DNA-functionalized Nanoparticles Research Articles

Naked-Eye LAMP Assay of M. tuberculosis in Sputum by In Situ Au Nanoprobe Identification: For the In Vitro Diagnostics of Tuberculosis.

In spite of the development of diagnostic tests for Mycobacterium tuberculosis (M. tuberculosis), the etiological agent of tuberculosis, there has remained a gap between the established methods and an easily accessible diagnostic test, particularly in developing and resource-poor areas. By combining isothermal amplification of IS6110 as the target gene and recognition by DNA-functionalized Au nanoparticles (DNA-AuNPs), we develop a colorimetric LAMP assay for convenient in vitro diagnostics of tuberculosis with a quick (≤50 min) "yes" or "no" readout. The DNA-AuNPs not only tolerate the interference in the complex LAMP system but also afford in situ identification of the amplicon, allowing for colloidal dispersion via steric effect depending on DNA grafting density. The target-induced stabilization and red appearance of the DNA-AuNPs contrast with the occurrence of gray aggregates in a negative sample. Furthermore, the DNA-AuNPs demonstrate excellent performance after long-term (≥7 months) storage while preserving the unsacrificed sensitivity. The high specificity of the DNA-AuNPs is further demonstrated in the naked-eye LAMP assay of M. tuberculosis in patients' sputum samples. Given the rapidity, cost-effectiveness, and instrument-free characteristics, the naked-eye LAMP assay is particularly beneficial for tuberculosis diagnosis in urgent situations and resource-limited settings and can potentially expedite patient care and treatment initiation.

Biomolecular interactions on densely coated nanoparticles: a single-molecule perspective.

DNA-modified gold nanoparticles (AuNPs) play a pivotal role in bio-nanotechnology, driving advancements in bio-sensing, bio-imaging, and drug delivery. Synthetic protocols have focused on maximizing the receptor density on particles by fine-tuning chemical conditions, particularly for DNA. Despite their significance, the understanding of hybridization kinetics on functionalized AuNPs is lacking, particularly how this kinetics depends on DNA density and to what extent it varies from particle-to-particle. This study explores the molecular mechanisms of DNA hybridization on densely coated AuNPs by employing a combination of single-molecule microscopy and coarse-grained molecular dynamics simulations providing a quantification of the molecular rate constants for single particles. Our findings demonstrate that DNA receptor density and the presence of spacer strands profoundly impact association kinetics, with short spacers enhancing association rates by up to ∼15-fold. In contrast, dissociation kinetics are largely unaffected by receptor density within the studied range. Single-particle analysis directly reveals variability in hybridization kinetics, which is analyzed in terms of intra- and inter-particle heterogeneity. A coarse-grained DNA model that quantifies hybridization kinetics on densely coated surfaces further corroborates our experimental results, additionally shedding light on how transient base pairing within the DNA coating influences kinetics. This integrated approach underscores the value of single-molecule studies and simulations for understanding DNA dynamics on densely coated nanoparticle surfaces, offering guidance for designing DNA-functionalized nanoparticles in sensor applications.

Transformations in crystals of DNA-functionalized nanoparticles by electrolytes.

Colloidal crystals have applications in water treatments, including water purification and desalination technologies. It is, therefore, important to understand the interactions between colloids as a function of electrolyte concentration. We study the assembly of DNA-grafted gold nanoparticles immersed in concentrated electrolyte solutions. Increasing the concentration of divalent Ca2+ ions leads to the condensation of nanoparticles into face-centered-cubic (FCC) crystals at low electrolyte concentrations. As the electrolyte concentration increases, the system undergoes a phase change to body-centered-cubic (BCC) crystals. This phase change occurs as the interparticle distance decreases. Molecular dynamics analysis suggests that the interparticle interactions change from strongly repulsive to short-range attractive as the divalent-electrolyte concentration increases. A thermodynamic analysis suggests that increasing the salt concentration leads to significant dehydration of the nanoparticle environment. We conjecture that the intercolloid attractive interactions and dehydrated states favour the BCC structure. Our results gain insight into salting out of colloids such as proteins as the concentration of salt increases in the solution.

