Messenger Materials Moving Forward: The Role of Functional Polymer Architectures as Enablers for Dynamic Nano‐to‐Macro Messaging

Identifying changes in the nanoscopic domain is a key challenge in the physicochemical sciences, where great interest is on sensing complex processes that involve cellular biochemical reactions, chemical heterogeneities, contact forces, and other interfacial phenomena. This has stimulated the development of diverse materials that allow subtle nanoscopic environments to be "seen". The challenge in the nano‐domain has always been the ability to sense changes on the minute scale and rapidly transduce the information out for macroscopical observation. Ideally, materials should inform when processes are occurring. Recently, new systems that leverage established concepts with fluorescence‐ and plasmonic‐based sensing have been devised, which has reinvigorated the domain, where functional polymers coupled in specific architectures to transducing motifs allow for a new basis of messenger materials to be realized. The key aspect in this regard is that the polymers allow for sensing to be achieved only when they are carefully coupled to the amplification system. In this perspective, the role of specific functional polymer architectures for the realization of nano‐to‐macro sensing of subtle nano‐messengers is discussed and where the exciting field of messenger materials is seen moving forward is pointed out.


Introduction
The concept of "smart" materials can be ambiguous, as this can include an extraordinarily wide range of substances with different functionalities, properties, and applications, making the concept somewhat difficult at times to define. [1] Broadly speaking, smart materials are materials that can undergo changes in one or more material properties due to some stimulus, [2] which have recently been leveraged in applications spanning drug delivery, [3] diagnostic sensors, [4] energy harvesting, [5] and microrobots, [6] DOI: 10.1002/adfm.202214915 amongst many others. Importantly, the ability of a material to change its properties due to a stimulus can also be used as a basis for transducing information on the local molecular environment if the materials are linked to amplification motifs (i.e., "messenger materials"). The more defined concept of messenger materials involves materials that can analyze local nanoscopic environments and process this information into a signal that can be transduced (messaged), or collected, macroscopically, where the amplification motif may be fluorescence-or plasmonic-based. A material that can say "hey, look at me, something is happening here", where the nano-/micro-scale nature of the reporting entity enables highly locally resolved signal collection and event communication. The utility of such communication can be seen in terms of reporting mechanical cues (damage, stress, strain, etc.) as well as changes in the physicochemical environments (pH, temperature, pressures, etc.), which is of interest across the biological and physical sciences. Particularly for spatially localizing these cues in high resolution. Research in this area of sensing such cues is by no means new, [7] but there are several recent strategies that have reinvigorated this domain. Of particular point are those that leverage established sensing concepts into new systems, targeted toward understanding subtle phenomena. The basis of recent work is the design of specific functional polymer architectures as the key component of the sensing system, where the polymer acts as the "messenger" by modulating the properties of the amplification motifs that send the "message".
This concept of nano-to-macro communication first requires materials that are capable of producing a signal that is well-above the background noise from local fluctuations in environmental conditions, and rotational-and diffusion-based processes. This is no easy challenge, where the actual conditions to be sensed can be very subtle, such as nano-buffering effects on pH, [8] minute temperature fluctuations, [9] solutes, [10] and applied pressure. [11] Sensing these stimuli are implicated in a range of different physiological and engineering areas, which requires motifs that exhibit changes in response to the stimuli, which is where functional polymers show outstanding potential. At this point in the polymer science community, polymers have been designed, Figure 1. Schematic describing amplification sources based on fluorescence (changing intensity, I, for FRET and quenching) and plasmonic coupling (modulation of UV/Visible/NIR extinction), where functional polymers are used to "hold" the amplification sources in place. I, intensity; Δ , change in wavelength; NIR, near infrared.
synthesized, and reported that are capable of responding to most imaginable real-world stimuli. [12] This includes general stimuli (temperature, pH, ionic strength, solvent, etc.) as well as extraordinarily specific stimuli (solutes, biomolecules, specific wavelengths of light, etc.), where the polymers typically change their conformation. These can be designed for singular or orthogonal responses to multiple stimuli. [13] This positions the field in a powerful place to leverage the responsiveness of functional polymers toward nano-to-macro messaging. That is, if the responsiveness can be intrinsically linked to a transduction source, which is a key challenge in moving the "messenger materials" field forward.
