Multi-level counterfeit protection using DNA

Abstract

There is a demand for reliable, durable, and accurate methods of authentication in markets of specialty goods, e.g., luxury items, and / or security-sensitive products. Currents methods of object authentication, including the incorporation of extrinsic and / or intrinsic markers, remain amenable to counterfeiting. This disclosure provides methods of object authentication and counterfeit protection using DNA sequences and associated spectroscopic markers, including DNA sequences encoding data strings, DNA sequences comprising multi-level authentication, and methods of synthesizing said DNA sequences.

Description

MULTI-LEVEL COUNTERFEIT PROTECTION USING DNAFIELD

[0001] The disclosure relates generally to the field of synthetic biology, and more specifically to methods of counterfeit protection using DNA sequences.BACKGROUND

[0002] We have previously described information storage using a charged polymer, for example DNA, comprising at least two distinct monomers or oligomers, wherein information is encoded in a machine-readable code, for example a binary code. For example, US 11505825, US 11655465, and U.S. Application No. 18 / 358,861, filed July 25, 2023, each incorporated herein by reference, describe, among other things, methods of synthesizing a DNA molecule using topoisomerase-mediated ligation, adding informational cassettes to a DNA strand in the 3' to 5' direction.

[0003] There is a demand for reliable, durable, and accurate methods of authentication in markets of specialty goods, e.g., luxury items, and / or security- sensitive products. Counterfeit goods lead to loss of revenue, damage to reputation, brand dilution, and circumvent safety and sustainability standards. Proper authentication methods allow for tracing of importation / exportation of goods and / or verification of object provenance. Previous approaches to address this demand include incorporation of extrinsic markers in product packaging or on the product itself. Extrinsic markers include watermarks, holograms, serialization marks, engravings, microprinting, smart labels (e.g., QR codes), specialty inks, guilloche patterns, and microscopic coatings (e.g., dust identification). However, extrinsic markers are still amenable to counterfeit. Alternatively, intrinsic markers, embedded in the product, have been developed to further increase the difficulty of counterfeiting efforts, such as radio frequency identification (RFID) tags, near field communication (NFC) tags, spectral and / or isotopic fingerprints, and blockchain tracking. Unfortunately, intrinsic markers are limited in their application and may become more vulnerable as technology develops.

[0004] DNA can prove a useful material for object authentication and object provenance, wherein data is encoded within one or more DNA sequence, incorporated into an object of interest, and is subsequently removed and analyzed. Published methods of DNA authentication, however, using a particular DNA sequence, are susceptible to forgery by replicating the DNA, using PCR or similar means.

[0005] Spectroscopic signature analysis has been suggested as a useful nascent tool in anticounterfeit technology. Embedding spectroscopic markers, or combinations thereof, provide methods of increasing anti-counterfeit resistance while often being unnoticeable. or minimally so, to the naked eye of the consumer. Such topics are explored in, e.g., Raman encoding for security labels: a review by Dong Yu, et al., Nanoscale Adv., 2023, 5, 6365, the contents of which are incorporated herein to the fullest extent permitted by applicable law.

[0006] There remains a need for improvement regarding methods of authentication and counterfeit protection of goods, particularly regarding efficient synthesis, encoding, and decoding of DNA markers incorporated within an object. Additionally, there remains a need for improvement regarding methods of analyzing and / or decoding of DNA markers outside of an analytical laboratory, e.g., field-deployable methods of analyzing and / or decoding of DNA markers.BRIEF DESCRIPTION

[0007] This disclosure is directed, in part, to the use of DNA polymers comprising multiple levels of authentication useful in the authentication of objects and for protection against counterfeiting. While in-depth analysis and exhaustive DNA sequencing methods are available, these approaches for analyzing and decoding DNA markers remain costly in terms of resources, finances, and time. Thus, this disclosure is directed, in part, to the use of DNA polymers comprising multiple levels of authentication, such that user-defined data is encoded into the DNA polymers and available for subsequent sequencing, decoding, and confirmation of authenticity, while also comprising a level of rapid authentication that does not require in-depth analysis and exhaustive DNA sequencing, e.g.. wherein object authenticity is supported and / or confirmed using field-deployable methods, e.g., spectroscopic methods, e.g., fluorescence and / or Raman spectroscopy.

[0008] In one aspect, this disclosure is directed to a population of deoxyribonucleic acid (DNA) molecules encoding data useful in the authentication of objects and for protection against counterfeiting, comprising nucleic acid data packets (“nackets”), wherein each nacket contains a plurality of synonymous DNA molecules having different sequences but encoding the same data, wherein the plurality of DNA polymers comprise, or are associated with, four or more authentication levels, comprisingi.) a Rapid Identification Level, wherein the DNA comprises or is associated with one or more detectable labels, e.g., labels providing a spectral fingerprint;ii.) a Nacket Data Level, wherein the DNA sequence encodes user-defined data, e.g., in a binary, ternary or other machine -readable code;iii.) a Production Lot Fingerprint Level, wherein the DNA has a specific percent abundance of different bases, sequence variants, and / or cassette variants; and iv.) an Object Fingerprint Level, comprising a unique population of different synonymous sequences.

[0009] For example, the nackets may be prepared by heterologous (or heterogeneous or varied) cassette data writing, wherein two or more cassette sequences are provided for (or associated with or indicative of) a single bit or combination of bits in a machine-readable code, e.g., a binary code, such that all or nearly all the DNA molecules in the nacket encode the same data, but the sequences of the individual molecules exhibit extremely high variation, e.g., due to the use of heterologous cassettes encoding the same bit or bits of data, e.g., wherein the percent abundance of the different cassette variants used in writing the nackets provides a unique and distinguishable feature of the nacket.

[0010] In some embodiments, the nackets are synthesized using one or more transferase enzymes, e.g., terminal deoxynucleotidyl transferase (TdT). For example, the nackets may be prepared by stepwise addition of non-identical nucleotides forming homopolymer extensions within the DNA sequence, wherein the transition from a first homopolymer extension to a second homopolymer extension comprises a transition between non-identical nucleotides, and wherein the transition(s) between non-identical nucleotides provide for (or are associated with or indicative of) a single bit or combination of bits in a machine-readable code, e.g., a ternary code, such that a population of DNA molecules encodes a desired data string. In some embodiments, the nackets are synthesized using topoisomerase mediated ligation. For example, synonymous cassettes, having different sequences but encoding the same information, can be added in each addition step, to build a set of DNA polymers, wherein each polymer has a series of informational cassettes encoding substantially the same information but wherein the polymers are heterogenous at a sequence level.

[0011] While portions of the linker sequences may be conserved, for example comprising complementary overhangs, topoisomerase recognition sequences and / or primer sequences, e.g., to facilitate joining and recovery of the cassettes, the sequences within the cassettes used to convey information need not be. For example, bit X may be encoded by different sequences XI, X2, X3, or X4, and bit Y may be encoded by Yl, Y2, Y3, or Y4. This permits heterogenous cassette datawriting, so that a very large number of different sequences can encode the same data. The number of permutations is approximately: (# of synonymous sequences or cassettes per addition) (# of rounds of cassette addition). For this example, with 10 rounds of addition (i.e., to make a nacket having 10 cassettes) with 4 synonymous sequences used in each round, there would be 410(i.e. more than one million) different synonymous DNA molecules corresponding to the nacket, which can be varied by varying the proportion of synonymous cassettes in each addition. Further information is provided by recording the percent abundance of different bases, sequence variants, and / or cassette variants in the final sample. Once the DNA is synthesized it may optionally be amplified by one or more rounds of PCR, providing a further random aspect to the sample, as not all strands will amplify with the same efficiency.

[0012] The population of DNA molecules synthesized in this way offers multiple layers of information and authentication. For example, the cassettes can be labeled with detectable markers (optionally a particular combination of markers), for example, spectroscopic markers, such as markers having distinct or unusual Raman spectra and / or fluorescence, so that the presence of the nackets can be rapidly detected in the field. Then, the machine-readable code information in the DNA lies on top of a more complex mixture of sequences, allowing layered data that lends itself to product identification. For example, in identifying a product, the first layer of data could be considered to be the associated markers (fairly easy to replicate), the second as the machine-readable code encoded by the series of cassettes (somewhat more difficult to replicate), the third as the chemical fingerprint of the molecule (relative percentage of bases or DNA cassettes), and finally, the precise mixture of sequences provided by the heterologous cassettes, which is random, highly variable, and nearly impossible to replicate. Amplification of the heterogeneous sequences by PCR would result in some sequences being amplified more than others, an error which would be magnified with each round of amplification. The use of the nackets comprising a large number of heterologous but synonymous sequences thus provides a far more unique and secure signature than the use of a homogeneous population of DNA molecules all having substantially the same sequence.

[0013] The nucleotide polymers or nackets may be incorporated into or associated with goods for purposes of identifying and authenticating the goods. In certain embodiments, the nucleotide polymers or nackets are encapsulated within micro-containers, e.g., encapsulated within silica beads or particles, which are optionally coated with polymer, and incorporated into goods, e.g., forpurposes of identification and authentication of the goods. Tn certain embodiments, the microcontainers, e.g., silica beads or particles, comprise spectroscopic markers, e.g., wherein the spectroscopic markers are covalently bound to the outer and / or inner surface of the microcontainers, adsorbed onto the inner and / or outer surface of the micro-containers, spin-coated, doped, annealed, or embedded onto or within the material of the micro-containers, e.g., onto or within the silica of silica beads or particle, or a combination thereof. In certain embodiments, the nucleotide polymers or nackets, optionally encapsulated within micro-containers, are added to an ink, e.g., a water-soluble ink, optionally comprising a polymer, e.g., for purposes of identification and authentication of signatures, documents, and prints.

[0014] In another aspect, the disclosure is directed to methods of marking, identifying, and authenticating goods, comprising (i) marking the goods by incorporating or associating the nackets described herein with the goods to be identified or authenticated, and (ii) identifying and authenticating the goods thus marked, by analyzing and / or sequencing the nackets, identifying the goods based on one or more level of authentication (e.g., spectroscopic signature and / or DNA sequence(s) and / or the encoded data encrypted therein) associated with the nackets, and authenticating the goods through analysis of the DNA sequence(s), e.g., a DNA “fingerprint” and / or sequencing and decoding the coded data.

[0015] In certain embodiments, analysis of the DNA polymers comprising a first level of authentication, e.g., a Rapid Identification Level, e.g., “Level 1 Authentication”, e.g., “LI” authentication, is accomplished using methods that are non-contact, non-destructive, rapid, field-deployable, or a combination thereof. For example, in some embodiments, the first level of authentication, e.g., Rapid Identification Level, is analyzed using spectroscopic methods, e.g., comprising absorption spectroscopy, infrared (e.g., FTIR) or near-infrared spectroscopy, fluorescence spectroscopy, emission spectroscopy, reflectance spectroscopy, luminescence spectroscopy. X-ray absorption spectroscopy, ultraviolet / visible light (UV / Vis) spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), or a combination thereof. Detectable labels, for example spectroscopic markers, i.e., molecular moieties amenable to detection using spectroscopic methods, may be associated with the DNA polymers or nackets, e.g., via direct conjugation onto the DNA polymers or nackets (e.g., onto one of the DNA strands within a DNA polymer or nacket, e.g., onto both of the DNA strands within a DNA polymer or nacket), adsorption onto the DNA polymers or nackets (e.g., onto one of the DNA strands within a DNApolymer or nacket, e.g., onto both of the DNA strands within a DNA polymer or nacket), or spatially constrained near the DNA polymers or nackets, e.g., within micro-containers, e.g., encapsulated within silica beads or particles with the DNA polymers or nackets, covalently bound to the inner and / or outer surface of the silica beads or particles, adsorbed onto the inner and / or outer surface of the silica beads or particles, spin-coated, doped, annealed, or embedded onto or within the material of the micro-containers, e.g.. onto or within the silica of silica beads or particle, or a combination thereof.

[0016] In certain embodiments, spectroscopic analysis of the DNA polymers or nackets incorporated into or onto an object may be completed prior or following production of the object, shipping of the object, sale of the object, offer for sale of the object, importation of the object, or exportation of the object.

[0017] In certain embodiments, spectroscopic analysis of the DNA polymers is completed in less than 30 minutes, e.g., in less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 12 minutes, less than 10 minutes, less than 8 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 90 seconds, less than 75 seconds, less than 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 18 seconds, less than 15 seconds, less than 12 seconds, less than 10 seconds, less than 8 seconds, less than 5 seconds.

[0018] In certain embodiments, the spectroscopic markers associated with the DNA polymers or nackets comprise inherent characteristics of the DNA polymers or nackets comprising one or more identifiable spectroscopic signature. For example, in some embodiments, the nackets comprise natural or canonical nucleotide bases. The spectroscopic signature of DNA polymers comprising canonical nucleotide bases may comprise spectroscopic signals indicating the presence and / or relative abundance of each constituent nucleotide or group of nucleotides. Alternatively, in some embodiments, the nackets comprise, or further comprise, non-natural, non-canonical. mutated, unusual, modified, or synthetic nucleotides. The spectroscopic signature of DNA polymers comprising such non-canonical or modified nucleotides may comprise spectroscopic signals indicating the presence and / or relative abundance of each constituent nucleotide or group of nucleotides, and / or the presence and / or relative abundance of non-canonical nucleotides to canonical nucleotides or groups / cassettes thereof. In certain embodiments, the spectroscopic markers associated with the DNA polymers or nackets comprise spectroscopically-active moietiesor fluorophores, e.g., molecules conjugated, adsorbed, spatially constrained near, or otherwise associated with the DNA polymers or nackets yielding detectable spectroscopic signals indicating the presence and / or relative abundance of each spectroscopic marker when evaluated using spectroscopic means, e.g., absorption spectroscopy, infrared (e.g., FTIR) or near-infrared spectroscopy, fluorescence spectroscopy, emission spectroscopy, reflectance spectroscopy, luminescence spectroscopy, X-ray absorption spectroscopy, ultraviolet / visible light (UV / Vis) spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), or a combination thereof.

[0019] In certain embodiments, the DNA polymers are further modified to comprise “molecular tags”, epigenetic tags, or combinations thereof. In some embodiments, molecular tags include nucleotide modifications via click reactions (e.g., azide-alkyne cycloaddition), metal-chelating or coordination complexes (e.g., platinum compounds, e.g., cisplatin), fluorescent proteins linked to a pH-reversible DNA-binding motif (e.g., KWKWKKA), or combinations thereof. Molecular tags may be permanent or reversible modifications or signals. Molecular tags may be added before or after incorporation of the nucleotide into a DNA polymer. Molecular tags and / or epigenetic tags may be used in the evaluation of a first level of authentication comprising a Production Lot Fingerprint, e.g., wherein the molecular tags and / or epigenetic tags comprise spectroscopic markers, and / or used in the evaluation of a second level of authentication comprising a Molecular Fingerprint.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 depicts spectral “fingerprints” of exemplary nucleobases detected using Raman spectroscopy, in accordance with embodiments of the present disclosure.

