Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells

This paper reports the creation of Au nanoparticles (AuNP) that are soluble in aqueous solution over a broad range of pH and ionic strength values and that are capable of selective uptake by folate receptor positive (FR+) cancer cells. A novel poly(ethylene glycol) (PEG) construct with thioctic acid and folic acid coupled on opposite ends of the polymer chain was synthesized for targeting the AuNP to FR+ tumor cells via receptor-mediated endocytosis. These folic acid-PEG-thioctic acid conjugates were grafted onto 10-nm-diameter Au particles in aqueous solution. The resulting folate-PEG-coated nanoparticles do not aggregate over a pH range of from 2 to 12 and at electrolyte concentrations of up to 0.5 M NaCl with particle concentrations as high as 1.5 x 10(13) particles/mL. Transmission electron microscopy was used to document the performance of these coated nanoparticles in cell culture. Selective uptake of folate-PEG grafted AuNPs by KB cells, a FR+ cell line that overexpress the folate receptor, was observed. AuNP uptake was minimal in cells that (1) do not overexpress the folate receptor, (2) were exposed to AuNP lacking the folate-PEG conjugate, or (3) were co-incubated with free folic acid in large excess relative to the folate-PEG grafted AuNP. Understanding this process is an important step in the development of methods that use targeted metal nanoparticles for tumor imaging and ablation.     

Colorimetric detection of mercury, lead and copper ions simultaneously using protein-functionalized gold nanoparticles

A simple, cost-effective and rapid colorimetric method for any or all of Hg(2+), Pb(2+) and Cu(2+) detection using papain-functionalized gold nanoparticles (P-AuNPs) has been developed. Papain is a protein with seven cystein residues, which can selectively bind with Hg(2+), Pb(2+) and Cu(2+). We functionalized gold nanoparticles with papain. The P-AuNPs could be used to simultaneously detect Hg(2+), Pb(2+) and Cu(2+), and showed different responses to the three ions in an aqueous solution based on the aggregation-induced color change of gold nanoparticles. The P-AuNPs displayed the most obvious response to mercury ions in water in contrast to lead and copper ions, and the real water sample analysis verified the conclusion. The sensitivity of the detection system was influenced by the pH of the P-AuNPs solution, the concentration of P-AuNPs and the size of gold nanoparticles, and we found that larger gold nanoparticles contributed to more sensitive results. The detection system can detect as low as 200 nM Hg(2+), Pb(2+) or Cu(2+) using 42 nm gold nanoparticles. We expect our approach to have wide-ranging applications in the developing region for monitoring water quality in some areas.     

Real-time colorimetric detection of target DNA using isothermal target and signaling probe amplification and gold nanoparticle cross-linking assay

We describe a facile gold nanoparticle (AuNP)-mediated colorimetric method for real-time detection of target DNA in conjugation with our unique isothermal target and signaling probe amplification (iTPA) method, comprising novel ICA (isothermal chain amplification) and CPT (cycling probe technology). Under isothermal conditions, the iTPA simultaneously amplifies the target and signaling probe through two displacement events induced by a combination of four specially designed primers, the strand displacement activity of DNA polymerase, and the RNA degrading activity of RNase H. The resulting target amplicons are hybridized with gold nanoparticle cross-linking assay (GCA) probes having a DNA-RNA-DNA chimeric form followed by RNA cleavage by RNase H in the CPT step. The intact GCA probes were designed to cross-link two sets of DNA-AuNPs conjugates in the absence of target DNA, inducing aggregation (blue color) of AuNPs. On the contrary, the presence of target DNA leads to cleavage of the GCA probes in proportion to the amount of amplified target DNA and the solution remains red in color without aggregation of AuNPs. Relying on this strategy, 10(2) copies of target Chlamydia trachomatis plasmid were successfully detected in a colorimetric manner. Importantly, all the procedures employed up to the final detection of the target DNA were performed under isothermal conditions without requiring any detection instruments. Therefore, this strategy would greatly benefit convenient, real-time monitoring technology of target DNA under restricted environments.     

A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification

A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs.     

