Self-assembled low-molecular-weight gelator injectable microgel beads for delivery of bioactive agents

We report the preparation of hybrid self-assembled microgel beads by combining the low molecular weight gelator (LMWG) DBS-CONHNH₂ and the natural polysaccharide calcium alginate polymer gelator (PG). Microgel formulations based on LMWGs are extremely rare due to the fragility of the self-assembled networks and the difficulty of retaining any imposed shape. Our hybrid beads contain interpenetrated LMWG and PG networks, and are obtained by an emulsion method, allowing the preparation of spherical gel particles of controllable sizes with diameters in the mm or μm range. Microgels based on LMWG/alginate can be easily prepared with reproducible diameters <1 μm (ca. 800 nm). They are stable in water at room temperature for many months, and survive injection through a syringe. The rapid assembly of the LMWG on cooling plays an active role in helping control the diameter of the microgel beads. These LMWG microbeads retained the ability of the parent gel to deliver the bioactive molecule heparin, and in cell culture medium this enhanced the growth of human mesenchymal stem cells. Such microgels may therefore have future applications in tissue repair. This approach to fabricating LMWG microgels is a platform technology, which could potentially be applied to a variety of different functional LMWGs, and hence has wide-ranging potential.

We report microgel beads with diameters of ca. 800 nm based on interpenetrating networks of a low-molecular-weight gelator and a polymer gelator, and demonstrate their use as heparin delivery vehicles to enhance stem cell growth.     

Double diffusion for the programmable spatiotemporal patterning of multi-domain supramolecular gels

To achieve spatial resolution of a multi-component gel, a double diffusion approach is used which enables the precise programming of self-assembled patterned domains with well-defined shapes and sizes. The low-molecular-weight gelators (LMWGs) used in this study are pH-responsive DBS-CO₂H and thermally-responsive DBS-CONHNH₂ (both based on 1,3:2,4-dibenzylidenesorbitol, DBS). A DBS-CONHNH₂ gel was initially assembled in a tray, and then loaded at carefully-selected positions with either basified DBS-CO₂H (i.e. DBS-carboxylate) or an acid. These soluble components subsequently diffuse through the pre-formed gel matrix, and in the domains when/where they mix, protonation of the DBS-carboxylate induces self-assembly of the DBS-CO₂H network, leading to a patterned gel-in-gel object with well-defined shape and dimensions. Using a strong acid achieves fast gelation kinetics, creating smaller, better-defined macroscale objects but with less nanoscale order. Using a weak acid source with slow kinetics, gives slightly larger objects, but on the nanoscale the DBS-CO₂H network formation is better controlled, giving more homogeneous nanoscale structures and stiffer objects. The patterned objects can be further reinforced by the presence of agarose polymer gelator. The shape of the patterning is programmed by both the shape of the central reservoir and the starting geometry in which the reservoirs are organised, with the balance between factors depending on assembly kinetics, as dictated by the choice of acid. This simple methodology therefore enables programming of patterned gels with spatiotemporal control and emergent patterning characteristics.

To achieve spatial resolution of a multi-component gel, a double diffusion approach is used which enables the precise programming of emergent self-assembled patterned domains with well-defined shapes and sizes.     

Sodium caseinate as a particulate emulsifier for making indefinitely recycled pH-responsive emulsions

pH-responsive emulsions are one of the simplest and most readily implementable stimuli-responsive systems. However, their practical uses have been greatly hindered by cyclability. Here, we report a robust pH-responsive emulsion prepared by utilizing pure sodium caseinate (NaCas) as the sole emulsifier. We demonstrate that the emulsification/demulsification of the obtained NaCas-stabilized emulsion can be triggered by simply changing the pH value over 100 cycles, which has never been observed in any protein-stabilized emulsion system. The NaCas-stabilized emulsion maintains its pH-responsive properties even in a saturated salt solution (NaCl ∼ 6.1 M) or seawater. We illustrate how NaCas functions in pH-responsive emulsions and show that when conventional nanoparticles such as zein protein or bare SiO₂ particles were coated with a layer of NaCas, the resulting formulated emulsions could be switched on and off over 10 cycles. The unique properties of NaCas thus enable the engineering of conventional Pickering emulsions to pH-responsive Pickering emulsions. Finally, we have integrated catalytically active gold (Au) nanoclusters (NCs) into the NaCas protein and then utilized them to produce emulsions. Remarkably, these NaCas–Au NCs assembled at the oil–water interface exhibited excellent catalytic activity and cyclability, not only in aqueous solution, but also in complicated seawater environments.