[0021] Figure 2 A provides a block diagram of an exemplary process of initially authenticating an item at the Rapid Identification Level, wherein the DNA comprises or is associated with one or more labels providing a spectral fingerprint, in accordance with embodiments of the present disclosure. The labels are excited using the light source and the emission spectra are detected using an optical sensor / detector, analyzed for the presence or absence of the specific spectral signal associated with the DNA using the signal processing logic / computer, and the result displayed on a user device, such as a laptop or smartphone. Optionally, the spectral data can be further transmittedto a data storage server for further analysis, in accordance with embodiments of the present disclosure.

[0022] Figure 2B depicts exemplary screen illustration showing criteria for Rapid Identification Level (LI) identification of an object, such as a handbag, in accordance with embodiments of the present disclosure.

[0023] Figure 3 depicts two approaches to encoding data in DNA. The first uses cassettes that can be ligated by topoisomerase-mediated ligation, wherein the cassettes can have different sequences, but encoding the same machine-readable information, which is similar to Fig. 4 of International Application No. PCT / US2024 / 046372, referenced herein. For example, Cassette “A”, Cassette “a”, and Cassette “a” could have different base sequences but represent the same bits of information. Similarly for Cassette “B”, Cassette “b”, and Cassette “|3.” If these cassettes are added to a DNA receptor strand in multiple rounds, strands of DNA having different sequences but the same information are provided, e.g.. A-B-A, A-B-a, A-B-a, A-b-A, A-b-a. A-b-a, A-[3-A, A-|3-a, A-P-a, etc. The number of different strands is Xnwhere X is the number of cassettes available in each round of addition and n is the number of cassettes in the strand. Thus, with a strand of 3 and 3 alternatives at each position, there are 33or 27 possible sequences. It will be readily apparent that longer strands will result in very diverse DNA populations all encoding the same information, so in this case for example, a strand of 20 cassettes could have about 3.5 billion (320) different DNA sequences. This provides opportunities to create unhackable (or attack resistant) molecular tags, as discussed in commonly-owned International Application No. PCT / US2024 / 046372, referenced herein. The example shown in Fig. 3 shows 16 unique cassettes (Cl -Cl 6), with 4 cassettes associated with each two-bit binary code (00,01,10,11), providing 2-bit multi-base encoding. A hacker or counterfeiter that substitutes a single base (or if the DNA has damage to a single base) will change the underlying binary code represented, thus requiring making easy to detect counterfeits.

[0024] Figure 4 provides a further depiction of the heterologous cassette approach, which is similar to Fig. 5 of commonly-owned International Application No. PCT / US2024 / 046372, referenced herein.

[0025] Figure 5 provides a further depiction of the heterologous cassette approach, which is similar to Fig. 26 of International Application No. PCT / US2024 / 046372, referenced herein.

[0026] Figure 6 shows a portion of the diagram of Figure 4, for Rapid Identification Level, the cassettes or DNA strands can be labeled with tags (shown as stars) that can be detected using spectroscopic means, e.g., fluorescent or Raman tags, having distinctive excitation / emission frequencies, in accordance with embodiments of the present disclosure.

[0027] Figure 7 depicts a Rapid Identification system to detect distinctive Raman optical signatures, in accordance with embodiments of the present disclosure.

[0028] Figure 8 depicts a method to make DNA chains by topoisomerase-mediated cassette ligation, where at least one strand of the cassettes is labeled with a moiety having a distinctive optical spectra (such as Raman or fluorescent). As long as the label is near the center of the cassette, it should not interfere with ligation of the cassettes. By labeling one DNA strand, but not the other, it is possible to amplify and sequence the unlabeled strand if desired, in accordance with embodiments of the present disclosure.

[0029] Figure 9 depicts a method of labeling the DNA strands post-synthesis, wherein the cassettes have synthesized chemical “Click” handles, e.g., such as azide moieties, which can be attached to larger molecules post-synthesis (i.e., after the cassette chains representing a desired digital code are formed) using “click” chemistry, such as strain-promoted azide-alkyne cycloaddition (SPAAC) reactions, in accordance with embodiments of the present disclosure.

[0030] Figure 10 depicts an example of how authentication tags as described herein can be deployed at specified locations on an object, optionally together with decoy tags to make detection and counterfeiting more difficult for an unauthorized person, in accordance with embodiments of the present disclosure.

[0031] Figure 11 depicts an embodiment where the authentication tags are deployed in a PC board, in accordance with embodiments of the present disclosure.

[0032] Figure 12 depicts an example of layering the spectral signal, in accordance with embodiments of the present disclosure.

[0033] Figure 13 depicts marking an object to be identified or authenticated using a of a system for multi-tiered identification, e.g. a process whereby digital codes written in DNA are placed in silica beads and put in ink or paint, which may be used in a paint pen for signing memorabilia and also used for ink in QR codes, in accordance with embodiments of the present disclosure.

[0034] Figures 14A and 14B are diagrams showing that an item (e.g., shirt) may be signed using a paint pen having a molecular NFT (Figure 14A), then a layer of UV / spectral ink may be applied to the item (Figure 14B), in accordance with embodiments of the present disclosure.

[0035] Figures 15A, 15B, 15C, and 15D are diagrams showing different levels of authentication, having Level 0 or first level authentication, where the QR code fluoresces in UV light, which may be detected using a smart phone (Figure 15 A), Level 1 authentication, where a spot pattern is illuminated by a light that excites the spectral inks in the spot patterns, which may be detected using a smart phone (Figure 15B), Level 2 authentication, where each spot has a unique spectral fingerprint, which may be detected using a known optical instrument designed for reading the spectral fingerprint (Figure 15C), and Level 3 authentication, where the DNA (or polymer) is swabbed to obtain ink from the QR code / spots and / or from the signature and the DNA is provided to a DNA reader to determine authentication, in accordance with embodiments of the present disclosure.

[0036] Figure 15 A shows using UV light to detect a QR code printed using ink containing UV dyes and silica nanobeads containing the DNA.

[0037] Figure 15B shows using light to detect spectral ink printed in random dot patterns, wherein the ink optionally contains silica nanobeads containing the DNA.

[0038] Figure 15C shows using a different light sources to detect a spectral fingerprint of inks printed in random dot patterns, wherein the ink optionally contains silica nanobeads containing the DNA.

[0039] Figure 15D shows retrieving and analyzing the DNA from the object.

[0040]

[0041] Figure 16 summarizes an example of a multi-tiered authentication approach, in accordance with embodiments of the present disclosure..

[0042] Figure 17 depicts an example of implementation of a multi-tiered system, specifically a process diagram having five steps including witnessed signing, session documentation (NFT of pens is recorded), hologram application (QR Code and spot pattern), certificate creation matching the QR code with NFT molecules, and customer packaging, in accordance with embodiments of the present disclosure.

[0043] Figure 18 depicts application of a multi-tiered authentication system to framed memorabilia, specifically an approach for using the present disclosure to authenticate framed memorabilia by using peelable hologram / QR code stickers on the back of the item, and showing that they can be removed and physically sent to an authentication service which can perform Level 3 (DNA) testing on the stickers, and confirmation of authentication may be provided by a physical certificate and / or by an electric communication to a computer based user device, such as a smart phone, and the like, in accordance with embodiments of the present disclosure.

[0044] Figure 19 shows a top level block diagram of a system which allows a creator (e.g., athlete) and / or dealer to get compensated for their work / signature, by allowing a user to buy authorization credits from an authentication clearinghouse (or the like) which credits are used to authenticate items, using an app (e.g., Dealer App) running on a user device, such as a smart phone or other computer based device, to perform Level 1 or Level 2 authentication of the item, and provide the results back to the user, the app may also provide the results to the clearinghouse to report results back to the creator and / or dealer, and the clearinghouse may also provide the creator and / or dealer with compensation for each authentication, whether valid or invalid (counterfeit), and the clearinghouse may store the data from the authentications on a data server, and the authentication provider (who mints the NFT and prints the spectral codes on the items) may also receive compensation for an authentication, in accordance with embodiments of the present disclosure.

[0045] Figures 20A,20B,20C are flow diagrams for the print system, authentication app, and clearinghouse, discussed herein, in accordance with embodiments of the present disclosure.

[0046] Fig. 21 shows 3 tables 2100, 2102, 2104 that can be used to hold data for multilevel printing, DNA synthesis, and authentication that may be used by the QR / Spot Data Server, in accordance with embodiments of the present disclosure.DETAILED DESCRIPTION OF THE INVENTION

[0047] The following description of different embodiments is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.

[0048] We have previously described information storage using a charged polymer, for example DNA, comprising at least two distinct monomers or oligomers, wherein information is encoded in a machine-readable code, for example a binary code. For example, US 11505825, US 11655465, and U.S. Application No. 18 / 358,861, filed July 25, 2023, each incorporated herein by reference,describe, among other things, methods of synthesizing a DNA molecule using topoisomerase-mediated ligation, adding informational cassettes to a DNA strand in the 3' to 5' direction.

[0049] The following commonly- owned issued patents and patent applications contain subject matter related to that described herein, each of which are hereby incorporated by reference in their entirety to the fullest extent permitted by applicable law: US Patent 10,438,662; and US Patent 10,640.822. The aforementioned commonly-owned patents discuss approaches for writing (or storing) data in a charged polymer, e.g., DNA, using Add "0" and Add "1" enzymes and a deblock enzyme, as described therein.

[0050] The following commonly-owned US Patent Application Ser. Nos. 18 / 358,861 and 18 / 444,662 contain subject matter related to that described herein, and are hereby incorporated by reference in their entirety to the fullest extent permitted by applicable law. The aforementioned commonly-owned patent applications discuss other approaches for writing (or storing) data in a charged polymer, e.g., DNA, such as, using an AB Adapter instead of a deblock enzyme and using "A0B" and "A IB" for the Add "0" and Add "1" reagents, as described therein, and methods of writing strands of DNA cassettes using inkjet reaction formats.

[0051] The following commonly-owned unpublished US Patent Application Nos. 63 / 582,199 and 63 / 623,085, and unpublished International Application No. PCT / US2024 / 046372, contain subject matter related to that described herein, and are hereby incorporated by reference in their entirety to the fullest extent permitted by applicable law. The aforementioned commonly-owned patent applications discuss other approaches for counterfeit protection using DNA.

[0052] As discussed herein, the present disclosure provides a novel system of storing (or writing or printing) information (or data) using a charged polymer, e.g., DNA, the monomers of which correspond to a machine-readable code, e.g., a binary, ternary, or other base code, and which can be synthesized in various ways, including using a piezo-electric inkjet printer system, such as that discussed in US Patent Application No. 18 / 444,662, filed Feb. 17. 2023, which is incorporated herein by reference in its entirety to the fullest extent permitted by applicable law.

[0053] This disclosure is directed, in one aspect, to a novel population of nucleotide polymers, e.g., deoxyribonucleic acid (DNA) polymers, encoding data useful in the authentication of objects and for protection against counterfeiting, comprising nucleic acid data packets (“nackets”), wherein each nacket contains a plurality of DNA molecules encoding the same data, wherein thepolymer or nackets comprise multiple authentication levels or layers of data, e.g., two or more, e.g., three or more layers of data.

[0054] In one embodiment, the nackets comprise four authentication levels: a Rapid Identification Level, wherein the DNA comprises or is associated with one or more labels providing a spectral fingerprint; a Nacket Data Level, wherein the DNA sequence encodes user-defined data, e.g., in a binary, ternary or other machine-readable code; a Production Lot Fingerprint Level, wherein the DNA has a specific percent abundance of different bases, sequence variants, and / or cassette variants; and an Object Fingerprint Level, comprising a unique population of different synonymous sequences. This presents a number of advantages:• All four layers of data are associated with the same population of DNA polymers - they are inseparable.• The DNA sequence may be tied to a block chain. The block chain may contain encrypted information to validate the other two levels. Use of public block chain will survive even if the company synthesizing the DNA goes out of business.• There are many techniques to sequence DNA. Other systems may come and go, but DNA will always be readable.

[0055] In some embodiments, the Rapid Identification Level comprises a first layer of data, or level of authentication, allowing for authentication without DNA sequencing. In some embodiments, the Rapid Identification Level comprises a spectroscopic fingerprint or signature. For example, the nucleotide polymer may be labeled with spectroscopic markers or spectroscopically-active moieties, such that a spectroscopic signature, or combination of spectroscopic signatures, can support and / or verify object authenticity. Spectroscopic signatures may include read-outs via absorption spectroscopy, infrared (e.g., FTIR) or near-infrared spectroscopy, fluorescence spectroscopy, emission spectroscopy, reflectance spectroscopy, luminescence spectroscopy, X-ray absorption spectroscopy, ultraviolet / visible light (UV / Vis) spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), or a combination thereof. Spectroscopic signatures may be read using traditional means, e.g., table-top spectrometer, or may be read using mobile spectrometer units, e.g., hand-held devices, e.g., such as those described in U.S. Patent No. US 11,879,777, the contents of which are incorporated herein by reference. In some embodiments, spectroscopic analysis is capable of Z-scanning focusing, e.g., wherein the spectroscopic marker may be detected (or read) by focusing the spectrometer onto aspecific layer or point of a substrate of interest, e.g., a micro-container, e.g., wherein the spectroscopic marker is detectable while not being on the outer surface of a micro-container, e.g., wherein the spectroscopic marker is detected through the surface of a micro-container.

[0056] In the present disclosure, the novel population of nucleotide polymers, e.g., deoxyribonucleic acid (DNA) polymers, or nackets, may be synthesized using any one or more method as taught by US Patent Application Nos. 63 / 582,199 and 63 / 623.085, and International Application No. PCT / US2024 / 046372. For example, the nucleotide polymers or nackets may be enzymatically synthesized, for example, using one or more topoisomerase, transferase, and / or ligase enzyme. In some embodiments, the nucleotide polymers or nackets may be synthesized on a surface, in solution (e.g., droplet or bulk solution, e.g., with or without mixing), under flow conditions, or a combination thereof. In some embodiments, the nucleotide polymers or nackets may be synthesized in an organic solvent, water, buffer, or a combination thereof.

[0057] In some embodiments, analysis of a spectroscopic fingerprint and / or detection of spectroscopic markers, such as on an object surface or in solution, can provide a rapid, non-contact, non-destructive, in-field authentication method. Analysis of a spectroscopic fingerprint or signature, e.g., Rapid Identification Level, may additionally provide information regarding inherent characteristics of the nucleotide polymers or nackets, or regarding characteristics added onto or associated with the nucleotide polymers for authentication purposes, e.g., Production Lot Fingerprint Level. For example, inherent characteristics of the nucleotide polymers or nackets include the ratio of nucleotide bases, the percent composition of each constituent polynucleotide block / cassette, the presence and relative amounts of unusual / nonnatural / synthetic nucleobases, or combinations thereof. Examples of characteristics added onto or associated with the nucleotide polymers for authentication purposes include spectroscopic markers, epigenetic tags, molecular tags, or combinations thereof. Molecular tags may include the modification of nucleotides via click reactions (e.g., azide-alkyne cycloaddition, for example a strain-promoted azide-alkyne cycloaddition (SPAAC), e.g., between an azide and a cyclooctyne, e.g., a dibenzocyclooctyne (DBCO) moiety or aza-dibenzocyclooctyne (ADIBO) moiety), coordination complexes (e.g., metal complexes, e.g., platinum compounds such as cisplatin), covalent bond conjugation, e.g., halide reactions, carboxyl reactions, ester reactions, amide reactions, amine-based reactions (e.g., N-hydroxysuccimide conjugation, phosphoramidite conjugation, etc.), or combinations thereof. Molecular tags may be permanent or reversible modifications or signals. Molecular tags may beadded before or after incorporation of the nucleotide into a DNA polymer. Molecular tags and / or epigenetic tags may be used in the evaluation of a first level of authentication comprising a Rapid Identification Level, e.g.. wherein the molecular tags and / or epigenetic tags comprise spectroscopic markers.

