Evaluation of gold nanoparticles as the additive in real-time polymerase chain reaction with SYBR Green I dye

Gold nanoparticles (AuNPs) have been proven to be able to improve the specificity or increase the efficiency of a polymerase chain reaction (PCR) when a suitable amount of AuNPs was used. However, there is still a lack of systematic evaluation of AuNPs in real-time PCR. In this study, DNA degradation and the fluorescence quenching effect of AuNPs were first tested in real-time PCR. Then two different kinds of Taq DNA polymerase, native and recombinant Taq polymerase, were employed to evaluate the AuNPs' effect on the threshold cycle (C(T)) values, standard curves and melting curves in real-time PCR. Different ratios of the amount of native Taq DNA polymerase to the amount of AuNPs were also tested. It was found that AuNPs could be applied in real-time PCR with correlation coefficient R(2)>0.989. The combination of 2.09?nM AuNPs with 3.75?U of native Taq DNA polymerase could make the amplification curves shift to the left and enhance the efficiency of the real-time PCR (0.628?39 without AuNPs compared with 0.717?89 with 2.09?nM AuNPs), thus enabling faster detection in comparison with those of control samples. However, no improvement ability of AuNPs was found in real-time PCR based on recombinant rTaq DNA polymerase. Besides, the results suggest that a complex interaction exists between AuNPs and native Taq DNA polymerase.     

Magnetic Bead/Gold Nanoparticle Double‐Labeled Primers for Electrochemical Detection of Isothermal Amplified Leishmania DNA

A novel methodology for the isothermal amplification of Leishmania DNA using labeled primers combined with the advantages of magnetic purification/preconcentration and the use of gold nanoparticle (AuNP) tags for the sensitive electrochemical detection of such amplified DNA is developed. Primers labeled with AuNPs and magnetic beads (MBs) are used for the first time for the isothermal amplification reaction, being the amplified product ready for the electrochemical detection. The electrocatalytic activity of the AuNP tags toward the hydrogen evolution reaction allows the rapid quantification of the DNA on screen‐printed carbon electrodes. Amplified products from the blood of dogs with Leishmania (positive samples) are discriminated from those of healthy dogs (blank samples). Quantitative studies demonstrate that the optimized method allows us to detect less than one parasite per microliter of blood (8 × 10^?3 parasites in the isothermal amplification reaction). This pioneering approach is much more sensitive than traditional methods based on real‐time polymerase chain reaction (PCR), and is also more rapid, cheap, and user‐friendly.     

Magnetic Plasmon Networks Programmed by Molecular Self‐Assembly

Nanoscale manipulation of magnetic fields has been a long‐term pursuit in plasmonics and metamaterials, as it can enable a range of appealing optical properties, such as high‐sensitivity circular dichroism, directional scattering, and low‐refractive‐index materials. Inspired by the natural magnetism of aromatic molecules, the cyclic ring cluster of plasmonic nanoparticles (NPs) has been suggested as a promising architecture with induced unnatural magnetism, especially at visible frequencies. However, it remains challenging to assemble plasmonic NPs into complex networks exhibiting strong visible magnetism. Here, a DNA‐origami‐based strategy is introduced to realize molecular self‐assembly of NPs forming complex magnetic architectures, exhibiting emergent properties including anti‐ferromagnetism, purely magnetic‐based Fano resonances, and magnetic surface plasmon polaritons. The basic building block, a gold NP (AuNP) ring consisting of six AuNP seeds, is arranged on a DNA origami frame with nanometer precision. The subsequent hierarchical assembly of the AuNP rings leads to the formation of higher‐order networks of clusters and polymeric chains. Strong emergent plasmonic properties are induced by in situ growth of silver upon the AuNP seeds. This work may facilitate the development of a tunable and scalable DNA‐based strategy for the assembly of optical magnetic circuitry, as well as plasmonic metamaterials with high fidelity.     

Visualizing Human Telomerase Activity with Primer‐Modified Au Nanoparticles

Telomerase is over‐expressed in over 85% of all known human tumors. This renders the enzyme a valuable biomarker for cancer diagnosis and an important therapeutic target. The most widely used telomeric repeat amplification protocol (TRAP) assay has been questioned for telomerase detection. It is reported that human telomerase activity can be visualized by using primer‐modified Au nanoparticles. The working principle is based on the elongated primers conjugated to the gold nanoparticle (AuNP) surface, which can fold into a G‐quadruplex to protect the AuNPs from the aggregation. The developed simple and sensitive colorimetric assay can measure telomerase activity down to 1 HeLa cell µL^?1. More importantly, this assay can be easily extended to high‐throughput and automatic format. The AuNP‐TS method is PCR‐free and therefore avoids the amplification‐related errors and becomes more reliable to evaluate telomerase activity. This assay has also been used for initial screening of telomerase inhibitors as anticancer drug agents.     

