N A N O P R O B E S     E - N E W S

Vol. 10, No. 8          August 31, 2009

Features of NTA-Ni(II)-Nanogold include:

• Higher labeling resolution. The NTA-Ni(II) chelate is much smaller than an antibody or protein: when it is bound, the gold is within about 1.5 nm of the target. This makes NTA-Ni(II)-Nanogold ideal for localizing subunits in protein complexes or specific sites in macromolecular assemblies at molecular resolution.

• Better penetration: because it is so small, NTA-Ni(II)-Nanogold can more easily penetrate into specimens and access sterically restricted sites within specimens. In some systems it may be used with stronger fixation or less permeabilization, enabling labeling with better ultrastructural preservation.

• Smaller size means less ultrastructural perturbation: it is ideal for labeling proteins in their natural configuration.

• Binding constants for Ni(II)-NTA are very high due to the combination of the chelate effect of multiple histidine binding, and target binding of multiple Ni(II)-NTA groups on each gold particle. Dissociation constants are estimated to be between 10^-7 to 10^-13 M^-1, comparable to antibodies for many applications.

• NTA-Ni(II)-Nanogold is prepared using a modified gold particle, with very high solubility and stability. At 1.8 nm in size, it is readily visualized by electron microscopy.

Calcium entry through voltage-gated calcium channels affects a wide range of physiological processes. Neuronal excitability, muscle excitation-contraction coupling, and secretion are all influenced by calcium flux, and therefore the mechanisms by which calcium is distributed are important targets for study. Using single particle analysis methods, Walsh and group have determined the first three-dimensional structure, at 23 Å resolution, of one member of the low voltage-activated voltage-gated calcium channel family, recombinant Ca_v3.1, a T-type channel.

Recombinant Ca_v3.1 was expressed in Sf9 cells: Ca_v3.1 containing a C-terminal c-Myc and decahistidine (His10) tag was subcloned into the baculovirus vector pFastBac-HTa. Sf9 insect cells were cultured in TNM-FH medium supplemented with 10% (v/v) fetal bovine serum and split when confluent. For protein expression, 7080% confluent Sf9 cells were infected with recombinant baculoviruses at a multiplicity of infection of 12, then incubated at 27°C for 72 hours. Virus-infected Sf9 cells expressing the Ca_v3.1 were centrifuged at 1000 x g for 5 minutes, resuspended in 10 mL of 20 mM Tris, pH 8.0, with 150 mM NaCl and 1 mM DTT plus protease inhibitor, then lysed by sonication for 3 x 30 seconds. After centrifugation at 1000 x g for 5 minutes to remove large particulate matter, the Sf9 cell membranes were pelleted by centrifugation at 50,000 x g for 1 hour. The pellet was resuspended in 10 mL of 20 mM Tris, pH 8.0, containing 150 mM NaCl, 20 mM imidazole, 1 mM DTT and 1% (w/v) CHAPS with protease inhibitors, incubated for 1 hour on a rolling platform at 4°C. Unsolubilized material was removed by centrifugation at 20,000 x g for 30 minutes at 4°C, the resulting supernatant was diluted 1:1 with 20 mM Tris buffer, pH 8.0, with 150 mM NaCl, 20 mM imidazole, and 1 mM DTT, and the His-tagged protein recovered chromatographically using a 1 mL Ni-NTA column pre-equilibrated with 20 mM Tris buffer, pH 8.0, with 150 mM NaCl, 20 mM imidazole, 1 mM DTT and 0.5% (w/v) CHAPS. The protein solution was eluted by the addition of 1 column volume of equilibration buffer containing 350 mM imidazole.

For the gold labeling, excess Ni-NTA-Nanogold was added to the purified Ca_v3.1 and incubated for 3 hours. Aliquots were then stained with uranyl acetate. Both unlabeled and labeled Ca_v3.1 were examined using the same electron microscopic procedure, and analyzed using the same initial start model.

Unlabeled and labeled Ca_v3.1 samples were examined using standard negative staining electron microscopic techniques with 2% (w/v) uranyl acetate. Images were recorded between a 0.651.05 µm defocus range at 100 kV, at a calibrated magnification of x 47,800. Micrographs were scanned at 3.6 Å/pixel at the specimen level. The contrast transfer function (CTF) was determined for each image using CTFIT (part of the EMAN software); images were corrected for phase effects only. The resolution of the final three-dimensional volumes were assessed using established procedures: each data set was divided into two, with volumes calculated for the two subsets, and the resolution was estimated by the Fourier shell correlation (FSC) coefficient with the resolution limit taken to be where the FSC value fell below 0.5. All models were generated using the UCSF Chimera program.