One of the most promising amplification sources are those based on fluorescence and/or plasmonic coupling (Figure 1), which can both produce outputs that are dependent on proximity of the sources, either to themselves or to other materials. For fluorescence, this can be given in terms of resonance coupling (e.g., Förster resonance energy transfer (FRET)) and/or quenching effects between organic fluorophores. For plasmonic effects, metallic nanoparticles can support dimension-and shape-controlled plasmon resonance modes, which can be excited with electromagnetic radiation of a suitable (typically vis/NIR) frequency. This excitation results in a localized near-field in proximity to the nanoparticles, making them highly sensitive nanoscale probes of their environment. [14] The role of the polymer in this context with amplification sources, is to simply act as arms that modulate the proximity based on stimuli. For this to occur efficiently, a preorganization of individual macromolecules is often at times necessary. These macromolecules can be carefully engineered into surface polymer brush films, hydrogels, and nanomaterial systems (Figure 2), which can be leveraged toward probing a variety of subtle cues. Herein, we discuss selected examples of recent progress in this space and offer our perspective on where we see significant potential moving forward in probing new sensing processes with these materials. We purposefully maintain focus on dynamic and reversible messenger materials, which are a class of smart materials, with high resolution messaging, and not on larger scale soft robotic systems or irreversible materials (e.g., which includes various mechanophores, mechanchromic, and mechanoluminescent materials), [15] which we consider as sepa- rate but equally exciting materials. Most of these systems are classified as self-reporting materials, which have mainly been leveraged in systems for reporting damage and forces [16] irreversibly, often by bond-cleavage and/or chemical reactions. [17] We focus on polymer materials (messengers) that dynamically modulate amplified signals (messages), which can be considered as a subclass of self-reporting materials.

Surfaces
Conjugation of end-tethered polymers to surfaces at a high grafting density, known as polymer brushes, has recently attracted renewed interest for use in sensing technologies, and for good reason (Figure 2A). Polymer brushes exhibit outstanding switching capabilities due to the high grafting density that leads to conformational coupling between the chains. This means that a single chain is not independent in its conformation with respect to its neighbors, so if some chains are forced to change conformation due to a stimulus, the change in conformation is then locally communal around the stimulus. The switching behavior due to stimuli leads to changes in the average distance over which polymer density exists away from the anchoring surface -this is generally termed the "height" of the brush. This can be typically accessed by atomic force microscopy (AFM) or spectroscopic ellipsometry. Significant work has gone into developing polymer brushes that respond to numerous singular or collective stimuli, but the mechanism of transducing the information from conformational transitions has largely been limited to 1D data transmission (i.e., a single parameter for the transitions averaged over a macroscopic surface). This has placed a limit on the ability of these materials to be leveraged as "messenger materials", as it requires significant work for us to "go in" and extract the message. Recent work has overcome this dimensionality problem by integrating fluorophores into specific polymer brush architectures for conformational fluorescence (fluorescence output that depends on the conformation of the polymer chains).
Fluorescent properties can readily be integrated into polymer brushes, either by copolymerization, post-polymerization monomer functionalization, or end-group substitution. It is not always straight-forward, but it can be done with care. This has been shown in many systems by a variety of authors. [18] Particularly notice must be paid to integration methods that do not hinder the native response of the main backbone monomers toward a stimulus. However, simply integrating fluorescence into poly-  [19] Copyright 2021 Wiley-VCH GmbH. Schematic (C) and fluorescence decay profiles with fitted lifetimes (D) of single fluorophore-integrated polymer brushes in the self-quenching regime. Reprinted with permission from. [22] Copyright 2022, American Chemical Society. Schematic (E), evolution of the (coupled) plasmon resonance wavelength maximum upon humidity changes (F), and schematic and digital photograph of the change in film color due to plasmonic coupling (G) for hybrid gold nanoparticle/polymer films. Reproduced with permission. [23] Copyright 2019, The Royal Society of Chemistry. mer brushes does not allow for conformational fluorescence. For this, the integrated architecture must be carefully designed.
