Nanogold Probe Research Articles

ExoSloNano: Multi-Modal Nanogold Tags for identification of Macromolecules in Live Cells & Cryo-Electron Tomograms.

In situ cryo-Electron Microscopy (cryo-EM) enables the direct interrogation of structure-function relationships by resolving macromolecular structures in their native cellular environment. Tremendous progress in sample preparation, imaging and data processing over the past decade has contributed to the identification and determination of large biomolecular complexes. However, the majority of proteins are of a size that still eludes identification in cellular cryo-EM data, and most proteins exist in low copy numbers. Therefore, novel tools are needed for cryo-EM to identify the vast majority of macromolecules across multiple size scales (from microns to nanometers). Here, we introduce and validate novel nanogold probes that enable the detection of specific proteins using cryo-ET (cryo-Electron Tomography) and resin-embedded correlated light and electron microscopy (CLEM). We demonstrate that these nanogold probes can be introduced into live cells, in a manner that preserves intact molecular networks and cell viability. We use this system to identify both cytoplasmic and nuclear proteins by room temperature EM, and resolve associated structures by cryo-ET. We further employ gold particles of different sizes to enable future multiplexed labeling and structural analysis. By providing high efficiency protein labeling in live cells and molecular specificity within cryo-ET tomograms, we establish a broadly enabling tool that significantly expands the proteome available to electron microscopy.

A Monoclonal Antibody-Based Immunochromatographic Test Strip and Its Application in the Rapid Detection of Cucumber Green Mottle Mosaic Virus

Two specific monoclonal antibodies (mAbs) were screened, and an immunochromatographic strip (ICS) test for rapid and specific detection of cucumber green mottle mosaic virus (CGMMV) was developed. The coat protein of CGMMV was heterologously expressed as an immunogen, and specific capture mAb 2C9 and the detection mAb 4D4 were screened by an uncompetitive immunoassay. The test and control lines on the nitrocellulose membrane were coated with the purified 2C9 and a goat anti-mouse IgG, respectively, and a nanogold probe combined with 4D4 was applied to the conjugate pad. Using these mAbs, a rapid and sensitive ICS was developed. Within the sandwich mode of 2C9-CGMMV-4D4, the test line showed a corresponding positive relationship with CGMMV in infected samples. The ICS test had a detection limit of 1:5000 (w/v) for CGMMV in samples and was specific for CGMMV, with no observed cross-reaction with TMV or CMV.

In situ strategy for biomedical target localization via nanogold nucleation and secondary growth

Immunocytochemistry visualizes the exact spatial location of target molecules. The most common strategy for ultrastructural immunocytochemistry is the conjugation of nanogold particles to antibodies as probes. However, conventional nanogold labelling requires time-consuming nanogold probe preparation and ultrathin sectioning of cell/tissue samples. Here, we introduce an in situ strategy involving nanogold nucleation in immunoenzymatic products on universal paraffin/cryostat sections and provide unique insight into nanogold development under hot-humid air conditions. Nanogold particles were specifically localized on kidney podocytes to target synaptopodin. Transmission electron microscopy revealed secondary growth and self-assembly that could be experimentally controlled by bovine serum albumin stabilization and phosphate-buffered saline acceleration. Valuable retrospective nanogold labelling for gastric H+/K+-ATPase was achieved on vintage immunoenzymatic deposits after a long lapse of 15 years (i.e., 15-year-old deposits). The present in situ nanogold labelling is anticipated to fill the gap between light and electron microscopy to correlate cell/tissue structure and function.

Differential diagnosis of Plasmodium falciparum and Plasmodium vivax in mixed infection by colorimetric nanogold probes

Malaria is an infectious disease reported mostly in the tropical region. The most severe human malaria is Plasmodium falciparum since it can cause cerebral malaria. Therefore, the presence of P. falciparum either in single or mixed infection needs accurate diagnosis. In some mixed infections, the presence of P. falciparum may be cryptic which cannot be detected by microscopic examination. The molecular diagnosis is required in these cases. Many methods based on amplification of malaria parasite genes have been developed but most of them need sophisticated instruments. Here, we created a colorimetric method using probe immobilized gold nanoparticles (AuNPs) to detect the malaria parasite gene. Color changes rely on salt-induced aggregation of AuNPs in the presence or absence of DNA hybridization. Color changes could be observed either by a naked eye or UV–vis spectrophotometer. By this approach, single infection by the most common malaria parasite, P. falciparum or P. vivax could be differentially identified. Mixed infection of these two malaria species could also be clearly diagnosed including cases of cryptic P. falciparum. The novel nanogold based molecular malaria diagnosis is sensitive, specific, rapid and cheap ($0.94). The prepared nanogold malaria probes are stable for up to 3 months indicating their filed application in remote areas.