An unprecedented pH-responsive emulsion is shaped by utilizing pure sodium caseinate (NaCas) as the sole emulsifier for recyclable interfacial catalysis. This emulsion could be reversibly switched on and off over 100 cycles.     

A pH-responsive ultrathin Cu-based nanoplatform for specific photothermal and chemodynamic synergistic therapy

Noninvasive tumor therapy requires a new generation of bionanomaterials towards sensitive response to the unique tumor microenvironment to achieve accurate and effective treatment. Herein, we have developed a tumor therapy nanoplatform by immobilizing natural glucose oxidase (GOD) onto Cu-based layered double hydroxide (CuFe-LDH) nanosheets, which for the first time integrates acid-enhanced photothermal therapy (PTT), and pH-responsive and heat-facilitated chemodynamic therapy (CDT) simultaneously. As demonstrated by EXAFS and HRTEM, CuFe-LDH nanosheets possess a considerable number of defects caused by different acid conditions, resulting in a significantly acid-enhanced photothermal conversion efficiency (83.2% at pH 5.4 vs. 46.0% at pH 7.4). Moreover, GOD/CuFe-LDH nanosheets can convert a cascade of glucose into hydroxyl radicals (˙OH) under tumor acid conditions, which is validated by a high maximum velocity (V_max = 2.00 × 10⁻⁷ M) and low Michaelis–Menten constant (K_M = 12.01 mM). With the combination of PTT and CDT, the tumor tissue in vivo is almost eliminated with low-dose drug injection (1 mg kg⁻¹). Therefore, this novel pH-responsive Cu-based nanoplatform holds great promise in tumor-specific CDT/PTT synergistic therapy.

A pH-responsive multifunctional nanosystem was synthesized by loading glucose oxidase (GOD) onto CuFe-layered double hydroxide (LDH) nanosheets, which exhibited synchronous acid-enhanced/responsive photothermal and chemodynamic synergistic therapy.     

Engineering bio-mimicking functional vesicles with multiple compartments for quantifying molecular transport

Controlled design of giant unilamellar vesicles under defined conditions has vast applications in the field of membrane and synthetic biology. Here, we bio-engineer bacterial-membrane mimicking models of controlled size under defined salt conditions over a range of pH. A complex bacterial lipid extract is used for construction of physiologically relevant Gram-negative membrane mimicking vesicles whereas a ternary mixture of charged lipids (DOPG, cardiolipin and lysyl-PG) is used for building Gram-positive bacterial-membrane vesicles. Furthermore, we construct stable multi-compartment biomimicking vesicles using the gel-assisted swelling method. Importantly, we validate the bio-application of the bacterial vesicle models by quantifying diffusion of chemically synthetic amphoteric antibiotics. The transport rate is pH-responsive and depends on the lipid composition, based on which a permeation model is proposed. The permeability properties of antimicrobial peptides reveal pH dependent pore-forming activity in the model vesicles. Finally, we demonstrate the functionality of the vesicles by quantifying the uptake of membrane-impermeable molecules facilitated by embedded pore-forming proteins. We suggest that the bacterial vesicle models developed here can be used to understand fundamental biological processes like the peptide assembly mechanism or bacterial cell division and will have a multitude of applications in the bottom-up assembly of a protocell.

Giant vesicle functional models mimicking a bacterial membrane under physiological conditions are constructed.     

Biocatalytic reversible control of the stiffness of DNA-modified responsive hydrogels: applications in shape-memory,self-healing and autonomous controlled release of insulin

The enzymes glucose oxidase (GOx), acetylcholine esterase (AchE) and urease that drive biocatalytic transformations to alter pH, are integrated into pH-responsive DNA-based hydrogels. A two-enzyme-loaded hydrogel composed of GOx/urease or AchE/urease and a three-enzyme-loaded hydrogel composed of GOx/AchE/urease are presented. The biocatalytic transformations within the hydrogels lead to the dictated reconfiguration of nucleic acid bridges and the switchable control over the stiffness of the respective hydrogels. The switchable stiffness features are used to develop biocatalytically guided shape-memory and self-healing matrices. In addition, loading of GOx/insulin in a pH-responsive DNA-based hydrogel yields a glucose-triggered matrix for the controlled release of insulin, acting as an artificial pancreas. The release of insulin is controlled by the concentrations of glucose, hence, the biocatalytic insulin-loaded hydrogel provides an interesting sense-and-treat carrier for controlling diabetes.