Regulating phase behavior of nanoparticle assemblies through engineering of DNA-mediated isotropic interactions.

Self-assembly of isotropically interacting particles into desired crystal structures could allow for creating designed functional materials via simple synthetic means. However, the ability to use isotropic particles to assemble different crystal types remains challenging, especially for generating low-coordinated crystal structures. Here, we demonstrate that isotropic pairwise interparticle interactions can be rationally tuned through the design of DNA shells in a range that allows transition from common, high-coordinated FCC-CuAu and BCC-CsCl lattices, to more exotic symmetries for spherical particles such as the SC-NaCl lattice and to low-coordinated crystal structures (i.e., cubic diamond, open honeycomb). The combination of computational and experimental approaches reveals such a design strategy using DNA-functionalized nanoparticles and successfully demonstrates the realization of BCC-CsCl, SC-NaCl, and a weakly ordered cubic diamond phase. The study reveals the phase behavior of isotropic nanoparticles for DNA-shell tunable interaction, which, due to the ease of synthesis is promising for the practical realization of non-close-packed lattices.

Designing Multimodal ON-OFF Nanoswitches of DNA-Functionalized Nanoparticles by Stimuli-Responsive Polymers.

It is a challenging task to realize highly reversible ON-OFF nanoswitches over a wide range of temperatures, which emerge as a versatile toolbox for use in nanobiotechnology. Herein, nanoparticles (NPs) bifunctionalized by DNA strands and stimuli-responsive polymers are proposed to construct multimodal ON-OFF nanoswitches by the coarse-grained model. The successful achievement of multimodal ON-OFF nanoswitches for bifunctionalized NPs at lower temperatures is attributed to the synergistic effects of the contraction and expansion configurations of stimuli-responsive polymers, combined with the hybridization-dehybridization event of DNA strands. Importantly, our simulations isolate the conditions of programmable self-assembly of bifunctionalized NPs to realize the multimodal ON-OFF nanoswitches by the changes of temperature and chain rigidity. In addition, it is found that the bifunctionalized NPs in the ON state display anisotropic and patchy features due to an introduction of stimuli-responsive polymers. Our simulation results provide fundamental insights on qualitative predictions of ON/OFF states of DNA-based NPs, which can aid in realizing a set of ON-OFF nanoswitches by the rational design of functionalization molecules.

Hints of Growth Mechanism Left in Supercrystals.

Supercrystals of DNA-functionalized nanoparticles are visualized in three dimensions using X-ray ptychographic tomography, and their reciprocal spaces are mapped with small-angle X-ray scattering in order to better understand their internal defect structures. X-ray ptychographic tomography reveals various types of defects in an assembly that otherwise exhibits a single crystalline diffraction pattern. On average, supercrystals composed of smaller nanoparticles are smaller in size than supercrystals composed of larger particles. Additionally, supercrystals composed of small nanoparticles are typically aggregated into larger "necklace-like" structures. Within these larger structures, some but not all pairs of connected domains are coherent in their relative orientations. In contrast, supercrystals composed of larger nanoparticles with longer DNA ligands typically form faceted crystals. The combination of these two complementary X-ray techniques reveals that the crystalline assemblies grow by aggregation of smaller assemblies followed by rearrangement of nanoparticles.

Shape-induced crystallization of binary DNA-functionalized nanocubes.

