Single-walled carbon nanotubes as near-infrared fluorescent probes for bio-inspired supramolecular self-assembled hydrogels.

Hydrogels derived from fluorenylmethoxycarbonyl (Fmoc)-conjugated amino acids and peptides demonstrate remarkable potential in biomedical applications, including drug delivery, tissue regeneration, and tissue engineering. These hydrogels can be injectable, offering a minimally invasive approach to hydrogel implantation. Given their potential for prolonged application, there is a need for non-destructive evaluation of their properties over extended periods. Thus, we introduce a hydrogel characterization platform employing single-walled carbon nanotubes (SWCNTs) as near-infrared (NIR) fluorescent probes. Our approach involves generating supramolecular self-assembling hydrogels from aromatic Fmoc-amino acids. Integrating SWCNTs into the hydrogels maintains their structural and mechanical properties, establishing SWCNTs as optical probes for hydrogels. We demonstrate that the SWCNT NIR-fluorescence changes during the gelation process correlate to rheological changes within the hydrogels. Additionally, single particle tracking of SWCNTs incorporated in the hydrogels provides insights into differences in hydrogel morphologies. Furthermore, the disassembly process of the hydrogels can be monitored through the SWCNT fluorescence modulation. The unique attribute of SWCNTs as non-photobleaching fluorescent sensors, emitting at the biologically transparent window, offers a non-destructive method for studying hydrogel dynamics over extended periods. This platform could be applied to a wide range of self-assembling hydrogels to advance our understanding and applications of supramolecular assembly technologies.

Single-Walled Carbon Nanotube Sensor Selection for the Detection of MicroRNA Biomarkers for Acute Myocardial Infarction as a Case Study.

MicroRNAs (miRNAs) are single-stranded non-coding short ribonucleic acid sequences that take part in many cellular and biological processes. Recent studies have shown that altered expression of miRNAs is involved in pathological processes, and they can thus be considered biomarkers for the early detection of various diseases. Here, we demonstrate a selection and elimination process of fluorescent single-walled carbon nanotube (SWCNT) sensors for miRNA biomarkers based on RNA-DNA hybridization with a complementary DNA recognition unit bound to the SWCNT surface. We use known miRNA biomarkers for acute myocardial infarction (AMI), commonly known as a heart attack, as a case study. We have selected five possible miRNA biomarkers which are selective and specific to AMI and tested DNA-SWCNT sensor candidates with the target DNA and RNA sequences in different environments. Out of these five miRNA sensors, three could recognize the complementary DNA or RNA sequence in a buffer, showing fluorescence modulation of the SWCNT in response to the target sequence. Out of the three working sensors in buffer, only one could function in serum and was selected for further testing. The chosen sensor, SWCNT-miDNA208a, showed high specificity and selectivity toward the target sequence, with better performance in serum compared to a buffer environment. The SWCNT sensor selection pipeline highlights the importance of testing sensor candidates in the appropriate environment and can be extended to other libraries of biomarkers.

Monitoring the Formation of Fibrin Clots as Part of the Coagulation Cascade Using Fluorescent Single-Walled Carbon Nanotubes.

Blood coagulation is a critical defense mechanism against bleeding that results in the conversion of liquid blood into a solid clot through a complicated cascade, which involves multiple clotting factors. One of the final steps in the coagulation pathway is the conversion of fibrinogen to insoluble fibrin mediated by thrombin. Because coagulation disorders can be life-threatening, the development of novel methods for monitoring the coagulation cascade dynamics is of high importance. Here, we use near-infrared (NIR)-fluorescent single-walled carbon nanotubes (SWCNTs) to image and monitor fibrin clotting in real time. Following the binding of fibrinogen to a tailored SWCNT platform, thrombin transforms the fibrinogen into fibrin monomers, which start to polymerize. The SWCNTs are incorporated within the clot and can be clearly visualized in the NIR-fluorescent channel, where the signal-to-noise ratio is improved compared to bright-field imaging in the visible range. Moreover, the diffusion of individual SWCNTs within the fibrin clot gradually slows down after the addition of thrombin, manifesting a coagulation rate that depends on both fibrinogen and thrombin concentrations. Our platform can open new opportunities for coagulation disorder diagnostics and allow for real-time monitoring of the coagulation cascade with a NIR optical signal output in the biological transparency window.