The three-dimensional reconstruction of recombinant Ca_v3.1 was generated using the common line projection matching methods employed in EMAN software, with no symmetry applied (C1). A wide variety of starting models were tested including Gaussian blobs, transmembrane domain of KV1.2 (PDB entry 2R9R, edited), and the full KV1.2 crystal structure (2R9R) filtered to an appropriate resolution. In all reconstructions, the principal structural features were consistent, indicating the fidelity of the final three-dimensional reconstruction. For the unlabeled protein, approximately 11,500 particles were selected interactively using the graphical interface boxer (EMAN image processing software) into a box of 64 x 64 pixels (230 x 230 Å); approximately 4,500 gold-labeled Ca_v3.1 particles were selected, and a good sampling of different orientations of labeled Ca_v3.1 was observed.

Particles were band-pass-filtered and centered, and a preliminary three-dimensional model was calculated from a set of unbiased reference-free class averages showing different orientations of the complex (refine2d.py program under EMAN). The three-dimensional model was iteratively refined at each step using a projection matching routine, whereby projections with uniformly distributed orientations of the preliminary three-dimensional model were used as references for classification of the raw data set, with the class averages from this step used to construct a new three-dimensional model. Refinement was carried out with an angular coverage of 7° generating 388 classes with between 10 and 48 particles per class. Final reconstruction included 86% of the selected images. Convergence (stabilization of the three-dimensional structure), was monitored by examining the FSC of the three-dimensional models generated from each iteration.

Ca_v3.1 has dimensions of ~115 x 85 x 95 Å. It is composed of two distinct segments. The cytoplasmic densities form a vestibule below the transmembrane domain with the C terminus, which was unambiguously identified by the presence of a His tag and attached gold label: this was ~65 Å long, and curls around the base of the structure. The cytoplasmic assembly has a large exposed surface area, which may serve as a signaling hub, where the C terminus acts as a "fishing rod" to bind regulatory proteins. A three-dimensional structure was also determined at a resolution of 25 Å for the monomeric form of the cardiac L-type voltage-gated calcium (high voltage-activated) channel with accessory proteins beta and alpha₂delta bound to the ion channel polypeptide Ca_v1.2. Comparison with the skeletal muscle isoform finds a good match particularly with respect to the conformation, size, and shape of the domain identified as that formed by alpha₂. Modeling of the Ca_v3.1 structure (analogous to Ca_v1.2 at these resolutions) into the heteromeric L-type voltage-gated calcium channel complex volume reveals multiple interaction sites for beta-Ca_v1.2 binding and for the first time identifies the size and organization of the alpha₂delta polypeptides.

Reference:

• Walsh, C. P.; Davies, A.; Butcher, A. J.; Dolphin, A. C., and Kitmitto, A.: Three-dimensional structure of CaV3.1: comparison with the cardiac L-type voltage-gated calcium channel monomer architecture. J. Biol. Chem., 284, 22310-22321 (2009).
  • Abstract (courtesy of the Journal of Biological Chemistry).
  • Reprint (PDF) (courtesy of the Journal of Biological Chemistry).

We will shortly introduce 5 nm NTA-Ni(II)-Gold. This probe provides new features, improved performance, and extends NTA-Ni(II) targeting technology to larger gold. It has the same high resolution as Ni-NTA-Nanogold, and the entire probe will still be smaller than an IgG molecule: however, the larger gold particles may be clearly visualized by standard TEM without silver or gold enhancement, even in wider views such as thick sections and whole cells. Blot sensitivity will be even higher due to the larger gold.

Features and advantages:

• High visibility: 5 nm gold is clearly visualized at TEM resolution without silver or gold enhancement. Labeling is simplified and more monodisperse: you can use this probe for multiple labeling studies in conjunction with gold particles of different sizes, or with silver or gold-enhanced Nanogold.

• Precise labeling resolution: when the probe is bound, the gold is much closer to its target: about 1.5 nm. This makes NTA-Ni(II)-Nanogold ideal for localizing molecular sites in protein complexes or other macromolecular assemblies.

• High solubility and stability: 5 nm Ni-NTA-Gold is prepared using a modified gold particle with a stable, highly hydrophilic surface functionalization.

