Werasak Surareungchai

Sub-Femtomolar Electrochemical Detection of DNA Hybridization Based on Latex/Gold Nanoparticle-Assisted Signal Amplification

We report a relatively simple electrostatic method for modifying submicrometer-size latex spheres with gold nanoparticles (AuNPs) based on layer-by-layer modification of the latex by polyelectrolytes. The AuNP coverages for 343- and 501-nm-diameter spheres were 4.0 x 10 (10) +/- 1.3 x 10 (10) and 8.2 x 10 (10) +/- 2.7 x 10 (10) particles cm (-2), respectively, which is an increase of 1 order of magnitude on the previously reported coverage at latex-AuNPs using streptavidin-biotin binding (Kawde, A.N.; Wang, J. Electroanalysis 2004, 16, 101-107). Due to the fact that the AuNPs used here are also of a larger size (mean diameter 15.5 +/- 1.6 nm, cf. 5 nm), this represents an increase of 2 orders of magnitude in the number of Au atoms delivered per sphere. The spheres were attached to DNA probes specific to E. coli and used to detect probe hybridization by dissolution of the AuNPs, followed by measurement of Au (3+) ions by anodic stripping voltammetry (ASV). Use of differential pulse voltammetry for the stripping step, along with optimization of the ASV conditions, enabled a detection limit of 0.5 fM, which is, to the best of our knowledge, equal or lower than previous voltammetric nanoparticle methods for detection of DNA hybridization.

Immunoassay based on carbon nanotubes-enhanced ELISA for Salmonella enterica serovar Typhimurium.

Among the methods used to detect pathogenic bacteria, enzyme linked immunosorbent assay (ELISA) is one of the most widely used techniques in routine sample analysis. For Salmonella enterica serovar Typhimurium detection, a typical ELISA yields a sensitivity of 10(6)-10(7)CFU/ml. To enhance the detection sensitivity, single-walled carbon nanotubes (SWCNTs) was employed in this study as a labelling platform for antibody and horseradish peroxidase (HRP) co-immobilizing. With high proteins recovery after the coupling process, the resulting Ab/SWCNTs/HRP bioconjugate was used in the proof-of-concept ELISA experiments. Limit of detection (LOD) was found to be 10(3) and 10(4)CFU/ml for direct and sandwich ELISA, respectively, when Ab/HRP at 1:400 ratio was used. This figure accounts for 1000-time greater in detection sensitivity when compared to a commercial Ab-HRP conjugate. The Ab/SWCNTs/HRP bioconjugate was tested further in real samples and found a superior activity over the commercial Ab-HRP by showing 100-time greater detection limit.

Study of the underlying electrochemistry of polycrystalline gold electrodes in aqueous solution and electrocatalysis by large amplitude fourier transformed alternating current voltammetry.

Polycrystalline gold electrodes of the kind that are routinely used in analysis and catalysis in aqueous media are often regarded as exhibiting relatively simple double-layer charging/discharging and monolayer oxide formation/removal in the positive potential region. Application of the large amplitude Fourier transformed alternating current (FT-ac) voltammetric technique that allows the faradaic current contribution of fast electron-transfer processes to be emphasized in the higher harmonic components has revealed the presence of well-defined faradaic (premonolayer oxidation) processes at positive potentials in the double-layer region in acidic and basic media which are enhanced by electrochemical activation. These underlying quasi-reversible interfacial electron-transfer processes may mediate the course of electrocatalytic oxidation reactions of hydrazine, ethylene glycol, and glucose on gold electrodes in aqueous media. The observed responses support key assumptions associated with the incipient hydrous oxide adatom mediator (IHOAM) model of electrocatalysis.

