DNA-functionalized Gold Nanoparticles Research Articles

The effects of polyethylene glycol on the nucleation and growth of DNA-functionalized gold nanoparticles crystals

This study presents findings on the crystal growth of DNA-functionalized gold nanoparticles (DNA-AuNPs) in the presence of polyethylene glycol (PEG), serving as an additive. Utilizing optical microscopy, UV–Vis spectroscopy, dynamic light scattering and small angle x-ray scattering, we observed enhanced crystal nucleation and growth facilitated by PEG. This effect is attributed to PEG’s promotion of the overall crystallization process of DNA-AuNPs. Furthermore, we examined the influence of PEG on short-range interactions between DNA-AuNPs, where DNA binding occurs, and long-range interactions in colloidal solutions. The results suggest that PEG plays a crucial role in both promoting crystal nucleation and influencing the interactions between DNA-AuNPs at various distances, shedding light on its potential impact on DNA-AuNP crystal formation.

Sensing of gene expression in live cells using electrical impedance spectroscopy and DNA-functionalized gold nanoparticles

A novel electrical impedance spectroscopy-based method for non-destructive sensing of gene expression in living cells is presented. The approach used takes advantage of the robustness and responsiveness of electrical impedance spectroscopy and the highly specific and selective nature of DNA hybridization. The technique uses electrical impedance spectroscopy and gold nanoparticles functionalized with single-stranded DNA complementary to an mRNA of interest to provide reliable, real-time, and quantifiable data on gene expression in live cells. The system was validated by demonstrating specific detection of the uidA mRNA, which codes for the β-glucuronidase (GUS) enzyme, in Solanum lycopersicum MsK8 cells. Gold nanoparticles were functionalized with single-stranded DNA oligonucleotides consisting of either a sequence complementary to uidA mRNA or an arbitrary sequence. The DNA-functionalized gold nanoparticles were mixed with cell suspensions, allowing the gold nanoparticles to penetrate into the cells. The impedance spectra of suspensions of cells with gold nanoparticles inserted within them were then studied. In suspensions of uidA-expressing cells and gold nanoparticles functionalized with the complementary single-stranded DNA oligonucleotide, the impedance magnitude in the frequency range of interest was significantly higher (146 %) in comparison to all other controls. Due to its highly selective nature, the methodology has the potential to be used as a precision agricultural sensing system for accurate and real-time detection of markers of stress, viral infection, disease, and normal physiological activities.

Differential local ordering of mixed crowders determines effective size and stability of ss-DNA capped gold nanoparticle.

Understanding the influence of a crowded intracellular environment on the structure and solvation of DNA functionalized gold nanoparticles (ss-DNA AuNP) is necessary for designing applications in nanomedicine. In this study, the effect of single (Gly, Ser, Lys) and mixture of amino acids (Gly+Ser, Gly+Lys, Ser+Lys) at crowded concentrations is examined on the structure of the ss-DNA AuNP using molecular dynamics simulations. Using the structural estimators such as pair correlation functions and ligand shell positional fluctuations, the solvation entropy is estimated. Combining the AuNP-solvent interaction energy with the solvation entropy estimates, the free energy of solvation of the AuNP in crowded solutions is computed. The solvation entropy favours the solvation free energy which becomes more favourable for larger effective size of AuNP in crowded solutions relative to that in water. The effective size of AuNP depends on the different propensity of the crowders to adsorb on Au surface, with the smallest crowder (Gly) having the highest propensity inducing the least effective AuNP size as compared to other single crowder solutions. In mixed crowded solutions of amino acids of variable size and chemistry, distinctive local adsorption of the crowders on the gold surface is observed that controls the additive or non-additive crowding effects which govern an increase (in Gly+Ser) or decrease (in Gly+Lys) in nanoparticle effective size respectively. The results shed light into the fundamental understanding of the influence of intracellular crowding on structure of ss-DNA AuNP and plausible employability of crowding as a tool to design programmable self-assembly of functionalized nanoparticles.