[0058] In one embodiment, for example, the DNA polymers are synthesized comprising two or more populations of DNA cassettes, wherein each cassette comprises one or more spectroscopically-active nucleotide species, e.g., adenosine triphosphate (e.g., dATP), guanosine triphosphate (e.g., dGTP), thymidine triphosphate (e.g., dTTP), cytidine triphosphate (e.g., dCTP), 2’0me-ATP, 5-Formyl-dc, 5-Methyl-dc, 5-hydroxymethyl-dCTP, 6-Methyl-dATP, 5-Hydroxy-UTP, and 5-Carboxy-dc in varying ratios. These DNA polymers all comprise the same encoded data, i.e., Nacket Data Layer, while each DNA polymer has a unique sequence of nucleotides, and the entire population of DNA polymers comprise a Production Lot Fingerprint that may be spectroscopically identifiable. These DNA polymers, optionally encapsulated within a microcontainer, may be subsequently incorporated into or onto an object. Analysis for Rapid Identification and / or Production Lot Fingerprint may be achieved using, for example, Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), absorbance spectroscopy, IR or near-IR spectroscopy, and / or fluorescence spectroscopy, e.g. through direct spectroscopic analysis of the intact object, e.g., focusing a Raman and / or fluorescence spectrometer laser onto the object surface. Alternatively, analysis of the Production Lot Fingerprint may comprise removing one or more pieces of the object, either temporarily or permanently, to make the sample available for spectroscopic analysis. Analysis using Raman, SERS, and / or fluorescence spectroscopy, may be accomplished using the presence and relative ratio of spectroscopic markers, e.g., spectroscopically-active nucleotide species, within each DNA cassette, and / or the presence and relative ratio compared between DNA cassettes, providing a spectroscopic signature or fingerprint to support or verify object authenticity. For example, a Rapid Identification or Production Lot Fingerprint spectroscopic signature may comprise any combination of one or more of the Raman spectra presented in Figure 1.

[0059] In another embodiment, for example, the DNA polymers or nackets, during or after synthesis, are associated with one or more spectroscopic marker, e.g., phenthiophenol, 4-mercaptobenzoic acid (4-MBA), rhodamine 6G (R6G), methylene blue (MB), crystal violet (CV), melamine, 4-aminothiophenol (4- ATP), 4-mercaptopyridine (4-Mpy), and malachite green (MG),or a combination thereof. In another embodiment, the one or more spectroscopic markers comprise (or further comprise) a photonic crystal, e.g., such as those taught in U.S. Patent Application No. US / 2015 / 0076412, the contents of which are incorporated herein, or substantially similar thereto, e.g., wherein the photonic crystal is on a length-scale sufficient for encapsulation with a microcontainer. e.g., a silica bead. Subsequent analysis may be accomplished using spectroscopy, e.g., Raman, SERS, absorbance, IR or near-IR, and / or fluorescence spectroscopy. In some embodiments, the spectroscopic markers are co-encapsulated with the DNA polymers or nackets within a micro-container, e.g., a silica bead or particle. In some embodiments, the spectroscopic markers are covalently bound to the inner and / or outer surface of the micro-container, adsorbed onto the inner and / or outer surface of the micro-container, spin-coated, doped, annealed, or embedded onto or within the material of the micro-containers, e.g., onto or within the silica of silica beads or particle, or a combination thereof. In some embodiments, the DNA polymers or nackets are encapsulated within a first population of micro-containers, e.g., silica beads or particles, and the spectroscopic markers are encapsulated (or bound, absorbed, spin-coated, doped, annealed, embedded, or otherwise associated with) within a second (or third or fourth, etc.) population of micro-containers, wherein the spectroscopic markers are associated with the DNA polymers or nackets by mixing the first and second (and third and fourth, etc.) population of microcontainers. In some embodiments, two or more spectroscopic markers are co-encapsulated within a micro-container, such that the two or more spectroscopic markers demonstrate Forster resonance energy transfer or fluorescence resonance energy transfer (FRET) pairing behavior. Alternatively, in some embodiments, one or more spectroscopic markers are co-encapsulated or spatially constrained within a micro-container, such that the one or more spectroscopic markers demonstrate quenching (e.g., self-quenching or FRET-quenching) when encapsulated, but are “activated” (i.e., increase in fluorescent activity / detectability, or wherein the quenching behavior is reduced) upon release of the one or more spectroscopic markers from the micro-container, e.g.. wherein the micro-container is dissolved or broken, such that the one or more spectroscopic markers are no longer co-encapsulated or spatially constrained to sufficiently induce quenching.

[0060] In some embodiments, a portion of the DNA molecules are labeled, and a portion is not. For example, in some embodiments, to incorporate an additional level of authentication, two or more populations of DNA polymers may be incorporated into or onto an object such that a first surface or material of the object comprises a population of positively authenticating DNApolymers, i.e., polymers encoding data that support or verify object authenticity, while a second surface or material of the object comprises a population of negatively authenticating DNA polymers, or “poison” polymers, i.e., polymers encoding data indicating analysis done by a nonauthorized entity. For example, both positively and negatively authenticating DNA polymers may be incorporated into an object, wherein the location of the positively and negatively authenticating DNA polymers is privately-held knowledge known only by authorized entities.

[0061] In some embodiments, one strand of the DNA molecules is labeled, and the other strand is not. This approach can facilitate amplification of the unmodified strand, for example. In some embodiments, the analysis comprises identifying the sequence(s), or key parts of the sequence(s), of the DNA polymers incorporated into or onto an object. Analysis may comprise amplifying the DNA polymers using PCR, e.g„ before sequencing. In some embodiments wherein unusual / non-natural / synthetic nucleotide species are present in the DNA polymer, sequence amplification may comprise denaturing of the DNA polymer comprising the usual / non-natural / synthetic nucleotide species, synthesis of a complimentary strand comprising common nucleotide species, e.g., adenosine, guanine, cytosine, thymine, and subsequent amplification of said complimentary strand. Said complimentary strand may provide information regarding the original DNA polymer and accompanying encoded data such that the object can be authenticated. In addition to said embodiments, or alternative embodiments, the analysis may comprise detection and identification of the DNA polymers using specific complimentary overhangs or sequences that adhere to unique capture sequences or sequence caps present in the DNA polymers, e.g., wherein the DNA polymers can be subsequently amplified using PCR or identified (i.e., bound) using antibodies. In such embodiments wherein the DNA polymers and nackets are encapsulated within a micro-container, the DNA polymers and nackets are removed or extracted from the micro-container before PCR amplification, optionally wherein any spectroscopic markers associated therewith are removed or not removed from the DNA polymers and nackets.

[0062] In some embodiments, analysis of the Nacket Data Level is achievable using a deciphering code and / or algorithm, for example, a deciphering code and / or algorithm only known to authorized entities. In some embodiments, the Nacket Data Level comprises encoded data that requires a private key to identify and / or decode (i.e., unencrypt) the DNA sequence(s). In some embodiments, the encoded data is linked or stored on a blockchain, e.g., a public-access blockchain. In some embodiments, the encoded data correspond to a unique data sequence, e.g., a non-fungible token(NFT). Tn some embodiments, the DNA polymers for authentication, and optionally the corresponding encoded data, are used for authentication of a class of object, e.g., a brand of objects, and / or for authentication of a sub-class of object, e.g., a specific or limited release of said objects, and / or for authentication of a specific individual object.

[0063] This disclosure is directed, in part, to the synthesis of DNA sequences or polymer(s) encoding data useful in the authentication of objects for protection against counterfeiting. This method involves first synthesizing one or more DNA sequences, incorporating said DNA sequences into an object, optionally extracting said DNA sequences from the object when necessary for authentication purposes, and analyzing the DNA sequences for confirmation of object authenticity and / or object provenance.

[0064] Thus, this disclosure provides methods of confirming object authenticity and / or provenance through incorporation of DNA sequences; said DNA sequences may be analyzed while incorporated into or onto an object, or said DNA sequences may be extracted from the object and analyzed / identified .

[0065] In some embodiments, the nackets may encode a non-fungible token (NFT), which is a unique digital identifier that is recorded on a blockchain and is used to certify ownership and authenticity. It cannot be copied, substituted, or subdivided.

[0066] DNA is a relatively stable molecule and can be readily incorporated into or associated with goods for purposes of identifying and authenticating the goods. In certain embodiments, the nackets are adsorbed to or encapsulated within micro-containers, e.g., silica beads or particles, which are optionally coated with polymer, and incorporated into goods, e.g.. for purposes of identification and authentication of the goods. For example, the DNA nackets can be incorporated into silica beads, e.g., using methods as described in Koch J, et al., “A DNA-of-things storage architecture to create materials with embedded memory” Nat. Biotechnol. (2020)38(1 ) :39-43, the contents of which are incorporated herein by reference.

[0067] In certain embodiments, the nucleotide polymers or nackets are incorporated into an object by direct surface conjugation. In alternative embodiments, the nucleotide polymers or nackets are encapsulated into micro-containers or molecular assemblies. In certain embodiments, these encapsulated DNA sequences are incorporated into constituent parts or materials used in the production of an object, such as textiles, fabrics, leather, biomaterial products, polymers, plastics,wood, metals, inks, paints, solutions, suspensions, and raw materials. In certain embodiments, the nucleotide polymers or nackets are inserted into a cell or cells, or inserted into a larger DNA construct and / or genome, such as into yeast, bacteria, fungi, plant, or animal cells, for example wherein the cells are used in the production of foods, drinks, biologies, or materials, e.g., cheese, beer, wine, vegan leather, pharmaceuticals.

[0068] In certain embodiments, the nucleotide polymers or nackets incorporated into an object, e.g., by direct surface conjugation and / or encapsulation into micro-containers or molecular assemblies, are incorporated into the entire object. In alternative embodiments, the nucleotide polymers or nackets incorporated into an object are incorporated into select parts or materials of an object, e.g., wherein analysis of a specific part or material, or analysis of a combination of specific parts or materials, is necessary for object authentication. In some embodiments, one or more select parts or materials comprise a “poison” sequence, wherein the poison sequence is one or more nucleotide polymers, e.g., DNA, encoding information indicative of analysis of a “wrong” or “unauthorized” part or material, indicating an unauthorized analysis.

[0069] In certain embodiments, nucleotide polymers or nackets incorporated into an object are extracted from the object; this extraction may be completed prior to or following production of the object, shipping of the object, sale of the object, offer for sale of the object, importation of the object, or exportation of the object. In certain embodiments, this extraction is completed for identification, authentication, and / or valuation of the object.

[0070] In certain embodiments, nucleotide polymers or nackets incorporated into an object are analyzed while the nucleotide polymers or nackets are still incorporated into or onto said object. For example. DNA polymers, along with any associated spectroscopic markers, may be analyzed using spectroscopic methods, comprising absorption spectroscopy, infrared (e.g., FTIR) or nearinfrared spectroscopy, fluorescence spectroscopy, emission spectroscopy, reflectance spectroscopy, luminescence spectroscopy. X-ray absorption spectroscopy, ultraviolet / visible light (UV / Vis) spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), or a combination thereof. Spectroscopic analysis of the nucleotide polymers or nackets incorporated into or onto an object may be completed prior to or following production of the object, shipping of the object, sale of the object, offer for sale of the object, importation of the object, or exportation of the object. In certain embodiments, spectroscopic analysis is completed without extracting any part or material of the object. In certain embodiments, spectroscopic analysis of the DNA polymersis completed in less than 30 minutes, e.g., in less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 12 minutes, less than 10 minutes, less than 8 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 90 seconds, less than 75 seconds, less than 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 18 seconds, less than 15 seconds, less than 12 seconds, less than 10 seconds, less than 8 seconds, less than 5 seconds.

[0071] In certain embodiments, the nucleotide polymers or nackets incorporated into an object is extracted from the object through physical and / or chemical means, such as cutting, grinding, scoring, chipping, shredding, pulverizing, dissolving, or cleaving the nucleotide polymers or nackets from one or more pieces of the object.

[0072] In certain embodiments, the nucleotide polymers or nackets extracted from an object are isolated and / or purified; this may be accomplished by chromatography, electrophoresis, centrifugation, or combinations thereof. In certain embodiments, nucleotide polymers or nackets extracted from an object are analyzed using mass spectrometry and / or high-throughput DNA sequencing.

[0073] In certain embodiments, the analyzed DNA sequences are compared to a database of object identification codes, wherein matching an object identification code to an extracted DNA sequence confirms the identity, authenticity, provenance, and / or security of the object. In certain embodiments, an analysis of the DNA sequences may be compared with results from a previous analysis of the DNA sequences from the same or similar object. In certain embodiments, analysis of the DNA sequences may comprise analysis and authentication corresponding to a spectroscopic signature, e.g., corresponding to a Production Lot Fingerprint, and / or a sequence identification, e.g., corresponding to a Molecular Fingerprint.

[0074] In one aspect, the disclosure thus provides a method of object authentication (Method 2), comprising:i. synthesizing nackets encoding data;ii. incorporating said nackets into or onto an object;iii. optionally, extracting said nackets from the object; andiv. analyzing the nackets;v. optionally, comparing the analyzed nackets to a database of DNA sequences and / or spectroscopic signatures;vi. optionally, confirming object authenticity;wherein the nackets comprise or are associated with detectable markers (e.g. spectroscopically detectable markers) for rapid identification.

[0075] In certain embodiments, one or more DNA sequences are synthesized to encode data designed as an identification code for the object. In certain embodiments, this identification code is written manually. In alternative embodiments, this identification code is a machine-generated and / or randomly generated number or numbers.

[0076] In certain embodiments, the nucleotide polymers or nackets are synthesized from a connection point on a surface, or are synthesized in solution. In certain embodiments, the nucleotide polymers or nackets are synthesized in well plates, droplets, or chambers, wherein each well / droplet / chamber is used to synthesize a unique DNA sequence or sequences, wherein the DNA has a unique sequence profile but retains the data (e.g. ternary code) encoded in the nacket. In certain embodiments, the nucleotide polymers or nackets are amplified and / or replicated, optionally wherein amplification bias is used to further make the population of DNA polymers or sequences unique. In alternative embodiments, the one or more DNA sequences are not amplified and / or replicated, and thus are directly used in incorporation into an object.