Electrophoretic Build‐Up of Alternately Multilayered Films and Micropatterns Based on Graphene Sheets and Nanoparticles and their Applications in Flexible Supercapacitors

Graphene nanosheets and metal nanoparticles (NPs) have been used as nano‐building‐blocks for assembly into macroscale hybrid structures with promising performance in electrical devices. However, in most graphene and metal NP hybrid structures, the graphene sheets and metal NPs (e.g., AuNPs) do not enable control of the reaction process, orientation of building blocks, and organization at the nanoscale. Here, an electrophoretic layer‐by‐layer assembly for constructing multilayered reduced graphene oxide (RGO)/AuNP films and lateral micropatterns is presented. This assembly method allows easy control of the nano‐architecture of building blocks along the normal direction of the film, including the number and thickness of RGO and AuNP layers, in addition to control of the lateral orientation of the resultant multilayered structures. Conductivity of multilayered RGO/AuNP hybrid nano‐architecture shows great improvement caused by a bridging effect of the AuNPs along the out‐of‐plane direction between the upper and lower RGO layers. The results clearly show the potential of electrophoretic build‐up in the fabrication of graphene‐based alternately multilayered films and patterns. Finally, flexible supercapacitors based on multilayered RGO/AuNP hybrid films are fabricated, and excellent performance, such as high energy and power densities, are achieved.     

Interaction of gold nanoparticles with Pfu DNA polymerase and effect on polymerase chain reaction

The interaction of gold nanoparticles with Pfu DNA polymerase has been investigated by a number of biological, optical and electronic spectroscopic techniques. Polymerase chain reaction was performed to show gold nanoparticles' biological effect. Ultraviolet-visible and circular dichroism spectra analysis were applied to character the structure of Pfu DNA polymerase after conjugation with gold nanoparticles. X-ray photoelectron spectroscopy was used to investigate the bond properties of the polymerase-gold nanoparticles complex. The authors demonstrate that gold nanoparticles do not affect the amplification efficiency of polymerase chain reaction using Pfu DNA polymerase, and Pfu DNA polymerase displays no significant changes of the secondary structure upon interaction with gold nanoparticles. The adsorption of Pfu DNA polymerase to gold nanoparticles is mainly through Au-NH(2) bond and electrostatic interaction. These findings may have important implications regarding the safety issue as gold nanoparticles are widely used in biomedical applications.     

Optical Detection of Human Papillomavirus Type 16 and Type 18 by Sequence Sandwich Hybridization With Oligonucleotide-Functionalized Au Nanoparticles

The importance of detecting and subtyping human papillomaviruses (HPVs) in clinical and epidemiological studies has been well addressed. In detecting the most common types of HPV, type 16 (HPV-16) and type 18 (HPV-18), in the cervical mucous of patients in a simple and rapid manner, the assay of a label-free colorimetric DNA sensing method based on sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles (AuNPs) was fabricated in this study. Specific oligonucleotide probes were designed for the sequence detection within the L1 gene of HPV-16 and HPV-18, and the probes were capped onto AuNPs, as AuNP probes. The target HPV sequences in clinical specimens were obtained by an asymmetric polymerase chain reaction (PCR) with universal primers, which can amplify the target sequences from several HPV serotypes, including HPV-16 and HPV-18. The DNA sandwich hybridization between the target sequences and the specific AuNP probes was performed at a temperature closer to the theoretical melting temperature of the DNA hybridization. Next, the procedure of increasing salt concentration and cooling the hybridizing solution was immediately utilized to discriminate the target sequences of HPV-16 or HPV-18. If the target sequences were not complementary to sequences of AuNP probes, the AuNPs would aggregate because no duplex DNA formation occurred such that the color of the reaction solution changed from red to purple. If the AuNP probes were a perfect match to the target sequences and a full DNA sandwich hybridization occurred, the reaction solution maintained its red color. A total of 70 mucous specimens from patients with cervical intraepithelial neoplasia were tested by the AuNP probes sandwich hybridization.      

