm), erythrosine (Red No. 3, 30 mm), and Allura red AC (Red No. 40, 100 mm) color dye solutions were used as the color inks.Food-grade polysorbate 80, food-grade propylene glycol, and ethyl alcohol were added.Polysorbate 80 acts as a dispersing agent and propylene glycol serves as a carrier to maintain the desired viscosity and prevent the inks from drying.The color dye-dissolved water (90%) was formulated along with ethyl alcohol (5%), propylene glycol (4.9%), and polysorbate 80 (0.1%) for color inks: Brilliant blue FCF for cyan, tartrazine for yellow, and a mixture of erythrosine and Allura red AC (1:3 ratio) for magenta.The prepared ink solutions were injected into refillable ink cartridges, which were subsequently installed in a commercial inkjet printer.
Fabrication of QR Code Taggants Using Inkjet Bioprinting: Representative static QR code images were designed using a commercially available online QR code generator, which allowed to generate URL hyperlinks. [38]he generated QR code was stored in a PowerPoint file for transfer to a commercially available inkjet printer (Canon PIXMA TS8220; Japan).Thereafter, the QR codes were printed on the silk fibroin print sheets using food coloring inks and IR820 inks via the inkjet printer.
Image Capture of QR Codes: A cut-off NIR optical filter in a smartphone camera was removed (referred to as NIR-imaging-enabled camera, SAM-SUNG Galaxy S21 Ultra) to capture images of QR codes composed of RGB colors and IR820.For comparison, an unmodified smartphone camera (referred to as visible camera, SAMSUNG Galaxy S21) was used.For white light illumination, a CIE Standard Illuminant LED light source with a color temperature of 6500 K (WaveForm Lighting, WA, USA), commonly known as D65, was employed.For NIR light illumination, two LED light sources with peaks of 855 nm (portable and mountable light source with a smartphone; Eigen Imaging, USA) and 860 nm (M850LP1, Thorlabs, USA) were used (Figure S10, Supporting Information).
Hemocompatibility Test: Hemocompatibility tests were conducted for IR820, color dyes, and silk fibroin in sheep erythrocytes.A solution (100 μL) of sheep red blood cells (sheep red blood cells packed 100%, Innovative Research, Inc., Novi, MI, USA) was added to a 1 mL PBS (pH 7.2) solu-tion, and it was centrifuged at 13 000 rpm for 10 min.Isolated red blood cells were diluted in 1 mL of PBS.As a negative control, a diluted solution (800 μL) of red blood cells was added with a PBS solution (200 μL, a total amount of 1000 μL).The positive control was prepared by adding a diluted solution (800 μL) of red blood cells to a solution (200 μL) of Triton X-100 (0.1%).The IR820 was solubilized in a PBS solution to create solutions of 1, 10, and 100 μm, and each solution (1 μL) was added to a diluted solution (800 μL) of red blood cells with additional PBS solutions for a total amount of 1000 μL.Solutions of color dyes (1 μm for Blue No. 1, Yellow No. 5, Red No. 3, and Red No. 40) and silk fibroin solution (1 mL, 5% w v −1 ) were solubilized, and 5 μL of each solution was added to a diluted solution (800 μL) of red blood cells with additional PBS solutions for a total amount of 1000 μL.After incubation at 37 °C for 10 min, the mixture was centrifuged at 13 000 rpm for 10 min.Finally, the hemolysis efficiency was calculated with the optical absorption values at  = 580 nm for the samples, positive control, and negative control, respectively.The hemolysis test was performed under ambient conditions in the dark: 23 ± 2 °C and 30−40% relative humidity.
Enzymatic Digestibility of QR Code Taggants: The enzymatic denaturation and degradation of QR code taggants were examined using the gastric proteolytic enzymes of pepsin (P7000, Sigma) and trypsin (15090046, Gibco).QR code taggants with a size of 10 × 10 mm 2 and a thickness of 50 ± 5 μm were submerged in pH 2.2 PBS containing urea (4 m), guanidine HCl (3 m), and pepsin (0.25%), and pH 7.2 PBS with trypsin (0.25%).For comparison, PBS solutions without proteolytic enzymes at the same pH values of 2.2 and 7.2 were also utilized, respectively.Prior to the digestibility tests, all solutions were prewarmed to 37 °C for 10 min.Photographs of the QR code taggants were captured at various time intervals using a smartphone camera (SAMSUNG Galaxy S21).
Photostability of QR Code Taggants Under Light Illumination: The photostability of QR code taggants was investigated using a CIE Standard Illuminant LED light source with a color temperature of 6500 K (D65).The size of QR codes is 10 × 10 mm 2 and the thickness of taggants is 50 ± 5 μm.The photobleaching test of the QR codes was conducted at an intensity of 500 lx, which corresponded to a recommended light level for a typical office workspace. [64]The optical intensity of the white LED light source was measured using a commercial optical meter (LX1330B-V; Dr. Meter).To examine the effect of light exposure on QR codes, the photodegradation tests of cyan, magenta, and yellow inks and IR820 ink were conducted by printing red (R), green (G), blue (B) colored and IR820 patterns on silk fibroin print sheets.The scan success rate was evaluated for 12 different QR code taggants printed with various concentrations of IR820 inks (10, 20, and 30 mm) (Figure S19, Supporting Information).The optical absorption of RGB colors and IR820 was also measured (Figure S20, Supporting Information).To quantitively examine the photodegradation of printed inks, the specific absorption wavelengths of RGB colors and IR820 were selected to be 450 nm for blue, 520 nm for green, 630 nm for red, and 850 nm for IR820, respectively.
Scanning Electron Microscopy (SEM): The structural morphologies of the silk fibroin print sheets were characterized using an SEM system (FEI Quanta 3D FEG) at 5 kV.
Viscosity Measurements of Inks: The viscosity of inks was measured using a rotational viscometer (NDJ-8S, SHengwin, China) at 22 °C with a speed of 60 rpm.
Surface Wettability of Silk Fibroin Print Sheets: Water contact angles was measured using a contact angle measurement system (Phoenix-300, SEO Co., Ltd., Republic of Korea) with a droplet (5 μL) of deionized water.The measured values were averaged at three different positions on the surface of the silk fibroin print sheets.
Characterization of Optical Properties: The optical properties (reflectance and transmittance) of the samples were measured using a fiber bundle-coupled spectrometer (VS140 VIS-NIR; Horiba Jobin Yvon Inc., Edison, NJ, USA) coupled with an integrating sphere.The absorption spectra were derived from the reflectance and transmittance spectra (i.e., 1−reflectance−transmittance).
Statistical Analysis: For the hemocompatibility test, an ANOVA test for hemolysis efficiency values of the test samples was conducted using Stata 14.2 statistical software (College Station, TX, USA).The number of samples is presented in the figure legend of each dataset.A significant level of 5% was considered statistically significant.The error bars in all data represent standard deviations.