[0077] In certain embodiments, the one or more DNA polymer is incorporated into an object by direct surface conjugation. In alternative embodiments, the one or more DNA polymer is encapsulated into micro-containers, such as microspheres, beads, or particles, such as silica microspheres, silica beads, or silica particles. In certain embodiments, these micro-containers are incorporated into constituent parts or materials used in the production of an object, optionally wherein the constituent parts or materials are textiles, fabrics, leather, biomaterial products, polymers, plastics, wood, metals, inks, paints, solutions, suspensions, and raw materials. In certain embodiments, the one or more DNA polymer is inserted into a cell or cells, optionally inserted into a larger DNA construct and / or genome, optionally inserted into yeast, bacteria, fungi, plant, or animal cells, optionally wherein the cells are used in the production of foods, drinks, biologies, or materials, e.g., cheese, beer, wine, vegan leather, pharmaceuticals.

[0078] In certain embodiments, one or more of the nucleotides polymers (i.e., sequences) incorporated into an object is extracted from the object. In certain embodiments, this extraction is completed prior to or following production of the object, shipping of the object, sale of the object, offer for sale of the object, importation of the object, or exportation of the object. In certain embodiments, this extraction is completed for identification, authentication, and / or valuation of the object.

[0079] In certain embodiments, one or more of the DNA polymers incorporated into an object are extracted from the object through physical means, such as cutting, grinding, scoring, chipping, shredding, or pulverizing one or more pieces of the object. In further embodiments, one or more of the DNA polymers incorporated into an object are extracted from the object through chemical means, such as dissolving or cleaving the DNA sequences from one or more pieces of the object.

[0080] In certain embodiments, nucleotide polymers or nackets incorporated into an object are analyzed while the DNA polymers are still incorporated into or onto said object. For example, DNA polymers, optionally comprising associated spectroscopic markers, may be analyzed using spectroscopic methods, comprising absorption spectroscopy, infrared (e.g., FTIR) or near-infrared spectroscopy, fluorescence spectroscopy, emission spectroscopy, reflectance spectroscopy, luminescence spectroscopy, X-ray absorption spectroscopy, ultraviolet / visible light (UV / Vis) spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), or a combination thereof. Spectroscopic analysis of the DNA polymers, optionally comprising associated spectroscopic markers, incorporated into or onto an object may be completed prior to or following production of the object, shipping of the object, sale of the object, offer for sale of the object, importation of the object, or exportation of the object. In certain embodiments, spectroscopic analysis is completed without extracting any part or material of the object. In certain embodiments, spectroscopic analysis of the DNA polymers, optionally comprising associated spectroscopic markers, is completed in less than 30 minutes, e.g., in less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 12 minutes, less than 10 minutes, less than 8 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 90 seconds, less than 75 seconds, less than 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 18 seconds, less than 15 seconds, less than 12 seconds, less than 10 seconds, less than 8 seconds, less than 5 seconds.

[0081] In certain embodiments, the nucleotide polymers or nackets incorporated into or onto an object is analyzed to determine authenticity using a first level of authentication, or layer of data, comprising a Production Lot Fingerprint. In certain embodiments, the Production Lot Fingerprint is analyzed using spectroscopic methods, e.g., Raman spectroscopy. In certain embodiments, a unique spectral signature, e.g., combination of spectral peaks useful for identification, for example, the spectra shown in Figure 1, provides a unique “fingerprint” allowing for support or verification of object authenticity.

[0082] In certain embodiments, one or more of the DNA polymers or nackets extracted from an object are isolated and / or purified, e.g., to yield a Molecular Fingerprint. In certain embodiments, one or more of the DNA sequences extracted from an object is isolated and / or purified using chromatography, for example ion exchange chromatography, size exclusion chromatography, normal-phase or reverse-phase high-performance liquid chromatography (HPLC), antibody affinity chromatography, or combinations thereof. In certain embodiments, one or more of the DNA polymers or nackets extracted from an object are isolated and / or purified using electrophoresis, for example polyacrylamide gel electrophoresis, two-dimensional electrophoresis, pulsed field electrophoresis, Southern blotting, or combinations thereof. In further embodiments, one or more of the DNA polymers or nackets extracted from an object are isolated and / or purified using centrifugation. In certain embodiments, one or more of the DNA polymers or nackets extracted from an object are isolated and / or purified using a combination of chromatography, electrophoresis, and / or centrifugation.

[0083] In certain embodiments, one or more of the nucleotide polymers or nackets extracted from an object is analyzed using mass spectrometry and / or high-throughput DNA sequencing. In certain embodiments, the analyzed DNA polymers or sequences are compared to a database of object identification codes, wherein matching an object identification code to an extracted DNA sequence confirms the identity, authenticity, provenance, and / or security of the object. In certain embodiments, an analysis of the DNA sequences may be compared with results from a previous analysis of the DNA sequences from the same or similar object.

[0084] In certain embodiments, analysis of the extracted DNA sequences yields a “fingerprint”, wherein the specific DNA sequence, the incidence rate of each individual nucleotide, the relative incidence rates of nucleotides, and / or the specific molecular mass of the DNA sequence and / or its degradation products may be compared with a database of object identification codes. In furtherembodiments, the sequence of DNA may be analyzed and used to determine the object identification code. In alternative embodiments, the sequence of nucleotides within heterogeneous DNA sequences may be analyzed and used to determine the object identification code.

[0085] In one embodiment, the disclosure provides a population of deoxyribonucleic acid (DNA 1) molecules encoding data useful in the authentication of objects and for protection against counterfeiting, comprising nucleic acid data packets (“nackets”), wherein each nacket contains a plurality of synonymous DNA molecules having different sequences but encoding the same data, wherein the plurality of DNA polymers comprise, or are associated with, four or more authentication levels, comprisingi.) a Rapid Identification Level, wherein the DNA comprises or is associated with one or more detectable labels, e.g., labels providing a spectral fingerprint; ii.) a Nacket Data Level, wherein the DNA sequence encodes user-defined data, e.g., in a binary, ternary or other machine -readable code;iii.) a Production Lot Fingerprint Level, wherein the DNA has a specific percent abundance of different bases, sequence variants, and / or cassette variants; and iv.) an Object Fingerprint Level, comprising a unique population of different synonymous sequences.

[0086] For example, the disclosure provides:1.1. DNA 1, wherein the DNA encodes data that function as an identification code for the object.1.2. Any foregoing DNA, wherein the encoded data is computer generated, e.g., not manually- defined, e.g.. randomly and / or algorithmically computer generated.1.3. Any foregoing DNA, wherein the data is in binary code.1.4. Any foregoing DNA, wherein the data is in ternary code.1.5. Any foregoing DNA, wherein the data useful in object authentication comprises a lot number, batch number, production number, data code, client number, etc., or a combination thereof.1.6. Any foregoing DNA, wherein the DNA comprises one or more canonical nucleotide, e.g., adenosine, guanosine, thymidine, and cytosine.1.7. Any foregoing DNA, wherein the DNA comprises the canonical nucleotides adenosine, guanosine, thymidine, and cytosine.Any foregoing DNA, wherein the DNA comprises one or more non-natural, synthetic, modified, mutated, or non-canonical nucleotide.Any foregoing DNA, wherein the DNA comprises modifications, e.g., polyadenylation, e.g., conjugation with small molecule and / or polymer moieties.. Any foregoing DNA, wherein the DNA comprises modifications, e.g., moieties conjugated via click chemistry, coordination chemistry, covalent conjugation chemistry, or a combination thereof.. Any foregoing DNA, wherein the DNA is single- stranded.. Any foregoing DNA, wherein the DNA is double-stranded.. Any foregoing DNA, wherein the DNA is linear.. Any foregoing DNA, wherein the DNA is branched.. Any foregoing DNA, wherein the DNA is cyclic and / or cyclized.. Any foregoing DNA, wherein the data carried by the DNA is a nonfungible token (NFT).. Any foregoing DNA, wherein the DNA is incorporated into or associated with goods for purposes of identifying and authenticating the goods.. Any foregoing DNA, wherein the DNA is adsorbed onto, incorporated into, or encapsulated by a micro-container, e.g., silica bead or particle.. Any foregoing DNA, wherein the DNA is adsorbed onto, incorporated into, or encapsulated by silica beads or particles and embedded or incorporated into goods, e.g., for purposes of identification and authentication of the goods.. Any foregoing DNA, wherein the DNA is associated with one or more spectroscopic markers.. Any foregoing DNA, wherein the DNA is associated with one or more spectroscopic markers co-encapsulated within a micro-container.. Any foregoing DNA, wherein the DNA is associated with one or more spectroscopic marker covalently bound to the outer and / or inner surface of a microcontainer.. Any foregoing DNA, wherein the DNA is associated with one or more spectroscopic marker adsorbed onto the inner and / or outer surface of a micro-container.. Any foregoing DNA, wherein the DNA is associated with one or more spectroscopic marker spin-coated, doped, annealed, or embedded onto or within the material of the micro-containers.. Any foregoing DNA, wherein the one or more spectroscopic marker is detectable using spectroscopy, optionally absorption spectroscopy, infrared (e.g., FTIR) or nearinfrared spectroscopy, fluorescence spectroscopy, emission spectroscopy, reflectance spectroscopy, luminescence spectroscopy, X-ray absorption spectroscopy, ultraviolet / visible light (UV / Vis) spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), or a combination thereof.. Any foregoing DNA, wherein the one or more spectroscopic marker comprises one or more spectroscopically-active nucleotide species.. Any foregoing DNA, wherein the one or more spectroscopic marker comprises a photonic crystal.. Any foregoing DNA, wherein the one or more spectroscopic marker comprises one or more of adenosine triphosphate (e.g., dATP), guanosine triphosphate (e.g., dGTP), thymidine triphosphate (e.g., dTTP), cytidine triphosphate (e.g., dCTP), 2’0me-ATP, 5-Formyl-dc, 5-Methyl-dc, 5-hydroxymethyl-dCTP, 6-Methyl-dATP, 5-Hydroxy-UTP, and 5-Carboxy-dc.. Any foregoing DNA, wherein the one or more spectroscopic markers comprises one or more of phenthiophenol, 4-mercaptobenzoic acid (4-MBA), rhodamine 6G (R6G), methylene blue (MB), crystal violet (CV), melamine, 4-aminothiophenol (4-ATP), 4-mercaptopyridine (4-Mpy), and malachite green (MG).. Any foregoing DNA, wherein the one or more spectroscopic markers comprises an IR or near IR dye, e.g. a dye selected from cyanines, phthalocyanines, rhodamine analogues and BODIPY dyes (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene).. Any foregoing DNA wherein some portion of the DNA is conjugated to an amine-binding dye, e.g., a dye comprising an NHS moiety, e.g.,. Any foregoing DNA wherein some portion of the DNAis conjugated to a dye via click chemistry, e.g., wherein the DNA is functionalized with an azide moiety and conjugated to a DBCO functionalized dye, e.g.,or. Any foregoing DNA, wherein some portion of the DNA is conjugated to a phosphoramidite-functionalized dye, e.g.,. Any foregoing DNA, wherein the DNA is double-stranded and comprises one or more spectroscopic markers covalently bound to one strand of the DNA, but not the other,e.g., such that the strand that is not covalently bound to one or more spectroscopic markers can be amplified in a polymerase chain reaction.1.35. Any foregoing DNA, wherein the DNA is associated with two or more different spectroscopic markers, e.g., such that the presence of the DNA can be readily detected by detecting the presence of the two or more different spectroscopic markers.1.36. Any foregoing DNA, wherein the DNA is synthesized by sequential addition of DNA cassettes to DNA receptor strands, wherein in each sequential addition step the cassettes comprise a heterologous population of synonymous cassettes, such that the cassettes have at least two different sequences encoding the same data in a machine- readable code (e.g., binary or ternary code).1.37. The foregoing DNA, wherein the sequential addition of DNA cassettes to DNA receptor strands uses a topoisomerase enzyme.1.38. The foregoing DNA, wherein the sequential addition of DNA cassettes to DNA receptor strands is a topoisomerase mediated ligation using DNA cassettes with complimentary overhangs, and 5’ phosphate and phosphatase for blocking and deblocking, to permit controlled, single cassette additions.1.39. Any foregoing DNA, formulated in an ink comprising an IR dye.1.40. Any foregoing DNA, formulated in an ink comprising a UV dye.1.41. Any foregoing DNA, formulated in an ink comprising an IR dye and a UV dye.1.42. Any foregoing DNA, incorporated in silica beads containing or coated with an IR dye or pigment.1.43. Any foregoing DNA, incorporated in silica beads containing or coated with a UV- protectant compound, e.g., cerium oxide (CeO2).1.44. Any foregoing DNA, wherein a portion of the DNA is labeled with a fluorescent dye or pigment, e.g., IR NHS Dye Formula 1 (as described in Example 4) or ethidium monoazide.1.45. Any foregoing DNA for use in any of Method 2, el seq.. Method 3, el seq., and / or Method 4, et seq., infra.

[0087] In another embodiment, the disclosure provides a method of object authentication (“Method 2”), comprising:i. synthesizing DNA polymers comprising nackets encoding data;ii. incorporating said nackets into or onto an object;iii. optionally, extracting said nackets from the object; andiv. analyzing the nackets;v. optionally, comparing the analyzed nackets to a database of DNA sequences and / or spectroscopic signatures;vi. optionally, confirming object authenticity;wherein the nackets comprise or are associated with detectable markers (e.g. spectroscopically detectable markers) for rapid identification.