Leveraging the anisotropic shape of DNA-functionalized nanoparticles holds potential for shape-directed crystallization of a wide collection of superlattice structures. Using coarse-grained molecular dynamics simulations, we study the self-assembly of a binary mixture of cubic gold nanoparticles, which are functionalized by complementary DNA strands. We observe the spontaneous self-assembly of simple cubic (SC), plastic body-centered tetragonal (pBCT), and compositionally disordered plastic body-centered tetragonal (d-pBCT) phases due to hybridization of the DNA strands. We systematically investigate the effect of length, grafting density, as well as rigidity of the DNA strands on the self-assembly behavior of cubic nanoparticles. We measure the potential of mean force between DNA-functionalized nanocubes for varying rigidity of the DNA strands and DNA lengths. Using free-energy calculations, we find that longer and flexible DNA strands can lead to a phase transformation from SC to the pBCT phase due to a gain in entropy arising from the orientational degrees of freedom of the nanocubes in the pBCT phase. Our results may serve as a guide for self-assembly experiments on DNA-functionalized cubic nanoparticles.

Catalytic-assembly of programmable atom equivalents

Escape from metastable states in self-assembly of colloids is an intractable problem. Unlike the commonly adopted approach of thermal annealing, the recently developed enthalpy-mediated strategy provided a different option to address this dilemma in a dynamically controllable manner at room temperature. However, it required a complex catalytic-assembly DNA strand-displacement circuitry to mediate interaction between multiple components. In this work, we present a simple but effective way to achieve catalytic-assembly of DNA-functionalized colloidal nanoparticles, i.e., programmable atom equivalents, in a far-from-equilibrium system. A removable molecule named "catassembler" that acts as a catalyst was employed to rectify imperfect linkages and help the system escape from metastability without affecting the assembled framework. Notably, catalytic efficiency of the catassembler can be effectively improved by changing the seesaw catassembler in toehold length design or numbers of the repeat units. Leveraging this tractable catalytic-assembly approach, different ordered architectures were easily produced by directly mixing all reactants, as in chemical reactions. By switching bonding identities, solid-solid phase transformations between different colloidal crystals were achieved. This work opens up an avenue for programming colloid assembly in a far-from-equilibrium system.

Self-Assembling Systems for Optical Out-of-Plane Coupling Devices.

Micromirrors are used in integrated photonics to couple extraplanar light into the planar structure of a device by redirecting light via specular reflection. Compared with grating or prism-based couplers, micromirrors allow for coupling of light over a broader range of wavelengths, provided that the micromirror is fabricated with a specific 3D shape to ensure proper reflection angles. In principle, self-assembly methods could enable reliable, parallelizable fabrication of such devices with a high degree of precision by designing self-assembling components that produce the desired microscale geometry as their thermodynamic products. In this work, we use DNA-functionalized nanoparticles to assemble faceted crystallites with predetermined crystal shapes, and demonstrate with microscale retroreflectance measurements that these self-assembled nanoparticle arrays do indeed behave like optically flat mirrors. Furthermore, we show that the tilt angle of the micromirrors can be intentionally controlled by altering the crystallographic symmetry and preferred crystal orientations as a function of the self-assembly process, thereby altering the resulting specular angle in a programmable manner. Measurements of optical coupling from normal incidence into the substrate plane via an optical fiber confirm that the faceted structures can function as optical out-of-plane coupling devices, and coating these structures with reflective materials allows for high efficiency of light reflection in addition to the angular control. Together, these experiments demonstrate how self-assembled nanoparticle materials can be used to generate optically relevant architectures, enabling a significant step in the development of self-assembly as a materials fabrication tool for integrated optical devices.