Figure 1 .
Figure 1.Camouflaged biosafe QR code taggants with color and invisible dual patterns for securing medicines at the dosage level.a) Schematic of the bioinkjet printing process for color and invisible dual-patterned QR codes using biologically safe materials (i.e., FDA-approved coloring dyes, IR820, and silk fibroin).b) QR code taggant integrated with a solid-type medicine (e.g., tablet or capsule) for on-dose authentication by the pharmaceutical manufacturer or pharmacist, and c) machine-readable authentication of the QR code upon visible and NIR light illuminations.

Figure 3 .
Figure 3. Optimizations of camouflaged biosafe QR code printing on protein print sheets.a,b) Photographs (a) and scan success rate (b) of QR codes printed on silk fibroin print sheets with different opacity levels of the RGB colors in a range of 20−100%.The concentration of IR820 ink is kept at 10 mm.c,d) Photographs (c) and scan success rate (d) of QR codes printed on silk fibroin print sheets with different concentrations of IR820 inks in a range of 1−30 mm.Scanning tests of five different QR codes are performed, and each QR code is read 10 times repeatedly.The size of the QR code taggants is 9 × 9 mm 2 .

Figure 4 .
Figure 4. Biological safety, biocompatibility, and digestibility of camouflaged biosafe QR code taggants.a) Hemocompatibility test of food colorings (Blue No.1, Yellow No.5, Red No.3, and Red No.40) and IR820 dissolved in a phosphate-buffered saline (PBS) solution.Silk fibroin solution (5% w v −1 ), Triton X-100 (0.1%, positive control: hemolysis efficiency of 100%), and PBS (negative control: hemolysis efficiency of 0%) are also tested.The hemolysis efficiency values of the main materials used in the QR code taggant are not statistically significantly different from those of the negative control (ANOVA p-value = 0.10 with n = 4 in each group), supporting the general nontoxicity of the constituent materials.b) Photographs of QR code taggants immersed in pepsin (pH 2.2) enzyme or trypsin (pH 7.2) enzyme solutions as a function of elapsed time for enzymatic digestibility.Further, QR code taggants are tested in buffer solutions with the same pH values without enzymes.The concentration of IR820 ink is 10 mm and the opacity of printed RGB colors is 80%.The QR code taggant size and thickness are 10 × 10 mm 2 and 50 μm, respectively.

Figure 5 .
Figure 5. Overall reliability and unclonability of camouflaged biosafe QR code taggants.a) Printing reproducibility test of 200 different taggants with the same QR codes using bioinkjet printing.A total of 199 QR codes succeed to read, which results in a printing reproducibility of 99.5%.b,c) Scan success rates of 12 different QR code taggants printed at different concentrations of IR820 ink for photostability b) and long-term stability c) tests.The photobleaching test of QR code taggants is conducted at an intensity of 500 lx, which corresponds to the recommended light level for a typical office workspace, using a CIE Standard Illuminant LED light source with a color temperature of 6500 K (also referred to as D65).For the longterm reliability test, the samples are stored in conditions of ambient dark environment (i.e., 22 ± 2 °C and 20 ± 10% relative humidity).d) Photographs of original and simulated (scan-print copy) QR code taggants.The scan-printed QR code is not readable by a QR code scanner because of the invisibility of IR820 ink patterns in the QR code.The concentration of IR820 ink is 10 mm and the opacity of printed RGB colors is 80%.The QR code taggant size is 10 × 10 mm 2 .

Figure 6 .
Figure 6.On-dose authentication demonstration of camouflaged biosafe QR code taggants.Simulated authentication process of a QR code taggant affixed to an oral-dosage tablet-type medicine.The onboard QR code scanning application in a smartphone consists of the following steps (Movie S2, Supporting Information): Launch the QR code scanner app, focus the QR code taggant on a medicine, and turn on the portable NIR LED flashlight (Figure S10, Supporting Information).The scanner app detects the QR code and further opens the encoded hyperlink to a webpage for confirming the verification and authentication and for providing the medicine information.The QR code taggant size is 10 × 10 mm 2 .