• Strong binding: binding constants for Ni(II)-NTA-5 nm Gold are very high due to the combination of the chelate effect of multiple histidine binding, and binding of multiple Ni(II)-NTA functionalities on each gold. Dissociation constants are estimated to be between 10^-7 to 10^-13 M^-1, comparable to antibodies.

• High penetration: 5 nm Ni-NTA-Gold is smaller than an unlabeled primary antibody, penetrates better, and accesses more sterically restricted interior sites. It may be used with stronger fixation or less permeabilization, enabling labeling with better ultrastructural preservation.

• Super sensitivity: the larger gold particle provides higher sensitivity with virtually no background when used to detect His-tagged targets on blots. 10 ng of His-tagged ATF-1 was detected without silver or gold enhancement. Gold enhancement allows the detection of 0.5 ng, with no visible binding to an E Coli extract control.

Applications of Ni-NTA-Gold probes include:

• High-resolution labeling of proteins, protein complexes or organelles containing recombinant His-tagged proteins for TEM or STEM localization.
• "Universal" pre-embedding labeling of His-tagged proteins in tissue sections for electron microscopic observation.
• identifying His-tagged proteins in fractions during Ni-NTA-column purifications.
• Detection of recombinant His-tagged proteins on blots and in gels.
• Heavy atom labeling of regular structures for image analysis and structure solution.

Product details:

Reference:

• Reddy, V.; Lymar, E.; Hu, M., and Hainfeld, J. F.: 5 nm Gold-Ni-NTA binds His Tags. Microsc. Microanal., 11, (Suppl. 2: Proceedings)(Proceedings of Microscopy and Microanalysis 2005); Price, R.; Kotula, P.; Marko, M.; Scott, J. H.; Vander Voort, G. F.; Nanilova, E.; Mah Lee Ng, M.; Smith, K.; Griffin, P.; Smith, P., and McKernan, S. (Eds.); Cambridge University Press, New York, NY, 1216CD (2005).

More information:

• 5 nm Ni-NTA-Gold: our paper from Microscopy and Microanalysis 2005
• Ni-NTA-Nanogold: catalog and general information
• Ni-NTA-Nanogold: product information and instructions
• Technical help: Ni-NTA-Nanogold
• References sorted by application: Ni-NTA-Nanogold
• Nickel-NTA-Nanogold Binds his-Tagged Proteins: our paper from Microscopy and Microanalysis 2002

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Should I Use Nanogold®-Fab' or IgG?

The difference refers to the antibody or fragment to which the Nanogold label is conjugated. Because Nanogold is covalently linked using different chemical reactions, it may be conjugated to specific chemical sites in a variety of antibody fragments instead of whole IgG molecules, including Fab' fragments, Fab fragments, and even ScFv fragments. Of these, we offer a range of secondary antibody conjugates comprising Nanogold attached to either whole IgG antibody molecules, or Fab' fragments. These conjugates have different properties, and these may make them more or less appropriate for different applications.

Nanogold-Fab' fragments are smaller, can label more densely, and penetrate better into cells and tissue sections, and access antigens that are more hindered. Since they also do not contain Fc, they therefore cannot be targeted by Fc-specific tertiary probes, or interact with specimens through Fc-specific mechanisms. This may be better for double labeling, since one mechanism for cross-reactivity or non-specific interaction is removed.

Nanogold-IgG, since it includes the whole IgG, can provide stronger binding since it has two antigen binding sites. Since it contains Fc, it can also be targeted by Fc-specific tertiary probes.

Both reagents will provide effective immunoelectron microscopic staining with silver enhancement; your choice should be dictated by which of the sets of properties above fits better with your experiment and with the system in which the probes will be used.

The key features and differences between these two Nanogold probes are summarized below:

Still have questions? Let us advise. Call us at 1-877-447-6266 (US and Canada) or +1 (631) 205-9490, or e-mail us.

Reference:

• Takizawa, T., and Robinson, J. M.: Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections. J. Histochem. Cytochem., 42, 1615-1623. (1994).
  • Abstract (courtesy of the Journal of Histohemistry and Cytochemistry).
  • Reprint (PDF) (courtesy of the Journal of Histohemistry and Cytochemistry).

More information:

• Nanogold-antibodies: catalog information
• Nanogold antibody conjugates: instructions and product information
• Technical help: Nanogold conjugates
• Nanogold labeling references, sorted by application

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Inexpensive Conductimetric Biochips with EnzMet^TM

Having demonstrated proof of principle, the group then pursued ways to fabricate this type of biochip affordably, and in the current issue of Biosensors and Bioelectronics, report an inexpensive microfluidic format that may make this method rapid and cost-effective for many DNA detection applications. By enclosing the chip within an inexpensive microfluidic flow cell, the operation of the detection system was simplified and the operator intervention minimized.