Enhancing Crystallization of DNA-Functionalized Nanoparticles by Polymer Chains

Enhancing Crystallization of DNA-Functionalized Nanoparticles by Polymer Chains

Symmetry-Breaking Dendrimer Synthons in Colloidal Crystal Engineering with DNA

Breaking symmetry in colloidal crystals is challenging due to the inherent chemical and structural isotropy of many nanoscale building blocks. If a non-particle component could be used to anisotropically encode such building blocks with orthogonal recognition properties, one could expand the scope of structural and compositional possibilities of colloidal crystals beyond what is thus far possible with purely particle-based systems. Herein, we report the synthesis and characterization of novel DNA dendrimers that function as symmetry-breaking synthons, capable of programming anisotropic and orthogonal interactions within colloidal crystals. When the DNA dendrimers have identical sticky ends, they hybridize with DNA-functionalized nanoparticles to yield three distinct colloidal crystals, dictated by dendrimer size, including a structure not previously reported in the field of colloidal crystal engineering, Si2Sr. When used as symmetry-breaking synthons (when the sticky ends deliberately consist of orthogonal sequences), the synthesis of binary and ternary colloidal alloys with structures that can only be realized through directional interactions is possible. Furthermore, by modulating the extent of shape anisotropy within the DNA dendrimers, the local distribution of the nanoparticles within the crystals can be directed.

Aggregation of amphiphilic nanocubes in equilibrium and under shear.

We investigate the self-assembly of amphiphilic nanocubes into finite-sized aggregates in equilibrium and under shear, using molecular dynamics (MD) simulations and kinetic Monte Carlo (KMC) calculations. These patchy nanoparticles combine both interaction and shape anisotropy, making them valuable models for studying folded proteins and DNA-functionalized nanoparticles. The nanocubes can self-assemble into various finite-sized aggregates ranging from rods to self-avoiding random walks, depending on the number and placement of the hydrophobic faces. Our study focuses on suspensions containing multi- and one-patch cubes, with their ratio systematically varied. When the binding energy is comparable to the thermal energy, the aggregates consist of only few cubes that spontaneously associate/dissociate. However, highly stable aggregates emerge when the binding energy exceeds the thermal energy. Generally, the mean aggregation number of the self-assembled clusters increases with the number of hydrophobic faces and decreases with increasing fraction of one-patch cubes. In sheared suspensions, the more frequent collisions between nanocube clusters lead to faster aggregation dynamics but also to smaller terminal steady-state mean cluster sizes. The results from the MD and KMC simulations are in excellent agreement for all investigated two-patch cases, whereas the three-patch cubes form systematically smaller clusters in the MD simulations compared to the KMC calculations due to finite-size effects and slow aggregation kinetics. By analyzing the rate kernels, we are able to identify the primary mechanisms responsible for (shear-induced) cluster growth and breakup. This understanding allows us to tune nanoparticle and process parameters to achieve desired cluster sizes and shapes.

Erythrocyte membrane-camouflaged DNA-functionalized upconversion nanoparticles for tumor-targeted chemotherapy and immunotherapy.

A synergistic combination of treatment with immunogenic cell death (ICD) inducers and immunoadjuvants may be a practical way to boost the anticancer response and successfully induce an immune response. The use of HR@UCNPs/CpG-Apt/DOX, new biomimetic drug delivery nanoparticles generated to combat breast cancer, is reported here as a unique strategy to produce immunogenicity and boost cancer immunotherapy. HR@UCNPs/CpG-Apt/DOX (HR-UCAD) consists of two parts. The core is composed of an immunoadjuvant CpG (a toll-like receptor 9 agonist) fused with a dendritic cell-specific aptamer sequence (CpG-Apt) to decorate upconversion nanoparticles (UCNPs) with the successful intercalation of doxorubicin (DOX) into the consecutive base pairs of Apt-CpG to construct an immune nanodrug UCNPs@CpG-Apt/DOX. The targeting molecule hyaluronic acid (HA) was inserted into a red blood cell membrane (RBCm) to form the shell (HR). HR-UCAD possessed a strong capacity to specifically induce ICD. Following DOX-induced ICD of cancer cells, sufficient exposure to tumor antigens and UCNPs@CpG-Apt (UCA) activated the tumor-specific immune response and reversed the immunosuppressive tumor microenvironment. In addition, HR-UCAD has good biocompatibility and increases the active tumor-targeting effect. Furthermore, HR-UCAD exhibits excellent near-infrared upconversion luminescence emission at 804 nm under irradiation with a 980 nm laser, which has great potential in biomedical imaging. Thus, the RBCm-camouflaged drug delivery system is a promising targeted chemotherapy and immunotherapy nanocomplex that could be used for effective targeted breast cancer treatment.