Single-Walled Carbon Nanotubes as Fluorescent Probes for Monitoring the Self-Assembly and Morphology of Peptide/Polymer Hybrid Hydrogels.

Hydrogels formed via supramolecular self-assembly of fluorenylmethyloxycarbonyl (Fmoc)-conjugated amino acids provide excellent scaffolds for 3D cell culture, tissue engineering, and tissue recovery matrices. Such hydrogels are usually characterized by rheology or electron microscopy, which are invasive and cannot provide real-time information. Here, we incorporate near-infrared fluorescent single-walled carbon nanotubes (SWCNTs) into Fmoc-diphenylalanine hydrogels as fluorescent probes, reporting in real-time on the morphology and time-dependent structural changes of the self-assembled hydrogels in the transparency window of biological tissue. We further demonstrate that the gelation process and structural changes upon the addition of cross-linking ions are transduced into spectral modulations of the SWCNT-fluorescence. Moreover, morphological differences of the hydrogels induced by polymer additives are manifested in unique features in fluorescence images of the incorporated SWCNTs. SWCNTs can thus serve as optical probes for noninvasive, long-term monitoring of the self-assembly gelation process and the fate of the resulting peptide hydrogel during long-term usage.

Super-Resolution Radial Fluctuations (SRRF) nanoscopy in the near infrared.

Super resolution microscopy methods have been designed to overcome the physical barrier of the diffraction limit and push the resolution to nanometric scales. A recently developed super resolution technique, super-resolution radial fluctuations (SRRF) [Nature communications, 7, 12471 (2016)10.1038/ncomms12471], has been shown to super resolve images taken with standard microscope setups without fluorophore localization. Herein, we implement SRRF on emitters in the near-infrared (nIR) range, single walled carbon nanotubes (SWCNTs), whose fluorescence emission overlaps with the biological transparency window. Our results open the path for super-resolving SWCNTs for biomedical imaging and sensing applications.

In vivo imaging of fluorescent single-walled carbon nanotubes within C. elegans nematodes in the near-infrared window.

Caenorhabditis elegans (C. elegans) nematodes serve as a model organism for eukaryotes, especially due to their genetic similarity. Although they have many advantages like their small size and transparency, their autofluorescence in the entire visible wavelength range poses a challenge for imaging and tracking fluorescent proteins or dyes using standard fluorescence microscopy. Herein, near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) are utilized for in vivo imaging within the gastrointestinal track of C. elegans. The SWCNTs are biocompatible, and do not affect the worms' viability nor their reproduction ability. The worms do not show any autofluorescence in the NIR range, thus enabling the spectral separation between the SWCNT NIR fluorescence and the strong autofluorescence of the worm gut granules. The worms are fed with ssDNA-SWCNT which are visualized mainly in the intestine lumen. The NIR fluorescence is used in vivo to track the contraction and relaxation in the area of the pharyngeal valve at the anterior of the terminal bulb. These biocompatible, non-photobleaching, NIR fluorescent nanoparticles can advance in vivo imaging and tracking within C. elegans and other small model organisms by overcoming the signal-to-noise challenge stemming from the wide-range visible autofluorescence.

Dendron-Polymer Hybrids as Tailorable Responsive Coronae of Single-Walled Carbon Nanotubes.

Functional composite materials that can change their spectral properties in response to external stimuli have a plethora of applications in fields ranging from sensors to biomedical imaging. One of the most promising types of materials used to design spectrally active composites are fluorescent single-walled carbon nanotubes (SWCNTs), noncovalently functionalized by synthetic amphiphilic polymers. These coated SWCNTs can exhibit modulations in their fluorescence spectra in response to interactions with target analytes. Hence, identifying new amphiphiles with interchangeable building blocks that can form individual coronae around the SWCNTs and can be tailored for a specific application is of great interest. This study presents highly modular amphiphilic polymer-dendron hybrids, composed of hydrophobic dendrons and hydrophilic polyethylene glycol (PEG) that can be synthesized with a high degree of structural freedom, for suspending SWCNTs in aqueous solution. Taking advantage of the high molecular precision of these PEG-dendrons, we show that precise differences in the chemical structure of the hydrophobic end groups of the dendrons can be used to control the interactions of the amphiphiles with the SWCNT surface. These interactions can be directly related to differences in the intrinsic near-infrared fluorescence emission of the various chiralities in a SWCNT sample. Utilizing the susceptibility of the PEG-dendrons toward enzymatic degradation, we demonstrate the ability to monitor enzymatic activity through changes in the SWCNT fluorescent signal. These findings pave the way for a rational design of functional SWCNTs, which can be used for optical sensing of enzymatic activity in the near-infrared spectral range.