[0088] For example, in particular embodiments the disclosure provides:2.1. Method 2, wherein the DNA polymers or nackets comprise any of DNA 1, et seq., supra.2.2. Method 2 or 2.1, wherein the DNA polymers or nackets are associated with one or more spectroscopic marker.2.3. Any previous method, wherein the DNA polymers or nackets are incorporated into an object by direct surface conjugation of the one or more DNA sequence onto the object. 2.4. Any previous method, wherein the DNA polymers or nackets are incorporated into a constituent part or material of an object used in production of said object, optionally into textiles, fabrics, leather, biomaterial products, polymers, plastics, wood, metals, inks, paints, solutions, suspensions, and raw materials.2.5. Any previous method, wherein the DNA polymers or nackets are encapsulated into a micro-container, optionally a microsphere, bead, or particle, optionally a silica microsphere, silica bead, or silica particle, prior to incorporation into the object.2.6.Any previous method, wherein the DNA polymers or nackets are encapsulated into a molecular assembly, such as a lipid nanoparticle, protein complex or aggregate, or crystal lattice.2.7. Any previous method, wherein the DNA polymers or nackets are inserted into a cell or cells, optionally inserted into a larger DNA construct and / or genome, optionally inserted into yeast, bacteria, fungi, plant, or animal cells, optionally wherein the cells are used in the production of foods, drinks, biologies, or materials, e.g., cheese, beer, wine, vegan leather, pharmaceuticals, etc.Any previous method, wherein the incorporated DNA polymers or nackets are extracted from the object through physical means, optionally cutting, grinding, scoring, chipping, shredding, or pulverizing one or more pieces of the object.Any previous method, wherein the incorporated DNA polymers or nackets are extracted from the object through chemical means, optionally dissolving or cleaving the DNA polymers and / or one or more pieces of the object.. Any previous method, wherein the extracted DNA polymers or nackets are isolated and / or purified, optionally by chromatography, ion exchange chromatography, size exclusion chromatography, normal-phase and / or reverse-phase high-performance liquid chromatography (HPLC), antibody affinity chromatography, or combinations thereof.. Any previous method, wherein the extracted DNA polymers or nackets are isolated and / or purified, optionally by electrophoresis, polyacrylamide gel electrophoresis, two-dimensional electrophoresis, pulsed field electrophoresis. Southern blotting, or combinations thereof.. Any previous method, wherein the extracted DNA polymers or nackets are isolated and / or purified, optionally by centrifugation, dialysis, or a combination thereof. . Any previous method, wherein the extracted DNA polymers or nackets are analyzed using mass spectrometry and / or high-throughput DNA sequencing.. Any previous method, wherein the extracted DNA polymers or nackets are compared to a database containing the object identification codes as originally synthesized for said object.. Any previous method, wherein the extracted DNA polymers or nackets are compared to results from one or more previous analysis of extracted DNA polymers or nackets from the same or similar object.. Any previous method, wherein the incorporated DNA polymers or nackets are not extracted from the object, and wherein the incorporated DNA polymers or nackets are analyzed while incorporated into or onto said object.. Any previous method, wherein the incorporated DNA polymers or nackets comprise a first level of authentication comprising a spectroscopic signature.2.18. Any previous method, wherein the incorporated DNA polymers or nackets are analyzed using non-contact, non-destructive, rapid, and / or field-deployable methods. 2.19. Any previous method, wherein the incorporated DNA polymers or nackets are analyzed using spectroscopy, optionally absorption spectroscopy, infrared (e.g., FTIR) or near-infrared spectroscopy, fluorescence spectroscopy, emission spectroscopy, reflectance spectroscopy, luminescence spectroscopy. X-ray absorption spectroscopy, ultraviolet / visible light (UV / Vis) spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), or a combination thereof.2.20. Any previous method, wherein the incorporated DNA polymers or nackets are analyzed with support or confirmation of authenticity provided in less than 30 minutes of analysis, optionally, in less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 12 minutes, less than 10 minutes, less than 8 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 90 seconds, less than 75 seconds, less than 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 18 seconds, less than 15 seconds, less than 12 seconds, less than 10 seconds, less than 8 seconds, less than 5 seconds.

[0089] In some embodiments, the disclosure provides an ink comprising a DNA population according to any of DNA 1, et seq. (for example a water-based ink, optionally comprising one or more pigments (for example carbon black or other pigment), binders (for example a polymer, oil, or resin), solvents (water and optionally an alcohol or organic solvent) and / or additives (e.g. drying or chelating agents)) comprising a DNA population according to any of DNA 1, et seq. Such as ink, for example, can be used to authenticate signatures, documents or prints. In certain embodiments, a DNA population according to any of DNA 1, et seq., in the ink encodes a non-fungible token (NFT) linked to a blockchain. Preliminary experiments suggest that the DNA will survive well in ink and paper. DNA stored on FTA cards and even dried blood spots collected on Guthrie filter cards permit accurate analysis after many years of storage without special precautions. In certain embodiments, the ink comprises a fluorescent dye, e.g., a near IR or IR dye. This can allow, for example, detection of which page or pages in a book or document are written with ink containing the authenticating DNA.

[0090] In another aspect, the disclosure provides methods of marking, identifying and authenticating goods, for example (i) methods marking the goods by incorporating or associating the DNA comprising nackets as described herein, e.g., any of DNA 1, et seq.. with the goods to be identified or authenticated, and (2) methods of identifying and optionally authenticating the goods thus marked by retrieving and sequencing the nackets, identifying the goods based on the data, e.g.. binary or ternary code data, encrypted in the nackets thus retrieved and sequenced.

[0091] In an embodiment, the disclosure provides a method (Method 3) for providing high-level authentication using optical signals for an attack-resistant digital code using DNA that uniquely defines a physical object to be authenticated, without requiring DNA sequencing, the digital code having at least four predetermined mixtures of a predetermined number of unique DNA cassette strings, the unique DNA cassette strings of each mixture being different from the DNA cassette strings in the other mixtures, the method comprising:attaching unique optically responsive molecules or particles to at least one of the cassette strings used to make up each mixture, as unique optically labelled cassettes; depositing a droplet of the mixture associated with a given n-bit binary code to be written onto a substrate to add a DNA cassette to an encoded DNA string being written, the droplet comprising the predetermined mixture of the unique cassettes associated with the n-bit binary code and including the at least one unique optically labelled cassette; and repeating the depositing until the desired digital code is written onto the encoded DNA string, andwherein the optically responsive molecules or particles emit an output light when illuminated by an incident light, the output light being indicative of a spectral fingerprint that uniquely defines at least one of: the attack-resistant digital code and the physical object to be authenticated.For example, the disclosure provides3.1. Method 3 wherein the optically responsive molecules or particles comprises at least one of:Raman particles or molecules and fluorescent molecules.3.2.Any foregoing method wherein the incident light comprises a laser light having a wavelength that causes the optically responsive molecules or particles emit the output light.3.3. Any foregoing method wherein the incident light comprises the wavelength of the incident light comprises at least one of: IR, near IR, and Raman excitation.3.4.Any foregoing method wherein the substrate has an acceptor DNA strand having one end attached to the substrate and an opposite end being available to attach to one of the unique DNA cassettes to be added.3.5. Any foregoing method wherein the predetermined number of unique DNA cassettes for one of the mixtures is a different from at least one other of the mixtures.3.6. Any foregoing method wherein the encoded DNA string is embedded in the physical object to be authenticated.3.7. Any foregoing method wherein the encoded DNA string is embedded in a silica bead which is made part of or attached to the physical object to be authenticated.3.8. Any foregoing method wherein the DNA is any of DNA 1, et seq., supra.

[0092] In an embodiment, the disclosure provides a method (Method 4) for providing high-level authentication using optical signals for an attack-resistant digital code using DNA that uniquely defines a physical object to be authenticated, without requiring DNA sequencing, the digital code having at least four predetermined mixtures of a predetermined number of unique DNA cassette strings, the unique DNA cassette strings of each mixture being different from the DNA cassette strings in the other mixtures, the method comprising:assembling the digital code using the DNA cassette strings by depositing individual droplets of the mixture associated with a given n-bit binary code to be written onto a substrate to add a DNA cassette string to an encoded DNA string being written until the desired digital code is written onto the encoded DNA string, each of the droplets comprising a predetermined mixture of the unique cassettes associated with the n-bit binary code; andattaching unique optically responsive molecules or particles to at least one of the cassette strings on the encoded DNA string, as optically labelled string;wherein the optically responsive molecules or particles emit an output light when illuminated by an incident light, the output light being indicative of a spectral fingerprint that uniquely defines at least one of: the attack-resistant digital code and the physical object to be authenticated.For example, the disclosure provides4.1. Method 4 wherein the optically responsive molecules or particles comprises at least one of: Raman particles or molecules and fluorescent molecules.4.2. Any foregoing method wherein the incident light comprises a laser light having a wavelength that causes the optically responsive molecules or particles emit the output light.4.3. Any foregoing method wherein the incident light comprises the wavelength of the incident light comprises at least one of: IR, near IR, and Raman excitation.4.4.Any foregoing method wherein the substrate has an acceptor DNA strand having one end attached to the substrate and an opposite end being available to attach to one of the unique DNA cassettes to be added.4.5. Any foregoing method wherein the predetermined number of unique DNA cassettes for one of the mixtures is a different from at least one other of the mixtures.4.6. Any foregoing method wherein the encoded DNA string is embedded in the physical object to be authenticated.4.7.Any foregoing method wherein the encoded DNA string is embedded in a silica bead which is made part of or attached to the physical object to be authenticated.4.8.Any foregoing method wherein the DNA is any of DNA 1, et seq., supra.

[0093] In certain embodiments, the disclosure provides an ink comprising (i) silica nanoparticles comprising DNA molecules, e.g., of DNA 1, et seq., wherein the DNA encodes data useful in the authentication of objects and for protection against counterfeiting, comprising nucleic acid data packets (“nackets”), wherein each nacket contains a plurality of synonymous DNA molecules having different sequences but encoding the same data, and (ii) UV and / or IR dyes, which fluoresce in the visible range when illuminated with UV or IR light.

[0094] In certain embodiments, the disclosure provides a multi-tiered system (System 1) for object authentication and provenance comprising a first ink comprising (i) silica nanoparticles comprising DNA molecules, e.g., of DNA 1, et seq., wherein the DNA encodes data useful in the authentication of objects and for protection against counterfeiting, comprising nucleic acid data packets (“nackets”), wherein each nacket contains a plurality of synonymous DNA molecules having different sequences but encoding the same data, and (ii) one or more UV and / or IR dyes or pigments, which fluoresce in the visible range when illuminated with UV or IR light, wherein the ink is disposed on an object to be identified or authenticated in a quick response (QR) code pattern.

[0095] Optionally System 1 further comprises a second ink containing one or more UV and / or IR dyes or pigments, which fluoresce in the visible range when illuminated with UV or IR light, wherein the second ink is disposed on an object to be identified or authenticated in a random spot pattern and wherein the second ink may be the same as or different from the first ink, and wherein optionally the second ink may contain dyes or pigments that provide a detectable spectral profile for the random spots. Figure 13 depicts how such a system can be applied to an item , such as a collectable, for subsequent identification and authentication. In some embodiments, the inks are applied to the object using continuous inkjet printers, as depicted in Figure 14B.

[0096] For example, in the foregoing system, the object can be authenticated using a multi-tiered authentication method as follows:i. Level 0 (Figure 15 A): A UV or IR light is provided, causing a UV or IR dye disposed in a QR code to become visible, wherein the QR code can be read by a cell phone, and optionally transmitted to a server for authentication, thereby providing a first indication of authenticity;ii. Level 1 (Figure 15B): A UV or IR light is provided, which may be the same or different from the first light, depending on the system, causing a UV or IR dye disposed in a random dot pattern to become visible, so it can be detected and photographed by a cell phone, and optionally transmitted to a server for authentication, thereby providing a second indication of authenticity;iii. Level 2 (Figure 15C): The spectral profile of the dots in the dot pattern is detected, thereby providing a third indication of authenticity;iv. Level 3 (Figure 15D): The object is swabbed to retrieve sufficient silica nanoparticles comprising DNA, which is extracted and sequenced to provide a further indication of authenticity.This system allows authentication (or counterfeit detection) to be performed stepwise, with increasingly high levels of confidence, as each indication of authenticity is more difficult to counterfeit than the prior indication (Figure 16A). An example of such a system is depicted in Figures 13-20.

[0097] Although the disclosure has been described herein using exemplary techniques, algorithms, or processes for implementing the present disclosure, it should be understood by those skilled in the art that other techniques, algorithms and processes or other combinations and sequences of thetechniques, algorithms and processes described herein may be used or performed that achieve the same function(s) and result(s) described herein and which are included within the scope of the present disclosure.

[0098] It should be understood that, unless otherwise explicitly or implicitly indicated herein, any of the features, functions, characteristics, alternatives or modifications described regarding a particular embodiment herein may also be applied, used, or incorporated with any other embodiment described herein.

[0099] Conditional language, such as. among others, "can," "could," "might," or "may," unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments could include, but do not require, certain features, elements, or steps. Thus, such conditional language is not generally intended to imply that features, elements, or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without user input or prompting, whether these features, elements, or steps are included or are to be performed in any particular embodiment.

[0100] Referring to Figure 1, various spectral profiles (or “fingerprints”) of exemplary nucleobases detected using Raman spectroscopy are shown, in accordance with embodiments of the present disclosure. In particular, a graph 10 shows a family of curves 20, each curve 20A-20K is associated with a chemical compound 22 indicated to the right of each curve. The horizontal axis shows the wavelength shift in cm1, and the vertical axis has arbitrary units showing relative intensity for each of the family 20 of curves 20A-20K for each chemical compound 22.

[0101] Referring to Figure 2A, a top-level block diagram 200 is shown of an exemplary process and system for initially authenticating an item 201 at the Rapid Identification Level, wherein the DNA comprises or is associated with one or more spectral labels providing a spectral fingerprint, in accordance with embodiments of the present disclosure.

[0102] The spectral labels are excited using a light source 204 which provides an excitation light 202 and the emission spectra shown as return light 206 are detected using an optical sensor / detector 208, which may be part of a measurement instrument 209. The return light signal 214 is analyzed using a signal processing logic / computer 210, which may have a display 211, for the presence or absence of the specific spectral signal associated with a spectral tag attached to the DNA cassettes or DNA strings or chains as discussed herein. The result of the analysis may be displayed on a userdevice 220, such as a laptop computer or smartphone, having a display 222 and an OAP software application (or OAP App) 224, and / or on the display 211 in the measurement instrument 209. In some embodiments, the spectral data may be further transmitted to a data storage server 244 for further analysis, in accordance with embodiments of the present disclosure. The data server 244 may hold information or data regarding the lot number (or other manufacturing information) relating to the item to be authenticated. The user device 220 may run the software application program (e.g., Object Authentication and Provenance (OAP) App) 224, which determines whether the received spectral signal 214 matches the expected signal for the item 201 being authenticated, and may display the result on the display 222 of the user device 220 for the user 230 to evaluate the authenticity of the desired item, as shown by a line 228.

[0103] Referring to Figure 2B, an exemplary screen illustration 250 is shown which may be populated by the OAP App (Fig. 2A), and may provide criteria and / or results for Rapid Identification Level identification of an object such as a handbag, in accordance with embodiments of the present disclosure. In particular, there may be a field 252 for selecting a data file associated with the item 252, which when selected provides a pop-up window 254 having a list of files to select from. When a file is selected, the OAP App reads the file and populates various data fields associated with the item of interest in a separate window 256. The data may include manufacturing details, including an item description in field 256A, optical type or form of optical detection used (e.g., Raman, fluorescent, or other optical tag) in field 256B, optical material used as the optical tag (e.g., molecules, particles or other optical material) in field 256C, incident or source light wavelength in field 256D, and return or detected or response light wavelength(s) in fields 256E and 256F, which may include the expected value(s) and actual received value(s) for a given lot number, and may also include intensity or magnitude values. Such manufacturing details may also be linked to a manufacturing lot number for the specific item 252 being evaluated for authenticity. In some embodiments, the user device may read a QR code or bar code or the like and obtain the lot number or file name and the system may automatically pull up the associated file with the details for the item 252.

[0104] Also, in some embodiments, a window 260 may be provided that shows the actual spectra measures having spectral graphs 262A-262D, and may also show where the errors occurred. When the results have been evaluated, the system may provide a Pass / Fail indication for the RapidIdentification Level (or LI) test, such as LI Authentication PASSED 264, or L1 Authentication FAILED 266, as shown in Fig. 2B.