Gold nanoparticles with asymmetric polymerase chain reaction for colorimetric detection of DNA sequence

We developed a novel strategy for rapid colorimetric analysis of a specific DNA sequence by combining gold nanoparticles (AuNPs) with an asymmetric polymerase chain reaction (As-PCR). In the presence of the correct DNA template, the bound oligonucleotides on the surface of AuNPs selectively hybridized to form complementary sequences of single-stranded DNA (ssDNA) target generated from As-PCR. DNA hybridization resulted in self-assembly and aggregation of AuNPs, and a concomitant color change from ruby red to blue-purple occurred. This approach is simpler than previous methods, as it requires a simple mixture of the asymmetric PCR product with gold colloid conjugates. Thus, it is a convenient colorimetric method for specific nucleic acid sequence analysis with high specificity and sensitivity. Most importantly, the marked color change occurs at a picogram detection level after standing for several minutes at room temperature. Linear amplification minimizes the potential risk of PCR product cross-contamination. The efficiency to detect Bacillus anthracis in clinical samples clearly indicates the practical applicability of this approach.     

In vivo Gold Nanoparticle Delivery of Peptide Vaccine Induces Anti‐Tumor Immune Response in Prophylactic and Therapeutic Tumor Models

Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, it is hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16‐OVA tumor model. AuNP delivery of OVA (AuNP‐OVA) and of CpG (AuNP‐CpG) enhanced the efficacy of both agents and induced strong antigen‐specific responses. In addition, it is found that AuNP‐OVA delivery alone, without CpG, is sufficient to promote significant antigen‐specific responses, leading to subsequent anti‐tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy is likely due to the adjuvant effect of peptide coated AuNPs, as they induce inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, AuNP‐mediated OVA peptide delivery can produce significant therapeutic benefits without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination.     

Chemical alignment of DNA origami to block copolymer patterned arrays of 5 nm gold nanoparticles

We have used block copolymer patterned arrays of 5 nm gold nanoparticles (AuNPs) for chemically aligned surface attachment of DNA origami. Addition of single-stranded DNA-thiol to AuNPs allowed a base paired attachment of sticky end modified DNA origami. Results indicate a stable, selective attachment between the DNA origami and ssDNA modified AuNPs. Yield data showed 74% of AuNP binding sites forming an attachment with a DNA origami rectangle, and control surfaces showed less than 0.5% nonspecific adsorption.     

Quantum dots trigger hot-start effects for pfu-based polymerase chain reaction

Hot-start (HS) effects were investigated in pfu-based polymerase chain reaction (PCR), when water-soluble CdTe quantum dots (QDs) were introduced in the PCR system. The HS effects were demonstrated by the higher amplicon yields and excellent suppression of non-specific amplification after pre-incubation of PCR mix with QDs between 35°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 1?h at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the pfu polymerase concentration, suggesting that there was an interaction between QDs and pfu DNA polymerase. Moreover, control experiment indicated that HS effect is not primarily due to the reduced pfu polymerase concentration resulted from the above interaction. Fluorescence correlation spectroscopy (FCS), a single molecule detection method, was used to investigate the possible mechanism of HS PCR with QDs. Preliminary FCS results suggested that CdTe QDs may directly interact with pfu DNA polymerase, rather than other components in the PCR system. Furthermore, results demonstrated that the interaction between QDs and pfu resulted in a reduction in pfu polymerase concentration. This study provided a good start to investigate potential implications of QDs in other key molecular biology techniques.     

DNA‐Origami‐Based Assembly of Anisotropic Plasmonic Gold Nanostructures

Precise control over the assembly of anisotropic plasmonic gold nanostructures with relative spatial directionality and sequence asymmetry remains a major challenge and offers great fundamental insight and optical application possibilities. Here, a novel strategy is developed to anisotropically functionalize gold nanorods (AuNRs) by using a DNA‐origami‐based precise machine to transfer essential DNA sequence configurations to the surface of the AuNRs through an intentionally designed toehold‐initiated displacement reaction. Different AuNR products are examined via hybridization with DNA‐AuNPs that display distinct elements of regiospecificity. These assembled anisotropic plasmonic gold nanostructures based on the DNA‐origami precise machine inherit the encoded information from the parent platform with high fidelity and show fixed orientation and bonding anisotropy, thereby generating discrete plasmonic nanostructures with enhanced Raman resonance.     