FRET chemistry has been integrated into poly(Nisopropylacrylamide) (PNIPAM) polymer brushes as diblock random copolymers with a FRET donor in one block, and the corresponding acceptor in the other block, where both blocks are approximately the same length. [19] This design therefore linked the fluorophore proximity (and thereby FRET pairing) to the conformation of the polymer chains ( Figure 3A-B). Such a system represented a "soft" reporting system, whereby the chain conformations could be resolved spatially across a surface, reversibly (i.e., dynamic). The ability to resolve the conformations was found through confocal laser scanning microscopy (CLSM), which allowed analysis of separate fluorescence "windows" across the brush surface, toward optical resolution. Importantly, this method leveraged real-time analysis, which was reversible. This concept demonstrates a surface coating strategy that can readily report, or message, the presence of different stimuli, through the "eyes" of CLSM. The architectures here pertain to diblock polymer systems, with randomly distributed fluorophores in each block. Similar systems have been designed to understand solute-penetration into brushes by solving brushes in a liquid containing a FRET acceptor with the donor tethered within [20] or below [21] the brush. The permeability into the brushes is directly correlated to the brush conformation, which allowed CLSM reporting of the stimuli-induced changes in conformation.
In a similar sense, other recent systems have been reported that leveraged fluorophore-to-fluorophore self-quenching effects to report changes in conformation. This concept is based on changes in the effective concentration of brush-tethered fluorophores as the polymer brush conformational changes modulate the free volume in which the fluorophores are situated. These quenching effects have been observed from changes in the fluorescence intensity of conformational fluorescent brushes (from CLSM). [24] However, a more sensitive measure has been found in observing the changes in fluorescence lifetimes (e.g., the duration the fluorophore spends in the excited state, on average, before emitting light), as seen through fluorescence lifetime imaging microscopy (FLIM). Carefully designed copolymer brushes of NIPAM with a specific concentration of a monomer of rhodamine B (Rhod B), chosen so as to sample points of the Rhod B self-quenching regime with respect to changes in volume (concentration) due to polymer brushes conformational changes, have been reported. [22] The polymer backbone basically functioned to "hold" the Rhod B in place within a specific volume. These conformationally fluorescent brushes demonstrated strong sensitivity and allowed for high-resolution imaging of the brush conformational changes due to solvation ( Figure 3C-D). Importantly, like with the FRET systems, these self-quenching systems could rapidly transduce messages of solvation effects (solvents and non-solvents). These were used as a basis to probe and ultimately understand subtle solvation effects around the 3phase contact line of a pinned aqueous droplet, and at the oilwater interface on brush surfaces.
Swelling in polymer brushes can also be monitored upon integrating plasmonic nanoparticles into brush architectures. However, to realize such systems, several challenges need to be overcome, which includes optimization with respect to the grafting density of the polymer chains. There is an optimum grafting density of the polymer brush for maximum nanoparticle load, as for too high grafting density the repulsive barriers for nanoparticle incorporation become too large, whereas for too small grafting densities there are simply too little interaction sites for the nanoparticles. [25] Furthermore, the responsive features of the polymer brushes need to be maintained in the desired parameter window, which is in fact not always the case. For example, when citrate-coated gold nanoparticle were adsorbed onto poly(N,Ndimethylaminoethyl methacrylate) brushes, this cancelled the polymer's critical phase transition. [26] When all parameters are carefully considered, though, responsive polymer brushes with adsorbed plasmonic nanoparticle reporting entities can be used to study swelling transitions, as has been shown for PNIPAM brushes with incorporated gold nanoparticles. [27] Aside from polymer brushes, thin films of polymer materials can be integrated with amplification sources for messaging. A conceptually simple but versatile approach is to crosslink plasmonic nanoparticles by means of a (macro-)molecular linker. Upon deposition on a surface, this can lead to a quasi-2D layer of cross-linked nanoparticles. Cross-linking by small molecule , -dithiol functionalized alkanes provides access to thin films that enable electromechanical signal transduction, which is achieved because deformation of the thin film alters the electron tunneling barrier between constituent nanoparticles. [28] These pressure-reporting thin films can be transferred onto soft elastomer materials, which has potential for healthcare device fabrication. [29] When the nanoparticle cross-linker is replaced by a polymer, signal transduction mechanisms based on changes in the nanoparticle configuration at larger length scales become possible. For instance, humidity-induced reversible swelling of , -homotelechelic dithiol-functionalized poly(ethylene glycol) spacers that connect gold nanoparticles in quasi-2D networks can be used to create colorimetric humidity sensors based on interparticle distance changes and concomitant modulation of plasmon coupling ( Figure 3E-G). [23] These systems offer high potential for communicating subtle changes in local chemical environments, rapidly in real-time. Crucially, the inherent reversibility of the conformational transitions means the materials allows dynamic stimuli to be probed and reported.