[0105] Referring to Figure 3, a diagram 300 shows a plurality of unique DNA cassettes associated with a 2-bit binary code, which allows the creation of unhackable (or attack resistant) DNA cassette based digital codes similar to that described in commonly-owned International Application No. PCT / US2024 / 046372, referenced herein. The example shown in Fig. 3 shows 16 unique cassettes (C1-C16), with groups of 4 cassettes (C1-C4) 302, (C5-C8) 304, (C9-C12) 306, and (C13-C16) 308 associated with (or assigned to) each two-bit binary code (00,01,10,11), respectively, providing 2-bit multi-base encoding. A hacker or counterfeiter that substitutes a single base (or if the DNA has damage to a single base) will change the underlying binary code represented, thus requiring making it easy to detect counterfeits. This example is shown for 15 bases (G.C,A,T) that make up each cassette, each corresponding to a 2-bit binary code as shown.

[0106] Referring to Figure 4, a further depiction of the heterologous cassette approach 400 is shown where the assigned cassettes for each 2-bit binary code are mixed in predetermined proportions, e.g., mix 1 and mix 2, as described in commonly-owned International Application No. PCT / US2024 / 046372, referenced herein. In particular, is shows a cassette formulation 1 (or mix 1) 402 and a cassette formulation 2 (or mix 2) 404, and also shows the 2-bit multi-base encoding 406, but in this example, not only are the four cassettes (C1-C4; C5-C8, C9-C12, C13-C16) assigned to a specific base pair (00,01,10,11), but each group of four cassettes are mixed together in predetermine proportions (or mixes), which causes each resultant 2-bit code 00 (410), 01 (412), 10 (414), 11 (416), that is written will be the assigned cassettes, but will also occur in the resulting DNA strings in proportion to their concentration in the mixture (for large numbers) mixl, mix2, providing another way to validate the resulting DNA written string at the verification facility.

[0107] Referring to Figure 5, a diagram 500 of the heterologous cassette approach is shown, which shows the various random cassettes 502 that make up memory strings or DNA chains or nackets on a given spot and shows an individual string 508 as indicated by a line 504, showing the two-bit binary codes 506 and which cassettes were assigned to each base pair and a corresponding cassette proportions for a given example shown as shown by the line 512 and the corresponding table 510, as described in International Application No. PCT / US2024 / 046372, referenced herein.

[0108] Referring to Figure 6, a portion 600 of the diagram of Figure 4 is shown, for Rapid Identification Eevel (El) detection, where the cassettes (C1-C16) or DNA strands or DNA stringscan be labeled with optical or spectral tags (shown as stars or spectral decorations) 602A-602P corresponding to C1-C16, respectively, that can be detected using spectroscopic means, e.g., fluorescent or Raman tags, having distinctive excitation / emission frequencies, in accordance with embodiments of the present disclosure.

[0109] Referring to Figure 7, an example of a Rapid Identification system 700 is shown to detect distinctive Raman optical signatures, in accordance with embodiments of the present disclosure. In particular, it shows a measurement instrument 702, which reads spots 706 on an array or wafer 704, the spots 706 having been written as described herein, and shows sample Raman spectra that may be detected at a given spot. In particular, a blowup image 708 of a given spot 708 is shown, where at the spot 708 there is attached a plurality of DNA strands or strings 710 having an optical spectra shown in graph 712 (similar to the graph 20 Fig. 1 for a plurality of molecules or moieties 22). Each DNA string 710 may contain a plurality of cassettes (e.g., C1-C16), as discussed further hereinafter with Fig. 8 and Fig. 9, having four different optical curves 712A, 712B, 712C, 712D that appear as an aggregate optical spectrum (or “fingerprint”) 712. Spectral graph 714, having five unique optical spectral curves 714A-714E, and spectral graph 716, having five different unique spectral curves 716A-716E (even for the same code) will exist at different spots because each of the spots contain a random selection of selected cassettes some of which are optically labeled. Thus, the optical fingerprint for two different spots on the array 704 having the same digital code will have different unique optical signatures, and the digital code can only be determined by knowing which cassettes have which optical labels and which cassettes are assigned to (or associated with) which 2-bit binary code. Thus, each spot on the wafer 704 will have a unique distribution of molecules based on some version of the heterogeneous / heterologous cassettes DNA writing (HCDW). Each set of molecules will be comprised of differentially labeled Topo blocks (or cassettes). Those blocks themselves will also be a combination of natural base composition plus any specially labeled bases. The aggregate Raman signal from each spot will therefore be unique aggregation of Raman spectra. A signature can be a singular Raman spectra or a combination of sub-signatures of Raman spectra from one or more spots on a surface. Complex signatures can be deconvoluted via several standard or known Al-based algorithms.

[0110] Referring to Figure 8, a diagram 800 depicts a method to make DNA chains (or strands or strings) by topoisomerase-mediated cassette ligation, where at least one DNA strand of the cassettes (or DNA cassette) is labeled with a moiety having a distinctive optical spectra (such asRaman or fluorescent). As long as the label is near the center of the cassette, it should not interfere with ligation of the cassettes that make up the DNA chain or string. By labeling one DNA strand (or DNA cassette), but not the other, it is possible to amplify and sequence the unlabeled strand if desired, in accordance with embodiments of the present disclosure. This method may be used to create optically labelled DNA chains by labelling the topo cassettes (or DNA cassettes or cassettes) before synthesizing the DNA chains. An example is shown where cassettes C1-C16 are shown (on the left side of Fig. 8) having certain cassettes (C1,C4,C6,C11,C14,C16) are shown labeled with stars (802,804,806,808,810,812), respectively, and a three-cassette chain representing 2-bit codes 00,11,10 is shown at a given spot (on the right side of Fig. 8) and the starred cassettes are randomly located in each of the DNA chains or strings. In particular, for cassettes C1-C4, cassette Cl has attached optical label 802 and C4 has attached optical label 804, but cassettes C2, C3 have no optical label attached. So the 2-bit pair 00 will randomly have a cassette that has an optical label attached, as C1-C4 are used to build the 2-bit pair 00 randomly. Similarly, for 2-bit pair 01, where cassette C6 has attached optical label 806, but cassettes C5, C7, C8 have no optical label attached. Similarly, for 2-bit pair 10, where cassette Cll has attached optical label 808, but cassettes C9, CIO, C12 have no optical label attached. Similarly, for 2-bit pair 11, where cassette C14 has attached optical label 810 and C16 has attached optical label 812, but cassettes C13, C15 have no optical label attached. Thus, in some embodiments, the method includes, creating a pre-set palette of cassettes (or top blocks or topo cassettes), labeling the cassette with adducts (or spectral labels) that have unique signatures prior to DNA data chain synthesis. Use the topo to grab on to the labeled cassettes in the charging process and use the labeled cassettes to conduct HCDW synthesis as usual (as discussed in aforementioned patent application).

[0111] Referring to Figure 9, a diagram 900 depicts a method of labeling the DNA strands postsynthesis. wherein the cassettes have synthesized chemical “Click” handles, e.g., such as azide moieties, which can be attached to larger molecules post-synthesis (i.e., after the cassette chains representing a desired digital code are formed) using “click” chemistry, such as strain-promoted azide-alkyne cycloaddition (SPAAC) reactions, in accordance with embodiments of the present disclosure. This method shown in diagram 900 may be used to create optically labelled DNA chains by labelling the topo cassettes after synthesizing the DNA chains. The example shown is similar to Fig. 8, where a three-cassette chain representing 2-bit pairs 00,11,10 is shown at a given spot, however, in this case, the click handles are randomly located in each of the DNA chains orstrings. Then, after the DNA chain is formed, it may be dipped in or washed with a mixture or solution which has an optical label or tag for a single moiety or an optical label or tag formulation, e.g.. formulation 1 (optical mix 1) or formulation 2 (or optical mix 2) of a plurality of moieties combined into a given formulation . In that case, the method couples the molecular randomness of the DNA cassette chain writing with a randomness of optical labelling post-synthesis. It also provides the flexibility of changing which optical labels are attached to a given spot or group of spots. Thus, a portion of a chip having a plurality of spots may be optically labelled with a first moiety or first mix of moieties (e.g., optical mixl) and a different portion of the chip may be optically labelled with a second moiety or second mix (e.g., optical mix 2) of moieties. This provides another level of randomness to the resulting code. Using an optical proportion or mix adds a further level of authentication because the proportions of the different optical signals in the mix must also match the proportion of each optical moiety in the mix. For example, optical mix 1 is shown having the moieties associated with curves 20H, 20G, 20F, 20K in Fig. 1 having proportions shown in the pie chart for optical mix 1, which could be stored in a vial 920. Similarly, optical mix 2 is shown having the moieties associated with curves 20B, 20C, 20E in Fig. 1 having proportions shown in the pie chart for optical mix 2, which could be stored in a vial 922. Thus, in some embodiments, the method includes, creating a pre-set palette of cassettes (or top blocks or topo cassettes), on select or all cassettes representing various combinations of binary, incorporate specific bases with chemical “click handles” or other similar, specific and bio-orthogonal conjugation chemistries with which to attach labels (or adducts) that have specific optical properties. Then, after the DNA chain is synthesized (post-synthesis), expose the synthesized DNA chains with click handles to different labels / moieties or formulations of labels (or adducts or moieties) in specific ratios to label the DNA chains.

[0112] Referring to Figure 10, an item 1000 to be authenticated is shown having authentication tags 1002A-1002E as described herein disposed at specified locations on an item, optionally together with decoy tags 1004A-1004E disposed at specified locations on an item to make detection and counterfeiting more difficult for an unauthorized person, in accordance with embodiments of the present disclosure. In particular, if a person identifies the location of all the tags, not knowing which ones are decoys, a counterfeiter could put authentication tags on all locations even the decoys and it would be detected as a counterfeit item. Thus, in some embodiments, each location shown on the object could be a different Raman spectra with the same(or different) data. A software application run on a computer, laptop, or smartphone and a paired Raman spectra measurement device could prompt the user, which may be randomly each time, to scan specific sections / locations to validate on the object. There may be a different order and number of locations each time. There may also add decoy or fake positions 1004A-1004E, where if the scanner does not know the correct locations, it may poison (or invalidate) the ability to authenticate the object.

[0113] Referring to Figure 11, a top view of a printed circuit (PC) board 1100 is shown with authentication tags having an array of dots (or chips) 1103,1105,1107 discussed herein disposed on a PC board or on components or chips 1102,1105,1106, respectively, on the PC board to authenticate the components and / or the PC board itself, in accordance with embodiments of the present disclosure. Other components such as capacitors 1112 or chip 1108 or components (e.g., diodes or resistors) 1014, or mini-transformer 110 may not have such an array. Thus, in some embodiments, arrays of dots of unique Raman spectra could be printed / applied to positions on a PC board (or on the PC board itself) in one or more locations. The Raman detector would scan the array and validate that a given component or components are authentic. As described herein, the LI (Level 11) signature could be “randomly” created based on a molecular process of writing the underlying molecules and then either: (1) post-syntheses modified using side chemistries with optical labels or otherwise, or (2) reprint the array using homogeneous cassettes to create the array (e.g., a 32 color printer) and then the original DNA overlaid (or laced) into the marked molecules. Also, fake or decoy sports may be interspersed to swabbing (or partial swabbing) could be detected and invalidate the authentication.

[0114] Referring to Figure 12, a diagram 1200 depicts an example of layering the spectral signal having multiple arrays of spectra at different depths, in accordance with embodiments of the present disclosure. In that case, a series of layers 1202-1208 may be disposed on the surface to created multiple Raman patterns that are detectable, e.g., via known SORS (Spatially Offset Raman Spectroscopy) by focusing the light source at different depths, you can generate distinct spectra for each of the layers 1202-1208. This further adds to the complexity to determine the authenticity of the item. Thus, in some embodiments, one can layer different Raman / optical signatures in the same location, which makes replication prohibitive due to the large number of permutations with a large number of tags (or labels) and large number of layers, e.g., 1.5 kb / code x [# of tags] x [# of stacks or layers]. Also, a given Raman spectral layer may be scanned forconcertation of a particular Raman / optical signature. Then, also can binarize the pixels of the image and send to a commercial off-the-shelf (COTS) library, i.e., data is extracted using known data libraries, e.g.. Data Matrix, where up to 30% of error correction may be performed, and it typically reliable and can find and correct errors, and it can be done non-contact.

[0115] Referring now to Figure 13, a drawing 1300 or a process is shown which depicts marking an object to be identified or authenticated using a of a system for multi-tiered identification, in accordance with embodiments of the present disclosure. In particular, digital codes 1302, which may be or contain an NFT linked to the blockchain, written in DNA 1306 (or molecular NFT or DNA NFT or DNA-based NFT) are placed in silica beads 1308 (e.g., as discussed in the aforementioned patent application) and then put in ink or paint (DNA ink) 1310, which may be used in a paint pen (or DNA pen) 1312 for signing memorabilia or collectables and / or used by ink a printing system 1314 (like that shown in Fig. 14B), which may print an image 1316 on an object (not shown), which may be written with the DNA ink 1310 having the silica beads with DNA encapsulated therein (DNA beads), or, in some embodiments, solely the DNA strands or strings without the beads. In addition, the image 1316 may be a spectral image that may be transparent to the naked eye but becomes visible under the illumination by certain wavelengths of incident light (discussed more hereinafter). The spectral image 1316 may contain a spectral QR Code 1318 and / or one or more spectral spots 1320 having a predetermined shape, size or pattern (discussed more hereinafter). Thus, in some embodiments, paint pens with blockchain linked, unhackable (or attack resistant) data polymers in the paints / ink of pens allow for authenticity using this technology for signatures. The spectral inks with blockchain linked unhackable (or attack resistant) data polymers in the ink, QR codes can be viewed with cell phones and inexpensive readers as rapid, in-field checks QR Codes with spectral inks and patterns put on the item signed with special DNA pen provides multiple levels of authenticity.

[0116] Referring to Figures 14A and 14B are diagrams 1400.1450 showing that an item (e.g., shirt) may be signed using a DNA paint pen which has a digital code which may be an NFT (Figure 14A) (or molecular NFT), then a layer of UV / spectral ink may be applied to the item (Figure 14B), the resulting product does not look any different than the originally signed product, in accordance with embodiments of the present disclosure.

[0117] In particular, in Figure 14A, the drawing 1400 depicts application of ink containing DNA (DNA ink) for authenticating the signature itself, wherein the DNA ink may contain silicananobeads containing the DNA and may also contain fluorescent dyes or pigments, e.g. IR and / or UV fluorescent dyes or pigments. In addition, the DNA may contain Raman or other spectral markers or labels attached to the DNA itself, as discussed herein above. As discussed here, also the digital code may contain or may be an NFT, linked to the blockchain.