Rapid colorimetric loop-mediated isothermal amplification for hypersensitive point-of-care Staphylococcus aureus enterotoxin A gene detection in milk and pork products

Staphylococcus aureus strains carrying enterotoxin A gene (sea) causes food poisoning and cannot be distinguished from non-pathogenic strains by the culture method. Here, we developed a rapid, specific and sensitive visual detection of sea using loop-mediated isothermal amplification (LAMP) combined with nanogold probe (AuNP) or styryl dye (STR). LAMP-AuNP and LAMP-STR can detect as low as 9.7 fg (3.2 sea copies) and 7.2 sea copies, respectively, which were lower than PCR (97 fg or 32 sea copies). The excellent performance of these new assays was demonstrated in food samples using crude DNA lysates. While the culture method detected 104 CFU/g in ground pork and 10 CFU/mL in milk in 5–7 days, LAMP-AuNP could detect down to 10 CFU/g for both samples in 27 minutes. Analyzing 80 pork and milk samples revealed that the LAMP-AuNP showed 100% sensitivity, 97–100% specificity and 97.5–100% accuracy, which were superior to the culture method, and comparable to PCR but without requirement of a thermal cycler. Furthermore, our LAMP-AuNP detect sea at a range below the food safety control (<100 CFU/g). The LAMP-STR quantitated sea in 10–1,000 CFU (7.2–720 copies). Our crude DNA lysis combined with LAMP-AuNP/STR present effective point-of-care detection and facilitate appropriate control strategies.

Application of a Y-type-DNA-functionalized nanogold probe featuring specific telomerase recognition and doxorubicin release in cancer cells.

A Y-type-DNA-functionalized nanogold probe was synthesized to identify telomerase and trigger drug release in cancer cells. This system involved a DNA-functionalized nanogold probe centered on gold nanoparticles with a dense modification of Y-type DNA molecular beacons on the surface. The Y-type DNA molecular beacons consisted of telomerase primers (TPs), a FAM-labeled single-strand DNA (Mismatch-DNA), and a single-strand DNA of two templates (Linker-DNA1 and Linker-DNA2). Doxorubicin (Dox), an anti-cancer drug molecule, was inserted into DNA double strands. When telomerase existed, TP was extended and the Mismatch-DNA was released, leading to the emission of fluorescent light from FAM while releasing Dox. This probe specifically detected cancer cells and did not affect normal cells. This drug delivery system will reduce the tumour size and cause minimal injury to normal tissues.

A Rapid and Sensitive Aptasensor for Cyromazine Detection in Raw Milk Based on a Nanogold Probe and G-Quadruplex Formation

Herein, a rapid, facile, and colourimetric sensor for the detection of cyromazine in raw milk is reported using an aptamer based on gold nanoparticles (AuNPs). A sequence-specific aptamer for cyromazine called Tcyr1 is designed to absorb on the surface of AuNPs and electrostatically interacts with poly(diallyldimethylammonium chloride) (PDDA), which prevents AuNPs from aggregating. It can also self-assemble to form a G-quadruplex-CYR complex with cyromazine. Because of its specificity and stability, the introduction of cyromazine in raw milk would influence the protection thus the following cationic polymer could aggregate AuNPs and cause a remarkable change in colour. According to this, the presence of cyromazine can be determined by the naked eye and means of absorbance. This sensor is selective for the detection of cyromazine in raw milk and has a limit of detection of 200 ppb by the naked eye and of 5.8 ppb by spectrophotometer, and has a detection range from 0.1 to 1 ppm.

Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonasaeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is an important opportunistic pathogen that causes serious infections in humans, including keratitis in contact lens wearers. Therefore, establishing a rapid, specific and sensitive method for the identification of P. aeruginosa is imperative. In the present study, the uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification combined with nanogold labeled hybridization probe (UDG-LAMP-AuNP) was developed for the detection of P. aeruginosa. UDG-LAMP was performed to prevent carry over contamination and the LAMP reactions can be readily observed using the nanogold probe. A set of 4 primers and a hybridization probe were designed based on the ecfX gene. The UDG-LAMP reactions were performed at 65°C for 60 min using the ratio of 40% deoxyuridine triphosphate to 60% deoxythymidine triphosphate. The detection of UDG-LAMP products using the nanogold labeled hybridization probe, which appeared as a red-purple color, was examined at 65°C for 5 min with 40 mM MgSO4. The UDG-LAMP-AuNP demonstrated specificity to all tested isolates of P. aeruginosa without cross reaction to other bacteria. The sensitivity for the detection of pure culture was 1.6×103 colony-forming units (CFU) ml−1 or equivalent to 3 CFU per reaction while that of polymerase chain reaction was 30 CFU per reaction. The detection limit of spiked contact lenses was 1.1×103 CFU ml−1 or equivalent to 2 CFU per reaction. In conclusion, the UDG-LAMP-AuNP assay was rapid, simple, specific and was effective for the identification of P. aeruginosa in contaminated samples.

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

Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non-AHPND bacteria commonly found in shrimp ponds (including other Vibrio species). The new method significantly reduced the time, difficulty and cost for molecular detection of VPAHPND in shrimp hatchery and farm settings.

Comparison of tumor markers using different detection devices.

BackgroundWith the development of proteomics, tumor markers have attracted increasing attention for the early diagnosis and treatment of lung cancer. As biochip technology and nanotechnology continues to grow, rapid and highly sensitive joint detection of multi-tumor markers has become possible.MethodsEighty-six patients with lung cancer and 42 healthy controls were recruited for this study. Based on analysis of the detection results, we plotted four standard tumor marker graphs, and compared the results of the highly sensitive nanogold probe and protein chip detection with the results of electrochemiluminescence immunoassay and Dickkopf-1 (DKK1) detection used in the clinic. We then analyzed the relationship between the detection results and our clinical data.ResultsFour plotted standard protein graphs all had stages with sound linear relationships. It was found in a correlation analysis of the detection results that overall the two methods showed consistency.ConclusionWe developed a detection method for ultra-trace protein that can detect four tumor markers, namely carcinoembryonic antigen, cytokeratin-19 fragments, neuron-specific enolase, and DKK1 in a highly sensitive way within 1.5 hours by magnifying the signal of nanogold deposition based on protein chips and nanogold probes. By comparing the results from the different detection devices, we have developed an experimental basis for detection of tumor markers in the clinic.

An on-site immunosensor for ractopamine based on a personal glucose meter and using magnetic β-cyclodextrin-coated nanoparticles for enrichment, and an invertase-labeled nanogold probe for signal amplification

We report on an on-site ultrasensitive sandwich immunoassay for ractopamine (RAC) that is based on the use of a personal glucose meter. RAC is preconcentrated with the help of magnetic nanoparticles coated with β-cyclodextrin (Fe3O4@SiO2-CD), and a novel polymer tag labeled with invertase is employed to catalyze the hydrolysis of sucrose to form glucose which is detected with the glucose meter. Invertase-modified gold nanoparticles were prepared and immobilized, along with antibodies against RAC, on an Envision reagent (a polymer that contains many anti-IgG antibodies). Because of its high loading with the enzyme, the resulting copolymers strongly amplify the signal. The concentration of RAC is calculated on the basis of the linear relationship between the concentration of RAC and the amount of glucose formed. The method exhibits a detection limit of 0.34 μg kg−1 as determined for a sample of pork. This on-site testing method may also be applied to the detection of other low-molecular toxins by simply changing the antibody.