Biocatalytic control over the stiffness of pH-responsive hydrogels is applied to develop shape-memory, self-healing and controlled release matrices.     

Spontaneous emergence of membrane-forming protoamphiphiles from a lipid–amino acid mixture under wet–dry cycles

Dynamic interplay between peptide synthesis and membrane assembly would have been crucial for the emergence of protocells on the prebiotic Earth. However, the effect of membrane-forming amphiphiles on peptide synthesis, under prebiotically plausible conditions, remains relatively unexplored. Here we discern the effect of a phospholipid on peptide synthesis using a non-activated amino acid, under wet–dry cycles. We report two competing processes simultaneously forming peptides and N-acyl amino acids (NAAs) in a single-pot reaction from a common set of reactants. NAA synthesis occurs via an ester–amide exchange, which is the first demonstration of this phenomenon in a lipid–amino acid system. Furthermore, NAAs self-assemble into vesicles at acidic pH, signifying their ability to form protocellular membranes under acidic geothermal conditions. Our work highlights the importance of exploring the co-evolutionary interactions between membrane assembly and peptide synthesis, having implications for the emergence of hitherto uncharacterized compounds of unknown prebiotic relevance.

Synthesis of lipoamino acids via ester–amide exchange under prebiotically plausible wet-dry cycling conditions that results in vesicles at acidic pH.     

Sequential Assembly of Mutually Interactive Supramolecular Hydrogels and Fabrication of Multi-Domain Materials

A two-component self-sorting hydrogel based on acylhydrazide and carboxylic acid derivatives of 1,3:2,4-dibenzylidene-d -sorbitol (DBS-CONHNH₂ and DBS-COOH) is reported. A heating–cooling cycle induces the self-assembly of DBS-CONHNH₂, followed by the self-assembly of DBS-COOH induced by decreasing pH. Although the networks are formed sequentially, there is spectroscopic evidence of interactions between them, which impact on the mechanical properties and significantly enhance the ability of these low-molecular-weight gelators (LMWGs) to form gels when mixed. The DBS-COOH network can be switched “off” and “on” within the two-component gel through a pH change. By using a photo-acid generator, the two-component gel can be prepared combining the thermal trigger with photo-irradiation. Photo-patterned self-assembly of DBS-COOH within a pre-formed DBS-CONHNH₂ gel under a mask yields spatially controlled multi-domain gels. Different gel domains can have different functions, for example, controlling the rate of release of heparin incorporated into the gel, or directing gold nanoparticle assembly. Such photo-patterned multi-component hydrogels have potential applications in regenerative medicine or bio-nano-electronics.     

Long-lived lanthanide emission via a pH-sensitive and switchable LRET complex

Lanthanide-based luminescence resonance energy transfer (LRET) can be used as a tool to enhance lanthanide emission for time-resolved cellular imaging applications. By shortening lanthanide emission lifetimes whilst providing an alternative radiative pathway to the formally forbidden, weak lanthanide-only emission, the photon flux of such systems is increased. With this aim in mind, we investigated energy transfer in differently spaced donor–acceptor terbium–rhodamine pairs with the LRET “on” (low pH) and LRET “off” (high pH). Results informed the design, preparation and characterisation of a compound containing terbium, a spectrally-matched pH-responsive fluorophore and a receptor-targeting group. By combining these elements, we observed switchable LRET, where the targeting group sensitises lanthanide emission, resulting in an energy transfer to the rhodamine dye with an efficiency of E = 0.53. This strategy can be used to increase lanthanide emission rates for brighter optical probes.

A pH-sensitive luminescence resonance energy transfer (LRET) was explored as a method to increase photon flux in a terbium-rhodamine-receptor targeting group construct. At low pH, long-lived dye emission and shorter terbium lifetimes were observed.     

Separation of durum wheat proteins by ultrathin-layer isoelectric focusing: a new tool for the characterization and quantification of low molecular weight glutenins

An isoelectric focusing method capable of resolving all groups of storage protein of the wheat seed, including the most basic low molecular weight glutenin (LMWG), was developed. Ultrathin polyacrylamide gels were used after drying and rehydration with 8 M urea, 50 mM DTE and 2.4% carrier ampholytes (pH 4-9). Densitometric scanning of the isoelectric focusing gels permitted a more accurate and specific quantitation of LMWG components among various cultivars than patterns based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two main genetic types (i.e. 'gamma-42' and 'gamma-45') of durum wheats were separated on the basis of the proportion in LMWG in storage proteins, but no significant difference was found within these groups. Advantages of the system as regards reliability, high resolution, ability to abolish protein oxidation and preventing reaggregation of LMWG were also discussed.     