Optical control of hierarchical DNA-functionalized nanoparticle self-assembly on 2D surfaces

We investigated the effect of light on the DNA-driven self-assembly to form hierarchical patterns on two dimensional surfaces. We specifically focused on the self-assembly of DNA-functionalized quantum dots (QDs) onto DNA-functionalized glass substrate while illuminating the surface with a laser during coating process. The region illuminated with a green laser remained uncoated with red-emitting QDs while the not-illuminated region was successfully coated. Next, a red laser whose light cannot be absorbed by the red-emitting QDs did not avoid the DNA-driven self-assembly of the QDs onto glass substrate. Additionally, we demonstrated that silica nanoparticles that had been functionalized with DNA and are not able to absorb the light in the visible regime were again coated on the surface while being exposed to a green laser. These results prove that the absorption of the light is responsible for controlling the binding-unbinding process of QDs. Finally, we added DNA-functionalized green-emitting QDs onto the area that was not coated with red-emitting QDs under the green laser exposure. We observed successful coating of previously uncoated regions with the green QDs. This revealed the viability of our technique for building up hierarchical structures without using, sophisticated, or resource-intensive microfabrication methods.

Magnetic Relaxation Switch Sensor Based on Magnetophoresis and "T-Hg(II)-T" Signal Amplification.

In this study, we designed a magnetic relaxation switch (MRS) sensor combined with magnetophoresis technology (MS-MRS), which helps solve the problems of traditional MRS sensors. The sensor is based on a new combined magnet and is composed of small magnetic blocks and iron sheets that can rapidly separate magnetic nanoparticles of different sizes within 5 min. The MS-MRS sensor consists of aptamer-functionalized magnetic nanoparticles (diameter: 200 nm) (MNP200-Apt), complementary DNA-functionalized magnetic nanoparticles (diameter: 20 nm) (MNP20-cDNA), and a combined magnet ("M2" magnet). The MNP200-Apt probe could be separated by an "M2" magnet but the MNP20-cDNA probe could not. To further improve the sensitivity of the sensor, we successfully constructed an MS-MRS-Hg sensor based on the "T-Hg(II)-T" specific recognition that aggregated MNP20-cDNA probes to amplify the relaxation signal. The detection working range of the MS-MRS sensor is 0.5-100 ng/mL and that of the MS-MRS-Hg sensor is 0.05-100 ng/mL. Their limit of detection (LOD) values are 0.15 and 0.01 ng/mL, respectively. The relative recoveries of the MS-MRS and MS-MRS-Hg sensors are 95.2-119.5% and 93.1-113.1%, respectively. These results indicate that the proposed sensors have a high accuracy level.

DNA-Functionalized Silver Nanoparticles in an Alcoholic Solvent for Environment-Dictated Multimodal Actuation

DNA-Functionalized Silver Nanoparticles in an Alcoholic Solvent for Environment-Dictated Multimodal Actuation

Confined Growth of DNA-Assembled Superlattice Films.

We study the assembly of DNA-functionalized nanocubes under lateral confinement in microscale square trenches on a DNA-functionalized substrate. Microfocus small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) are used to characterize the superlattices (SLs). The results indicate that nanocubes form simple-cubic SLs with square-prism morphology and a (100) out-of-plane orientation to maximize DNA bonding. In-plane, SLs align with the template, exposing their {100} side facets, and the degree of alignment depends on trench size. Interestingly, the distribution of in-plane orientations determined from SAXS and SEM do not agree, indicating that the internal and external structures of the SLs differ. To understand this discrepancy, X-ray ptychography is employed to image the internal structures of the SLs, revealing that SLs which appear to be single-crystalline in SEM may have subsurface grain boundaries, depending on trench size. SEM reveals that the SLs grow via nucleation and growth of randomly oriented domains, which then coalesce; this mechanism explains the observed dependence of alignment and defect structure on size. Interestingly, crystallization occurs via an unusual growth mode, whereby continuous SL layers grow on top of several misoriented islands. Overall, this work elucidates the effect of lateral confinement on the crystallization of DNA-functionalized nanoparticles and shows how X-ray ptychography can be used to gain insight into nanoparticle crystallization.

