AuNP Probes Research Articles

Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased.

Janus PEGylated gold nanoparticles: a robust colorimetric probe for sensing nitrite ions in complex samples.

We presented a Janus PEGylated AuNP probe where PEGs and recognition ligands (e.g., 4-aminobenzenethiol, 4-ABT) were asymmetrically functionalized on an AuNP. With this design, the probes showed high colloidal stability, signal robustness, specificity, and sufficient sensitivity in the determination of NO2- in various complex samples.

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform.

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 µl in each operation, which is remarkably less than that of a macro system.

1,4-Benzenediboronic-Acid-Induced Aggregation of Gold Nanoparticles: Application to Hydrogen Peroxide Detection and Biotin-Avidin-Mediated Immunoassay with Naked-Eye Detection.

Hydrogen-peroxide (H2O2)-induced growth of small-sized gold nanoparticles (AuNPs) is often implemented for H2O2 sensing and plasmonic immunoassay. In contrast, there is little-to-no information in the literature regarding the application of H2O2-inhibited aggregation of citrate-capped AuNPs. This study discloses that benzene-1,4-diboronic acid (BDBA) was effective in driving the aggregation of citrate-capped AuNPs through an interaction between α-hydroxycarboxylate of citrate and boronic acids of BDBA. The H2O2-mediated oxidation of BDBA resulted in the conversion of boronic acid groups to phenol groups. The oxidized BDBA was incapable of triggering the aggregation of citrate-capped AuNPs. Thus, the presence of H2O2 prohibited BDBA-induced aggregation of citrate-capped AuNPs. The BDBA-induced aggregation of citrate-capped AuNPs can be paired with the glucose oxidase (GOx)-glucose system to design a colorimetric probe for glucose. Moreover, a H2O2·BDBA·AuNP probe was integrated with sandwich immunoassay, biotinylated antibody, and avidin-conjugated GOx for the selective naked-eye detection of rabbit immunoglobulin G (IgG) and human-prostate-specific antigen (PSA). The lowest detectable concentrations of rabbit IgG and human PSA by the naked eye were down to 0.1 and 4 ng/mL, respectively. More importantly, the proposed plasmonic immunoassay allowed the naked-eye quantification of 0-10 ng/mL PSA at an interval of 2 ng/mL in plasma samples.

Chip-based visual detection of microRNA using DNA-functionalized gold nanoparticles.

In the present study, we developed a highly sensitive and convenient biosensor consisting of gold nanoparticle (AuNP) probes and a gene chip to detect microRNAs (miRNAs). Specific oligonucleotides were attached to the glass surface as capture probes for the target miRNAs, which were then detected via hybridization to the AuNP probes. The signal was amplified via the reduction of HAuCl4 by H2O2. The use of a single AuNP probe detected 10 pmol L(-1) of target miRNA. The recovery rate for miR-126 from fetal bovine serum was 81.5%-109.1%. The biosensor detection of miR-126 in total RNA extracted from lung cancer tissues was consistent with the quantitative PCR (qPCR) results. The use of two AuNP probes further improved the detection sensitivity such that even 1 fmol L(-1) of target miR-125a-5p was detectable. This assay takes less than 1 h to complete and the results can be observed by the naked eye. The platform simultaneously detected lung cancer related miR-126 and miR-125a-5p. Therefore, this low cost, rapid, and convenient technology could be used for ultrasensitive and robust visual miRNA detection.

Tumor Angiogenesis Targeted Radiosensitization Therapy Using Gold Nanoprobes Guided by MRI/SPECT Imaging.

Gold nanoparticles (AuNPs) have recently garnered great interest as potential radiosensitizers in tumor therapy. However, major challenges facing their application in this regard are further enhancement of tumor accumulation of the particles in addition to enhanced permeability retention (EPR) effect and an understanding of the optimal particle size and time for applying radiotherapy after the particle administration. In this study, we fabricated novel cyclic c(RGDyC)-peptide-conjugated, Gd- and 99 mTc-labeled AuNPs (RGD@AuNPs-Gd99 mTc) probes with different sizes (29, 51, and 80 nm) and evaluated their potential as radiosensitization therapy both in vitro and in vivo. We found that these probes have a high specificity for αvβ3 integrin positive cells, which resulted in their high cellular uptake and thereby enhanced radiosensitization. Imaging in vivo with MRI and SPECT/CT directly showed that the RGD@AuNPs-Gd99 mTc probes specifically target tumors and exhibit greater accumulation within tumors than the RAD@AuNPs-Gd99 mTc probes. Interestingly, we found that the 80 nm RGD@AuNPs-Gd99 mTc probes exhibit the greatest effects in vitro; however, the 29 nm RGD@AuNPs-Gd99 mTc probes were clearly most efficient in vivo. As a result, radiotherapy of tumors with the 29 nm probe was the most potent. Our study demonstrates that RGD@AuNPs-Gd99 mTc probes are highly useful radiosensitizers capable of guiding and enhancing radiation therapy of tumors.