Aptamer-functionalized gold nanoparticles for mercury ion detection in a colorimetric assay based on color change time as signal readout.

Auniversal strategy for a rapid colorimetric method for Hg2+ in an aqueous solutionis described. The specific binding of Hg2+ (thymine-Hg2+-thymine) with thiolated DNA-functionalized gold nanoparticles (AuNPs) via Au-S bonds increases the spatial hindrance of the AuNP surface, resulting in a weakened catalytic ability of AuNPs to catalyze the reaction between p-nitrophenol and NaBH4. Therefore, the color change time (CCT) of the solution from yellow to colorless becomes longer. Based on the kinetic curve of absorbance over time measured by a UV spectrometer, the level of Hg2+ in aqueous solutionscan be easily quantified. A linear relationship between CCT and Hg2+ concentration was obtained in the 10-600-nM range with a detection limit of 0.20nM, which is much lower than the limit value (10nM) defined by the US Environmental Protection Agency for Hg2+ indrinking water. The excellent sensitivity comes from CCT as the signal output of the probe, rather than the absorbance or wavelength change used in traditional colorimetric probes as the signal output.

Transformation of nanoparticles via the transition of functional DNAs responsive to pH and vascular endothelial growth factor for photothermal anti-tumor therapy.

This study presents a novel approach for the development of DNA-functionalized gold nanoparticles (AuNPs) capable of responding to disease-specific factors and microenvironmental changes, resulting in an effective anti-tumor effect via photothermal therapy. The AuNPs are decorated with two types of DNAs, an i-motif duplex and a VEGF split aptamer, enabling recognition of changes in pH and VEGF, respectively. The formation of VEGF aptamers on the AuNPs induces their aggregation, further enhanced by VEGF ligands. The resulting changes in the optical properties of the AuNPs are detected by monitoring the absorbance. Upon irradiation with a near-infrared laser, the aggregated AuNPs generate heat due to their thermoplasmonic characteristic, leading to an anti-tumor effect. This study demonstrates the enhanced anti-tumor effect of DNA-functionalized AuNPs via photothermal therapy in both in vitro and in vivo tumor models. These findings suggest the potential utilization of such functional AuNPs for precise disease diagnosis and treatment by detecting disease-related factors in the microenvironment.

Devising a colorimetric aptasensor for detection of basal stem rot-associated RNA marker during early Ganoderma boninense infection in oil palm

Basal stem rot (BSR) is a detrimental disease in oil palm (Elaeis guineensis) due to the host's lack of symptoms during the early phase of infection. Early detection of BSR at non-symptomatic phase is feasible via molecular-based approaches and has become the major interest among researchers. Our previous study had identified a differentially expressed gene encoding auxin efflux carrier component 5-like (EgPIN5) at 11 day-post-inculation of Ganoderma boninense (symptomless early stage of infection) on oil palm seedlings. We further evaluated the gene expression on samples collected from an oil palm plantation at different BSR stages (symptomless, mild and severe). Our findings revealed that EgPIN5 was expressed in symptomless group but was not expressed in either mild or severe BSR groups. Next, we developed a sandwich-type aptamer-based lateral flow assay (LFA) against EgPIN5 RNA by adapting the versatile DNA-functionalized gold nanoparticles for colorimetric detection. The LFA strip demonstrated visible colorimetric signal of EgPIN5 RNA at 12.5 ng/μL with limit of detection at 2.45 ng/μL using a scanning device. Based on our consistent results, we presented EgPIN5 as a diagnostic biomarker for symptomless BSR infection in oil palm. Utilization of LFA assay strip for practical EgPIN5 RNA detection is hoped to encourage establishment of a fast, cheap, and sensitive point-of-care detection system in plant biology.