Optical Nanosensors for Real-Time Feedback on Insulin Secretion by ß-Cells.

Quantification of insulin is essential for diabetes research in general, and for the study of pancreatic ß-cell function in particular. Herein, fluorescent single-walled carbon nanotubes (SWCNT) are used for the recognition and real-time quantification of insulin. Two approaches for rendering the SWCNT sensors for insulin are compared, using surface functionalization with either a natural insulin aptamer with known affinity to insulin, or a synthetic lipid-poly(ethylene glycol) (PEG) (C16 -PEG(2000Da)-Ceramide), both of which show a modulation of the emitted fluorescence in response to insulin. Although the PEGylated-lipid has no prior affinity to insulin, the response of C16 -PEG(2000Da)-Ceramide-SWCNTs to insulin is more stable and reproducible compared to the insulin aptamer-SWCNTs. The SWCNT sensors successfully detect insulin secreted by ß-cells within the complex environment of the conditioned media. The insulin is quantified by comparing the SWCNTs fluorescence response to a standard calibration curve, and the results are found to be in agreement with an enzyme-linked immunosorbent assay. This novel analytical tool for real time quantification of insulin secreted by ß-cells provides new opportunities for rapid assessment of ß-cell function, with the ability to push forward many aspects of diabetes research.

Dissipative Constitutional Dynamic Networks for Tunable Transient Responses and Catalytic Functions.

Following the significance of dissipative, out-of-equilibrium biological processes controlling living systems, we introduce nucleic acid-based dissipative constitutional dynamic networks (CDNs) that exhibit tunable transient composition changes of the networks dictated by auxiliary fuel strands. CDN "X" composed of four equilibrated nucleic acid constituents, AA', AB', BA', and BB', and the accompanying "dormant" structures T1L1 and T2L2 and nicking enzyme Nt.BbvCI, undergoes dissipative orthogonal transitions to CDN "Y" and back or to CDN "Z" and back. In the presence of the fuel strand L1' or L2', the displacement of the respective "dormant" structure releases the trigger T1 or T2 that activates the reconfiguration of CDN "X" to CDN "Y" or CDN "X" to CDN "Z". The generated duplex L1L1' or L2L2' is designed to be nicked by Nt.BbvCI, leading to the regeneration of L1 or L2 that rebinds to T1 or T2, resulting in the dissipative cyclic recovery of CDN "X". Kinetic simulations of the dissipative processes allow us to predict the dissipative behavior of the systems under different auxiliary conditions. Subjecting CDN "X" to altering sets of the fuel strands L1' and L2' yields programmed reconfiguration patterns of dissipative reaction cycles. By engineering functional nucleic acid tethers on the constituents and the triggering strands, orthogonal dissipative emerging catalytic transformations dictated by the dissipative CDNs are demonstrated.

Stiffness-switchable DNA-based constitutional dynamic network hydrogels for self-healing and matrix-guided controlled chemical processes.

Constitutional dynamic networks (CDNs) attract interest as signal-triggered reconfigurable systems mimicking natural networks. The application of CDNs to control material properties is, however, a major challenge. Here we report on the design of a CDN consisting of four toehold-modified constituents, two of which act as bidentate units for chain-elongating, while the other two form a tetradentate structure acting as a crosslinking unit. Their hybridization yields a hydrogel of medium stiffness controlled by the balance between bidentate and tetradentate units. Stabilization of the tetradentate constituent by an auxiliary effector up-regulates the crosslinking unit, yielding a high-stiffness hydrogel. Conversely, stabilization of one of the bidentate constituents by an orthogonal effector enriches the chain-elongation units leading to a low-stiffness hydrogel. Using appropriate counter effectors, the hydrogels are reversibly switched across low-, medium- and high-stiffness states. The hydrogels are used to develop self-healing and controlled drug-release matrices and functional materials for operating biocatalytic cascades.