Influence of Plasmonic Nanoparticles on the Performance of Colorimetric Cell Viability Assays

The conventional colorimetric assays based on measurement of the metabolic activity are routinely used to evaluate the cytotoxicity of nanomaterials (NMs). However, due to the varying absorbance properties of plasmonic NMs in the visible region of the spectrum, obtained results can be misleading. In this study, MTT, MTS, and WST-1 colorimetric cell viability assays were evaluated in the presence of gold (AuNPs) or silver nanoparticles (AgNPs). Since a living cell a complex system containing many molecular and ionic species, the plasmonic AuNP and AgNPs may selectively interact with intracellular components possessing thiol, amino, and carboxyl group moieties change the aggregation behavior of the NMs and thus their absorbance. A series of UV/Vis and DLS experiments were conducted to understand the interference possibility of the tested plasmonic NMs. The results show that the AuNPs and AgNPs do not have absorption at the wavelength where MTT formazan is measured while the both NPs may interfere with absorbance of MTS and WST-1 formazan.The overall assessments show that MTT assay is more suitable for the cell viability evaluation of spherical AuNPs and AgNPs with an average diameter of 50 nm. This study also suggests that a preliminary ex situ evaluation of plasmonic nanoparticles can provide valuable information for the suitability of the assay.     

A gold nanoparticle-based strategy for label-free and colorimetric screening of DNA triplex binders

A label-free colorimetric assay, using non-crosslinking AuNP aggregation, has been developed for the screening of specific triplex DNA binders. The relative binding affinities can be simultaneously determined. Our novel assay is simple in design and fast in operation, avoiding either AuNPs modification or oligonucleotide labeling, and easy to implement for visual detection. This strategy may offer a new approach for developing low cost, sensitive and high-throughput screening platform that is likely to be highly useful in a wide range of applications.     

Protein kinase assay on peptide-conjugated gold nanoparticles

We demonstrate that protein kinase can be assayed with high sensitivity on peptide-conjugated gold nanoparticles (AuNPs). Phosphorylation of peptides on the AuNP-monolayers was detected by using an anti-phosphotyrosine antibody (alpha-pY) and Cy3-labeled secondary antibody (Cy3-alpha-mIgG) as a probing molecule. When compared to conventional self-assembled monolayers (SAMs), spherical and three-dimensional geometry of AuNPs led to high surface density of peptide substrate and easy accessibility to enzyme, and consequently the resulting AuNP monolayers gave rise to improved detection sensitivity. Blocking of peptide-conjugated AuNPs with a poly(ethylene glycol) (PEG) also contributed to a higher signal-to-background ratio in kinase and its inhibition assays. The use of AuNPs as the platform surface will enable highly sensitive detection of protein kinases in a high-throughput manner.     

基于纳米金复合探针的基因芯片膜转印检测法

建立了一种基于纳米金复合探针的基因芯片膜转印核酸检测新方法。首先,用纳米金颗粒同时标记检测探针P2和两种长短不同且生物素化的信号探针 (T10,T40),其中检测探针与靶DNA 5￠端互补,两种信号探针起信号放大作用。当靶DNA分子存在时,芯片表面捕捉探针P1 (与靶DNA分子3￠端互补) 通过碱基互补配对原则结合靶DNA分子,将其固定于芯片上,同时检测探针通过与靶DNA 5￠端互补配对将纳米金复合探针结合于芯片表面,结果在芯片表面形成“三明治”结构,后通过链霉亲和素-生物素反应,使芯片表面对应有靶DNA分子的部位结合上碱性磷酸酶,最后利用BCIP/NBT显色系统使芯片表面信号结果镜面转印至尼龙膜表面。当检测探针和信号探针摩尔比为1∶10,T10和T40摩尔比为9:1时可以检测1 pmol/L合成靶DNA分子或0.23 pmol/L结核分枝杆菌16S rDNA PCR扩增产物,检测结果通过普通的光学扫描仪读取或肉眼直接判读信号有无。本芯片检测系统灵敏度高,操作方法简单、快速,不需要特殊仪器设备,在生物分子的检测方面具有较高的应用价值。     

DNA‐based chemiluminescent nanoprobes for highly sensitive and selective detection of mercury(II) ion

