Optical detection and discrimination of cystic fibrosis-related genetic mutations using oligonucleotide–nanoparticle conjugates

Novel methods for application of oligonucleotide–gold nanoparticle conjugates to selective colorimetric detection and discrimination of cystic fibrosis (CF) related genetic mutations in model oligonucleotide systems are presented. Three-strand oligonucleotide complexes are employed, wherein two probe oligonucleotide–gold nanoparticle conjugates are linked together by a third target oligonucleotide strand bearing the CF-related mutation(s). By monitoring the temperature dependence of the optical properties of the complexes, either in solution or on silica gel plates, melting behaviors may be accurately and reproducibly compared. Using this approach, fully complementary sequences are successfully distinguished from mismatched sequences, with single base mismatch resolution, for F 508, M470V, R74W and R75Q mutations.     

Hybridization and Melting Behavior of Peptide Nucleic Acid (PNA) Oligonucleotide Chimeras Conjugated to Gold Nanoparticles

Peptide nucleic acids (PNA) and PNA–DNA chimeras carrying thiol groups were used for surface functionalization of Au nanoparticles. Conjugation of PNA to citrate‐stabilized Au nanoparticles destabilized the nanoparticles causing them to precipitate. Addition of a tail of glutamic acid to the PNA prevented destabilization of the nanoparticles but resulted in loss of interaction with complementary sequences. Importantly, PNA–DNA chimeras gave stable conjugates with Au nanoparticles. The hybridization and melting properties of complexes formed from chimera–nanoparticle conjugates and oligonucleotide–nanoparticle conjugates are described for the first time. Similar to oligonucleotide–nanoparticle conjugates, conjugates with PNA–DNA chimeras gave sharper and more‐defined melting profiles than those obtained with unmodified oligonucleotides. In addition, mismatch discrimination was found to be more efficient than with unmodified oligonucleotides.     

采用纳米金和双链探针比色检测单碱基突变

将链置换的高度特异性与纳米金凝聚变色的光学特性相结合,设计了一种新型的单碱基突变比色检测方法。本方法直接采用纳米金作为比色报告基团,以两个末端均带有巯基的双链DNA为特异捕获探针,利用互补序列和单碱基突变序列对双链探针置换能力的差异,实现了对单碱基突变的检测。本检测方法直观、快速、简便、成本低,pmol级的样品无需仪器就可以观察到颜色的变化。     

Colorimetric biosensors based on DNA-nanoparticle conjugates.

In this review, we present an overview of the technologies in colorimetric biosensors based on DNA-nanoparticle conjugates. Two types of DNA-nanoparticles aggregation assays are summarized. One of the methods relies on cross-linking of the gold nanoparticle (GNP) by hybridization. The crosslinking system was used not only to detect target DNA sequences, but also to detect metal ions or small molecules which were recognized by DNAzymes. The other method is the GNP non-crosslinking system. This approach shows high performance in the detection of single nucleotide polymorphisms. These methods do not need special equipment and open up a new possibility of point-of-care diagnoses.     

Preparation of Nanogapped Gold Nanoparticle Array for DNA Detection

A novel DNA detection technique using a gold nanoparticle array film electrode has been reported here. The gold nanoparticles molecularly linked with binder molecule (1,10‐decanedithiol) were separated 1.3 nm from each other, and the DNA conductivity change from single to double strand was measured by monitoring a voltage drop across the particles, between which a probe of a 12‐mer oligonucleotide was immobilized. In adding a complementary oligonucleotide on the nanoparticle film chip, an immediate decrease in the film resistance (ca. 1.4 Ω) due to a hybridization event occurred in a reproducible manner with this simple setup. In the paper, we have an interest in the primary sensing properties; effect of the film resistance on the sensor response, dependence of the resistance change on the DNA concentration, and the performance of the system for DNA detection including single nucleotide polymorphisms were described.     