Acid–base chemistry at the single ion limit

We present the results of acid–base experiments performed at the single ion (H⁺ or OH⁻) limit in ∼6 aL volume nanopores incorporating electrochemical zero-mode waveguides (E-ZMWs). At pH 3 each E-ZMW nanopore contains ca. 3600H⁺ ions, and application of a negative electrochemical potential to the gold working electrode/optical cladding layer reduces H⁺ to H₂, thereby depleting H⁺ and increasing the local pH within the nanopore. The change in pH was quantified by tracking the intensity of fluorescein, a pH-responsive fluorophore whose intensity increases with pH. This behavior was translated to the single ion limit by changing the initial pH of the electrolyte solution to pH 6, at which the average pore occupancy 〈n〉_pore ∼3.6H⁺/nanopore. Application of an electrochemical potential sufficiently negative to change the local pH to pH 7 reduces the proton nanopore occupancy to 〈n〉_pore ∼0.36H⁺/nanopore, demonstrating that the approach is sensitive to single H⁺ manipulations, as evidenced by clear potential-dependent changes in fluorescein emission intensity. In addition, at high overpotential, the observed fluorescence intensity exceeded the value predicted from the fluorescence intensity-pH calibration, an observation attributed to the nucleation of H₂ nanobubbles as confirmed both by calculations and the behavior of non-pH responsive Alexa 488 fluorophore. Apart from enhancing fundamental understanding, the approach described here opens the door to applications requiring ultrasensitive ion sensing, based on the optical detection of H⁺ population at the single ion limit.

Visualizing dynamic change in the number of protons during electroreduction of protons in attoliter volume zero-mode waveguides.     

High-efficiency dynamic sensing of biothiols in cancer cells with a fluorescent β-cyclodextrin supramolecular assembly

A unique fluorescent supramolecular assembly was constructed using coumarin-modified β-cyclodextrin as a reversible ratiometric probe and an adamantane-modified cyclic arginine–glycine–aspartate peptide as a cancer-targeting agent via host–guest inclusion complexation. Importantly, the coumarin-modified β-cyclodextrin not only showed higher sensitivity than the parent coumarin derivatives owing to the presence of numerous hydroxyl groups on the cyclodextrin but also provided a hydrophobic cavity for encapsulation of a cancer-targeting agent. The assembly showed a reversible and fast response to biothiols with a micromolar dissociation constant, as well as outstanding cancer cell permeability, which can be used for high-efficiency real-time monitoring of biothiols in cancer cells. This supramolecular assembly strategy endows the fluorescent probe with superior performance for dynamic sensing of biothiols.

A unique fluorescent supramolecular assembly was constructed from coumarin-modified β-cyclodextrin and an adamantane-modified cyclic arginine–glycine–aspartate peptide for high-efficiency real-time monitoring of biothiols in cancer cells.     

Hybrid Self-Assembled Gel Beads for Tuneable pH-Controlled Rosuvastatin Delivery

This article describes the fabrication of new pH-responsive hybrid gel beads combining the polymer gelator calcium alginate with two different low-molecular-weight gelators (LMWGs) based on 1,3 : 2,4-dibenzylidene-d -sorbitol: pH-responsive DBS-COOH and thermally responsive DBS-CONHNH₂, thus clearly demonstrating that different classes of LMWG can be fabricated into gel beads by using this approach. We also demonstrate that self-assembled multicomponent gel beads can be formed by using different combinations of these gelators. The different gel bead formulations exhibit different responsiveness – the DBS-COOH network can disassemble within those beads in which it is present upon raising the pH. To exemplify preliminary data for a potential application for these hybrid gel beads, we explored aspects of the delivery of the lipid-lowering active pharmaceutical ingredient (API) rosuvastatin. The release profile of this statin from the hybrid gel beads is pH-dependent, with greater release at pH 7.4 than at pH 4.0 – primary control of this process results from the pK_a of the API. The extent of pH-mediated API release is also significantly further modified according to gel bead composition. The DBS-COOH/alginate beads show rapid, highly effective drug release at pH 7.4, whereas the three-component DBS-COOH/DBS-CONHNH₂/alginate system shows controlled slow release of the API under the same conditions. These initial results indicate that such gel beads constitute a promising, versatile and easily tuned platform suitable for further development for controlled drug-delivery applications.     