The microarray measurement platform is a microstructured glass chip with screen printed electrodes and 42 measurement points (electrode gaps). The capture DNA molecules are immobilized in the gap between the electrodes. The DNA chips were chemically modified with (3-glycidyloxypropyl)-trimethoxysilane (GOPS) in order to bind amino-modified single stranded (ss) capture-DNA molecules. First, the chips were cleaned by sonication for 5 minutes each with acetone, ethanol, and water. After drying with nitrogen, the chips were modified in a 10mM GOPS solution in dry toluene for 7 hours at 70°C, then finally washed 2 X 5 minutes each with toluene, ethanol, and water. The deposited capture DNA was modified at the 5' or 3' end with a C₆-Aminolink, which was then attached to the epoxy (introduced by GOPS) modified surface. After the specific binding of biotinylated target molecules, a streptavidin-horseradishperoxidase-polymer was bound in the electrode gap. EnzMet^TM-induced deposition of silver nanoparticles leads to bridging the electrode gap by a conductive silver layer: the increase of the conductivity over the electrode gap can be measured by a straightforward DC measurement, managed using an attached PC; a heating foil and thermocouple are also incorporated into the polycarbonate flow cell cover.

Slightly differently to the conventional (non-microfluidic) system, the best specificity for the hybridization of the biotin-labeled target-DNA sequences was achieved in 0.5 X SSC buffer and 0.1% SDS hybridization buffer at 42°C. Hybridization was performed in the microfluidic device using a tubing pump with a planetary drive at a flow rate of 600 µL/min in 3 s intervals for 3 minutes. Subsequent washing in 0.2 X SSC for 1 minutes (in continuous flow) removed unbound DNA to avoid false positive signals. The streptavidin-horseradishperoxidase-polymer was diluted 1:1000 in PBS buffer and 0.05% Tween-20. This buffer was also used for the washing step, which was applied after a 3 minute incubation step. Enzyme binding was also carried out at the same pump parameters as those used for the hybridization. After binding of the streptavidin-enzyme conjugate, the EnzMet^TM solution was mixed and introduced to the chip surface. Reaction was stopped after 3 minutes to avoid non-specific silver deposition. Before enzyme-induced silver deposition, the flow cell was rinsed with deionized water to remove any chloride from the PBS buffer.

An important factor for a bioanalytical tool is the total analysis time. In this system, the active movement of the different solutions by interval pumping means that binding processes are no longer solely diffusion limited, which should lead to faster reactions and higher binding efficiencies. To investigate this, different hybridization times were tested using a 500 nM concentration of biotin modified target DNA. A weak signal was already detectable for 30 second hybridization time. The signal intensity was found to increase up to 5 minutes, but with longer hybridization times, up to 15 minutes, the signal intensity approaches in saturation. Binding was confirmed by optical measurements. In all, a complete hybridization experiment could be completed within one hour using the flow cell, but required 4 hours in its absence. The detection limit of 50 pM biotinylated target DNA was comparable with other methods. Overall, this approach provides a robust, fast and cost-effective method for NA detection which promises to be readily applicable to use in the field.

References:

• Schüler, T.; Kretschmer, R.; Jessing, S.; Urban, M.; Fritzsche, W.; Möller, R., and Popp, J.: A disposable and cost efficient microfluidic device for the rapid chip-based electrical detection of DNA. Biosens. Bioelectron., 15-21 (2009).
  • Abstract (courtesy of Science Direct).

• Möller, R.; Schüler, T.; Günther, S.; Carlsohn, M. R.; Munder, T., and Fritzsche, W.: Electrical DNA-chip-based identification of different species of the genus Kitasatospora. Appl. Microbiol. Biotechnol., 77, 11811188 (2008).
  • Abstract (courtesy of SpringerLink).