Redox-Switchable Binding Properties of the ATP-Aptamer.

In this study, we report on a redox-controllable and reversible complete "ON"/"OFF"-switchable aptamer binding to ATP. A series of methylene blue-modified ATP-aptamers was synthesized, revealing improved binding affinities toward ATP as compared to the nonmodified aptamer. These binding affinities were dependent on the conjugation site of the redox label on the aptamer scaffold. Importantly, we find that the oxidized methylene blue-modified aptamers bind to ATP with micromolar affinity, while the reduced form lacks binding affinity toward ATP, resulting in an unprecedented complete "ON"/"OFF" redox-controllable aptamer switch. We demonstrate the cyclic "ON"/"OFF" binding of ATP to the methylene blue-functionalized aptamer through cyclic oxidation and reduction of the redox label using both chemical and electrochemical means. Molecular dynamics and docking simulations were performed to account for the redox-switchable properties of the conjugated aptamers and to rationalize the enhanced binding affinities of the different aptamer designs.

Molecularly Imprinted Sites Translate into Macroscopic Shape-Memory Properties of Hydrogels.

The polymerization of acrylamide, dopamine methacrylamide, and bis-acrylamide in the presence of one of the electron acceptors, N,N'-dimethyl-4,4'-bipyridinium, (1), N,N'-dimethylbipyridinium-4,4'-ethylene, (2), or bipyridinium dithienylethene, (3), yields hydrogel matrices of high stiffness that are cooperatively cross-linked by bis-acrylamide and electron donor (dopamine)-acceptor complexes. Washing off the diffusional electron acceptor units yields molecularly imprinted matrices of lower stiffness, stabilized only by the bis-acrylamide bridges that include specific binding sites for the selective association of the electron acceptor (1), (2), or (3). These imprinted hydrogel matrices show selective recovery of the stiff properties upon binding the respective electron acceptor units to the imprinted sites. The control over the stiffness properties enables the development of shape-memory, molecularly imprinted hydrogels and stiffness-based sensors. The results show how molecularly imprinted sites translate into macroscopic shape-memory properties of hydrogels.

Evolution of Nucleic-Acid-Based Constitutional Dynamic Networks Revealing Adaptive and Emergent Functions.

The evolution of networks is a fundamental unresolved issue in developing the area of systems chemistry. We introduce a versatile rewiring mechanism that leads to the emergence of nucleic-acid-based constitutional dynamic networks (CDNs). A two-component constituent AA' functionalized with a Mg2+ -ion-dependent DNAzyme activator unit forms a complex with an intact hairpin HBB' composed of B and B' sequences. Cleavage of HBB' leads to the two-component constituent BB', and its rewiring with AA' yields CDN X composed of the equilibrated constituents AA', AB', BA', and BB'. In analogy, subjecting AA' to an intact hairpin HCC' leads to the formation of CDN Y consisting of AA', AC', CA', and CC'. Subjecting AA' to the mixture of HBB' and HCC' evolves the [3×3] CDN Z, composed of nine constituents, thus demonstrating hierarchical adaptive properties. Furthermore, the DNAzyme units associated with the constituents are applied to tailor emerging catalytic functions from the different CDNs.

DNA-Based Hydrogels Loaded with Au Nanoparticles or Au Nanorods: Thermoresponsive Plasmonic Matrices for Shape-Memory, Self-Healing, Controlled Release, and Mechanical Applications.

Gold nanoparticles (AuNPs) or gold nanorods (AuNRs) are loaded in polyacrylamide hydrogels cooperatively cross-linked by bis-acrylamide and nucleic acid duplexes or boronate ester-glucosamine and nucleic acid duplexes. The thermoplasmonic properties of AuNPs and AuNRs are used to control the stiffness of the hydrogels. The irradiation of the AuNP-loaded (λ = 532 nm) or the AuNR-loaded (λ = 808 nm) hydrogels leads to thermoplasmonic heating of the hydrogels, the dehybridization of the DNA duplexes, and the formation of hydrogels with lower stiffness. By ON/OFF irradiation, the hydrogels are switched between low- and high-stiffness states. The reversible control over the stiffness properties of the hydrogels is used to develop shape-memory hydrogels and self-healing soft materials and to tailor thermoplasmonic switchable drug release. In addition, by designing bilayer composites of AuNP- and AuNR-loaded hydrogels, a reversible thermoplasmonic, light-induced bending is demonstrated, where the bending direction is controlled by the stress generated in the respective bilayer composite.