A simple and sensitive DNA‐stablized gold nanoparticle (AuNP)‐based chemiluminescent (CL) probe for detecting mercury ion (Hg²⁺) in aqueous solution has been developed. The CL strategy relies upon the catalytic activity of unmodified AuNPs on the luminol–H₂O₂ CL reaction, and the interaction of unmodified AuNPs with DNA. The unmodified AuNPs can effectively differentiate unstructured and folded DNA. The DNA desorbs from AuNPs in the presence of Hg²⁺, leading to the increase in CL signal. By rationally varying the number of thymine in single‐strand oligonucleotides, the detection range could be tuned. Employing single‐strand oligonucleotides with 14 thymine in the detecting system, a sensitive linear range for Hg²⁺ ions from 5.0 × 10^–10 to 1.0 × 10^–7 mol/L and a detection limit of 2.1 × 10^–10 mol/L are obtained. Changing the number of thymine to 10 and 6, it leads to a narrow detection range but a high sensitivity. Besides, DNA‐based CL nanoprobes exhibit a remarkable selectivity for Hg²⁺ ions over a variety of competing metal ions. Copyright © 2012 John Wiley & Sons, Ltd.     

Direct synthesis of Rev peptide-conjugated gold nanoparticles and their application in cancer therapeutics

We have developed a simple approach for generating peptide-conjugated gold nanoparticles (AuNPs) from the Rev peptide and gold aqueous solution. The peptide functions as both a reducing agent and a capping molecule. AuNPs of various sizes (20-300 nm) and shapes (spheres, triangular plates, and polygons) can be obtained upon modulating the ratio of gold ions to the Rev peptide. Transmission electron microscopy, X-ray diffraction, and UV-vis spectroscopy were utilized to characterize these nanoparticles. Fourier-transform infrared and X-ray photoelectron spectroscopy measurements were performed to investigate chemical interactions between the Rev peptide and AuNPs. Lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed that the Rev peptide-AuNP nanocomposites exhibited exceptionally high cytotoxic effects toward mouse ovarian surface epithelial cell lines, relative to the effects of equal doses of the free Rev peptide. Our study suggests a new way of utilizing biomolecule-conjugated AuNPs as potentially effective anticancer drugs.     

Colorimetric Sensor of Cobalt Ions in Aqueous Solution Using Gold Nanoparticles Modified with Glycyrrhizic Acid

Plasmonics - The present work describes simple and green method for the preparation of gold nanoparticles (AuNPs) in aqueous medium under ambient condition and their use in colorimetric detection...     

Quantum dots‐based fluorescence resonance energy transfer biosensor for monitoring cell apoptosis

The development of advanced methods for accurately monitoring cell apoptosis has extensive significance in the diagnostic and pharmaceutical fields. In this study, we developed a rapid, sensitive and selective approach for the detection of cell apoptosis by combining the site‐specific recognition and cleavage of the DEVD–peptide with quantum dots (QDs)‐based fluorescence resonance energy transfer (FRET). Firstly, biotin‐peptide was conjugated on the surface of AuNPs to form AuNPs‐pep through the formation of an Au‐S bond. Then, AuNPs–pep–QDs nanoprobe was obtained through the connection between AuNPs–pep and QDs. FRET is on and the fluorescence of QDs is quenched at this point. The evidence of UV–vis spectra, transmission electron microscopy (TEM), and Fourier transform infrared (FT‐IR) spectroscopy revealed that the connection was successful. Upon the addition of apoptosis cell lysis solution, peptide was cleaved by caspase‐3, and AuNPs was dissociated from the QDs. At this time, FRET is off, and thus the QDs fluorescence was recovered. The experimental conditions were optimized in terms of ratio of peptide to AuNPs, buffer solution, and the temperature of conjugation and enzyme reaction. The biosensor was successfully applied to distinguishing apoptosis cells and normal cells within 2 h. This study demonstrated that the biosensor could be utilized to evaluate anticancer drugs.     