More information:

• EnzMet^TM - catalog information
• Product information and instructions - EnzMet^TM for research applications
• EnzMet^TM - references by application
• Enzyme Metallography - a New Staining Method (our Microscopy & Microanalysis 2002 abstract)
• Enzyme Metallography for correlative labeling (our Microscopy & Microanalysis 2004 abstract)
• Enzyme Metallography for in situ hybridization

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Labeling for EM and LM with Silver-Enhanced Nanogold® and DAB

Regulator of G protein signaling 4 (RGS4) regulates intracellular signaling via G proteins and is markedly reduced in the prefrontal cortex (PFC) of patients with schizophrenia. Therefore, characterizing the expression of RGS4 within individual neuronal compartments is important to understanding its actions on individual G protein - coupled receptors (GPCRs), and ultimately the nature of schizophrenia and related disorders. The authors address this challenge by presenting an ultrastructural reference map of RGS4 protein in macaque PFC, based on immunogold electron microscopic analysis. In order to better identify specific targets, the authors used a tertiary labeling protocol in order to deposit 'clusters,' or groups of gold labels, at each target site, and to verify the distribution at the light and EM level, compared enzymatic staining with silver-enhanced Nanogold.

3 adult rhesus macaques were used for this study: the primates were deeply anesthetized prior to transcardial perfusion of artificial cerebrospinal fluid, followed by 4% paraformaldehyde/0.05 - 0.1% glutaraldehyde in 100 mM phosphate-buffered saline. Brains were sectioned coronally at 60 µm, cryoprotected, and stored at -80°C. Sections of the dorsolateral PFC (Walkers area) were processed for RGS4 immunocytochemistry. In order to facilitate penetration of immunoreagents, all sections went through 3 freeze-thaw cycles in liquid nitrogen. Nonspecific reactivity was blocked with 10% normal donkey serum (NDS) and 2% bovine serum albumin (BSA) in 50 mM Tris-buffered saline (TBS). Immunoaffinity purified RGS4 antibody (IgY) was raised in chicken against amino acids 127-205 of the RGS4 protein C-terminus: this antibody recognizes human and rodent RGS4 based on sequence homology.

Immunocytochemistry for light microscopy was conducted with both DAB and silver-enhanced Nanogold. For enzymatic staining, sections were incubated for 36 hours in 1 : 1000 anti-RGS4 in Tris-buffered saline (TBS) with 0.05% Triton X-100, 2% normal serum (NDS), and 1% bovine serum albumin (BSA), then for a further 3 hours in 1 : 500 biotinylated F(ab')₂. A 1 : 100 dilution of avidin-biotin-peroxidase complexes (Vector Laboratories) was then applied for one hour. For Nanogold-silver staining, a 1 : 100 Nanogold-labeled anti-biotin IgG was applied for 2 hours. RGS4 was then visualized either with diaminobenzidine as an enzyme chromogen, or by silver enhancement of Nanogold after glutaraldehyde fixation using HQ Silver. For immunoelectron microscopy, sections were placed in 1 : 2000 anti-RGS4 for 48 hours, followed by 1 : 800 biotinylated F(ab')₂, both diluted in TBS with 2% NDS, 0.1% acetylated BSA-C (Aurion), and 0.01% Tween-20 (gold buffer). Next, a 1 : 200 dilution of Nanogold-IgG anti-biotin in gold buffer was applied, followed by silver enhancement (autometallography) to visualize the Nanogold particles. As there are multiple gold probes per primary antibody, this 3-layer immunoprocedure was expected to produce multiparticle aggregates, or 'particle clusters.' Omission of the anti-RGS4 or substitution with normal chicken serum abolished all reactivity. When bridging biotinylated antibodies were excluded, both Nanogold and peroxidase signals were eliminated. Similarly, peroxidase labeling was abolished when blocking the biotinylated probes with avidin/biotin. To control for self-nucleation of the metallographic developer, Nanogold was omitted and the sections processed for silver enhancement for 15 minutes at room temperature. In RGS4 labeling, enhancement time never exceeded 8 min at 4°C. All controls were evaluated under the electron microscope. At the light microscopic level, a faint diffuse precipitate remained in control sections treated with diaminobenzidine, but was not detectable ultrastructurally. Matching sections were processed in parallel for gold immunocytochemistry against antigens with known distribution patterns to test the validity of nuclear labeling for RGS4.

For electron microscopy, layers I-III and IV-VI of PFC were sampled for resectioning and analysis under the transmission electron microscope at 80 kV. Twenty plastic blocks of each brain were examined using only the 4th to 12th surface-most sections of each block (i.e., 200-600 nm): signal sharply declined with depth from the tissue surface, possibly due to the larger size of the IgY primary antibody. RGS4 structures were digitally captured at X 25,000 to X 160,000 magnification and individual panels adjusted for brightness and contrast. Quantitative assessments were performed on series of low magnification micrographs of supragranular PFC, each covering a field of 30 µm² captured from the 4th and the 12th thin section to avoid recounting a single structure.