Hierarchical Flowerlike Gold Nanoparticles Labeled Immunochromatography Test Strip for Highly Sensitive Detection of Escherichia coli O157:H7

Gold nanoparticles (AuNPs) labeled lateral-flow test strip immunoassay (LFTS) has been widely used in biomedical, feed/food, and environmental analysis fields. Conventional ILFS assay usually uses spherical AuNPs as labeled probes and shows low detection sensitivity, which further limits its widespread practical application. Unlike spherical AuNP used as labeled probe in conventional ILFS, in our present study, a hierarchical flowerlike AuNP specific probe was designed for LFTS and further used to detect Escherichia coli O157:H7 (E. coli O157:H7). Three types of hierarchical flowerlike AuNPs, such as tipped flowerlike, popcornlike, and large-sized flowerlike AuNPs were synthesized in a one-step method. Compared with other two kinds of Au particles, tipped flowerlike AuNPs probes for LFTS particularly exhibited highly sensitive detection of E. coli O157:H7. The remarkable improvement of detection sensitivity of tipped flowerlike AuNPs probes can be achieved even as low as 10(3) colony-forming units (CFU)/mL by taking advantages of its appropriate size and hierarchical structures, which is superior over the detection performance of conventional LFTS. Using this novel tipped flower AuNPs probes, quantitative detection of E. coli O157:H7 can be obtained partially in a wide concentration range with good repeatability. This hierarchical tipped flower-shaped AuNPs probe for LFTS is promising for the practical applications in widespread analysis fields.

Horseradish peroxidase and antibody labeled gold nanoparticle probe for amplified immunoassay of ciguatoxin in fish samples based on capillary electrophoresis with electrochemical detection.

This paper describes a new amplified immunoassay with horseradish peroxidase (HRP) and antibody (Ab) labeled gold nanoparticles (AuNPs) probe hyphenated to capillary electrophoresis (CE) with electrochemical (EC) detection for ultrasensitive determination of ciguatoxin CTX1B. AuNPs were conjugated with HRP and Ab, and then incubated with limited amount of CTX1B to produce immunocomplex. The immunoreactive sample was injected into capillary for CE separation and EC detection. Enhanced sensitivity was obtained by adopting the AuNPs as carriers of HRP and Ab at high HRP/Ab molar ratio. The calibration curve of CTX1B was in the range of 0.06-90ng/mL. The detection limit was 0.045ng/mL, which is 38-fold lower than that of HPLC-MS method for CTX1B analysis. The proposed method was successfully applied for the quantification of CTX1B in contamined fish samples by simultaneously labeling Ab and HRP on AuNPs. The amplified IA with HRP and Ab labeled AuNPs probe hyphenated to CE and EC detection provides a sensitive analytical approach for the determination of trace ciguatoxin in complex samples.

Gold nanoparticles biosensor of Brucella spp. genomic DNA: Visual and spectrophotometric detections

IntroductionThe causative agents of brucellosis from genus Brucella can infect a variety of animal species and human. However, conventional methods for the detection of Brucella are time-consuming and the handling of microorganism is hazardous for laboratory personnel. In this study, a nanobiosensor based on gold nanoparticles (AuNPs) and oligonucleotide probe was designed for the visual detection of Brucella spp. MethodsA specific oligonucleotide probe from IS711 gene region was functionalized with AuNPs (AuNP-probe). Then, AuNP-probe was exposed to target (complementary) and non-target (non-complementary) DNA for the hybridization. Also, hybridization conditions were analyzed in the presence of Brucella genomic DNA and extracted DNA from clinical samples. ResultsUpon acid addition, a red color for the samples containing complementary DNA was observed, whereas in the samples without complementary DNA, AuNP-probe turned blue-purple. The results were investigated visually and also by UV–vis spectroscopic measurements. This method detected up to 1.09pgμL−1 of unamplified Brucella genomic DNA. Since Brucella is a facultative intracellular bacteria, clinical samples were amplified by PCR, and colorimetric detections were performed. The results showed high sensitivity and selectivity for the amplified clinical samples. ConclusionTo the best of our knowledge, this developed assay is highly specific, and this is the first assay that can be used with low cost, and as a method of rapid, direct and visual detection of Brucella spp.