[0118] Referring to Figure 14B, a drawing 1450 depicts a printing system for a spectral QR code and spectral spots is shown, using ink containing silica nanobeads containing the DNA and fluorescent dyes or pigments, e.g. IR and / or UV fluorescent dyes or pigments, using a continuous inkjet printer 1454, which allows the printed image (QR code and spots) 1454 to be visible when illuminated by a particular wavelength or type of incident light. The marked or tagged article or item is marked so that the marking image (e.g., QR Code and / or spectral spots) are not evident or visible in visible light. In particular, the system 1450 may have as an input the DNA Pen serial number (or if no pen is used, as no signature, such as a statue), the general serial number or other identifying number of the item being tagged, is provided on a line 1451 to an instrument / computer 1452, which receives this input and determines from the QR / Spot data server 1453 specific information about the QR code and spots being written. For example, the QR / Spot data server 1453 may contain data for the QR code being written, such as: QR code image, QR code size (in), QR excitation color, and data for the spot being written, data such as: spot excitation color, spot pattern, spot location (coordinates from a calibration point on the item), spot shape (square, rectangle, oval, circle, triangle, and the like), spot size (dimensions for the selected shape). Such data may be stored in and retrieved from a lookup table of other technique on the QR / Spot data server 1453. The image parameters are provided to the printer 1454 on a line 1455 from the computer 1452. The printer 1454 provides the DNA ink 1456 from an output nozzle having the criteria specified, onto the item, e.g., collectible shirt 1460 with a signature on it that desires authentication. The resultant image is 1456 is printed on a desired area 1458 of the shirt 1460 which is shown on the number two 1464. The shirt may be laid upon a support 1462 which may be a moving conveyor belt or the like to facilitate movement of the item in the process. Image of the number 2 and the DNA ink imagel458 is shown blown up as indicated by 1466. The DNA ink image 1472 is similar to the image 1316 shown in Fig. 13 and includes a QR code 1470 (similar to the QR code 1318 in Fig 13) and a grouping of spectral spots 1474 (similar to the spots 1320 in Fig. 13), where some spots are shown written on top of the QR code 1470, which may be done if desired. Also, the signature 1476 is shown in the blow up, as the printing was performed after theitem (shirt) was signed. The printer 1454 may be a continuous inkjet printer which prints the NFT carrying beads in UV and / or spectral inks over a target area to apply codes the collectable item.

[0119] Referring to Figures 15A, 15B, 15C. and 15D which are diagrams 1500, 1550, 1570, 1590 showing different levels of authentication, having Level 0 or first level authentication (Figure 15A), where the QR code 1504 fluoresces in UV light 1508, which may be detected using a smart phone 1510, Level 1 authentication (Figure 15B), where a spot pattern 1556 is illuminated by a light 1554 that excites the spectral inks in the spot patterns, which may be detected using a smart phone 1510, Level 2 authentication (Figure 15C) where each spot has a unique spectral fingerprint, which may be detected using a known optical instrument 1576 designed for reading the spectral fingerprint, and Level 3 authentication (Figure 15D), where the DNA (or polymer) ink printed on the item is swabbed 1592 to obtain ink from the QR code / spots and / or from the signature and the DNA is provided to a DNA reader to determine authentication, in accordance with embodiments of the present disclosure.

[0120] In particular, referring to Figure 15 A (Level 0 detection) a UV excitation or incident light 1508 is provided by a ultra violet (UV) light source 1506 which illuminates the QR code 1504 printed using ink containing UV dyes and silica nanobeads containing the DNA. A smartphone 1510 may be used to take a picture of the QR code and determine if it matches the code associated with the signed item. The QR code (or the digital code it represents) may be linked to an NFT on the blockchain if desired. The NFT can be specific to the item (e.g., signed shirt, player cards, print run, or the like). Such a Level 0 detection may be done with solely a smartphone and provides a quick digital-only verification of authenticity, and may be used by a dealer or a consumer to quickly verify authenticity. The dealer app 1512 runs on the smartphone 1510 and provides a picture of the QR code 1504 on the smartphone display 1514 and also provides a “good” indicator 1518 and a “bad” indicator 1516, which indicates whether the authentication at this level is verified (“good” lights up green) or not verified (“bad” lights up red). The device 1510 may also communicate with the light source 1506 on a line 1501 to command to turn on and off the excitation light 1508 and may also set the intensity and communicate with the QR / Spot Data Server 1450 to obtain the needed information to determine pass or fail (or good or bad).

[0121] Referring to Figure 15B (Level 1 detection) an excitation or incident light 1554 is provided by a light source 1552 having a wavelength which illuminates the spots 1556 printed using ink containing dyes that are illuminated (or are fluorescent) at the excitation wavelength and silicananobeads containing the DNA. The smartphone 1510 may be used to take a picture of the spots and determine if it matches the proprietary spot color, pattern, size, location, and shape associated with the signed item, which proprietary spot information may be obtained from the QR / spot data server, or other server, discussed herein before. The spot pattern is unique to the individual item, e.g., player card, signed shirt, or other collectable item. Such a Level 1 detection may be done with solely a smartphone and provides a quick digital-only verification of authenticity, and may be used by a dealer or a consumer to quickly verify authenticity. The dealer app 1512 runs on the smartphone 1510 and provides a picture of the spots 1504 on the smartphone display 1514 and also provides a “good” indicator 1518 and a “bad” indicator 1516, which indicates whether the authentication at this level is verified (“good” lights up green) or not verified (“bad” lights up red). Thus, the Level 1 detection shows using light to detect spectral ink printed in random dot or spot patterns, wherein the ink optionally contains silica nanobeads containing the DNA. The app 1512 may also communicate with the light source 1506 on a line 1501 to command to turn on and off the excitation light 1508 and may also set the intensity and communicate with the QR / Spot Data Server 1450 to obtain the needed information to determine pass or fail (or good or bad), device 1510 may also communicate with the light source 1552 on aline 1551 to command to turn on and off the excitation light 1554 and may also set the intensity and wavelength and may communicate with the QR / Spot Data Server 1450 on a line 1517 to obtain the needed information to determine pass or fail (or good or bad).

[0122] Referring to Figure 15C (Level 2 detection) an excitation or incident light 1574 is provided by a light source 1572 having a wavelength which illuminates the spectra of the spots 1556 printed using ink containing moieties (such as those discussed herein above) that are illuminated at the excitation wavelength. The smartphone 1510 may be used to take a picture of the spots and determine if it matches the proprietary spot color, pattern, size, location, and shape associated with the signed item, which proprietary spot information may be obtained from the QR / spot data server, or other server, discussed herein before. The device 1510 may also communicate with the instrument on a line 1577 to command to turn on and off the excitation light 1574 and may also set the intensity and communicate with the QR / Spot Data Server 1450 to obtain the needed information to determine pass or fail (or good or bad).

[0123] / / / / The spot pattern is unique to the individual item, e.g., player card, signed shirt, or other collectable item. Such a Level 1 detection may be done with solely a smartphone and provides aquick digital-only verification of authenticity, and may be used by a dealer or a consumer to quickly verify authenticity. The dealer app 1512 runs on the smartphone 1510 and provides a picture of the spots 1504 on the smartphone display 1514 and also provides a “good” indicator 1518 and a “bad” indicator 1516, which indicates whether the authentication at this level is verified (“good” lights up green) or not verified (“bad” lights up red). Thus, the Level 1 detection shows using light to detect spectral ink printed in random dot or spot patterns, wherein the ink optionally contains silica nanobeads containing the DNA.

[0124] Figure 15C shows using a different light sources to detect a spectral fingerprint of inks printed in random dot patterns, wherein the ink optionally contains silica nanobeads containing the DNA.

[0125] Figure 15D shows retrieving and analyzing the DNA from the object.

[0126] Refering to Figure 16 are diagrams showing financial incentives for the collectible dealer and the content creator, where a multi-layer authentication stack is provided such that the shows with increasing item / product value, the value / cost to authenticate also increases. (Figure 16A), Also, Level 0, which is a basic level, is free to the user and provides data about the item but no monetary compensation / incentive, and for Level 1, where there is monetary compensation / incentive for both the dealer (or broker) and the content creator when an authentication is performed (using the App), and for Level 2, where there is monetary compensation / incentive for both the dealer and the content creator when authentication is performed (using the App), and for Level 3, where there is monetary compensation / incentive for both the dealer and the content creator when authentication is performed (using the App), in accordance with embodiments of the present disclosure.

[0127] Figure 17 depicts an example of implementation of a multi-tiered system, specifically a process diagram having five steps including witnessed signing, session documentation (NFT of pens is recorded), hologram application (QR Code and spot pattern), certificate creation matching the QR code with NFT molecules, and customer packaging, in accordance with embodiments of the present disclosure, having the steps 1702,1704,1708,1710,1712.

[0128] Figure 18 depicts application of a multi-tiered authentication system to framed memorabilia, specifically an approach for using the present disclosure to authenticate framed memorabilia by using peelable hologram / QR code stickers on the back of the item, and showing that they can be removed and physically sent to an authentication service which can perform Level3 (DNA) testing on the stickers, and confirmation of authentication may be provided by a physical certificate and / or by an electric communication to a computer based user device, such as a smart phone, and the like, in accordance with embodiments of the present disclosure.

[0129] Figure 19 shows a top level block diagram of a system which allows a creator (e.g., athlete) and / or dealer to get compensated for their work / signature, by allowing a user to buy authorization credits from an authentication clearinghouse (or the like) which credits are used to authenticate items, using an app (e.g., Dealer App) running on a user device, such as a smart phone or other computer based device, to perform Level 1 or Level 2 authentication of the item, and provide the results back to the user, the app may also provide the results to the clearinghouse to report results back to the creator and / or dealer, and the clearinghouse may also provide the creator and / or dealer with compensation for each authentication, whether valid or invalid (counterfeit), and the clearinghouse may store the data from the authentications on a data server, and the authentication provider (who mints the NFT and prints the spectral codes on the items) may also receive compensation for an authentication, in accordance with embodiments of the present disclosure.

[0130] Figures 20A,20B,20C are flow diagrams for the print system, authentication app, and clearinghouse, discussed herein.

[0131] Fig. 21 shows 3 tables 2100, 2102,2104 that can be used to hold data for multilevel printing, DNA synthesis, and authentication that may be used by the QR / Spot Data Server.EXAMPLE 1: OB JECT AUTHENTICATION USING FOUNTAIN PEN INK (General method)

[0132] To exemplify one embodiment of the present disclosure, six commercially-available fountain pen inks of various colors are acquired. Each ink is labeled Ink #1 through Ink #6, and each ink is serially diluted 10-fold four times. Separately, a 32-byte NFT, along with accompanying meta-data and error correcting features, is encoded into DNA strands synthesized using topoisomerase-mediated heterologous DNA cassette data writing, with said DNA strands comprising 51 nackets each. The DNA is added to each of the ink samples (i.e., Ink #1 through Ink #6, across four dilutions each) at a concentration of 0.3 ng / pL. As an initial evaluation, the DNA is added to the ink samples, mixed thoroughly, and immediately aliquoted for DNA analysis. The DNA is subsequently isolated and amplified to verify that introduction into the ink is not deleterious in the process of object (i.e., ink) authentication.

[0133] Next, Ink #4 and Ink #5 are selected for further evaluation, since both inks are black inks, though color does not seem to impact the DNA based on the above experiment. NFT-encoding DNA is incorporated into the fountain pen inks as described above, the inks are used in fountain pens to write on commercially-available printer paper and are subsequently analyzed after 7 days to evaluate the stability of the DNA in both the liquid ink and when written / dried on the paper. The DNA is subsequently isolated and amplified. When sampling directly from the ink solution, an aliquot of the ink solution is diluted and then directly amplified via PCR. When sampling from the ink dried on paper, a wetted cotton swab is lightly brushed over the dried ink, dipped in a small volume of water, and then amplified via PCR. Alternatively, the ink dried on paper may be sampled by pipetting a small volume of water (e.g., IOUL) onto the dried ink, solubilizing part of the dried ink and retrieving it via the pipette, and then amplifying via PCR. In these examples the resulting liquid is typically diluted substantially, e.g., >1 / 1000, before PCR.

[0134] It is observed via gel electrophoresis of the amplified DNA that the DNA in Ink #4 remains stable after 7 days. Surprisingly, the DNA amplified from the liquid ink of Ink #5 yields a markedly lower DNA concentration compared to Ink #4. In contrast to the liquid ink samples, the DNA in both Ink #4 and Ink #5 used to write on paper on day 0 is observed to be stable at the day 7 timepoint. Notably, the DNA in Ink #4 is further observed to have similar recovery of DNA from both the liquid ink sample and from the sample written on paper. Due to the observed stability, Ink #4 is used for subsequent evaluation.

[0135] The Ink #4 samples are next used in deep sequencing analysis of the NFT-encoding DNA. More specifically, the NFT is encoded into the DNA using 51 nackets, and the heterologous DNA cassette writing method used in the synthesis of the NFT-encoding DNA strands provides a collection of approximately 109unique DNA sequences. PCR analysis of aliquots taken directly from this collection of synthesized DNA sequences yields identification of approximately 106unique DNA sequences (i.e., 1,623.092 unique DNA sequences). This collection of NFT-encoding DNA is incorporated into Ink #4, as above, used in the ink when writing on paper, as above, and subsequently analyzed from the dried ink samples on said paper. Two dried ink samples written on paper are analyzed using PCR and deep sequencing, which are labeled Ink Sample #1 and Ink Sample #2. During analysis, it is observed that Ink Sample #1 has 5,160 unique DNA sequences (1,311 of which are shared with the original DNA sequences identified from the collection previously analyzed) and Ink Sample #2 has 6,218 unique DNA sequences (2,615 of which areshared with the original DNA sequences identified from the collection previously analyzed). Additionally, Ink Sample #1 and Ink Sample #2 share 442 unique DNA sequences amongst each other. Thus, this shows that the heterologous DNA cassette data writing produces a significant amount of heterogeneity among the DNA sequences, though each DNA strand is ultimately synonymous with all other DNA strands from the same original collection of DNA strands.

[0136] The protocols described above are repeated to further evaluate the stability of the NFT-encoding DNA in ink written on paper over time. More specifically, the DNA in ink written on paper is extracted and analyzed at 2 weeks and 6 weeks post-writing on paper. Notably, the stability at both 2 and 6 weeks are remarkably similar, with no significant difference between time points. Additionally, during deep sequencing of the recovered DNA strands, full length nackets of each of the 51 nacket positions were readily identifiable, indicating the absence of any significant breakage in the DNA strands. Moreover, the sequenced nackets yielded consensus sequences for each nacket position, wherein the consensus sequences are useful in the decoding of the DNA sequence back into the original NFT code. By decoding as described herein, the original NFT code is reliably recoverable and the object (i.e., ink) is amenable to authentication.

[0137] The protocols described above are further repeated to evaluate the stability of the NFT-encoding DNA in ink written on paper at 8 weeks. In this example, 3 replicates (labeled Replicate #1 through Replicate #3) of writing samples are evaluated at 8 weeks post-writing on paper. When comparing nacket analysis, the DNA samples recovered from each of the 3 replicates display remarkable similarity to one another, and are notably similar to the nacket analysis at weeks 2 and 6. After deep sequencing of the 3 replicates of writing samples after 8 weeks, each sample is compared to the other two. In the first analysis, Replicate #1 is observed to have 8.033 unique nackets, while Replicate #2 is observed to have 9,965 unique nackets. Between Replicate #1 and Replicate #2, 36 nackets are shared. When comparing the number of recovered nackets for each nacket ID, the comparison between Replicate #1 and Replicate #2 yields a linear trend line with R2= 0.954. In the next analysis, Replicate #2 is observed to have 9,690 unique nackets, while Replicate #3 is observed to have 10,160 unique nackets. Between Replicate #2 and Replicate #3, 311 nackets are shared. When comparing the number of recovered nackets for each nacket ID, the comparison between Replicate #2 and Replicate #3 yields a linear trend line with R2= 0.969. In the third analysis, Replicate #1 is observed to have 8,045 unique nackets. while Replicate #3 is observed to have 10,447 unique nackets. Between Replicate #1 and Replicate #3, 24 nackets areshared. When comparing the number of recovered nackets for each nacket ID, the comparison between Replicate #1 and Replicate #3 yields a linear trend line with R2= 0.954. These data demonstrate, inter alia, that between different samples of nacket populations, the majority of synonymous nacket sequences are unique, though a small degree of overlap is possible.

