Endonuclease Controlled Aggregation of Gold Nanoparticles for the Ultrasensitive Detection of Pathogenic Bacterial DNA

The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNAse H enzymatic cleavage of the RNA probe, allowing the target DNA to liberate and hybridize with another RNA probe. This continuously happens until all of the RNA probes are cleaved, leaving the nanoparticles unprotected and thus aggregated upon exposure to a high electrolytic medium. The assay is ultrasensitive, allowing the detection of target DNA at femtomolar level by simple spectroscopic analysis (40.7 fM and 2.45 fM as measured by UV-vis and dynamic light scattering (DLS), respectively). The target DNA spiked food matrix (chicken meat) is also successfully detected at a concentration of 1.2 pM (by UV-vis) or 18.0 fM (by DLS). In addition to the ultra-high sensitivity, the total analysis time of the assay is less than 3 hours, thus demonstrating its practicality for food analysis.

Laser ablation-based one-step generation and bio-functionalization of gold nanoparticles conjugated with aptamers

Background: Bio-conjugated nanoparticles are important analytical tools with emerging biological and medical applications. In this context, in situ conjugation of nanoparticles with biomolecules via laser ablation in an aqueous media is a highly promising one-step method for the production of functional nanoparticles resulting in highly efficient conjugation. Increased yields are required, particularly considering the conjugation of cost-intensive biomolecules like RNA aptamers. Results: Using a DNA aptamer directed against streptavidin, in situ conjugation results in nanoparticles with diameters of approximately 9 nm exhibiting a high aptamer surface density (98 aptamers per nanoparticle) and a maximal conjugation efficiency of 40.3%. We have demonstrated the functionality of the aptamer-conjugated nanoparticles using three independent analytical methods, including an agglomeration-based colorimetric assay, and solid-phase assays proving high aptamer activity. To demonstrate the general applicability of the in situ conjugation of gold nanoparticles with aptamers, we have transferred the method to an RNA aptamer directed against prostate-specific membrane antigen (PSMA). Successful detection of PSMA in human prostate cancer tissue was achieved utilizing tissue microarrays. Conclusions: In comparison to the conventional generation of bio-conjugated gold nanoparticles using chemical synthesis and subsequent bio-functionalization, the laser-ablation-based in situ conjugation is a rapid, one-step production method. Due to high conjugation efficiency and productivity, in situ conjugation can be easily used for high throughput generation of gold nanoparticles conjugated with valuable biomolecules like aptamers.