Highly responsive glutathione functionalized green AuNP probe for precise colorimetric detection of Cd2+ contamination in the environment

Precise colorimetric detection of Cd2+ using a glutathione functionalized phytosynthesized AuNP probe provides an ecofriendly approach to heavy metal detection.

Colorimetric detection of influenza A virus using antibody-functionalized gold nanoparticles.

Early and accurate diagnosis is considered the key issue to prevent the further spread of viruses and facilitate influenza therapy. Herein, we report a colorimetric immunosensor for influenza A virus (IAV) based on gold nanoparticles (AuNPs) modified with monoclonal anti-hemagglutinin antibody (mAb). The immunosensor allows for a fast, simple, and selective detection of IAV. In this assay, influenza-specific antibodies are conjugated to AuNPs to create mAb-AuNP probes. Since IAV has multiple recognition sites for probes on the surface, the mAb-AuNP probes can be specifically arranged on the virus surface due to their very specific antigen recognition. In this case, this aggregation of the mAb-AuNP probes produces a red shift in the absorption spectrum due to plasmon coupling between adjacent AuNPs, and it can be detected with the naked eye as a color change from red to purple and quantified with the absorption spectral measurements. The aggregate formation is also confirmed with transmission electron microscopy (TEM) imaging and dynamic light scattering (DLS). Under the optimal conditions, the present immunoassay can sensitively measure H3N2 IAV (A/Brisbane/10/2007) with a detection limit of 7.8 hemagglutination units (HAU). This proposed immunosensor revealed high specificity, accuracy, and good stability. Notably, it is a single-step detection using AuNP probes and UV-vis spectrophotometer for readout, and no additional amplification, e.g., enzymatic, is needed to read the result. This assay depends on an ordered AuNP structure covering the virus surface and can be applied to any virus pathogen by incorporating the appropriate pathogen-specific antibody.

Aptamer biosensor for highly sensitive and selective detection of dopamine using ubiquitous personal glucose meters

In this paper a highly sensitive and selective aptamer biosensor for quantitative detection of a model target, dopamine, was developed by using a ubiquitous personal glucose meter (PGM). Dopamine aptamer was combined on the gold nanoparticles (AuNPs) modified 96-well plate. The signal probe, dopamine aptamer complementary strand (DNA2)–invertase–AuNPs, which was prepared with DNA2, invertase and AuNPs was immobilized onto the 96-well plate through the hybridization of aptamer and DNA2. Upon the addition of dopamine, aptamer would interact with it, and the hybridization of aptamer and DNA2 was destroyed and the DNA2–invertase–AuNPs probe was forced away from the surface of the 96-well plate. The invertase carried on AuNPs catalyzed the hydrolysis of sucrose to produce a large amount of glucose for quantitative readout by the PGM. PGM signal versus the concentration of dopamine was linear in the range from 0.08 to 100μmolL−1. The detection limit was 0.03μmolL−1 in 2μL (∼60femtomol), and the relative standard deviation (RSD) was 3.7% at 5μmolL−1 (n=7). The developed method is successfully demonstrated for dopamine determination in human blood serum. Due to its simplicity, sensitivity and selectivity, this biosensor offers a promising approach for the detection of dopamine and other biomolecules.