Hydrogels
Hydrogels, which consist of strongly solvated cross-linked polymer chains, can allow the concept of messenger materials to become macroscopic ( Figure 2B). Hydrogels can be prepared on mm dimensions (and greater), or down to micro-to-nano domain particles. Importantly, hydrogels strongly depart from the dimensional limitations of surface polymer brushes (i.e., the materials are macroscopic in all 3Ds, whereas polymer brushes are typically nanoscopic in the dimension normal to the tethered surface). Hydrogels can be designed as messenger materials in a similar manner to polymer brushes, where fluorophores can be integrated in such a way so as to non-invasively monitor the conformation of the gel-forming polymer chains in the cross-linked network. [30] In this space, there are several different strategies that can be pursued so that the hydrogels can message information on strain, damage, and/or compression. In this sense, significant work has been done with hydrogels within the concept of "mechanosensing".
Examples of recent strategies that have been pursued include those that polymerize from a DNA hairpin structure that contains a fluorophore and its corresponding quencher on either side of the hairpin (Figure 4A,B). [31] The functional polymer architecture, in this sense, pertains to the breakable/repairable hairpin structure on DNA. The resulting materials exhibited fluorescence intensity that scaled with the elongation (stretching) of the hydrogels due to the rupturing of the hairpin, due to greater distances between the fluorophore and its quencher ( Figure 4C). Other DNA-based mechanophores have been designed that contain sacrificial duplexes of different lengths, [32] leading to different fluorescent outputs depending on the force required to "unzip" the duplexes. Importantly, these recently reported systems provide intense (and reference corrected) reporting of strain, where the DNA can quickly close back and recover the unstretched configuration upon stress release, highlighting the reversibility of the process. In other work, FRET-integrated PEG hydrogel microparticles have been reported [33] that consisted of FRET acceptor and donor motifs covalently tethered separately within the hydrogel matrix ( Figure 4D,E). The concept in this space is that deformation of the hydrogel microparticles simply changes the distance between the fluorophores, leading to FRET coupling. This system is therefore inherently reversible, could be used to monitor contact pressures exerted by colloidal probe atomic force microscopy, in a unique combination with CLSM that provided real-time collection of deformation messages induced by the AFM (Figure 4F). Furthermore, due to its basis on only fluorophore proximity, the system could also be used as a sensor of relative humidity (swelling/deswelling due to atmospheric water).
Other recent strategies for messenger hydrogels have leveraged plasmonic particle-based nanosensors at the macroscopic scale, creating skin-implantable hydrogel sensors with integrated gold nanorods as resonant refractive index sensing entities ( Figure 4G,H). [34] The hydrogel system contained three gold nanorod containing strips separated at specific distances, each containing different nanorod types. Together with a special signal referencing approach, this enabled nano-to-macro messaging, where the plasmon resonance shift signal could be www.advancedsciencenews.com www.afm-journal.de conveniently read with a CMOS camera. The choice of the chemical composition of the embedding hydrogel is critical for the described functionality. The requirements include nanoparticle dispersion within the hydrogel, biocompatibility, and minimal undesired interaction of the hydrogel with the target analyte and analyte receptor entities at the nanoparticles. For a studied case involving aptamer-functionalized gold nanorods for kanamycin sensing, [35] hydrogels of copolymers of 2-hydroxyethyl methacrylate and poly(ethylene glycol)diacrylate provided an adequate solution to the mentioned requirements.