Effective delivery of anti-miRNA DNA oligonucleotides by functionalized gold nanoparticles

MicroRNAs (miRNAs) are gaining recognition as essential regulators involved in many biological processes, and they are emerging as therapeutic targets for treating disease. Here, we introduce a method for effective delivery of anti-miRNA oligonucleotides (AMOs) using functionalized gold nanoparticles (AuNPs). To demonstrate the ability of AMOs to silence miRNA, we selected miR-29b, which is known to downregulate myeloid cell leukemia-1 (MCL-1), a factor responsible for promoting cell survival. We first generated AuNPs coated with cargo DNA, which was then coupled to complementary DNA linked to an antisense miR-29b sequence. When the AuNPs were delivered into HeLa cells, MCL-1 protein and mRNA levels were increased significantly. Furthermore, apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was inhibited, proving that AMOs targeting miR-29b were effectively delivered by our innovative AuNP. In addition, we provided evidence that AuNP could deliver other AMOs against miR-21 into two independent cell lines, KGN and 293T, suggesting that the AuNP conjugates can be versatile for any AMO and cell type.     

Aptamer–nanoparticle-based chemiluminescence for p53 protein

A simple colorimetric biosensing technique based on the interaction of gold nanoparticles (AuNPs) with the aptamer was developed for detection of p53, a tumor suppressor protein, in the current study. Aggregation of AuNPs was induced by desorption of the p53 binding RNA aptamer from the surface of AuNPs as a result of the aptamer target interaction leading to the color change of AuNPs from red to purple. The detection limit of p53 protein by the colorimetric approach was 0.1 ng/ml after successful optimization of the amount of aptamer, AuNPs, salts, and incubation time. Furthermore, the catalytic activity of the aggregated AuNPs was greatly enhanced by chemiluminescence (CL) reaction, where the detection limit was enhanced to 10 pg/ml with a regression coefficient of R² = 0.9907. Here the sensitivity was increased by 10-fold compared with the AuNP-based colorimetric method. Hence, the sensitivity of detection was increased by employing CL, by using the catalytic activity of aggregated AuNPs, on the luminol–hydrogen peroxide reaction. Thus, the combination of colorimetric and CL-based aptasensor can be of great advantage in increasing the sensitivity of detection for any target analyte.     

Design, synthesis, and evaluation of peptidyl fluorescent probe for Zn2+ in aqueous solution

A new fluorescent peptide probe for the detection of Zn(2+) was synthesized on the basis of zinc binding ligands in zinc enzymes. The peptide that has a unique amino acid sequence displayed a great selectivity for Zn(2+) in the presence of several transition metal ions in aqueous solution. The reversibility, binding stoichiometry, binding affinity, and pH sensitivity of the sensor were studied. Further, on-bead application of the peptide as chemosensors was demonstrated.     

Aptamer-based colorimetric biosensing of Ochratoxin A using unmodified gold nanoparticles indicator

This work presents an aptasensor for Ochratoxin A (OTA) using unmodified gold nanoparticles (AuNPs) indicator. The assay method is based on the conformation change of OTA's aptamer in phosphate buffered saline (PBS) containing Mg(2+) and OTA, and the phenomenon of salt-induced AuNPs aggregation. A single measurement took only five minutes. Circular dichroism spectroscopic experiments revealed for the first time that upon the addition of OTA, the conformation of OTA's aptamer in PBS buffer changed from random coil structure to compact rigid antiparallel G-quadruplex structure. This compact rigid G-quadruplex structure could not protect AuNPs against salt-induced aggregation, and thus the color change from red to blue could be observed by the naked eye. The linear range of the colorimetric aptasensor covered a large variation of OTA concentration from 20 to 625 nM and the detection limit of 20 nM (3σ) was obtained.     