Genotyping of α-thalassemias by the colorimetric nanogold probes

BackgroundThe novel colorimetric nanogold probe was created to genotype subgroups of the mostly found α-thalassemias. They are α-thalassemia 1 (SEA and THAI deletion) and α-thalassemia 2 (3.7-kb and 4.2-kb deletion). MethodsThe genotyping was performed by two-steps hybridizations. First step was hybridization of target DNA with the nanogold mixed probes of either α-thalassemia 1 or α-thalassemia 2. No hybridization in both reactions showing blue color indicated absence of abnormal genes causing these α-thalassemias. Positive reaction showing either red or purple color was further analyzed in second hybridization with the nanogold single probe. Positive of α-thalassemia 1 was genotyped with the single probes of both SEA and THAI deletion while those of α-thalassemia 2 were genotyped with both 3.7-kb and 4.2-kb deletion. ResultsGenotypic potency of the nanogold mixed and single probes was evaluated using both known diagnosed and suspected clinical samples. The results by naked eye were consistence with those analyzed by standard agarose gel electrophoresis. ConclusionsPotency of the colorimetric nanogold α-thalassemia probes was accurate, precise, sensitive, specific, simple, cheap and field applicable. Color reaction was simply visualized by naked eye. This development is an example of colorimetric molecular diagnosis which can be applied in any genetic detection.

Resonance Rayleigh scattering detection of trace PDGF based on catalysis of an aptamer-modified nanogold probe in the Fehling reaction

Gold nanoparticles (GN) were modified by a platelet-derived growth factor (PDGF) aptamer to obtain stable aptamer-nanogold probes (Apt-GN). The probes specifically combined with PDGF-AA to form Apt-GN–PDGF-AA clusters that exhibited a resonance Rayleigh scattering (RRS) peak at 550 nm. The RRS intensity ΔI550nm was linear to the PDGF-AA concentration in the range of 0.33–40 ng mL−1. The probes exhibit strong catalysis of the Fehling reagent–glucose Cu2O particle reaction that can be monitored by the RRS technique at 610 nm, but the cluster is very weak. When PDGF-AA concentration increased, the Apt-GN decreased, and the RRS intensity at 610 nm decreased. The decreased RRS intensity ΔI610nm was linear to PDGF-AA concentration in the range of 0.03–26.67 ng mL−1. Accordingly, two new aptamer-nanogold RRS methods were established.

Molecular diagnosis of α-thalassemias by the colorimetric nanogold

A new application of gold nanoparticles (AuNPs) as a colorimetric method for gene detection of α-thalassemia1 (SEA deletion) is reported here for the first time. This technique is based on color changes from salt-induced aggregation of un-hybridized nanogold probes after hybridization with the target DNA. Specific DNA probes were synthesized, thiol modified and conjugated on the surface of AuNPs. The target DNA was amplified and hybridized with the AuNPs-immobilized probe. Salt solution (NaCl) was added to induce aggregation of the un-hybridized nanogold probes. The color changes were visualized either by the naked eye or by UV-vis spectrophotometry at 520 nm. By this nanogold colorimetric method samples carrying normal α-globin genes could be successfully identified from samples carrying α-globin genes causing α-thalassemia1 (SEA deletion), either as a carrier or disease form. Results demonstrated that the new colorimetric nanogold method is a definite gene diagnosis of α-thalassemia. It is accurate, simple, rapid, specific, sensitive, and cost effective. It is also a promising point-of-care testing (POCT) method for thalassemias and other genetic disorders. The new colorimetric nanogold is a method of choice for areas where access to sophisticated molecular diagnosis is limited.

A simple and sensitive resonance Rayleigh scattering method for determination of As(III) using aptamer‐modified nanogold as a probe

A simple and selective aptamer (ssDNA)-modified nanogold probe (AussDNA) was prepared for the determination of trace As(III) in HEPES buffer solution (pH 8.2) containing 0.05 mol/L NaCl. The method coupled the aptamer reaction of AussDNA-As(III) and the resonance Rayleigh scattering (RRS) of nanogold aggregations at 278 nm. When the As(III) concentration increased, the RRS intensity at 278 nm increased to form more nanogold aggregation and a stable As(III)-ssDNA complex. Under selected conditions, the increased RRS intensity (ΔI) was linear to the concentration of As(III) in the range 3.8-230.4 ng/mL, with a detection limit of 1.9 ng/mL. This RRS method was applied to detect As(III) in water samples, with simplicity, sensitivity and selectivity.