A key benefit of moving ahead with hydrogel messenger materials is that the physical properties of the materials can be readily adjusted through cross-linking degree. The ability to tune these parameters is not so easy for polymer brush or polymer film surfaces, where varying the surface grafting density in a controlled manner can pose difficulties. Furthermore, integrating conformational fluorescence and/or plasmonic coupling into hydrogel matrices can often be simpler than for surfaces, where the architecture can be chosen between co-monomer, cross-link, or postfunctionalization of a coupling monomer. Such systems have less hindrances in achieving this goal due to the relatively large changes in the dimensions of the hydrogels under strain, deformation, and/or damage. We anticipate many exciting applications for conformationally fluorescent and plasmonic hydrogels ahead, especially outside of strictly "mechanosensing" (i.e., for probing subtle stimuli that change the conformation of polymer chains, rather than physical pressures). The spark for such future developments is already there in the form of additive manufacturing techniques for creating highly integrated, hierarchically structured hydrogel materials. [36]

Nanomaterials
Nanomaterials, which we here classify as in-solution systems, opposed to the macroscopic materials discussed above, can possess a variety of properties that make them predestined for nanoto-macro messaging, particularly for in vivo or in vitro studies ( Figure 2C). In these contexts, nanomaterial messengers need to be of dimensions significantly smaller than the biological machinery and processes to be probed. A key requirement for the material is to produce a signal that is above and distinct from background biological processes. The biological milieu is incredibly complex, with numerous multi-pathway events occurring on time-scales that are difficult to monitor with current technologies. However, some recent works investigating intracellular physical quantities have provided a strong basis to move forward toward greater complexity. These have involved designing simple yet elegant architectures of polymers to enable messeng amplification. Whilst significant work has been done on developing nanomaterials for monitoring specific cellular processes by disassembly mechanisms of fluorophores [15a] or plasmonicnanoparticle/quantum-dot conjugates, [37] as well as other material systems, [38] here we remain focused on polymeric materials that modulate the message response, reversibly.
Nanomaterials can be readily decorated with polymer brushes, chains, and films, in similar strategies to those discussed above for both surface coatings and hydrogels. This includes coupling FRET chemistry into conjugated diblock copolymers, [39] and into cross-linked polymeric nanoparticles, [40] which can exhibit photoswitching capabilities within complex biological environments. [41] Recent strategies that leverage nanoprecipitation methods for the polymer conjugations have shown great potential. [42] Polymeric nanoparticles composed of poly(methyl methacrylate)(PMMA) and poly(ethyl methacrylate)(PEMA) have recently been reported, which contained strongly confined FRET donors. These particles were functionalized with oligonucleotide sequences with bound FRET acceptor labeled sequences ( Figure 5A). [43] Upon binding and displacement by target DNA/RNA, high resolution amplification of FRET loss were observed, allowing for real-time and reversible detection of single oligonucleotide hybridization processes ( Figure 5B). This results from end-tethering the oligonucleotide sequences onto azidefunctionalized nanoparticles, where the polymer controls the FRET through binding with the target, and the dislocation events reduce the fluorophore proximities required for FRET. For example, nanoparticles containing an oxygen-sensitive porphyrin acceptor molecule in the polymer matrix, were used to monitor oxygen concentrations (due to diffusion processes into the nanoparticles) in cells exposed to abundant oxygen and subjected to hypoxia. [44] Importantly, the basis of the method on diffusion meant the process was inherently reversible as the environment was switched. These systems are not based on polymers that "change" their properties (like those discussed above), but the choice of polymers in these contexts would influence the diffusive processes that occur through them, and thereby the access to fluorescence processes. Other recent unique architectures have included fluorescence cage-based PNIPAM nanoparticle systems that have FRET and cascade-FRET associated fluorophores inside and outside of the cage nanoparticle, leading to temperatureinduced changes (reversible) in tricolor FRET based on PNIPAM conformations ( Figure 5C). [45] This system is based on a change in material size due to the cage polymer structure, and thereby proximity between embedded and associated fluorophores that have been used as temperature probes in living cells, with temperature spatially localized by CLSM ( Figure 5D).
