Detection of DNA using gold nanoparticle-coated silica nanoparticles.

We report a sensitive lateral flow assay (LFA) in which the assay colour change originated from reporter labels constructed from silica spheres (radius = 450 nm) coated with approximately 3.9 × 103 gold nanoparticles (radius = 8.5 nm). These reporter labels were modified with DNA and deposited in the conjugation area of an LFA device assembled on wax-patterned Fusion 5 paper. Test and control zones of the device were pre-loaded with capture probe formed by avidin-coated mesoporous silica nanoparticles attached with biotin-tagged DNA sequences. Proof-of-concept was demonstrated by the detection of a partial sequence of the actin gene of Colletotrichum truncatum. The DNA target could be detected with an LOD of 46 pM, which was 5 times lower than a comparative assay using gold nanoparticles alone. The assay showed good selectivity against the Colletotrichum species C. scovillei and C. gloeosporioides, as well as against DNA from the fungal genera Aspergillus niger and Alternaria alternata. There was negligible change in sensor response over storage for one month at room temperature. The LFA was used to detect PCR products following extraction from mycelium.

Label-free ultra-sensitive colorimetric detection of hepatitis E virus based on oxidase-like activity of MnO2 nanosheets.

Hepatitis E virus (HEV) is an evolving infectious entity that causes viral hepatitis infections worldwide. Current routine methods of identifying and diagnosing HEV are someway laborious and costly. Based on the biomimicking oxidase-like activity of MnO2 nanosheets, we designed a label-free, highly sensitive colorimetric sensing technique for HEV detection. The prepared MnO2 catalyst displays intrinsic biomimicking oxidase-like catalytic activity and efficiently oxidizes the 3,3',5,5'-tetramethylbenzidine (TMB) substrate from colorless to blue colored oxidized TMB (oxTMB) product which can be measured at 652 nm by UV-visible spectrum. When the HEV-DNA was added, DNA adsorbed easily on MnO2 surface through physical adsorption and electrostatic interaction which hinders the oxidase-like catalytic activity of MnO2. Upon the introduction of target, the HEV target DNA binds with its complementary ssDNA on the surface of MnO2, the hybridized DNA releases from the surface of MnO2, which leads to recovery of oxidase-like catalytic activity of MnO2. This strategy was applied to construct a colorimetric technique for HEV detection. The approach works in the linear range of 1 fM-100 nM DNA concentration with the limit of detection (LOD) of 3.26 fM (S/N = 3) and quantitative limit (LOQ) of 36.08 fM. The TMB-MnO2 platform was highly selective for HEV target DNA detection when compared with potential interferences. Result of serum sample analysis demonstrates that this sensing system can be used for clinical diagnostic applications.

Aptamer-functionalised magnetic particles for highly selective detection of urinary albumin in clinical samples of diabetic nephropathy and other kidney tract disease.

We report a new highly selective detection platform for human albumin (HA) in urine based on aptamer-functionalised magnetic particles. Magnetic separation and re-dispersion was utilised to expose the HA-bound particles to a methylene blue solution. A second magnetic collection step was then used to allow the methylene blue supernatant to be reduced at an unmodified screen-printed electrode. Since methylene blue adsorbs to HA, the reduction current fell in proportion to HA concentration. There was no interference from compounds such as dopamine, epinephrine, vanillylmandelic acid, normetanephrine, metanephrine and creatinine in artificial urine at the concentrations at which they would be expected to appear. A calibration equation was derived to allow for the effect of pH on the response. This enabled measurement to be made directly in clinical urine samples of varying pH. After optimisation of experimental parameters, the total assay time was 40 min and the limit of detection was between 0.93 and 1.16 µg mL-1, depending on the pH used. HA could be detected up to 400 µg mL-1, covering the range from normoalbuminuria to macroalbuminuria. Analysis of urine samples of patients, with diabatic nephropathy, type I & II diabetes mellitus and chronic kidney disease, from a local hospital showed good agreement with the standard urinary human albumin detection method.

Photostable methylene blue-loaded silica particles used as label for immunosorbent assay of Salmonella Typhimurium.

Salmonella Typhimurium is a major cause of food poisoning. To solve the limitations of the routine enzyme linked immunosorbent assay such as laborious assay procedure, lack of long-term enzyme stability, and insufficient sensitivity, we provided a non-enzymatic colorimetric immunosorbent assay platform to overcome these problems. The highly photostable redox dye particles was constructed by silica particles (diameter = 598 ± 14.4 nm) loaded with methylene blue (Si-MB) and applied to be a label for immunoassay of S. Typhimurium. The sandwich assay format involved incubation of an analyte in a microplate wells modified with monoclonal anti-Salmonella, followed by exposure to a polyclonal anti-Salmonella/Si-MB bioconjugate and then measurement of absorbance at 598 nm. The platform had an assay time of 20 min, could detect heat-killed Salmonella with a limit of detection of 48 CFU mL-1 , and gave good recoveries in milk. The labels could be stored at 4 °C for 70 days without any deterioration.