Chemical and photochemical DNA "gears" reversibly control stiffness, shape-memory, self-healing and controlled release properties of polyacrylamide hydrogels.

A new class of stimuli-responsive DNA-based polyacrylamide hydrogels is described. They consist of glucosamine-boronate ester-crosslinked polyacrylamide chains being cooperatively bridged by stimuli-responsive nucleic acids. The triggered closure and dissociation of the stimuli-responsive units lead to switchable stiffness properties of the hydrogel. One hydrogel includes glucosamine-boronate esters and K+-ion-stabilized G-quadruplex units as cooperative crosslinkers. The hydrogel bridged by the two motifs reveals high stiffness, whereas the separation of the G-quadruplex bridges by 18-crown-6-ether yields a low stiffness hydrogel. By cyclic treatment of the hydrogel with K+-ions and 18-crown-6-ether, it is reversibly cycled between high and low stiffness states. The second system involves a photo-responsive hydrogel that reveals light-induced switchable stiffness functions. The polyacrylamide chains are cooperatively crosslinked by glucosamine-boronate esters and duplex nucleic acid bridges stabilized by trans-azobenzene intercalator units. The resulting hydrogel reveals high stiffness. Photoisomerization of the trans-azobenzene units to the cis-azobenzene states results in the separation of the duplex nucleic acid bridges and the formation of a low stiffness hydrogel. The control over the stiffness properties of the hydrogel matrices by means of K+-ions/crown ether or photoisomerizable trans-azobenzene/cis-azobenzene units is used to develop shape-memory, self-healing, and controlled drug-release hydrogel materials.

Consecutive feedback-driven constitutional dynamic networks.

Cellular transformations are driven by environmentally triggered complex dynamic networks, which include signal-triggered feedback processes, cascaded reactions, and switchable transformations. We apply the structural and functional information encoded in the sequences of nucleic acids to construct signal-triggered constitutional dynamic networks (CDNs) that mimic the functions of natural networks. Using predesigned hairpin structures as triggers, the network generates functional strands, which stabilize one or the other of the constituents of the network, leading to feedback-driven reconfiguration and time-dependent equilibration of the networks. Using structurally designed hairpins, positive-feedback or negative-feedback mechanisms operated by the CDNs are demonstrated. With two predesigned hairpins, the coupled consecutive operations of negative/positive- or positive/positive- feedback cascades are accomplished. The time-dependent composition changes of the networks are well reproduced by chemical kinetics simulations that provide predictive behaviors of the network, under variable auxiliary conditions. Beyond mimicking natural network properties and functions by means of the synthetic nucleic-acid-based CDNs, the systems introduce versatile perspectives for the design of amplified sensors (sensing of miRNA-376a) and the development of logic gate circuits.

Nucleoapzymes: catalyst-aptamer conjugates as enzyme-mimicking structures.

The conjugation of catalytic sites to sequence-specific, ligand-binding nucleic acid aptamers yields functional catalytic ensembles mimicking the catalytic/binding properties of native enzymes. These catalyst-aptamer conjugates termed 'nucleoapzymes' reveal structural diversity, and thus, vary in their catalytic activity, due to the different modes of conjugation of the catalytic units to the nucleic acid aptamer scaffold. The concept of nucleoapzymes is introduced with the assembly of a set of catalysts consisting of the hemin/G-quadruplex DNAzyme (hGQ) conjugated to the dopamine aptamer. The nucleoapzymes catalyze the oxidation of dopamine by H2O2 to yield aminochrome. The catalytic processes are controlled by the structures of the nucleoapzymes, and chiroselective oxidation of l-DOPA and d-DOPA by the nucleoapzymes is demonstrated. In addition, the conjugation of a Fe(III)-terpyridine complex to the dopamine aptamer and of a bis-Zn(II)-pyridyl-salen-type complex to the ATP-aptamer yields hybrid nucleoapzymes (conjugates where the catalytic site is not a biomolecule) that catalyze the oxidation of dopamine to aminochrome by H2O2 and the hydrolysis of ATP to ADP, respectively. Variable, structure-controlled catalytic activities of the different nucleoapzymes are demonstrated. Molecular dynamic simulations are applied to rationalize the structure-catalytic function relationships of the different nucleoapzymes. The challenges and perspectives of the research field are discussed.