Nanochannels for diagnostic of thrombin-related diseases in human blood

A high sensitive voltammetric method for rapid determination of thrombin spiked in whole blood by taking advantage of both aptamer-based recognition and the use of a nanoporous membrane has been developed. The nanoporous membrane not only acts as platform for the thrombin recognition but also as filter of the micrometric components such as white and red blood cells, consequently minimizing matrix effects. The protocol involves a sandwich format in the inner walls (200 nm diameter) of an anodized alumina oxide filter membrane (AAO). The analytical signal, by DPV oxidation of [Fe(CN)(6)](4-), is based on the blockage in the pores which affects the diffusion of [Fe(CN)(6)](4-) to the screen-printed carbon electrotransducer (SPCEs) modified with the membrane. By labeling the anti-thrombin IgG with AuNPs followed by silver enhancement a greater passive signal enhancement in comparison to the membrane blockage has been observed. The contribution of both electrostatic/steric effects in this blockage due to the subsequent formation of the aptamer-thrombin complex and the final sandwich assay is investigated. The efficiency of the system is also monitored by microscopic techniques. The resulted biosensing system allows detecting thrombin spiked in whole blood at very low levels (LOD 1.8 ng mL(-1)) which are within the range of clinical interest for the diagnostic of coagulation abnormalities as well as pulmonary metastasis.

Femtomolar Electrochemical Detection of DNA Hybridization Using Hollow Polyelectrolyte Shells Bearing Silver Nanoparticles

The preparation, and use as electrochemical labels, of polyelectrolyte shells bearing Ag nanoparticles is described. Their potential for highly sensitive detection is demonstrated. The shells are prepared by layer-by-layer self-assembly around templates (500 nm diameter) which are then dissolved. The shells can be opened and closed by adjustment of solution pH, and this process is utilized to encapsulate Ag nanoparticles, chiefly by adsorption to the inner walls of the capsules. Based on absorbance, TEM and voltammetric measurements, the highest loading achieved is approximately 78 Ag particles per capsule. The Ag capsules are used via biotin-avidin binding as labels for the detection of DNA hybridization, following acid dissolution and then detection of the Ag (+) by ASV. A 30-mer sequence specific to Escherichia coli is measured at DNA-modified screen-printed electrodes with a detection limit of approximately 25 fM, which corresponds to the detection of 4.6 fg ( approximately 3 x 10 (5) molecules) in the 20 microL analyte sample. A 200 fM target containing a single mismatch gives a significantly (<74%) lower response than 200 fM of complementary target; 60 pM of noncomplementary target gives a negligible response.

Double-Walled Carbon Nanotube Electrodes for Electrochemical Sensing

We report on the electrochemical properties of electrodes fabricated using spun fibers of purified double-walled nanotubes (DWNTs). These electrodes show reversible and faster electron-transfer kinetics in electrochemical reactions compared to multiwalled nanotubes (MWNTs) and standard glassy carbon electrodes (GCE). The detection capabilities of the DWNT electrodes for chemical species such as hydrogen peroxide, ascorbic acid (AA), and electroactive neurotransmitters such as dopamine are presented and compared with the detection abilities for these species using GCE and MWNT. These electrodes also show excellent selective sensing properties (with observed detection limits as low as 20 μM of DA in 1 mM of AA) and can be developed for active components in electrochemical sensors.

Scano-magneto immunoassay based on carbon nanotubes/gold nanoparticles nanocomposite for Salmonella enterica serovar Typhimurium detection

To improve sensitivity of S. enterica serovar Typhimurium detection, multiwalled carbon nanotubes (MWCNTs) and gold nanoparticles (AuNPs) were combined and used as a label to amplify signal in a scanometric based assay. In this study, the MWCNTs/AuNPs nanocomposite was fabricated by directly assemble of Au(3+) to MWCNTs and allowed growing of AuNPs along the MWCNTs surface. This MWCNTs/AuNPs nanocomposite was then attached to anti-S. typhimurium antibody (MWCNTs/AuNPs/Ab(1)) and used as a detecting molecule. Upon binding to Salmonella, they were pre-concentrated by magenetic beads/antibody (MBs/Ab(2)) forming a sandwich immuno-complex which is later spotted on a nitrocellulose membrane coated slide. Silver reduction was applied to amplify signal. The detection limit of 42CFU/ml was achieved when 2% BSA was used as a blocking agent. Given different types of real samples testing, chicken broth was found to give lowest detection limit, followed by orange juice low fat and whole milk. Selectivity testing was performed by using Escherichia coli as interference and found slightly cross-reactivity which could be due to specificity of the Ab used. By virtue of using a slide for multi-samples spotting and a flatbed scanner for signal-read out acquisition, this scano-magneto immunoassay could enable low-cost detection as well as high throughput screening.