Programmed catalysis within stimuli-responsive mechanically unlocked nanocavities in DNA origami tiles

The assembly of reversible stimuli-responsive locked DNA origami tiles being unlocked, in the presence of appropriate triggers, to form nanocavities in the origami rafts, is introduced. In the presence of ATP, K⁺-ion-stabilized G-quadruplexes or pH-responsive T-A·T triggers and appropriately engineered “helper units”, the origami rafts are unlocked to form nanocavities. By the application of appropriate counter-triggers, the nanocavities are relocked, thus establishing the switchable and reversible “mechanical” opening and closure mechanism of the nanocavities. The interconnection of the stimuli-responsive origami tiles into dimer structures enables the programmed triggered unlocking of each of the origami tiles, or both of the origami tiles, to yield dictated nanocavity-containing tiles. In addition, the functionalization of the opposite faces of the origami tiles with Mg²⁺-ion-dependent DNAzyme subunits leads, upon the triggered unlocking of the nanocavities, to the self-assembly of the active DNAzymes in the confined cavities. By the cyclic opening and closure of the cavities the reversible “ON”/“OFF” activation of the Mg²⁺-ion-dependent DNAzyme is demonstrated. Furthermore, upon the tethering of different Mg²⁺-ion-dependent subunits to the opposite faces of stimuli-responsive dimer origami tiles, the triggered programmed catalytic operation of different Mg²⁺-ion-dependent DNAzymes in the confined nanocavities, associated with the origami tiles, is demonstrated.

Programmed unlocking of nanocavities in origami dimer structures using different auxiliary triggers.     

Exploring long-range proton conduction,the conduction mechanism and inner hydration state of protein biopolymers

Proteins are the main proton mediators in various biological proton circuits. Using proteins for the formation of long-range proton conductors is offering a bioinspired approach for proton conductive polymers. One of the main challenges in the field of proton conductors is to explore the local environment within the polymers, along with deciphering the conduction mechanism. Here, we show that the protonic conductivity across a protein-based biopolymer can be hindered using straightforward chemical modifications, targeting carboxylate- or amine-terminated residues of the protein, as well as exploring the effect of surface hydrophobicity on proton conduction. We further use the natural tryptophan residue as a local fluorescent probe for the inner local hydration state of the protein surface and its tendency to form hydrogen bonds with nearby water molecules, along with the dynamicity of the process. Our electrical and spectroscopic measurements of the different chemically-modified protein materials as well as the material at different water–aprotic solvent mixtures result in our fundamental understanding of the proton mediators within the material and gaining important insights on the proton conduction mechanism. Our biopolymer can be used as an attractive platform for the study of bio-related protonic circuits as well as a proton conducting biopolymer for various applications, such as protonic transistors, ionic transducers and fuel cells.

Post formation modification of protein-based materials can attenuate the proton conduction efficiency resulting from change in conduction mechanism, charge carrier mobility, carrier concentrations and inner hydration layer.     

Surface-controlled spatially heterogeneous physical properties of a supramolecular gel with homogeneous chemical composition

Controlling supramolecular self-assembly across multiple length scales to prepare gels with localised properties is challenging. Most strategies concentrate on fabricating gels with heterogeneous components, where localised properties are generated by the stimuli-responsive component. Here, as an alternative approach, we use a spiropyran-modified surface that can be patterned with light. We show that light-induced differences in surface chemistry can direct the bulk assembly of a low molecular weight gelator, 2-NapAV, meaning that mechanical gel properties can be controlled by the surface on which the gel is grown. Using grazing incidence X-ray diffraction and grazing incidence small angle X-ray scattering, we demonstrate that the origin of the different gel properties relates to differences in the architectures of the gels. This provides a new method to prepare a single domain (i.e., chemically homogeneous) hydrogel with locally controlled (i.e., mechanically heterogeneous) properties.

A mechanical pattern is created in a hydrogel film by pre-patterning the underlying surface chemistry. This allows spatial variation of the viscous component of the gel, controlling dissipative forces in the gel film without altering gel chemistry.     