Nanogold Resonance Rayleigh Scattering Method for Trace NO&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; Based on &lt;i&gt;P&lt;/i&gt;-Aminobenzene Sulfonic Acid-&lt;i&gt;Α&lt;/i&gt;-Naphthylamine Colored Reaction

In acidic condition, NO2- combined with p-aminobenzene sulfonic acid by diazotization, and then reacted with α-naphthylamine to form purple azo dye. The resonance Rayleigh scattering (RRS) signal of the color system was detected by nanogold probe. Under the chosen conditions, the decreased RRS intensity ΔI (ΔI= Ib - I) at 500 nm was linear to the NO2- concentration in the range of 7.0×10-2-42.0μmol/L, with a regression equation of ΔI=6.72 C - 6.93, a correlation coefficient of 0.9982 and a detection limit of 2.6×10-4 mol/L NO2-.

Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp.

Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop-mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite. A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 in at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA-labelled nanogold probe, followed by salt-induced AuNP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0·02 fg total DNA) than a conventional nested PCR (0·2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP-AuNP assay was specific for E. hepatopenaei. Without sacrificing sensitivity or specificity, the new LAMP-AuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp. The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.

Resonance scattering spectrum detection of trace using nanogold probe as catalyst of Cu(II)-glucose reaction

exhibited catalytic effect on the cracking reaction of double-stranded DNA (dsDNA) to form a short single-stranded DNA (ssDNA) that protected the nanogold (NG) to produce stable NGssDNA conjugate. The un-protected NG was aggregated to form NG aggregates (NGA) that appeared a resonance scattering (RS) peak at 542 nm. Unlike NGA, the NGssDNA exhibited a strong catalytic activity on the Cu2O particle reaction of Fehling reagent-glucose that can be monitored by RS technique at 608 nm. When the concentration increased, the NGssDNA increased, and the RS intensity at 608 nm increased. The increased RS intensity ΔI 608 nm was linear to the concentration in the range of 15–200 pmol L−1, with a regression equation of ΔI 608 nm = 1.24 C + 4.6, and a detection limit of 5 pmol L−1. This new RS assay was applied to assay in water sample, with satisfactory results.

Sensitive colorimetric detection of melamine in milk with an aptamer-modified nanogold probe

We report a rapid and facile colorimetric sensing method for melamine detection in milk using aptamer based nanoparticles (AuNPs). Aptamers can absorb on the surface of AuNPs and electrostatically interact with poly(diallyldimethylammonium chloride) (PDDA), which protects the AuNPs from aggregation. The introduction of melamine causes a melamine–aptamer complex to form via hydrogen bonding, thus the resulting cationic polymer can aggregate the AuNPs and cause a remarkable change in color. As a result of this, the presence of melamine can be determined by the naked eye and by measuring absorbance. This sensor is selective for the detection of melamine in milk samples and has a limit of detection of 34 nM. Since aptamer-modified AuNPs are more stable and selective than unmodified AuNPs in the sensing of melamine, we hope that this type of detection method will be helpful for the detection of other small molecules.

A highly sensitive aptamer method for Ag+sensing using resonance Rayleigh scattering as the detection technique and a modified nanogold probe

An aptamer was modified using a gold nanoparticle (AuNP) to form a stable aptamer–AuNP probe that was not aggregated in the pH 7.2 2-4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer solution in the presence of NaCl. The Ag+ reacted with the aptamer–AuNP probe to form a hairpin structure complex of Ag+–aptamer and released the AuNPs that were aggregated to large particles, which led to a resonance Rayleigh scattering (RRS) peak at 596 nm enhancement. The enhanced value ΔI596 nm varies linearly with the Ag+ concentration in the range 6.7–133.3 × 10−8 M. The probe in the aptamer reaction solution has a strong catalytic effect on the Cu2O particle reaction of Fehling reagent and glucose, and the particle products exhibited a strong RRS peak at 610 nm. However, catalysis of AuNP aggregation is very weak, which means it can not be removed from the aptamer reaction solution. When the concentration of Ag+ increased, the RRS peak intensity at 610 nm decreased. The decreased value ΔI610 nm is linear to the Ag+ concentration in the range of 3.3–666.7 × 10−9 M. Thus, two new RRS methods were proposed for the detection of Ag+, with high sensitivity, good selectivity and simplicity. A highly sensitive resonance Rayleigh scattering method was proposed for trace Ag+ sensing using the aptamer-modified nanogold as a probe.