Probing and modulating the interactions of the DNAzyme with DNA-functionalized nanoparticles

DNA-functionalized gold nanoparticles are one of the most versatile bionanomaterials for biomedical and clinical diagnosis. Herein, we discovered that the performance of DNAzyme cleaving the substrate is highly related to its length. This intriguing phenomenon only appears at the interfaces of DNA-functionalized gold nanoparticles. We systematically investigated the causes of this phenomenon. We conjectured that the DNAzyme with extended nucleotides that do not match its substrate strand is vulnerable to non-specific adsorption, electrostatic repulsion, and steric hindrance. Based on our improved understanding of this phenomenon, we have successfully developed a highly sensitive and specific amplifiable biosensor to detect human apurinic/apyrimidinic endonuclease 1.

Upconversion Nanoprobes Based on a Horseradish Peroxidase-Regulated Dual-Mode Strategy for the Ultrasensitive Detection of Staphylococcus aureus in Meat.

Staphylococcus aureus (S. aureus) is one of the foodborne pathogens that can cause infectious diseases and food poisoning. Herein, colorimetric and fluorescent dual-mode nanoprobes were developed for ultrasensitive detection of S. aureus to immediately respond to public health emergencies, reduce false positives, and improve measurement accuracy and persuasiveness. The nanoprobe consists of aptamer-labeled magnetic nanoparticles (apt-MNPs) as the capture signal probe and horseradish peroxidase and complementary DNA-functionalized upconversion nanoparticles (HRP-UCNPs-cDNA) as the chromogenic signal probe. In the absence of S. aureus, the probe forms an immune complex through base complementation with an observable signal. When S. aureus is introduced to this system, it preferentially binds to the apt-MNPs, releasing HRP-UCNPs-cDNA from the apt-MNPs and restoring the chromogenic probe signal. Under optimum conditions, an ultrasensitive assay of S. aureus was obtained, with limits of detection of 22 CFU mL-1 for fluorescence and 20 CFU mL-1 for colorimetry in a linear range of 56-5.6 × 106 CFU mL-1. Additionally, the standard plate counting method confirmed the reliability and accuracy of the established nanoprobe with an insignificant difference. Hence, the developed dual-mode platform has extensive application prospects for speedy and specific determination of S. aureus in meat.

Formulation of DNA Nanocomposites: Towards Functional Materials for Protein Expression.

DNA hydrogels are an emerging class of materials that hold great promise for numerous biotechnological applications, ranging from tissue engineering to targeted drug delivery and cell-free protein synthesis (CFPS). In addition to the molecular programmability of DNA that can be used to instruct biological systems, the formulation of DNA materials, e.g., as bulk hydrogels or microgels, is also relevant for specific applications. To advance the state of knowledge in this research area, the present work explores the scope of a recently developed class of complex DNA nanocomposites, synthesized by RCA polymerization of DNA-functionalized silica nanoparticles (SiNPs) and carbon nanotubes (CNTs). SiNP/CNT–DNA composites were produced as bulk materials and microgels which contained a plasmid with transcribable genetic information for a fluorescent marker protein. Using confocal microscopy and flow cytometry, we found that the materials are very efficiently taken up by various eukaryotic cell lines, which were able to continue dividing while the ingested material was evenly distributed to the daughter cells. However, no expression of the encoded protein occurred within the cells. While the microgels did not induce production of the marker protein even in a CFPS procedure with eukaryotic cell lysate, the bulk composites proved to be efficient templates for CFPS. This work contributes to the understanding of the molecular interactions between DNA composites and the functional cellular machinery. Implications for the use of such materials for CFPS procedures are discussed.