CRISPR-powered optothermal nanotweezers: Diverse bio-nanoparticle manipulation and single nucleotide identification

Optothermal nanotweezers have emerged as an innovative optical manipulation technique in the past decade, which revolutionized classical optical manipulation by efficiently capturing a broader range of nanoparticles. However, the optothermal temperature field was merely employed for in-situ manipulation of nanoparticles, its potential for identifying bio-nanoparticles remains largely untapped. Hence, based on the synergistic effect of optothermal manipulation and CRIPSR-based bio-detection, we developed CRISPR-powered optothermal nanotweezers (CRONT). Specifically, by harnessing diffusiophoresis and thermo-osmotic flows near the substrate upon optothermal excitation, we successfully trapped and enriched DNA functionalized gold nanoparticles, CRISPR-associated proteins, as well as DNA strands. Remarkably, we built an optothermal scheme for enhancing CRISPR-based single-nucleotide polymorphism (SNP) detection at single molecule level, while also introducing a novel CRISPR methodology for observing nucleotide cleavage. Therefore, this innovative approach has endowed optical tweezers with DNA identification ability in aqueous solution which was unattainable before. With its high specificity and feasibility for in-situ bio-nanoparticle manipulation and identification, CRONT will become a universal tool in point-of-care diagnosis, biophotonics, and bio-nanotechnology.

Towards rational design: Developing universal freezing routes for anchoring DNA onto gold nanoparticles

DNA-functionalized gold nanoparticles (AuNPs), also known as spherical nucleic acids, are widely used in the development of biosensors, resulting in anchoring DNA onto AuNPs being a crucial preparation step and a popular research topic. The latest freeze-anchoring method is a simple and time-saving alternative to traditional salt aging; however, its universal applicability remains limited. In this study, we explored the interfacial interaction between DNA and the AuNP surface and proposed various universal routes for promoting freezing anchoring. Among them, rational design has been considered as the core idea to overcome these limitations, particularly using non-thiolated DNA anchoring, which offers significant advantages such as being unmodified, cost-effective, and easily accessible. We emphasize the importance of sequence structure and preparation process optimization, which mainly considers differences in DNA conformation and electrostatic repulsion. Additionally, the prepared DNA-functionalized AuNPs exhibited complete biological hybridization capability, and the extreme limiting conditions for non-thiolated DNA freeze anchoring were clarified. In summary, this study enhances our understanding of the interfacial relationship between DNA and AuNPs in the freeze-anchoring process and can significantly advance the applications of DNA-functionalized AuNP-based biosensors.

MNAzymes and gold nanoparticles as isothermal signal amplification strategy for visual detection of miRNA

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that are considered a novel emerging class of disease biomarkers in a variety of afflictions. Sensitive detection of miRNA is typically achieved using hybridization-based methods coupled with genetic amplification techniques. Although their sensitivity has improved, amplification techniques often present erroneous results due to their complexity. In addition, the use of these techniques is usually linked to the application of protein enzymes, the activity of which is dependent on the temperature and pH of the medium. To address these drawbacks, an alternative genetic enzyme for the highly sensitive detection of miRNAs is proposed in this work. Multicomponent nucleic acid enzymes (MNAzymes), coupled with the use of DNA-functionalized gold nanoparticles (AuNPs), were used in this study to develop an isothermal signal amplification strategy for visual genetic detection. miR146a, a biomarker of bovine mastitis present in milk, was selected as a model analyte. The developed methodology is easily carried out in 80 min at 50 °C, generating a low visual limit of detection of 250 pM based on the observation of a color change. The methodology was successfully applied to the detection of miR146a in raw cow milk samples.Graphical

Avoiding Kinetic Trapping in the Self-Assembly of DNA-Functionalized Gold Nanoparticles by Using an Enthalpy-Mediated Strategy

Avoiding Kinetic Trapping in the Self-Assembly of DNA-Functionalized Gold Nanoparticles by Using an Enthalpy-Mediated Strategy

Retraction: Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.