Sub-attomolar electrochemical measurement of DNA hybridization based on the detection of high coverage biobarcode latex labels at PNA-modified screen printed electrodes.

We have constructed biobarcode labels based on 468nm diameter latex spheres. Modification with polyallylamine and then glutaraldehyde was used to attach a high DNA loading, consisting of aminated probe DNA (approx. 1.01×102 molecules per sphere) and biobarcode DNA (approx. 1.66×104 molecules per sphere). Detection of the biobarcodes was performed by application of a Ag enhancer solution, causing association of the Ag+ ions with the phosphate groups of the DNA. The deposited Ag was detected by differential pulse voltammetry. A 30 mer sequence from the BL21 strain of E. coli was detected with an LOD of 2.6fM (calibration range 10 aM to 0.1pM, r2=0.91, n=45). The LOD was lowered to 0.56aM (calibration range 100zM to 0.1nM, r2=0.991, n=50) by utilizing a sandwich assay with PNA-modified screen printed electrodes, which lowered the Ag background current. The sandwich assay platform was used to calibrate E. coli strain BL2(DE3) with an LOD of 17.0 CFU mL-1 (calibration range 10 to 106 CFU mL-1, r2=0.99, n=33) with good discrimination against Salmonella.

Highly sensitive electrochemical detection of genomic DNA based on stem loop probes structured for magnetic collection and measurement via metalised hollow polyelectrolyte shells.

We report a highly sensitive method for the electrochemical detection of genomic DNA, based on the employment of two sub-micron oligonucleotide labels - one for magnetic collection and the other for voltammetric detection - and their incorporation onto a stem loop DNA probe. The magnetic label consists of a latex particle of mean diameter 441±6 nm, bearing magnetic Fe3O4 particles and approx. 3.5×10(5) anti-DIG antibodies. The voltammetric label is a hollow polyelectrolyte shell containing approx. 1.0×10(11) Au atoms in the form of well dispersed Au nanoparticles. A DIG tag on one arm of the stem loop enables binding to the magnetic label, while a thiol tag on the other arm enables attachment to the Au nanoparticles. Due to steric hindrance from the two relatively large labels, attachment of both moieties is dependant on target-probe hybridisation straightening the loop. Once attached, sensitive DNA measurement is facilitated by magnetic collection of the DNA into a small volume and by the high quantity of Au atoms available for detection. Using differential pulse anodic stripping voltammetry we calibrated a 30 mer sequence common to 71 strains of Escherichia coli across the concentration range from 0.1 aM to 100 pM with a LOD of 1.8 aM. Three strains of E. coli, BL 21, ATCC8739, O157:H7, when spiked into UHT milk and fermented palm juice, could be detected with LODs of approx. 2-4 CFU mL(-1) in an assay time of approx. 140 min.

Highly sensitive electrochemical detection of DNA hybridisation by coupling the chemical reduction of a redox label to the electrode reaction of a solution phase mediator.

We have described a highly sensitive method for detecting DNA hybridisation using a redox-labeled stem loop probe. The redox labels were poly(styrene-co-acrylic) (PSA) spheres of 454 nm diameter, modified by methylene blue (MB) deposited alternatively with poly(sodium 4-styrene sulphonate) (PSS) in a layer-by-layer process. Each PSA sphere carried approx. 3.7 × 10(5) molecules of MB, as determined optically. DIG-tagged stem loop probes were immobilised on screen printed electrodes bearing anti-DIG antibodies. Binding with the target enabled straightening of the stem loop, which made attachment to the MB-coated PSA spheres possible. For measuring the current from the direct reduction of MB by differential pulse voltammetry, a 30 mer DNA target common to 70 strains of Escherichia coli was calibrated across the range 1.0 fM to 100 pM (gradient = 3.2 × 10(-8) A (log fM)(-1), r(2) = 0.95, n = 60), with an LOD of ∼58 fM. By using Fe(CN)6(3-/4-) as a solution phase mediator for the MB reduction, we were able to lower the LOD to ∼39 aM (gradient = 5.95 × 10(-8) A (log aM)(-1), r(2) = 0.96, n = 30), which corresponds to the detection of 0.76 ag (∼50 molecules) in the 2 µL analyte sample. We hypothesise that the lowering of the LOD was due to the fact that not all the MB labels were able to contact the electrode surface.