Label-free colorimetric and quantitative detection of cancer marker protein using noncrosslinking aggregation of Au/Ag nanoparticles induced by target-specific peptide probe

Assays for non-enzyme protein based on peptide-protein interaction are few due to the fact that most of peptide-protein bindings do not produce easily measurable output signals. Here we report a homogenous assay for colorimetric and quantitative detection of a cancer marker and promising antitumor target, cyclin A(2), using noncrosslinking aggregation of unmodified AuNPs/AgNPs by utilizing the difference of coagulating ability of a cationic peptide probe (P1) and its binding form toward naked AuNPs/AgNPs. In the absence of cyclin A(2), P1 coagulates particles immediately, whereas cyclin A(2) binding prevents the interaction of P1 with metal particles surface, significantly reducing the magnitude of aggregation. The extent of aggregation is dependent on the concentration of the target protein cyclin A(2) and the difference in color can readily be distinguished by spectrometer and naked eyes. The assay is sensitive and selective. Cyclin A(2) assay using AuNPs as colorimetric indicator is more easily monitored by naked eyes owing to the distinct color change, and 40 nM cyclin A(2) can be detected without the aid of any instruments. Using inexpensive desktop spectrometer, cyclin A(2) assay using AgNPs as colorimetric indicator can detect as low as 30 nM cyclin A(2), which is 20 fold lower than that of cyclin A(2) assay using terbium-chelating peptide as the probe reported recently (Pazos et al., 2008, 130, 9652-9653). This strategy will shed light on developing of unlabeled peptide-based protein biosensors.     

Electrochemical sensor systems based on one-dimensional (1D) nanostructures for analysis of bioaffinity interactions

Modification of screen printed graphite electrodes (SPE) with gold nanoparticles (AuNPs) decorated Pb nanowires (PbNWs) enhances such analytical characteristics of the sensor as effective surface area, electrocatalytic properties and kinetics of heterogeneous electron transfer. The reason for such improvement may be associated with the synergistic effect of AuNPs and PbNWs. Nanowires ensembles on the electrode surface were employed for the detection of cardiac myoglobin in human blood, cytochrome P450 2B4, cytochrome c. Composite materials based on nanoparticles with different dimensions (three dimensional (3D) gold nanoparticles and one dimensional (1D) Pb nanowires) create a platform for electrochemical analysis of proteins with low detection limits.     

Affinity analysis of DNA aptamer-peptide interactions using gold nanoparticles

Gold nanoparticles (AuNPs) were used as colorimetric probe and fluorescence quencher for affinity analysis of DNA aptamers toward their target mucin 1 (MUC1) peptide. Single-stranded DNA (ssDNA) aptamer-coated AuNPs showed increased stability (i.e., more resistant to aggregation induced by NaCl) in the presence of their target peptide due to the increase in steric protection conferred by the ssDNA-peptide complexes formed on the AuNPs. Based on changes in the UV-vis extinction spectrum of AuNPs (a measure of AuNPs aggregation) and fluorescence restoration of CY5-ssDNA upon ssDNA-peptide complex formation, the formation of the complexes and ssDNA sequence-dependent dissociation constant (K(d)) were determined. Besides the UV-vis and fluorescence measurements, the hydrodynamic diameters, zeta potential measurements, and transmission electron microscopy (TEM) images of AuNPs after various coatings supported the assay principle. The methodology presented herein provides a rapid and sensitive alternative solution for the identification of high affinity binders from systematic evolution of ligands by exponential enrichment (SELEX).     

Caspase sensitive gold nanoparticle for apoptosis imaging in live cells

We developed a new apoptosis imaging probe with gold nanoparticles (AuNPs). A near-infrared fluorescence dye was attached to AuNP surface through the bridge of peptide substrate (DEVD). The fluorescence was quenched in physiological conditions due to the quenching effect of AuNP, and the quenched fluorescence was recovered after the DEVD had been cleaved by caspase-3, the enzyme involved in apoptotic process. The adhesion of DEVD substrates on AuNP surface was accomplished by conjugation of the 3,4-dihydroxy phenylalanine (DOPA) groups which are adhesive to inorganic surface and rich in mussels. This surface modification with DEVD substrates by DOPA groups resulted in increased stability of AuNP in cytosol condition for hours. Moreover, the cleavage of substrate and the dequenching process are very fast, and the cells did not need to be fixed for imaging. Therefore, the real-time monitoring of caspase activity could be achieved in live cells, which enabled early detection of apoptosis compared to a conventional apoptosis kit such as Annexin V-FITC. Therefore, our apoptosis imaging has great potential as a simple, inexpensive, and efficient apoptosis imaging probe for biomedical applications.