Retraction: Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.

Refreshing DNA-Based Nanoprobes by Alcohol for Heavy Metal Detection: Toward Sustainable Sensing Nanomaterials

Increasing the applications of disposable nanomaterials yet with potential environmental risk and the resulting nanowastes have become a problem of public concern worldwide. However, efficient recovery of useful materials from nanowastes is currently limited by their tiny size, high physicochemical activity, and unpredictable aggregation. We have developed a conceptually new strategy for recycling nanoprobes by alcoholic solvent “refreshing”. In specific, DNA-functionalized gold nanoparticles and nanorods are employed as colorimetric nanoprobes for Hg(II) and Ag(I) ions, respectively, both of which rely upon target-induced DNA terminal base pairing and the subsequent base pair stacking assembly of the nanoprobes. Upon the introduction of isopropanol to the “waste” aggregates, however, the DNAs are denaturalized to release heavy metal ions, leading to restoration of individual nanoprobes. The fine modulation of DNA hybridization not only affords sensitive detection but also addresses the recycling of the nanoprobes with high efficiency (>80%) while preserving the detection performance irrespective of the nanoprobe shape and surface-grafted DNA sequence. We further demonstrate the use of the recycled nanoprobes in the detection of Hg(II) species from salmon muscle tissue. With the generality of the recovering method and the combined features of the renewable nanoprobes, including high recycling efficiency and retained functionality, sustainable sensing nanomaterials are within reach.

Suppressed DNA base pair stacking assembly of gold nanoparticles in an alcoholic solvent for enhanced ochratoxin A detection in Baijiu.

The currently established DNA nanoprobes for the detection of mycotoxin from beverages have been limited by complicated sample pretreatment and uncontrollable nanoparticle flocculation in complex systems. We develop a rapid colorimetric approach for ochratoxin A (OTA) detection in Baijiu in a sample-in/"yes" or "no" answer-out fashion through target-modulated base pair stacking assembly of DNA-functionalized gold nanoparticles (DNA-AuNPs). The colorimetric signification of OTA relies on the competition of OTA with the AuNP surface-grafted DNA in binding with an OTA-targeted aptamer. The specific recognition of OTA by the aptamer prevents DNA duplex formation on the AuNP surface, thereby inhibiting the base pair stacking assembly of the DNA-AuNPs and giving rise to a "turn-on" color. By further suppressing DNA hybridization using a bulged loop design and an alcohol solution, the DNA-AuNPs exhibit an improved reproducibility for OTA sensing while maintaining excellent susceptivity to OTA. A detection limit of 88 nM was achieved along with high specificity towards OTA, which is lower than the maximum tolerated level of OTA in foodstuffs defined by countries worldwide. The entire reaction time, avoiding sample pretreatment, is less than 17 min. The DNA-AuNPs with anti-interference features and sensitive "turn-on" performance promise convenient on-site detection of mycotoxin from daily beverages.

A sandwich-type electrochemical immunosensor based on spherical nucleic acids-templated Ag nanoclusters for ultrasensitive detection of tumor biomarker

The accurate determination of tumor biomarkers in blood is of vital significance in the diagnosis and therapy of tumor disease. In this research, an innovative sandwich-type electrochemical immunosensor is designed for the ultrasensitive determination of tumor biomarker AFP using spherical nucleic acids-templated silver nanoclusters (AgNCs) sensing platform. For this purpose, on one hand, DNA functionalized gold nanoparticles (AuNPs@DNA) is selected not only as the cross-linker to immobilize the primary antibody (anti-AFP antibody 1, Ab1) to obtain AuNPs@DNA-Ab1, but also as the template for synthesizing AgNCs on AuNPs to form AuNPs@DNA-AgNCs. On the other hand, p-sulfonated calix[4]arene (pSC4) modified Au is chosen to immobilize the secondary antibody (anti-AFP antibody 2, Ab2) through host-guest recognition between Ab2 and pSC4. When AFP is encountered, the immunoreaction signal can be significantly amplified by the electrochemical reduction of AgNCs. Under optimal circumstances, the sandwich-type electrochemical immunosensor exhibits broad limit of linearity from 0.001 to 100 ng mL−1 (R2 = 0.997) and low detection limit of 7.74 fg mL−1 (S/N = 3). The immunosensor possesses excellent repeatability and selectivity, offering a novel method for sensitive clinical diagnosis of tumor markers in human hepatocellular carcinoma.