Thin functional conducting polymer films

In the present study, thin functional conducting polyaniline (PANI) films, either doped or undoped, patterned or unpatterned, were prepared by different approaches. The properties of the obtained PANI films were investigated in detail by a combination of electrochemistry with several other techniques, such as SPR, QCM, SPFS, diffraction, etc. The sensing applications (especially biosensing applications) of the prepared PANI films were explored. Firstly, the pure PANI films were prepared by the electropolymerisation method and their doping/dedoping properties in acidic conditions were investigated in detail by a combination of electrochemistry with SPR and QCM. Dielectric constants of PANI at different oxidation states were obtained quantitatively. The results obtained here laid a good foundation for the following investigations of PANI films in neutral pH conditions. Next, PANI multilayer films doped by a variety of materials were prepared by the layer-by-layer method in order to explore their biosensing applications, because of the loss of redox activity of pure PANI in neutral pH conditions. The dopants used include not only the traditionally used linear polyelectrolytes, but also, for the first tim, some other novel materials, like modified gold nanoparticles or modified carbon nanotubes. Our results showed that all the used dopants could form stable multilayer films with PANI. All the obtained PANI multilayer films showed good redox activity in a neutral pH environment, which makes them feasible for bioassays. We found that all the prepared PANI multilayer films can electrocatalyze the oxidation of NADH in neutral conditions at a low potential, although their catalytic efficiencies are different. Among them, PANI/carbon nanotube system showed the highest catalytic efficiency toward the oxidation of NADH, which makes it a good candidate as a NADH sensor. Besides, because some of the prepared PANI multilayer systems were end-terminated with –COOH groups (like PANI/Au nanoparticles system), which can be utilized to easily link biomolecules for biosensing applications. Here, we demonstrated, for the first time, to use the prepared PANI multilayer films for the DNA hybridisation detection. The detection event was monitored either by direct electrochemical method, or by enzyme-amplified electrochemical method, or by surface plasmon enhanced fluorescence spectroscopic method. All the methods can effectively differentiate non-complementary DNA from the complementary ones, even at the single-base mismatch level. It should also be noted that, our success in fabricating PANI multilayer films with modified Au nanoparticles or carbon nanotubes also offered another novel method for incorporating such novel materials into (conducting) polymers. Because of the unique electrochemical and optical properties of each component of the obtained PANI multilayer films, they should also find potential applications in many other fields such as microelectronics, or for electrochromic and photovoltaic devices. Finally, patterned PANI films were fabricated by the combination of several patterning techniques, such as the combination of electrocopolymerization with micromolding in capillaries (EP-MIMIC), the combination of microcontact printing with the layer-by-layer technique (µCP-LBL), and the polystyrene (PS) template induced electropolymerisation method. Using the obtained stripe-shaped PANI/PSS film, a redox-switchable polymer grating based on the surface-plasmon-enhanced mode was constructed and its application in the field of biosensing was explored. It was found that the diffraction efficiency (DE) of the grating was very sensitive to the applied potential (i.e. redox state of the film) as well as the pH environment of the dielectric medium. Moreover, the DE could also be effectively tuned by an electrocatalytic event, such as the electrocatalytic oxidation of NADH by the grating film. By using PS colloidal crystal assemblies as templates, well-ordered 3D interconnected macroporous PANI arrays (PANI inverse opals) were fabricated via electropolymerisation method. The quality of the obtained inverse opals was much higher than those reported by chemical synthesis method. By electrochemical method, the structures of the prepared inverse opals can be easily controlled. To explore the possible biosensing applications of PANI inverse opals, efforts were also done toward the fabrication of PANI composite inverse opals. By selecting proper dopants, high quality inverse opals of PANI composites were fabricated for the first time. And the obtained opaline films remained redox-active in neutral pH conditions, pointing to their possible applications for electrobioassays.

“Green Electrodes” Modified with Au Nanoparticles Synthesized in Glycerol, as Electrochemical Nitrite Sensor

A new environmentally friendly Au nanoparticles (Au NPs) synthesis in glycerol by using ultraviolet irradiation and without extra-added stabilizers is described. The synthesis proposed in this work may impact on the non-polluting production of noble nanoparticles with simple chemicals normally found in standard laboratories. These Au NPs were used to modify a carbon paste electrode (CPE) without having to separate them from the reaction medium. This green electrode was used as an electrochemical sensor for the nitrite detection in water. At the optimum conditions the green sensor presented a linear response in the 2.0×10−7–1.5×10−5 M concentration range, a good detection sensitivity (0.268 A L mol−1), and a low detection limit of 2.0×10−7 M of nitrite. The proposed modified green CPE was used to determine nitrite in tap water samples.

Noble metal nanoparticles - Au and Ag - for biodetection

Dissertation submitted for obtainment of the Master’s Degree in Biotechnology, by the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label-Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold-nanoparticles over graphene screen-printed electrode (SPE). The antibody was covalently bound to CS via its Fc-terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG-immunosensor displayed linear response against the logarithm-hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Gold nanoprobes for the detection of mutations associated with antibiotic resistance in Mycobacterium tuberculosis complex

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Study of gold nanoparticle assisted neuron stimulation