Self‐Assembly of Poly‐Adenine‐Tailed CpG Oligonucleotide‐Gold Nanoparticle Nanoconjugates with Immunostimulatory Activity

Synthetic unmethylated cytosine–guanine (CpG) oligodeoxynucleotides (CpG ODNs) possess high immunostimulatory activity and have been widely used as a therapeutic tool for various diseases including infection, allergies, and cancer. A variety of nanocarriers have been developed for intracellular delivery of CpG ODNs that are otherwise nonpermeable through the cellular membrane. For example, previous studies showed that gold nanoparticles (AuNPs) could efficiently deliver synthetic thiolated CpG ODNs into cultured cells and induce expression of proinflammatory cytokines. Nevertheless, the necessity of using thiolated CpG ODNs for the modification of AuNPs inevitably complicates the synthesis of the nanoconjugates and increases the cost. A new approach is demonstrated for facile assembly of AuNP‐CpG nanoconjugates for cost‐effective drug delivery. It is found that non‐thiolated, diblock ODNs containing a CpG motif and a poly‐adenine (polyA) tail can readily self‐assemble on the surface of AuNPs with controllable and tunable density. Such nanoconjugates are efficiently delivered into RAW264.7 cells and induce immune response in a Toll‐like receptor 9 (TLR9)‐dependent manner. Under optimal conditions, polyA‐CpG‐AuNPs show significantly higher immunostimulatory activity than their thiolated counterpart. In addition, the immunostimulatory activity of CpG‐AuNPs can be modulated by varying the length of the polyA tail. In vivo induction of immune responses in mice is demonstrated by using polyA‐tailed CpG‐AuNP nanoconjugates.     

3D nanometer images of biological fibers by directed motion of gold nanoparticles

Using near-infrared femtosecond pulses, we move single gold nanoparticles (AuNPs) along biological fibers, such as collagen and actin filaments. While the AuNP is sliding on the fiber, its trajectory is measured in three dimensions (3D) with nanometer resolution providing a high-resolution image of the fiber. Here, we systematically moved a single AuNP along nanometer-size collagen fibers and actin filament inside chinese hamster ovary K1 living cells, mapping their 3D topography with high fidelity.     

The Role of Temperature and Lipid Charge on Intake/Uptake of Cationic Gold Nanoparticles into Lipid Bilayers

Understanding the molecular mechanisms governing nanoparticle–membrane interactions is of prime importance for drug delivery and biomedical applications. Neutron reflectometry (NR) experiments are combined with atomistic and coarse‐grained molecular dynamics (MD) simulations to study the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes composed of a mixture of zwitterionic di‐stearoyl‐phosphatidylcholine (DSPC) and anionic di‐stearoyl‐phosphatidylglycerol (DSPG). MD simulations show that the interaction between AuNPs and a pure DSPC lipid bilayer is modulated by a free energy barrier. This can be overcome by increasing temperature, which promotes an irreversible AuNP incorporation into the lipid bilayer. NR experiments confirm the encapsulation of the AuNPs within the lipid bilayer at temperatures around 55 °C. In contrast, the AuNP adsorption is weak and impaired by heating for a DSPC–DSPG (3:1) lipid bilayer. These results demonstrate that both the lipid charge and the temperature play pivotal roles in AuNP–membrane interactions. Furthermore, NR experiments indicate that the (negative) DSPG lipids are associated with lipid extraction upon AuNP adsorption, which is confirmed by coarse‐grained MD simulations as a lipid‐crawling effect driving further AuNP aggregation. Overall, the obtained detailed molecular view of the interaction mechanisms sheds light on AuNP incorporation and membrane destabilization.     

Fe–Au Nanoparticle‐Coupling for Ultrasensitive Detections of Circulating Tumor DNA

Effectiveness of cancer therapy relies heavily on the efficient early diagnosis. Circulating tumor DNA (ctDNA) detection is one of the most clinically meaningful liquid biopsy approaches for the noninvasive cancer early diagnosis, which, unfortunately, cannot be applied as a routine diagnostic tool till a number of obstacles, for example, unsatisfactory specificity and sensitivity, and extremely high costs, are overcome. Here, the first paradigm of nanomaterial's application in the extremely specific, ultrasensitive, and yet economical ctDNA detections is reported based on a facile nanoparticle‐coupling strategy without amplification, with which polymerase chain reaction (PCR)‐introduced bias and other shortcomings are successfully circumvented. Aiming at seven Kirsten rat sarcoma‐2 virus (KRAS) point mutations, the present strategy exhibits high specificity and an ultrahigh sensitivity of detecting as low as 0.1 pg mL^?1 of KRAS point mutation without prior PCR amplification. Discriminating KRAS gene mutations in lung adenocarcinoma patients at an extremely low detection limit equivalent to 0.12% mutation relative to wild‐type gene is successful. It is envisioned that this nanoparticle‐coupling approach could be routinely applied clinically for ultra‐early diagnosis and monitoring of diverse malignant tumors, thus facilitating the fight against cancer.     