DNA-functionalized gold nanoparticles: Modification, characterization, and biomedical applications.

With the development of technologies based on gold nanoparticles (AuNPs), bare AuNPs cannot meet the increasing requirements of biomedical applications. Modifications with different functional ligands are usually needed. DNA is not only the main genetic material, but also a good biological material, which has excellent biocompatibility, facile design, and accurate identification. DNA is a perfect ligand candidate for AuNPs, which can make up for the shortcoming of bare AuNPs. DNA-modified AuNPs (DNA-AuNPs) have exciting features and bright prospects in many fields, which have been intensively investigated in the past decade. In this review, we summarize the various approaches for the immobilization of DNA strands on the surface of AuNPs. Representative studies for biomedical applications based on DNA-AuNPs are also discussed. Finally, we present the challenges and future directions.

Colorimetric Detection of Antimicrobial Resistance from Food Processing Facilities Using a CRISPR System

The overuse of antibiotics has led to considerable increases of antibiotic resistance genes (ARGs) that have spread throughout the entire food supply chain. ARGs often transfer from processing facilities to food, causing major public health concerns. The current standard for detecting ARGs typically depends on quantitative polymerase chain reaction (qPCR) and gene sequencing. However, the reliance on experienced personnel and complicated readout equipment substantially inhibits the expansion of ARGs testing in nonlaboratory settings. Improved on-site testing will help monitor the spread of ARGs contamination to ensure food safety and address public health concerns. Herein, we developed a CRISPR-Cas12a-based assay for the colorimetric detection of ARGs in washing water collected from a food processing plant. In our assay, DNA-functionalized gold nanoparticles (AuNPs) were cross-linked with a ssDNA cross-linker. Target-induced Cas12a trans-cleavage was utilized for degradation of the cross-linkers, shifting the optical properties of the AuNPs to produce a facile visual readout. Without DNA amplification, we were able to detect three representative ARGs with a detection limit of 5 nM or lower. In addition, our assay was extended to a more complex medium, where as few as 103 gene copies in washing water from a fruit washing facility were visually detected. Based on our results, our method is both highly specific and sensitive. Due to the affordability and simplicity of our assay, this method can improve ARGs detection to monitor and prevent the immense spread of antimicrobial resistance among the food supply chain.

Front Cover: A Simple Colorimetric Assay of Bleomycin‐Mediated DNA Cleavage Utilizing Double‐Stranded DNA‐Modified Gold Nanoparticles (ChemBioChem 1/2023)

A colorimetric assay of sequence-specific DNA cleavage by bleomycin (BLM) derivatives is a promising methodology for facilitating the development of DNA-associated drugs with antitumor activity. Double-stranded DNA-functionalized gold nanoparticles (dsDNA-AuNPs) possessing BLM-mediated DNA cleavage sites were designed. The front cover highlights a non-fluorescence-based analytical method that allows visual screening of potent antitumor agents using the DNA-protruding structure formed by BLM-mediated DNA cleavage on the outermost surfaces of the dsDNA-AuNPs. This study could provide a facile method for efficiently screening BLM-like drug candidates obtained from combinatorial synthesis. More information can be found in the Research Article by Y. Akiyama et al.

Effects of Amine Linkers with Different Carbon Chain Lengths at Guanine-Rich Polynucleotides on Chemical Interface Damping in Single Gold Nanorods.