The unique proprieties exhibited by nanoscale particles compared to their macro size counterparts allow for the creation of novel neural activity manipula-tion procedures. In this sense, gold nanoparticles (AuNPs) can be used to stimu-late the electrical activity of neuron by converting light into heat. During this dissertation, AuNPs are synthesized by the citrate reduction method, resulting in a hydrodynamic diameter of approximately 16 nm and an absorbance peak of 530 nm. A system to control a 532 nm laser and measure the temperature variation was custom built from scratch specifically for this project. Temperature is then measured with recourse to a thermocouple and through changes in impedance. The built system had in consideration the necessities pre-sented by in vivo tests. Trials were performed by measuring the temperature rise of colloidal AuNP solutions, having the temperature variation reached a maximum of ap-proximately 18 ºC relative to control trials; successfully showing that light is ef-fectively transduced into heat when AuNPs are present. This novel approach enables an alternative to optogenetics, which require the animal to be genetically modified in order to allow neuron stimulation.

Self-assembly of a Model Peptide incorporating a Hexa-Histidine sequence attached to an Oligo-Alanine sequence, and binding to Gold NTA/Nickel nanoparticles

Amyloid fibrils are formed by a model surfactant-like peptide (Ala)10-(His)6 containing a hexahistidine tag. This peptide undergoes a remarkable two-step self-assembly process with two distinct critical aggregation concentrations (cac’s), probed by fluorescence techniques. A micromolar range cac is ascribed to the formation of prefibrillar structures, whereas a millimolar range cac is associated with the formation of well-defined but more compact fibrils. We examine the labeling of these model tagged amyloid fibrils using Ni-NTA functionalized gold nanoparticles (Nanogold). Successful labeling is demonstrated via electron microscopy imaging. The specificity of tagging does not disrupt the β-sheet structure of the peptide fibrils. Binding of fibrils and Nanogold is found to influence the circular dichroism associated with the gold nanoparticle plasmon absorption band. These results highlight a new approach to the fabrication of functionalized amyloid fibrils and the creation of peptide/nanoparticle hybrid materials.

Supramolecular Approach to Gold Nanoparticle/Triruthenium Cluster Hybrid Materials and Interfaces

A systematic and comprehensive study of the interaction of citrate-stabilized gold nanoparticles with triruthenium cluster complexes of general formula [Ru(3)(CH(3)COO)(6)(L)](+) [L = 4-cyanopyridine (4-CNpy), 4,4`-bipyridine (4,4`-bpy) or 4,4`-bis(pyridyl)ethylene (bpe)] has been carried out. The cluster-nanoparticle interaction in solution and the construction of thin films of the hybrid materials were investigated in detail by electronic and surface plasmon resonance (SPR) spectroscopy, Raman scattering spectroscopy and scanning electron microscopy (SEM). Citrate-stabilized gold nanoparticles readily interacted with [Ru(3)O(CH(3)COO)(6)(L)(3)](+) complexes to generate functionalized nanoparticles that tend to aggregate according to rates and extents that depend on the bond strength defined by the characteristics of the cluster L ligands following the sequence bpe > 4,4`-bpy >> 4-CNpy. The formation of compact thin films of hybrid AuNP/[Ru(3)O(CH(3)COO)(6)(L)(3)](+) derivatives with L = bpe and 4,4`-bpy indicated that the stability/lability of AuNP-cluster bonds as well as their solubility are important parameters that influence the film contruction process. Fluorine-doped tin oxide electrodes modified with thin films of these nanomaterials exhibited similar electrocatalytic activity but much higher sensitivity than a conventional gold electrode in the oxidation of nitrite ion to nitrate depending on the bridging cluster complex, demonstrating the high potential for the development of amperometric sensors.

Ligand-Assisted Preparation of Palladium Supported Nanoparticles: a Step toward Size Control

Supported nanoparticles (SNPs) with narrow size distribution were prepared by H(2) reduction of Pd(2+) previously bound, to ligand-modified silica surfaces. Interestingly, the size of the Pd SNPs was tuned by the ligand grafted on the support surface. Amino- and ethylenediamino-functionalized supports formed Pd(0) SNPs of ca. 6 and 1 nm, respectively. The catalytic properties of both Pd(0) SNPs were investigated.