Thermo- and pH-responsive gels for application in colon delivery systems

Swelling-controlled drug delivery copolymer gels were synthesized by introducing thermo- and pH-responsive methacryloyl-glycine (MA-Gly) of pH-responsive methacrylic acid (MA-Ac) into thermo-responsive acryloyl- -proline ethyl ester (A-ProOEt). The threshold of swelling for copolymer gels consisting of A-ProOEt/MA-Gly and A-ProOEt/MA-Ac with a composition of 40/60 mol% was found to be pH 3.0 and pH 5.5, respectively, in buffer solutions at 37°C. The diffusion characteristics of 2-(3-benzoylphenyl)propionic acid (ketoprofen) from such copolymer gels were evaluated in buffer solutions at pHs more than 5.5, and it was found that A-ProOEt/MA-Gly gel possessed a Case II transport, while the A-ProOEt/MA-Ac gel exhibited a non-Fickian (or anomalous) diffusion behavior under the same conditions.     

Synthesis of 1,3-disubstituted bicyclo[1.1.0]butanes via directed bridgehead functionalization

Bicyclo[1.1.0]butanes (BCBs) are increasingly valued as intermediates in ‘strain release’ chemistry for the synthesis of substituted four membered rings and bicyclo[1.1.1]pentanes, with applications including bioconjugation processes. Variation of the BCB bridgehead substituents can be challenging due to the inherent strain of the bicyclic scaffold, often necessitating linear syntheses of specific BCB targets. Here we report the first palladium catalyzed cross-coupling on pre-formed BCBs which enables a ‘late stage’ diversification of the bridgehead position, and the conversion of the resultant products into a range of useful small ring building blocks.

Bicyclo[1.1.0]butanes (BCBs) are valuable precursors to four-membered rings and bicyclo[1.1.1]pentanes, and useful bioconjugation agents. We describe a versatile approach to access 1,3-disubstituted BCBs, which are otherwise challenging to prepare.     

Twist and sliding dynamics between interpenetrated frames in Ti-MOF revealing high proton conductivity

We report the design and synthesis of a titanium catecholate framework, MOF-217, comprised of 2,4,6-tri(3,4-dihydroxyphenyl)-1,3,5-triazine (TDHT) and isolated TiO₆ clusters, with 2-fold interpenetrated srs topology. The dynamics of the organic linker, breaking the C_3h symmetry, allowed for reversible twist and sliding between interpenetrated frames upon temperature change and the inclusion of small molecules. Introduction of 28 wt% imidazole into the pores of MOF-217, 28% Im-in-MOF-217, resulted in four orders of magnitude increase in proton conductivity, due to the appropriate accommodation of imidazole molecules and their proton transfer facilitated by the H-bond to the MOF structure across the pores. This MOF-based proton conductor can be operated at 100 °C with a proton conductivity of 1.1 × 10⁻³ S cm⁻¹, standing among the best performing anhydrous MOF proton conductors at elevated temperature. The interframe dynamics represents a unique feature of MOFs that can be accessed in the future design of proton conductors.

Twist and sliding dynamics observed in a titanium catecholate MOF induced by imidazole for efficient proton conduction.     

A facile combinatorial approach to construct a ratiometric fluorescent sensor: application for the real-time sensing of cellular pH changes

Realtime monitoring of the cellular environment, such as the intracellular pH, in a defined cellular space provides a comprehensive understanding of the dynamics processes in a living cell. Considering the limitation of spatial resolution in conventional microscopy measurements, multiple types of fluorophores assembled within that space would behave as a single fluorescent probe molecule. Such a character of microscopic measurements enables a much more flexible combinatorial design strategy in developing fluorescent probes for given targets. Nanomaterials with sizes smaller than the microscopy spatial resolution provide a scaffold to assemble several types of fluorophores with a variety of optical characteristics, therefore providing a convenient strategy for designing fluorescent pH sensors. In this study, fluorescein (CF) and tetramethylrhodamine (CR) were assembled on a DNA nanostructure with controlling the number of each type of fluorophore. By taking advantage of the different responses of CF and CR emissions to the pH environment, an appropriate assembly of both CF and CR on DNA origami enabled a controlled intensity of fluorescence emission and ratiometric pH monitoring within the space defined by DNA origami. The CF and CR-assembled DNA origami was successfully applied for monitoring the intracellular pH changes.

A combinatorial assembly of two types of intensity-based fluorophores on a DNA nanostructure provided a ratiometric pH probe with high emission intensity for monitoring intracellular pH changes.