Examination of the micrographs revealed that at the soma, all labeling was asynaptic and affiliated with subsurface cistern microdomains of pyramidal neurons. Most immunoreactivity was associated with the nucleus. RGS4 levels were particularly high along proximal apical dendrites, and markedly decreased with distance from the soma; clustered Nanogold labels were observed at the bifurcation into second-order branches. In distal dendrites and in spines, the protein was found flanking or directly facing the postsynaptic density of symmetric and asymmetric synapses. Axons also expressed RGS4, and the density and distribution of pre- and postsynaptic labeling was correlated with the axon ultrastructure and the type of established synapses. These data indicate that RGS4 is strategically positioned to regulate not only postsynaptic but also presynaptic signaling in response to synaptic and nonsynaptic GPCR activation. This gives it broad but highly selective influence on multiple aspects of PFC cellular physiology.

Reference:

• Paspalas, C. D.; Selemon, L. D., and Arnsten, A. F.: Mapping the regulator of G protein signaling 4 (RGS4): presynaptic and postsynaptic substrates for neuroregulation in prefrontal cortex. Cereb. Cortex., 19, 2145-2155 (2009).
  • Abstract (courtesy of Oxford Journals).

More information:

• Nanogold conjugates: Catalog information
• Nanogold conjugates: Product information, instructions and protocols
• Nanogold conjugates: Technical help
• References: Nanogold labeling, sorted by application
• Silver enhancement: Catalog and general information
• HQ Silver: Product information, instructions and protocols

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Nanoprobes Wins New SBIR Grant

We also welcome new Research Assistant Ping Lin to our company. Ms. Lin will assist with the development of gold as a labeling reagent and as a biomedical imaging reagent.

More information:

• Nanoprobes: about us - company information
• Our technology
• Company history
• Key personnel
• News

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Other Recent Publications

Reference:

• Ottenheijm, C. A.; Witt, C. C.; Stienen, G. J.; Labeit, S.; Beggs, A. H., and Granzier, H.: Thin filament length dysregulation contributes to muscle weakness in nemaline myopathy patients with nebulin deficiency. Hum. Mol. Genet., 18, 2359-2369 (2009).
  • Abstract (courtesy of Oxford Journals).

Reference:

• Odegard, A. L.; Kwan, M. H.; Walukiewicz, H. E.; Banerjee, M.; Schneemann, A., and Johnson, J. E.: Low endocytic pH and capsid protein autocleavage are critical components of Flock House virus cell entry. J. Virol., 83, 8628-8637 (2009).
  • Abstract (courtesy of Journal of Virology).

Gold nanoparticles can facilitate electron transfer, and therefore can be used to amplify redox reactions. One application of this is the use of gold nanoparticle-modified electrodes for the detection of analytes that can undergo an electrochemical reaction; an example was presented by Teresa Luczak in a recent paper in Electrochemica Acta. The author used mercaptopropionic acid (MPA), gold nanoparticles (Au-NPs) and cystamine (CA) modified gold bare electrodes as voltammetric sensors for simultaneous detection of epinephrine (EP), ascorbic (AA) and uric (UA) acids. Colloidal gold nanoparticles some 10 - 15 nm in diameter were prepared using a conventional citrate reduction of tetrachloroaurate, and an activated (polished) "2D" gold electrode was immersed in the colloidal gold solution for 24 hours at 4°C. Both the bare gold ("2D") and the nanoparticle-modified ("3D") electrodes were then coated with MPA Modification of the electrode surface by self-assembled layers (SAMs) by immersion in a 5mM ethanolic solution of MPA for 10 hours. The presence of the SAM was found to improve the reactivity of the gold electrode for EP oxidation remarkably. A linear relationship between the epinephrine concentration and the current response was obtained in the range of 0.1700 µM with a detection limit of 0.042 µM for gold electrodes, and in the range of 0.1800 µM with the detection limit of 0.040 µM for the electrodes incorporating gold nanoparticles. The results have shown that the overlapping voltammetric responses of epinephrine, ascorbic and uric acids are well resolved at modified electrodes. The modified SAMs electrodes show high selectivity, sensitivity, reproducibility and stability.

Reference:

• Luczak, T.: Comparison of electrochemical oxidation of epinephrine in the presence of interfering ascorbic and uric acids on gold electrodes modified with S-functionalized compounds and gold nanoparticles Electrochim. Acta, 54, 5863-5870 (2009).
  • Abstract (courtesy of Science Direct).

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