DNA-functionalized gold nanoparticles (AuNPs) are used for various bioapplications, such as biosensor development and drug delivery. Nevertheless, no study has reported the effect of polynucleotide chains on chemical interface damping (CID), the most recently proposed plasmon damping pathway in single AuNPs. In this study, we conducted total internal reflection scattering measurements of gold nanorods (AuNRs) to reveal the CID effect induced by amine (NH2)-linked polynucleotides (or DNA) with guanine-rich sequences through the interaction between nitrogen and Au surfaces. Additionally, we elucidated the effect of a linear hydrocarbon chain length between NH2 and DNA (NH2-Cn-DNA, n = 6, 12, 18, 24) on spectral changes in single AuNRs. The localized surface plasmon resonance (LSPR) linewidth increased with an increasing number of linear carbon, from 6 to 24, due to the increase in van der Waals forces. Second, the effect of the direction (5' or 3' ends) of DNA attachment to the AuNR surfaces on LSPR spectral changes was investigated, and there was no significant difference in LSPR wavelength and full linewidth at half-maximum shifts caused by the DNA attachment directions (5' or 3' ends). Third, guanine-rich DNA can fold into four-stranded secondary structures called G-quadruplexes (GQs). We demonstrated the effect of linear carbon chain length, between NH2 and GQs, on CID in single AuNRs. Lastly, a label-free detection of DNA hybridization events on single AuNRs was demonstrated for sensing applications. Thus, we provide an insight into the effect of amine-functionalized guanine-rich DNA with different carbon chains on LSPR spectral changes, including CID in single AuNRs.

G-Quadruplex-Functionalized Gold Nanoparticles for a Real-Time Biomolecule Sensor with On-Demand Tunable Properties.

G-quadruplex (G4) DNA-functionalized gold nanoparticles (AuNPs) were fabricated for a new sensing platform for a biomolecule, thrombin. Thrombin-binding aptamer (TBA), which forms a highly ordered G4 structure, was immobilized on AuNPs. The particles were induced to aggregate by binding of thrombin to G4 DNA. Thrombin was thus detected by the color change of the colloidal system from red to purple-blue. The aggregation was not due to the bridging between the particles through thrombin but to the reduction in steric repulsion attributable to the mobility and flexibility of G4 DNA. The change in the colloidal stability was quick and the bathochromic peak shift varied with the concentration of thrombin. The sensor showed a high specificity to the thrombin target over major proteins in human serum. The detection sensitivity and analytical performance were successfully tuned for an on-demand sensor with a linearity of 10.0-40.0 nM. The limits of detection and of quantification were 3.6 and 10.7 nM, respectively.

Freeze-Driven Adsorption of Poly-A DNA on Gold Nanoparticles: From a Stable Biointerface to Plasmonic Dimers.

Increasing attention is paid to poly-adenine (poly-A) DNA-functionalized gold nanoparticles due to the high cost of thiols. Freezing is an effective approach for immobilizing poly-A DNA on gold nanoparticles, but its mechanism remains elusive. To cope with this issue, in this paper, some experimental insights are provided. It is shown that (1) the DNA loading density is independent of the length of poly-A. (2) DNA is densely packed on gold nanoparticles, and the biointerface is peculiarly stable, which is not in line with the existing “wrapping” model. (3) Using a DNA-staining dye, thiazole orange, it is shown that poly-A duplex structures are formed on the surface of gold nanoparticles, with evidence given by fluorescence and Raman measurements. An alternative model involving stable poly-A duplexes anchored by finite terminal adenines is proposed. Based on it, a strategy for constructing plasmonic dimers is developed, using freeze-driven adsorption of a DNA sequence with poly-adenine at both ends. This work provides insights into the reaction between poly-A DNA and AuNPs upon freezing and is expected to facilitate related research in biosensor development and nanotechnology.