PolyA-tailed and fluorophore-labeled aptamer-gold nanoparticle conjugate for fluorescence turn-on bioassay using iodide-induced ligand displacement

Depending on the strong affinity of polyA sequence to gold (or silver) surface, applicability of polyA-tailed DNA–gold (or silver) nanoparticle conjugates in homogeneous and heterogeneous protein assays was first demonstrated. Interestingly, when using polyA-tailed, fluophore-labeled DNA–AuNP conjugate, it was found that iodide and thiosulfate anions could act as the ligand displacing reagent to detach polyA-tailed DNA strands from AuNP surface and simultaneously activate the AuNP-quenched fluorophores by destroying the polyA–AuNP interaction via a divide-and-conquer strategy. Based on this new discovery, we have developed a novel, cost-effective and sandwich-type fluorescence turn-on aptasensor for highly sensitive and specific thrombin detection, what took advantage of aptamer-conjugated magnetic beads (apt-MBs) for protein capture and separation, and iodide-induced fluorescence recovery of activatable polyA-based AuNP probes through ligand displacement for fluorescence turn-on detection. This proposed aptasensor could detect thrombin specifically with a detection limit as low as 89pM, which was better than or comparable to many existing fluorescent thrombin assays. Importantly, employment of such polyA-based AuNP conjugate not only avoids the use of thiolated oligonucleotides and thiol-containing displacing reagents, but also offers new possibilities for fabricating convenient and cost-effective bioanalytical applications.

Gold Nanoparticle–Dynamic Light Scattering Tandem for the Rapid and Quantitative Detection of the let7 MicroRNA Family

The discovery of miRNAs as key regulators of the cellular machinery and their implication in diseases has blossomed research in miRNA detection. Conventional techniques such as quantitative reverse transcriptase polymerase chain reaction (qRT–PCR), northern blotting and microarray technologies have contributed significantly to the detection of miRNAs. But, areas of unmet need remained and emerging nanotechnology platforms have the potential to make the biosensing process simpler while maintaining good sensitivity and selectivity. To this effect, a gold nanoparticle (AuNP) scattering technique is presented, which shows the controlled formation of defined nanoassemblies through the hybridization of uniquely designed AuNP probes to its targeted miRNA. The defined assemblies exhibit a distinct size shift that can be detected through dynamic light scattering (DLS). Thus, instead of using DLS as a characterization tool as it is conventionally used for, herein, its potential use is demonstrated as a bioanalytical system. Using the let7 family miRNA, which associates with increasingly many medical conditions, as the model of study, we are able to achieve a limit of detection of 100 fmol and a selectivity discriminating close members of the let7 family. Potentially, detection can be done in 5 min, which also makes this a rapid detection system.

A novel electrochemical sensing strategy for rapid and ultrasensitive detection of Salmonella by rolling circle amplification and DNA–AuNPs probe

A novel electrochemical sensing strategy was developed for ultrasensitive and rapid detection of Salmonella by combining the rolling circle amplification with DNA–AuNPs probe. The target DNA could be specifically captured by probe 1 on the sensing interface. Then the circularization mixture was added to form a typical sandwich structure. In the presence of dNTPs and phi29 DNA polymerase, the RCA was initiated to produce micrometer-long single-strand DNA. Finally, the detection probe (DNA–AuNPs) could recognize RCA product to produce enzymatic electrochemical signal. Under optimal conditions, the calibration curve of synthetic target DNA had good linearity from 10aM to 10pM with a detection limit of 6.76aM (S/N=3). The developed method had been successfully applied to detect Salmonella as low as 6CFUmL−1 in real milk sample. This proposed strategy showed great potential for clinical diagnosis, food safety and environmental monitoring.

Chronocoulometric biosensor for K-ras point mutation detection based on E. coli DNA ligase and AuNPs amplification effects

We report a novel method for the amplified detection of K-ras point mutation by using a chronocoulometric DNA sensor based on Escherichia coli DNA ligase and AuNPs amplification effects. Our method is based on a ‘sandwich’ detection strategy, involving capture probe immobilized on a gold electrode (GE) and reporter probe loaded on Au nanoparticles (AuNPs probe). Each probe flanks one of two fragments of the target DNA sequence. Successive hybridization event brings AuNPs, along with hundreds of reporter probes to form the ‘sandwich’ structure. After high-fidelity E. coli DNA ligase reaction and denaturing at an elevated temperature, We then employ chronocoulometry to detect [Ru(NH3)6]3+ electrostatically bounding to the DNA probe strands. If there is an allele mismatch, no ligation takes place. In this way, the novel approach had successfully distinguish the K-ras mutant DNA and the K-ras wild DNA. The charge variable (ΔQ) is proportional to the logarithmic K-ras mutant DNA concentration from 1.0nM to 0.1pM, with the detection limit of 0.01pM. Owing to its ease of operation and low detection limit, it is expected that the proposed approach may hold great promise in both research-based and clinical genomic assays.

Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

Selective determination of tobramycin in the presence of streptomycin through the visible light effect on surface plasmon resonance of gold nanoparticles

Tobramycin (TBR) is an antibiotic of the aminoglycoside class used for the treatment of bacterial infections. A careful study was made aiming to find conditions for the determination of TBR in the presence of streptomycin (STP) as well as in the presence of components used in ophthalmic solutions by taking advantage of the effect of the surface plasmon resonance of gold nanoparticles (AuNPs). The proposed modified Turkevich method for AuNPs synthesis as well as the stabilization of the aqueous dispersion under specific experimental conditions allowed a linear analytical response that did not require rationing of signal values to correct for linearity. The analytical curves, constructed by measuring transmitted light either at 529nm or at 681nm, covered three decades of concentration (from 4.0×10−9 to 1.6×10−7molL−1). In this concentration range, STP does not interact with the monosodium citrate capped AuNPs probe. The limit of detection for TBR was 3.8×10−9molL−1. The method was applied in ophthalmic solutions and in simulated samples containing STP. The results produced recoveries compatible to the ones achieved using a chromatographic method based on chemical derivatization. No significant interference was imposed by substances presents in ophthalmic solutions in their characteristic concentrations. Studies in saliva from volunteers under the treatment with TBR were made.

Sensitive Colorimetric Visualization of Perfluorinated Compounds Using Poly(ethylene glycol) and Perfluorinated Thiols Modified Gold Nanoparticles

In this work, we have developed a novel sensing strategy employing mixed poly(ethylene glycol)-terminated (PEG-thiols) and perfluoroalkyl-terminated (F-thiols) alkanethiols modified gold nanoparticles (Au@PEG-F NPs) as a probe to detect perfluorinated compounds (PFCs) from water samples. PEG-thiols with high density and long carbon chains make the Au NPs probe well-dispersed in solution and stable even in high concentration of salt solution; F-thiols provide specific fluorous-fluorous interactions to PFCs, which results in adsorption of PFCs on Au@PEG-F NPs. The adsorbed PFCs cause the aggregation of Au@PEG-F NPs probes and thus induce the insolubility of probes and precipitation directly from reaction solution due to the superhydrophobicity of perfluorocarbon monolayers, leading to color and absorbance response of the assay to PFCs. The preparation of the Au@PEG-F NPs probe is very simple, and the colorimetric assay based on this mechanism for the detection of PFCs is selective and convenient. Combined with UV-vis spectrophotometry, the assay demonstrates good sensitivities to PFCs with wide linear range. In the designed concentration range, the response of the colorimetric assay to long-chain PFCs (perfluoroalkyl chain ≥7) is discerned even as the concentration of these PFCs is as low as 10 μg L(-1). This low-cost and sensitive assay shows great potential to measure total PFCs in water samples. To the best of our knowledge, this is the first application of the specific fluorous-fluorous interactions and Au NPs based probes for colorimetric recognition for PFCs.

A rapid and sensitive immunoassay for detection of E. coli O157:H7 using multienzyme — Au nanoparticle complex

In this study, a novel design of gold nanoparticle (AuNP) probe containing large number of enzymes is reported for ultrasensitive detection of Escherichia coli (E. coli) O157:H7. The AuNP was functionalized with a polyclonal antibody (Pab) that has an affinity for E. coli O157:H7. The digoxigenin (DIG) incorporated oligonucleotides were covalently attached on the AuNP. And then Horseradish peroxidase (HRP) labeled anti-DIG was bound to the DIG incorporated oligonucleotide on the AuNP. Afterwards, magnetic microparticles (MMPs) were functionalized with monoclonal antibody (Mab) to E. coli O157:H7 and used to form a sandwich structure with target E. coli O157:H7 and AuNP probes via immune reaction. A magnetic field was used to effectively remove unbound AuNP probes. Finally, we exploited the reaction of HRP enzyme immobilized on AuNP probes with tetramethylbenzidine (TMB) that is a substrate of HRP, followed by stopping the reaction with 2 M H2SO4. The resulting end products were analyzed by UV-vis spectroscopy. Using this method, we could detect ca. 102 CFU/ mL of E. coli O157:H7 within an hour.