A new approach to amplified telomerase detection with polyvalent oligonucleotide nanoparticle conjugates

We report a new assay for human telomerase activity that relies on polyvalent oligonucleotide nanoparticle conjugates as diagnostic probes and amplification units. Gold nanoparticles functionalized with specific oligonucleotide sequences can efficiently capture telomerase enzymes and subsequently be elongated. Both the elongated and unmodified oligonucleotide sequences are simultaneously measured. The two strands not only serve as internal positive controls for each other but also provide a way of amplifying signal. At high concentrations, both elongated and unmodified strands exhibit measurable responses. At low telomerase concentrations (e.g., from 10 HeLa cells), elongated strands cannot be detected, but the unmodified sequences, which come from the same probe particles, can be detected because their concentration is higher, providing a novel form of amplification. This new assay rivals the sensitivity of the conventional PCR-based method of telomerase detection.     

Design of sugar–oligonucleotide conjugates installed gold nanoparticle for effective delivery to hepatic parenchymal cells

A novel oligonucleotide delivery system that is based on oligonucleotide–nanoparticle conjugates has been described. Installed oligonucleotides were modified with the carbohydrate at the 3′ terminus, accordingly, constructed nanoparticles display clustered carbohydrates on their outer layer for the targeted delivery of oligonucleotides. The method for the construction of ligand-functionalized nanoparticle was simple and reproducible. The stability of the nanoparticles displaying clustered carbohydrates greatly increased in serum compared to nanoparticles without carbohydrates. In order to investigate the targetability of oligonucleotide–nanoparticle conjugates into primary hepatic parenchymal cells, freshly isolated rat hepatocytes were incubated with nanoparticles and the amount of internalized gold nanoparticles was evaluated by an inductively coupled plasma mass spectroscopy analysis. Nanoparticles displaying clustered carbohydrates internalized more efficiently than nanoparticles without carbohydrate modifications. In particular, the cellular uptakes of oligonucleotide-conjugated gold nanoparticle increased 1.7 ～2.0-fold by galactose modification. Competition assay revealed that clustered galactose enhanced the internalization of the nanoparticle into primary hepatic parenchymal cells by a receptor-mediated process.

Synthesis of Branched Oligonucleotides as Templates for the Assembly of Nanomaterials

Branched oligonucleotides with symmetric arms 6 – 15 , which may contain biotin as a second recognition code, were prepared. These molecules are designed to be used for the directed assembly of nanomaterials. The branched structure of the desired oligonucleotides was confirmed by mass spectrometry on small branched oliognucleotides, by gel electrophoresis, and by hybridization with complementary oligonucleotide? nanoparticle conjugates, followed by visualization of the complexes by transmission electron microscopy.     

Simultaneous identification of point mutations via DNA ligase-mediated gold nanoparticle assembly

Multiplex single nucleotide polymorphisms analysis has found a great demand in human genetics and pharmacogenetics. The present study reports a novel approach for a genotyping assay that could achieve simultaneous identification of multiple point mutations via a ligase-mediated gold nanoparticle assembly. Based on the allelic specificity of DNA ligase, gold nanoparticles modified by oligonucleotide probes perfectly matched to the DNA targets were assembled into a thermally-stable aggregate, while a single-base mismatch would result in the dissociation of the gold nanoparticle assembly at high temperature. Then, DNA targets and their point mutations could be differentiated using a multi-step temperature elevation analysis monitored by ultraviolet-visible measurements. This approach offered a direct colorimetric discrimination of multiple point mutations without stringent temperature control. The proposed approach is demonstrated using a model system for the identification of single-base mutations in codon 17 and position -28 of the beta-thalassemia gene. The results reveal that the wild and the mutant types could be simultaneously determined successfully. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in both research-oriented and clinical genomic assays.     

Highly reproducible hybridization assay of zeptomole DNA based on adsorption of nanoparticle-bioconjugate

A nanoparticle-bioconjugate was formed by homogeneous hybridization of one polynucleotide target with two oligonucleotide probes labelled by thiol and a nanoparticle, respectively. Deposition of the nanoparticle-bioconjugate on a gold surface by thiol-gold reaction was monitored in situ by quartz crystal microbalance (QCM) and applied for flow analysis of zeptomole amounts of polynucleotide. The formation in solution and adsorption of thiolated conjugates on gold could be fast, uniform and effective, and has been successfully exploited to construct a highly reproducible and sensitive platform for detection of target sequences. Being more rapid, reproducible, sensitive and amenable to automation than previously reported microgravimetric hybridization assays, this technology has great promise for practical applications in molecular diagnostics.     