Intercommunication of DNA-Based Constitutional Dynamic Networks.

Intercommunication between dynamic chemical networks plays a major role in cellular transformations. Inspired by nature, we introduce the intercommunication between two constitutional dynamic networks, CDNs, "S" and "T" composed, each, of four equilibrated supramolecular constituents AA', AB', BA', and BB', and of CC', CD', DC', and DD', respectively. Each of the constituents is conjugated to a Mg2+-ion-dependent DNAzyme unit that acts as a reporter element for the concentration of the respective constituent via the catalyzed cleavage of the fluorophore/quencher-functionalized substrate associated with the respective DNAzyme reporter. Also, constituents BB' (in CDN "S") and CC' (in CDN "T") include Mg2+-ion-dependent DNAzymes acting as activator units for generating triggering signals between the networks. Subjecting CDNs "S" and "T" to the catalytically cleavable hairpin trigger Hdd' or Haa', respectively, yields input strands that intercommunicate the CDNs by affecting the time-dependent re-equilibration of the constituents of the counter CDN without affecting the dynamic equilibrium of the constituents of the CDN that generates the triggering strands. Treatment of CDNs "S" and "T" with hairpins Hdd' and Haa' (or Hba'), respectively, stimulates autonomous positive/positive or positive/negative feedback to the programmed time-dependent up-regulation or down-regulation of the equilibrated constituents in the two CDNs.

Application of DNA Machineries for the Barcode Patterned Detection of Genes or Proteins.

The study introduces an analytical platform for the detection of genes or aptamer-ligand complexes by nucleic acid barcode patterns generated by DNA machineries. The DNA machineries consist of nucleic acid scaffolds that include specific recognition sites for the different genes or aptamer-ligand analytes. The binding of the analytes to the scaffolds initiate, in the presence of the nucleotide mixture, a cyclic polymerization/nicking machinery that yields displaced strands of variable lengths. The electrophoretic separation of the resulting strands provides barcode patterns for the specific detection of the different analytes. Mixtures of DNA machineries that yield, upon sensing of different genes (or aptamer ligands), one-, two-, or three-band barcode patterns are described. The combination of nucleic acid scaffolds acting, in the presence of polymerase/nicking enzyme and nucleotide mixture, as DNA machineries, that generate multiband barcode patterns provide an analytical platform for the detection of an individual gene out of many possible genes. The diversity of genes (or other analytes) that can be analyzed by the DNA machineries and the barcode patterned imaging is given by the Pascal's triangle. As a proof-of-concept, the detection of one of six genes, that is, TP53, Werner syndrome, Tay-Sachs normal gene, BRCA1, Tay-Sachs mutant gene, and cystic fibrosis disorder gene by six two-band barcode patterns is demonstrated. The advantages and limitations of the detection of analytes by polymerase/nicking DNA machineries that yield barcode patterns as imaging readout signals are discussed.

Gold Nanorods as Plasmonic Sensors for Particle Diffusion.

Plasmonic gold nanoparticles are normally used as sensor to detect analytes permanently bound to their surface. If the interaction between the analyte and the nanosensor surface is negligible, it only diffuses through the sensor's sensing volume, causing a small temporal shift of the plasmon resonance position. By using a very sensitive and fast detection scheme, we are able to detect these small fluctuations in the plasmon resonance. With the help of a theoretical model consistent with our detection geometry, we determine the analyte's diffusion coefficient. The method is verified by observing the trends upon changing diffusor size and medium viscosity, and the diffusion coefficients obtained were found to reflect reduced diffusion close to a solid interface. Our method, which we refer to as NanoPCS (for nanoscale plasmon correlation spectroscopy), is of practical importance for any application involving the diffusion of analytes close to nanoparticles.