Lighting Up MicroRNA in Living Cells by the Disassembly of Lock‐Like DNA‐Programmed UCNPs‐AuNPs through the Target Cycling Amplification Strategy

Intracellular microRNAs imaging based on upconversion nanoprobes has great potential in cancer diagnostics and treatments. However, the relatively low detection sensitivity limits their application. Herein, a lock‐like DNA (LLD) generated by a hairpin DNA (H1) hybridizing with a bolt DNA (bDNA) sequence is designed, which is used to program upconversion nanoparticles (UCNPs, NaYF₄@NaYF₄:Yb, Er@NaYF₄) and gold nanoparticles (AuNPs). The upconversion emission is quenched through luminescence resonance energy transfer (LRET). The multiple LLD can be repeatedly opened by one copy of target microRNA under the aid of fuel hairpin DNA strands (H2) to trigger disassembly of AuNPs from the UCNP, resulting in the lighting up of UCNPs with a high detection signal gain. This strategy is verified using microRNA‐21 as model. The expression level of microRNA‐21 in various cells lines can be sensitively measured in vitro, meanwhile cancer cells and normal cells can be easily and accurately distinguished by intracellular microRNA‐21 imaging via the nanoprobes. The detection limit is about 1000 times lower than that of the previously reported upconversion nanoprobes without signal amplification. This is the first time a nonenzymatic signal amplification method has been combined with UCNPs for imaging intracellular microRNAs, which has great potential for cancer diagnosis.     

TGF‐β1 Conjugated to Gold Nanoparticles Results in Protein Conformational Changes and Attenuates the Biological Function

Gold nanoparticles (AuNPs) are widely used as carriers or therapeutic agents due to their great biocompatibility and unique physical properties. Transforming growth factor‐beta 1 (TGF‐β1), a member of the cysteine‐knot structural superfamily, plays a pivotal role in many diseases and is known as an immunosuppressive agent that attenuates immune response resulting in tumor growth. The results reported herein reflect strong interactions between TGF‐β1 and the surface of AuNPs when incubated with serum‐containing medium, and demonstrate a time‐ and dose‐dependent pattern. Compared with other serum proteins that can also bind to the AuNP surface, AuNP–TGFβ1 conjugate is a thermodynamically favored compound. Epithelial cells undergo epithelial–mesenchymal transition (EMT) upon treatment with TGF‐β1; however, treatment with AuNPs reverses this effect, as detected by cell morphology and expression levels of EMT markers. TGF‐β1 is found to bind to AuNPs through S–Au bonds by X‐ray photoelectron spectroscopy. Fourier transform infrared spectroscopy is employed to analyze the conformational changes of TGF‐β1 on the surface of AuNPs. The results indicate that TGF‐β1 undergoes significant conformational changes at both secondary and tertiary structural levels after conjugation to the AuNP surface, which results in the deactivation of TGF‐β1 protein. An in vivo experiment also shows that addition of AuNPs attenuates the growth of TGF‐β1‐secreting murine bladder tumor 2 cells in syngeneic C3H/HeN mice, but not in immunocompromised NOD‐SCID mice, and this is associated with an increase in the number of tumor‐infiltrating CD4⁺ and CD8⁺ T lymphocytes and a decrease in the number of intrasplenic Foxp3(+) lymphocytes. The findings demonstrate that AuNPs may be a promising agent for modulating tumor immunity through inhibiting immunosuppressive TGF‐β1 signaling.     

Hybrid Structures for Surface‐Enhanced Raman Scattering: DNA Origami/Gold Nanoparticle Dimer/Graphene

A combination of three innovative materials within one hybrid structure to explore the synergistic interaction of their individual properties is presented. The unique electronic, mechanical, and thermal properties of graphene are combined with the plasmonic properties of gold nanoparticle (AuNP) dimers, which are assembled using DNA origami nanostructures. This novel hybrid structure is characterized by means of correlated atomic force microscopy and surface‐enhanced Raman scattering (SERS). It is demonstrated that strong interactions between graphene and AuNPs result in superior SERS performance of the hybrid structure compared to their individual components. This is particularly evident in efficient fluorescence quenching, reduced background, and a decrease of the photobleaching rate up to one order of magnitude. The versatility of DNA origami structures to serve as interface for complex and precise arrangements of nanoparticles and other functional entities provides the basis to further exploit the potential of the here presented DNA origami–AuNP dimer–graphene hybrid structures.