[0138] Following the initial evaluation of DNA stability, heat is used to simulate accelerated aging of DNA sample. In these experiments, a quarter-inch punch of paper with 1 pL of ink is placed into a sealed microcentrifuge tube. The 1 pL of ink is estimated to comprise approximately 4 x 108molecules of NFT-encoding DNA and 1 x 108molecules of ddPCR tracer. The sealed microcentrifuge tube containing the ink-marked paper punch is placed in a 75°C oven for various lengths of time before transfer to a 4°C refrigerator for storage before analysis. It is estimated that storing the ink-marked paper at 75°C for 0, 1. 2, 3, 4, 5, 6. 7, 8, and 9 days will mimic the roomtemperature equivalent of approximately 0, 2.3, 4.6, 6.8, 9.1, 11.4, 13.7, 16.1, 18.3, and 20.5 years, respectively. As a control, an ink-marked paper is stored at -20°C throughout the experiment. After 9 days, wherein each day a sample is moved from the 75°C oven to the 4°C refrigerator, each sample is analyzed using digital PCR. In this case, samples from days 0 and 1 look substantially the same in concentration, while days 2 through 6 each display a steady reduction in DNA concentration after the same number of PCR amplification cycles, and days 7 through 9 display a low concentration of DNA. This likely indicates that the DNA is degrading over time under the accelerated aging conditions at 75 °C, though the extent of degradation is unclear. Next, the aged samples are amplified via PCR at varying cycle numbers to yield sufficient material for sequencing. In this case, the ddPCR tracer added to the NFT-encoding DNA in the ink marking the paper punch is used to amplify a 700 bp length of DNA. While quantifying the amplified DNA, it is observed that approximately 6.5% of the DNA is recovered in the day 0 sample. Next, approximately 3% of the DNA is recovered in the day 1 (approx. 2.3 year equivalence) sample, approximately 1% of the DNA is recovered in the day 2 (approx. 4.6 year equivalence) sample, and progressively less DNA is recovered in each subsequently aged sample. The results demonstrate a logarithmic decline in DNA recovery.

[0139] Continuing the PCR analysis of the DNA after accelerated aging, amplicons on each end of the 700 bp length of DNA targeted by the ddPCR tracer allow for analysis of double- stranded DNA breakage in the aged samples. Surprisingly, the DNA stays largely resistant to breakage throughout the evaluated time points, with less than 10% breakage observed for days 0, 1, and 2(approx. 0, 2.3, and 4.6 year equivalence), while days 3, 4, and 5 (approx. 6.8, 9.1, and 11.4 year equivalence) display 10-25% breakage. However, days 6 tiirough 9 display more notable DNA breakage, between 40-65% breakage.

[0140] Lastly, by directly comparing the sequenced DNA samples after undergoing accelerated aging, it is observed that the error rate of the DNA only slightly increases over time, while the sequence efficiency (i.e.. proportion of DNA that are “correct” reads or consensus sequences) decreases over time. This is emphasized by the sequence length distribution, wherein the sequence length shifts over time from a single prominent length of DNA to a series of shorter DNA strands. Thus, these results indicate that the DNA does sustain damage over time, but the error rate in the DNA sequence remains relatively stable and the DNA is still capable of decoding and recovery of consensus sequences, even after an equivalence of 20 years accelerated aging.EXAMPLE 2: ENCAPSULATION AND EXTRACTION OF DNA FROM SILICA BEADS (General method)

[0141] It is known that DNA can be encapsulated in nanometer silica beads, which can be fused into various materials that are used to print or cast objects in any shape and subsequently recovered. See, e.g., Koch J, et al., “A DNA-of-things storage architecture to create materials with embedded memory.'” Nat. Biotechnol. (2020)38(l):39-43; e.g., U.S. Patent No. 9,850,531, “Molecular code systems”,' e.g., Bossert, et al., “A hydrofluoric acid-free method to dissolve and quantify silica nanoparticles in aqueous and solid matrices” Sci. Rep. (2019)9:7938, the contents of each of which are incorporated herein by reference.

[0142] For example, a machine-readable code is converted into a collection of DNA strands using heterologous DNA cassette data writing, as described in Example 1. After synthesis, but before incorporating the DNA into a material or object, the DNA is encapsulated into silica beads, e.g., silica microspheres, together with fluorescent markers

[0143] Silica seed particles are mixed with a solution of the free DNA encoding the NFT together with the labeled DNA of Example 2. The DNA strands coat the seed particles. Optionally, the silica seed particles may be modified with amine-bearing functional groups to allow for enhanced interaction with DNA polymers. The DNA-coated seed particles are subsequently mixed with a solution of tetra ethoxy silane (TEOS) and base in ethanol to grow a SiOz layer around the DNA, yielding the silica beads with DNA encapsulated therein. More specifically, 5 pL of free DNA (at28 ng / pL) is mixed with 10 pL of silica seed particles (at 60 mg / mL) in 500 pL TE buffer. The resulting mixture is centrifuged (at 21,500 g) for 1 minute, the supernatant is removed, and the pellet is dispersed in 1 mL ethanol. To this suspension, 2 pL APTES is added with 20 pL TEOS and 20 pLTE buffer. The solution is allowed to react overnight at room temperature while shaking, after which the solution is again centrifuged and the precipitate is washed with ethanol and TE buffer before re-suspension.

[0144] Following encapsulation, a first extraction protocol is used. In this first extraction protocol, the DNA-encapsulating silica beads are dissolved in buffered oxide etch solution, wherein the oxide etch solution comprises an aqueous mixture of ammonium fluoride and hydrofluoric acid, which may be done in 0-50°C, though readily proceeds at room temperature. The beads readily dissolve within several seconds in the oxide etch solution, yielding the original free DNA within a high-salt solution (e.g., F‘, NHT, and SiFe2-), though it is thought that the relatively high pKa of hydrofluoric acid prevents damage to the DNA. More specifically, 5 pL of silica beads encapsulating DNA is added to 10 pL of a buffer oxide etch solution (0.34g NH4F and 10g HF (at 1%) in TE buffer), and shaken for 1 minute. The mixture transitions from a turbid to clear solution, and the resulting solution is dialyzed against 10 mL of water for 30 minutes. Following dialysis, the free DNA is analyzed via PCR, as described in Example 1.

[0145] A second extraction protocol is also useful as an alternative, particularly since the use of hydrofluoric acid is often undesirable. In this alternative extraction protocol, the etch solution used for dissolving the silica beads is composed of aqueous potassium hydroxide. In this case, 10 pg / mL of silica beads is mixed with IM KOH in an aqueous solution with a pH of 12, wherein the silica beads dissolve overnight at room temperature. Alternatively, 10 pg / mL of silica beads is mixed with 0.1M KOH in an aqueous solution with a pH of 12, wherein the silica beads dissolve within 15 minutes under 1500 W of microwave radiation. Following silica bead dissolution and extraction of the encapsulated DNA, the free DNA is dialyzed and analyzed as described above.EXAMPLE 3: LABELING OF DNA USING VISIBLE LIGHT LABEL

[0146] DNA strands synthesized using topoisomerase-mediated heterologous DNA cassette data writing, with said DNA strands comprising 51 nackets each, as described in the preceding example. An aliquot of this DNA is labeled with ethidium monoazide (EMA), which binds irreversibly to DNA in the presence of visible light. EMA has excitation / emission maxima of ~504 / 600 nm andcan be detected using a standard argon laser operating at 488 nm. The EMA-labeled aliquot is added to the ink comprising the unlabeled DNA of Example 1 or incorporated along with the unlabeled DNA into the silica beads of Example 2, to provide a detectible marker.EXAMPLE 4: LABELING OF DNA USING IR LABEL

[0147] IRDye 800RS NHS ester (Li-Cor Biosciences):) O(IR NHS Dye Formula 1) is used to label an aliquot of DNA. The N-hydroxysuccinimide (NHS) moiety binds to amines in the DNA. This dye absorbs at about 767nm and emits at 786nm. The labeled aliquot of DNA is added to the ink comprising the unlabeled DNA in Example 1 or incorporated along with the unlabeled DNA into the silica beads of Example 2, to provide a detectible label.EXAMPLE 5 : INK CONTAINING IR DYE

[0148] Indocyanine green (Sodium 4-(2-((lL,3L’,5L’,7Z)-7-(l,l-Dimethyl-3-(4-sulfonatobutyl)-1 ,3 -dihydro-2 / 7-benzo[e]indol-2-ylidene)hepta- 1 ,3 ,5-trien- 1 -yl)- 1 , 1 -dimethyl- 17 / -benzo[e]i ndol -3 -ium-3-yl)butane-l -sulfonate) is dissolved in the ink of Example 1 (10 mg / ml). When the ink is exposed to near IR light, it emits a signal detectible by an IR camera (excitation / emission 787 nm and 815 nm resp.), indicating that the ink is the ink comprising the DNA authentication tags.EXAMPLE 6: SURFACE-LABELED SILICA BEADS

[0149] The hydroxyl groups on the surface of the silica beads of Example 2 are activated by incubating the beads for one hour in a solution of 50% methanol, 47.5% 3-aminopropyltriethoxysilane (APTES), and 2.5% distilled water. The APTES functionalizes the silica beads with amino groups. The beads are then rinsed with methanol, and placed in solutionwith TRDye 800RS NHS ester (Li-Cor Biosciences)(see Example 4), which binds to the amino groups on the APTES, and rinsed again. The IRDye 800RS absorbs at about 767nm and emits at 786nm, permitting detection of the silica beads.

[0150] Although the above example(s) have been described using exemplary procedures, materials, objects, concentrations, or processes for implementing the present disclosure, it should be understood by those skilled in the art that alternative procedures, materials, objects, concentrations, or processes or other combinations and sequences of the procedures, materials, objects, concentrations, and processes described herein may be used or performed that achieve the same function(s) and result(s) described herein and which are included within the scope of the present disclosure. For example, beyond the examples and embodiments described above, additional exemplary embodiments have been developed with success, including applications of the present invention in latex paint (both free and encapsulated DNA), acrylic paint (both free and encapsulated DNA), industrial inkjet printer ink (free DNA), perfume (free DNA), oil paint (encapsulated DNA), permanent marker ink (free and encapsulated DNA), stamp-pad ink (free DNA), watercolor paint (free DNA), and 3D printing plastic (encapsulated DNA).

Claims

CLAIMSWhat is claimed is:

1. A population of deoxyribonucleic acid (DNA 1) molecules encoding data useful in the authentication of objects and for protection against counterfeiting, comprising nucleic acid data packets (“nackets”), wherein each nacket contains a plurality of synonymous DNA molecules having different sequences but encoding the same data, wherein the plurality of DNA polymers comprise, or are associated with, four or more authentication levels, comprisingi.) a Rapid Identification Level, wherein the DNA comprises or is associated with one or more labels providing a spectral fingerprint;ii.) a Nacket Data Level, wherein the DNA sequence encodes user-defined data, e.g., in a binary, ternary or other machine -readable code;iii.) a Production Lot Fingerprint Level, wherein the DNA has a specific percent abundance of different bases, sequence variants, and / or cassette variants; and iv.) an Object Fingerprint Level, comprising a unique population of different synonymous sequences,e.g., a population of deoxyribonucleic acid (DNA) sequences or polymers selected from any of DNA 1, et seq., supra.

2. A method of object authentication (e.g., according to any of Method 2, et seq., supra), comprising:i.) synthesizing DNA polymers comprising nackets encoding data;ii.) incorporating said nackets into or onto an object;iii.) optionally, extracting said nackets from the object; andiv.) analyzing the nackets;v.) optionally, comparing the analyzed nackets to a database of DNA sequences and / or spectroscopic signatures;vi.) optionally, confirming object authenticity;wherein the nackets comprise or are associated with detectable markers (e.g. spectroscopically detectable markers) for rapid identification;e.g., a method according to any of Method 2, et seq., supra.

3. A method for providing high-level authentication using optical signals for an attack-resistant digital code using DNA that uniquely defines a physical object to be authenticated, without requiring DNA sequencing, the digital code having at least four predetermined mixtures of a predetermined number of unique DNA cassette strings, the unique DNA cassette strings of each mixture being different from the DNA cassette strings in the other mixtures, the method comprising:attaching unique optically responsive molecules or particles to at least one of the cassette strings used to make up each mixture, as unique optically labelled cassettes; depositing a droplet of the mixture associated with a given n-bit binary code to be written onto a substrate to add a DNA cassette to an encoded DNA string being written, the droplet comprising the predetermined mixture of the unique cassettes associated with the n-bit binary code and including the at least one unique optically labelled cassette; and repeating the depositing until the desired digital code is written onto the encoded DNA string, andwherein the optically responsive molecules or particles emit an output light when illuminated by an incident light, the output light being indicative of a spectral fingerprint that uniquely defines at least one of: the attack-resistant digital code and the physical object to be authenticated,e.g., according to any of Method 3, et seq., supra.

4. A method for providing high-level authentication using optical signals for an attack-resistant digital code using DNA that uniquely defines a physical object to be authenticated, without requiring DNA sequencing, the digital code having at least four predetermined mixtures of a predetermined number of unique DNA cassette strings, the unique DNA cassette strings of each mixture being different from the DNA cassette strings in the other mixtures, the method comprising:assembling the digital code using the DNA cassette strings by depositing individual droplets of the mixture associated with a given n-bit binary code to be written onto a substrate to add a DNA cassette string to an encoded DNA string being written until thedesired digital code is written onto the encoded DNA string, each of the droplets comprising a predetermined mixture of the unique cassettes associated with the n-bit binary code; andattaching unique optically responsive molecules or particles to at least one of the cassette strings on the encoded DNA string, as optically labelled string;wherein the optically responsive molecules or particles emit an output light when illuminated by an incident light, the output light being indicative of a spectral fingerprint that uniquely defines at least one of: the attack-resistant digital code and the physical object to be authenticated,e.g., according to any of Method 4, et seq., supra.

5. A multi-tiered system for object authentication and provenance comprisinga first ink comprising (i) silica nanoparticles comprising DNA molecules according to claim 1, and (ii) one or more UV and / or IR dyes or pigments, which fluoresce in the visible range when illuminated with UV or IR light, wherein the ink is disposed on an object to be identified or authenticated in a quick response (QR) code pattern;and optionally further comprising a second ink containing one or more UV and / or IR dyes or pigments, which fluoresce in the visible range when illuminated with UV or IR light, wherein the second ink is disposed on an object to be identified or authenticated in a random spot pattern and wherein the second ink may be the same as or different from the first ink, and wherein optionally the second ink may contain dyes or pigments that provide a detectable spectral profile for the random spots.

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