Capillary electrophoresis,a method for the determination of nucleic acid ligands covalently attached to quantum dots representing a donor of Förster resonance energy transfer

The synthesis and determination of the structure of a Förster resonance energy transfer probe intended for the detection of specific nucleic acid sequences are described here. The probe is based on the hybridization of oligonucleotide modified quantum dots with a fluorescently labeled nucleic acid sample resulting in changes of the fluorescence emission due to the energy transfer effect. The stoichiometry distribution of oligonucleotides conjugated to quantum dots was determined by capillary electrophoresis separation. The results indicate that one to four molecules of oligonucleotide are conjugated to the surface of a single nanoparticle. This conclusion is confirmed by the course of the dependence of Förster resonance energy transfer efficiency on the concentration of fluorescently labeled complementary single‐stranded nucleic acid, showing saturation. While the energy transfer efficiency of the probe hybridized with complementary nucleic acid strands was 30%, negligible efficiency was observed with a noncomplementary strand.     

Sensitized photomodification of oligonucleotide-binding proteins displayed on the eucaryotic cell surface

Interactions of double-stranded nucleic acids with cell surface proteins, which are involved in binding and transport of extracellular nucleic acids, were studied by the photoaffinity modification with a binary system of oligonucleotide conjugates. The photoreactive double-stranded complex involved an oligonucleotide template and two complementary to adjacent sequences oligonucleotide conjugates. One conjugate contained a photoreagent, viz., 4-azido-2,3,5,6-tetrafluorobenzaldehyde N-(3-aminopropionyl)hydrazone, at the terminus located in proximity to the terminus of another conjugate containing the sensitizer, viz., 9-aminomethylanthracene. Binding of photoreagent and the sensitizer to a single-stranded template yields the photoreactive center. Upon irradiation with visible light (400—580 nm), this photoreactive double-stranded complex forms covalent cross-linkages with oligonucleotide-binding surface proteins of eucaryotic SPEV cells.     

Sorting fluorescent nanocrystals with DNA

Semiconductor nanocrystals with narrow and tunable fluorescence are covalently linked to oligonucleotides. These biocompounds retain the properties of both nanocrystals and DNA. Therefore, different sequences of DNA can be coded with nanocrystals and still preserve their ability to hybridize to their complements. We report the case where four different sequences of DNA are linked to four nanocrystal samples having different colors of emission in the range of 530-640 nm. When the DNA-nanocrystal conjugates are mixed together, it is possible to sort each type of nanoparticle by using hybridization on a defined micrometer-size surface containing the complementary oligonucleotide. Detection of sorting requires only a single excitation source and an epifluorescence microscope. The possibility of directing fluorescent nanocrystals toward specific biological targets and detecting them, combined with their superior photostability compared to organic dyes, opens the way to improved biolabeling experiments, such as gene mapping on a nanometer scale or multicolor microarray analysis.     

Self-assembled nanoparticle probes for recognition and detection of biomolecules

Colloidal gold nanocrystals have been used to develop a new class of nanobiosensors that is able to recognize and detect specific DNA sequences and single-base mutations in a homogeneous format. At the core of this biosensor is a 2.5-nm gold nanoparticle that functions as both a nano-scaffold and a nano-quencher (efficient energy acceptor). Attached to this core are oligonucleotide molecules labeled with a thiol group at one end and a fluorophore at the other. This hybrid bio/inorganic construct is found to spontaneously assemble into a constrained arch-like conformation on the particle surface. Binding of target molecules results in a conformational change, which restores the fluorescence of the quenched fluorophore. Unlike conventional molecular beacons with a stem-and-loop structure, the nanoparticle probes do not require a stem, and their background fluorescence increases little with temperature. In comparison with the organic quencher Dabcyl (4,4'-dimethylaminophenyl azo benzoic acid), metal nanoparticles have unique structural and optical properties for new applications in biosensing and molecular engineering.     