Similar changes can be thermally induced if plasmonic nanoparticles are joined into well-defined assembly structures, in which interparticle spacing can be changed with a targeted temperature trigger, resulting in interparticle coupling effects. In the case of assemblies of plasmonic nanoparticles, these coupling effects can result in a pronounced change in perceivable color. [46] A versatile structural motif that allows this kind of signal transduction is represented by planet-satellite nanostructures, in which smaller "satellite" nanoparticle surround a central "planet" nanoparticle, linked together by stimulus-responsive polymer. In such systems, reversible expansion and contraction of interparticle spacing becomes possible. [47] Using homopolymer linkers with a single (lower) critical temperature, this feature can be used to create temperature sensitive nanostructures that reversibly report heating events; [48] whereas diblock copolymers featuring both a lower and upper critical temperature can be employed to construct nanostructured temperature reporters that report both heating and cooling events. [49] Tantalizing messaging possibilities arise from combining distinct nanomaterials synergistically in a well-defined assembly structure. For example, decoration of gold nanorods with iron oxide "satellite" nanoparticles, renders the plasmonic gold nanorods responsive to external magnetic fields ( Figure 5E); in Figure 5. Schematic for the synthesis of polymeric nanoparticles with a single acceptor conjugated and its response to a target (A), along with widefield FRET images of the nanoprobe without and with DNA and RNA targets. Reproduced with permission. [43] Copyright 2020, Wiley VCH. Schematic describing the assembly of cage-based PNIPAM polymers into hybrid FRET nanoparticles (C) along with confocal images describing their application in HeLa cells for monitoring temperature. Reproduced with permission. [45] Copyright 2020, American Chemical Society. Schematic and digital photographs (taken with a horizontal polarizer) of core-nanoparticle clusters rotating in aqueous solution on a stirrer plate (E), along with digital photographs (taken with a horizontal (left) and vertical (right) polarizer) of the same systems placed between cube magnets. Reproduced with permission. [50] Copyright 2022, Wiley-VCH GmbH. www.advancedsciencenews.com www.afm-journal.de a homogeneous magnetic field, the nanorods will align parallel to the magnetic field direction ( Figure 5F). [50] This alignment brings the optical anisotropy of gold nanorods into effect at the macroscale, as light absorption and scattering will depend on the orientation of the nanorods with respect to the light propagation direction. This effect can be further enhanced using polarizer filters and enables the visualization and dynamic tracking of external magnetic fields.

Where to from here?
Materials that can inform, "messenger materials", is a simple concept that has been startlingly difficult to engineer. At least, broadly speaking, until now. At this stage in the polymer science community groups have engineered unique ways to leverage basic changes in functional polymer conformations due to specific stimuli, or as architectures that keep amplification motifs separated or linked until a stimulus is present, which have been used as a basis for understanding some minute, nanoscopic processes that are spatially localized in high resolution. The understanding of the distinct signal transduction mechanisms achieved forms a solid basis for future developments, where the scope of forming well-defined responsive polymers by methods of macromolecular chemistry [51] opens up a field rich with possibilities that are just now being realized. Most of the systems we have discussed are focused on proximity-based changes in a material that lead to reporting from amplification motifs. We have purposefully maintained focus on surface bound polymers, hydrogels, and nanomaterials, but there are other stimulating possibilities based on macroscopic polymeric materials with integrated messenger capabilities, such as mechanochromic donor-acceptor torsional springs, [52] which provide a wider scope.
From here on, we anticipate new strategies to emerge that build from multi-level responsivity, deriving, for instance, from multiblock-copolymer transducing elements. This may lead toward materials that can communicate with each other based on unique cascades of events inscribed into the materials. This basis would advance past simple nano-to-macro communication, and rather offer the potential for nano-to-nano discussion before macroscopic communication. We point out that such advancements should not be constrained within conceptual locks of fluorescence and plasmonic couplings, but rather should build toward greater complexity, which can be realized using the strategic combination of several distinct reporting, transducing, and additional modulating entities into highly integrated polymerbased messenger materials. Possibilities in this space can involve coupling between physical properties (e.g., electroluminescence), which can inform/control downstream processes with other functional polymers to pass the message along for multimodal processing. A key application for such material systems is toward probing cellular processes, which often contain complex overlapping cascade loops for information processing. We envisage new possibilities from material systems which process and output information upon, e.g., opening/closing of specific sites and/or additional internal or external interactions. Recent work on autonomous systems capable of actuation [53] may make this possible. Furthermore, the rapid advancement of super resolution fluorescence microscopy techniques can allow for highly localized messaging toward the nanoscale. For all this, the dis-cussed methods may or may not be used as tools along the communication pathway, but nonetheless establish the current status quo. It is our view that the use of messenger materials in future artificial intelligence systems will be a fruitful domain, where we anticipate exciting movement in the materials sciences going forward.