Investigation of mediated oxidation of ascorbic acid by ferrocenemethanol using large-amplitude fourier transformed ac voltammetry under quasi-reversible electron-transfer conditions at an indium tin oxide electrode

The ability of the technique of large-amplitude Fourier transformed (FT) ac voltammetry to facilitate the quantitative evaluation of electrode processes involving electron transfer and catalytically coupled chemical reactions has been evaluated. Predictions derived on the basis of detailed simulations imply that the rate of electron transfer is crucial, as confirmed by studies on the ferrocenemethanol (FcMeOH)-mediated electrocatalytic oxidation of ascorbic acid. Thus, at glassy carbon, gold, and boron-doped diamond electrodes, the introduction of the coupled electrocatalytic reaction, while producing significantly enhanced dc currents, does not affect the ac harmonics. This outcome is as expected if the FcMeOH (0/+) process remains fully reversible in the presence of ascorbic acid. In contrast, the ac harmonic components available from FT-ac voltammetry are predicted to be highly sensitive to the homogeneous kinetics when an electrocatalytic reaction is coupled to a quasi-reversible electron-transfer process. The required quasi-reversible scenario is available at an indium tin oxide electrode. Consequently, reversible potential, heterogeneous charge-transfer rate constant, and charge-transfer coefficient values of 0.19 V vs Ag/AgCl, 0.006 cm s (-1) and 0.55, respectively, along with a second-order homogeneous chemical rate constant of 2500 M (-1) s (-1) for the rate-determining step in the catalytic reaction were determined by comparison of simulated responses and experimental voltammograms derived from the dc and first to fourth ac harmonic components generated at an indium tin oxide electrode. The theoretical concepts derived for large-amplitude FT ac voltammetry are believed to be applicable to a wide range of important solution-based mediated electrocatalytic reactions.

Non-protein coding RNA-based genosensor with quantum dots as electrochemical labels for attomolar detection of multiple pathogens

The ability of a diagnostic test to detect multiple pathogens simultaneously is useful to obtain meaningful information for clinical treatment and preventive measures. We report a highly sensitive and specific electrochemical biosensor assay for simultaneous detection of three gene targets using quantum dots (QDs). The targets are novel non-protein coding RNA (npcRNA) sequences of Vibrio cholerae, Salmonella sp. and Shigella sp., which cause diarrheal diseases. QDs (PbS, CdS, ZnS) were synthesized and functionalized with DNA probes that were specific to each pathogen. Electrochemical detection of QDs was performed using square wave anodic stripping voltammetry (SWASV). The QDs gave distinct peaks at 0.5 V (PbS), 0.75 V (CdS) and 1.1 V (ZnS). There was no interference in signal response when all three QDs were mixed and detected simultaneously. The detection limits of single and multiplex assays with linear targets and PCR products were in the attomolar ranges. The high assay sensitivity, in combination with specific npcRNA sequences as novel diagnostic targets, makes it a viable tool for detecting pathogens from food, environment and clinical samples.

Electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere label for detection of Vibrio cholerae.

Vibrio cholerae is a Gram-negative bacterium that causes cholera, a diarrheal disease. Cholera is widespread in poor, under-developed or disaster-hit countries that have poor water sanitation. Hence, a rapid detection method for V. cholerae in the field under these resource-limited settings is required. In this paper, we describe the development of an electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere (AuNPs-PSA) reporter label. The reporter label mixture was prepared by lyophilization of AuNPs-PSA-avidin conjugate with different types of stabilizers. The best stabilizer was 5% sorbitol, which was able to preserve the dried conjugate for up to 30 days. Three methods of DNA hybridization were compared and the one-step sandwich hybridization method was chosen as it was fastest and highly specific. The performance of the assay using the lyophilized reagents was comparable to the wet form for detection of 1aM to 1fM of linear target DNA. The assay was highly specific for V. cholerae, with a detection limit of 1fM of PCR products. The ability of the sensor is to detect LAMP products as low as 50ngµl(-1). The novel lyophilized AuNPs-PSA-avidin reporter label with electrochemical genosensor detection could facilitate the rapid on-site detection of V. cholerae.