Surface plasmon resonance study on HIV-1 integrase strand transfer activity

Understanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the binding of ds long terminal repeats–IN complexes with the host DNA. HIV-1 integrase strand transfer activity was monitored in real time using a multichannel surface plasmon resonance biosensor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional gold nanoparticle-peptide complexes as cell-targeting agents

In this paper, we report a novel approach using peptide CALNN and its derivative CALNNGGRRRRRRRR (CALNNR(8)) to functionalize gold nanoparticles for intracellular component targeting. The translocation is effected by the nanoparticle diameter and CALNNR(8) surface coverage. The intracellular distributions of the complexes are change from the cellular nucleus to the endoplasmic reticulum by increasing the density of CALNNR(8) at a constant nanoparticle diameter. Additionally, increasing the nanoparticle diameter at a constant density of CALNNR(8) leads to less cellular internalization. These translocations of the complexes cause unique colorimetric expressions of the cell structure. The cell viability is affected by the internalized gold nanoparticle-peptide complexes in terms of quantities of particles per cell. In addition, the intracellular distribution of the fluorescence quenching is investigated by a fluorescent confocal scanning laser microscopy, which also gives further evidence of intracellular distribution of the gold nanoparticle-peptide complexes.     

Spherical Nucleic Acid Nanoparticle Conjugates Enhance G‐Quadruplex Formation and Increase Serum Protein Interactions

To understand the effect of three‐dimensional oligonucleotide structure on protein corona formation, we studied the identity and quantity of human serum proteins that bind to spherical nucleic acid (SNA) nanoparticle conjugates. SNAs exhibit cellular uptake properties that are remarkably different from those of linear nucleic acids, which have been related to their interaction with certain classes of proteins. Through a proteomic analysis, this work shows that the protein binding properties of SNAs are sequence‐specific and supports the conclusion that the oligonucleotide tertiary structure can significantly alter the chemical composition of the SNA protein corona. This knowledge will impact our understanding of how nucleic acid‐based nanostructures, and SNAs in particular, function in complex biological milieu.     

Synthesis and photophysical studies of phthalocyanine-gold nanoparticle conjugates

This work reports on the synthesis, characterization and photophysical studies of phthalocyanine-gold nanoparticle conjugates. The phthalocyanine complexes are: tris-(5-trifluoromethyl-2-mercaptopyridine)-2-(carboxy)phthalocyanine (3), 2,9,17,23-tetrakis-[(1, 6-hexanedithiol) phthalocyaninato]zinc(II) (8) and [8,15,22-tris-(naptho)-2(amidoethanethiol) phthalocyanato] zinc(II)(10). The gold nanoparticles were characterized using transmission electron microscopy, X-ray diffraction, atomic force microscopy and UV-vis spectroscopy where the size was confirmed to be ～5 nm. The phthalocyanine Au nanoparticle conjugates showed lower fluorescence quantum yield values with similar fluorescence lifetimes compared to the free phthalocyanines. The Au nanoparticle conjugates of 3 and 10 also showed higher triplet quantum yields of 0.69 to 0.71, respectively. A lower triplet quantum yield was obtained for the conjugate compared to free phthalocyanine for complex 8. The triplet lifetimes ranged from 70 to 92 μs for the conjugates and from 110 to 304 μs for unbound Pc complexes.     

DNA detection method based on the two-dimensional aggregation and selective desorption of nanoparticle probes

A label-free two-dimensional colorimetric DNA sensor is reported. This sensor is based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes induced by the molecular hybridization of single-stranded oligonucleotide probes and their complementary single-stranded DNA targets. To detect the aggregation, we have developed a new detection method based on the selective desorption of nonaggregated nanoparticles. We will show here that this detection method is highly specific and allows the quantification of the DNA targets.     

超顺磁性DNA纳米富集器应用于痕量寡聚核苷酸的富集

随着纳米技术的迅速发展 ,纳米材料逐渐被应用到细胞生物学和分子生物学研究领域 ,为生物医学的研究和发展提供了新的技术和手段 [1～ 4 ] .如超顺磁性纳米颗粒由于具有尺寸小、比表面积大、悬浮稳定性好及在外磁场作用下的磁导向性运输和富集等优良特性 ,使其在细胞和生物活性