Bacterial patterns invisible to the eye reveal hidden information only with correct biochemical triggers, creating anti-counterfeiting codes that are harder to copy or forge.
(Nanowerk Spotlight) A petri dish sits under ultraviolet light, revealing a glowing QR code formed entirely from living bacterial colonies. Scan it with a smartphone, and it directs to a website. Transfer the same bacterial film to a different growth medium containing a specific chemical trigger, wait a few hours, and the code transforms. The original information becomes unreadable while a new message emerges. This is not a conceptual art project but a working information storage system in which the medium is alive, responsive, and self-modifying.
Bacteria are not obvious candidates for data storage. They divide, migrate, die, and respond unpredictably to their environment. These behaviors seem antithetical to the stability information systems require. Yet these same properties, properly harnessed, could make bacterial patterns harder to counterfeit without knowledge of the correct strains and chemical triggers. A living code that changes over time or responds only to specific biochemical cues offers security features that static media cannot match.
The concept of bacterial cryptography has attracted interest, but practical implementation has remained elusive. Previous attempts relied heavily on genetic engineering to make bacteria produce visible signals, limiting applicability to specially modified strains. Techniques for arranging bacteria into patterns, such as microcontact printing and inkjet methods, have suffered from low resolution and cumbersome protocols. Most existing bacterial codes have been confined to agar-based substrates and limited to single-mode encoding, restricting both storage capacity and security.
The researchers developed a photodynamic patterning strategy using specially designed nanoparticles to selectively kill bacteria exposed to light, leaving living bacteria in unexposed regions to form high-resolution patterns. Specific substrates can then trigger metabolic reactions unique to each bacterial species that reveal these patterns, enabling information to remain hidden until the correct biochemical trigger is applied.
Schematic illustration of the aggregation-enhanced photosensitization nanosystem for high-resolution photodynamic patterning and subsequent biochemical reactions within bacteria. A) Construction of the aggregation-enhanced photosensitization nanosystem (AIE/NSFA@PVA/PAH). B) Mechanism of photodynamic performance enhancement via natural saturated fatty acids (NSFA) induced aggregation. C) Principle of the photodynamic patterning strategy and the subsequent biochemical responses within spatially confined bacterial architectures. (Image: Reproduced with permission from Wiley-VCH Verlag) (click on image to enlarge)
The core innovation lies in a photodynamic nanosystem built from four components. The first is a light-activated molecule called MeO-TSP that generates cell-damaging reactive oxygen species. The second consists of natural saturated fatty acids, specifically a mixture of lauric acid and stearic acid, that enhance this light-activated killing. The final two components are polymers called polyvinyl alcohol and poly(allylamine hydrochloride), which form a protective, positively charged shell around the nanoparticle.
The fatty acid matrix promotes tight clustering of the light-activated molecules, dramatically enhancing their ability to generate reactive oxygen species when exposed to white light. Computational modeling revealed that this clustering causes the molecules to adopt a twisted shape, reducing the singlet-triplet energy gap from 0.569 eV to just 0.007 eV. This shift facilitates intersystem crossing, a quantum mechanical process in which electrons transition between different spin states, essential for efficient reactive oxygen species production.
The positively charged polymer shell serves a crucial function. Bacteria carry negative surface charges, and the positive coating enables strong electrostatic attraction between nanoparticles and bacterial cells. This close contact shortens the distance reactive oxygen species must travel to damage bacterial membranes, significantly boosting antibacterial efficacy.
In laboratory tests, the nanosystem achieved 99.999% killing efficiency against methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive bacterium with a single-layer cell wall. Against wild-type Escherichia coli, a Gram-negative species with a protective double-membrane structure that typically resists photodynamic treatment, the system achieved 99.9% killing.
With this potent antibacterial capability established, the researchers turned to patterning. They spread bacteria coated with nanoparticles uniformly on mixed cellulose ester membranes, then exposed them to light through a photomask. In illuminated regions, reactive oxygen species killed the bacteria. In masked regions, bacteria survived.
The result was a living bacterial pattern faithfully reproducing the photomask design with a spatial resolution of 15.99 μm, surpassing most previously reported bacterial patterning techniques.
Importantly, the mixed cellulose ester membranes allow bacterial biofilms to transfer between different culture media. This represents a significant advantage over conventional agar-based systems, which cannot be moved without destroying the pattern.
The system derives its real power from metabolic differences between bacterial species. Different bacteria possess distinct enzymes and biochemical pathways. By introducing chromogenic substrates, compounds that change color when processed by specific enzymes, the researchers selectively revealed patterns formed by different strains.
MRSA reduces tellurite to produce black colonies, while E. coli cleaves a compound called X-Gal through an enzyme called β-galactosidase to produce cyan-colored precipitates. Live bacteria stain blue with Hoechst 33342, which binds to DNA, while dead bacteria with compromised membranes take up propidium iodide and fluoresce red.
These metabolic responses enabled construction of increasingly sophisticated encoding systems. The researchers demonstrated one-dimensional Morse codes using star and triangle patterns to represent dots and dashes. By incorporating both MRSA and E. coli, they created codes that displayed false information when exposed to one substrate but revealed true information with a different reagent.
They also fabricated two-dimensional QR codes that remained invisible under normal light but became readable after metabolic activation on specific media. The team even developed puzzle-based QR codes requiring physical assembly of multiple fragments alongside biochemical activation for successful decryption.
The complexity extends to three and four dimensions. Color serves as a third encoding dimension: different bacteria-substrate combinations yield black, red, bluish violet, or cyan signals. Smartphone applications can scan these multicolor arrays to retrieve encoded information such as website addresses.
Time functions as a fourth dimension through gradual color changes in certain bacteria. As specific colonies continue metabolizing substrates, their colors shift over hours, eventually obscuring the original pattern and rendering the code unreadable. Information encoded in such a time-gated bacterial code becomes invalid after a predetermined window, enabling automatic data protection.
A nested architecture embeds QR codes within three-dimensional color arrays, requiring sequential decryption steps. Scanning the outer code grants access credentials needed to unlock information in the inner code.
Fluorescent encoding adds another security layer. Patterns invisible under normal light become visible only under ultraviolet illumination, with different bacterial species and staining methods producing blue, red, or green fluorescence. The combination of spatial structure, bacterial species identity, biochemical response, fluorescence switching, and temporal evolution creates a framework where distinct decryption pathways coexist within the same physical medium.
This work represents the first demonstration of photodynamic patterning to engineer biochemically responsive bacterial encoding. The strains used, MRSA and wild-type E. coli, served as proof-of-concept models and could be replaced with non-pathogenic laboratory strains for practical applications. By integrating precise spatial control with the metabolic diversity of living organisms, the platform offers a fundamentally different approach to secure information storage and anti-counterfeiting, one where the living nature of the medium itself becomes a security feature.
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