Electrochemical immunoassay for Salmonella Typhimurium based on magnetically collected Ag-enhanced DNA biobarcode labels.

We describe a sensitive electrochemical immunoassay for Salmonella enterica serovar Typhimurium, a common foodborne pathogen which can cause infection at extremely small doses. The assay is based on the recognition of DNA biobarcode labels by differential pulse anodic stripping voltammetry (DPASV), following Ag enhancement. The biobarcodes consist of latex spheres (mean diameter 506 nm ± 22 nm) modified by ferromagnetic Fe3O4 particles. Each biobarcode is loaded by adsorption with approx. 27 molecules of mouse monoclonal antibody against S. Typhimurium and 3.5 × 10(5) molecules of 12 mer ssDNA. The assay is performed by adding the biobarcode, S. Typhimurium cells, and biotin-conjugated rabbit polyclonal antibody against Salmonella into well plates. After antigen-antibody binding, magnetic collection enables the excess polyclonal antibody to be washed off. Exposure to avidin-coated screen printed electrodes, and formation of the avidin-biotin bond, then enables the excess biobarcode to be removed. The biobarcode remaining on the electrode is quantified by DPASV measurement of Ag(+) ions following catalytic Ag deposition. The assay showed a negligible response to 10(7) CFU mL(-1)E. coli and had a limit of detection of 12 CFU mL(-1) in buffer, and 13 to 26 CFU mL(-1) for heat-killed and whole cell S. Typhimurium in plain milk, green bean sprouts and raw eggs. To the best of our knowledge, this is the lowest reported limit of detection for Salmonella by an electrochemical immunoassay not requiring sample pre-enrichment.

Thermodynamically unfavorable DNA hybridizations can be made to occur by a water to ice phase change.

In an apparent contradiction to Debye-Hückel theory, it was possible to hybridize DNA in solutions of Milli-Q water (resistivity>18MΩcm(-1)) containing no added ions. This was demonstrated by hybridizing four bi-complementary DNA sequences to form an 'X' shape, as indicated by acrylamide gel electrophoresis. The requirement for hybridization was that a water-to-ice phase change should occur. Comparative experiments, using freezing by liquid nitrogen and thawing at different temperatures, showed that hybridization could take place during either the freezing or thawing process provided either was slow enough. We speculate that the low solubility of DNA in ice creates liquid inclusions of extremely high DNA and counter-ion concentration prior to complete freezing, and that hence in these inclusions hybridization was actually in accordance with Debye-Hückel theory.

Use of 3-D plots to avoid mutual interference in bianalyte ASV determinations: application to cadmium and lead detection.

We have examined the anodic stripping voltammetry (ASV) of Cd and Pb at carbon screen printed electrodes modified by an in situ deposited Bi film, and have demonstrated significant cross talk between the stripping peaks of the two metals. A simple and generally applicable method for dealing with this problem is described, based on curve-fitting three-dimensional calibration plots using MATLAB. Non-linear fitting to the calibrations produced coefficients of determination R(2)>0.99 for both metals. We have illustrated use of the plots in conjunction with Bi-plated electrodes by measuring 15 randomly selected mixtures of Cd and Pb of known concentration.

Electrochemical immunoassay platform for high sensitivity protein detection based on redox-modified carbon nanotube labels.

We report a highly sensitive immunoassay protocol based on the use of redox-modified multi-walled carbon nanotubes (MWNTs) as electrochemical labels. The MWNTs were coated with methylene blue (MB) at an optically-determined loading of 3.41 × 10(-3) mol g(-1), and were then attached to secondary antibodies (Ab(2)) by adsorption. As a model analyte mouse IgG was collected by primary antibody (Ab(1))-coated magnetic beads. Following binding of the MB-MWNT-Ab(2) conjugates, IgG could be measured by MB reduction. Using differential pulse voltammetry for quantification, IgG was calibrated with a dynamic range of 0.1 pg mL(-1) to 100 pg mL(-1). Given the different possible Ab(1)-MB-MWNT-Ab(2) orientations on the magnetic beads, it was likely that not all the MB communicated with the electrode. A greater quantity of MB could be accessed by using the Fe(CN)(6)(3-/4-) redox couple as a solution phase mediator. This enabled us to lower the dynamic range down to 5 fg mL(-1) to 100 fg mL(-1).

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.

RGB colour coding of Y-shaped DNA for simultaneous tri-analyte solid phase hybridization detection.

We present a new concept for tri-analyte DNA detection based on the idea of a Y-shaped capture probe which, after tri-target and fluorescently labeled reporter probe binding, becomes colour-coded to generate images in an RGB colour scheme. Hence, the RGB value of the resulting secondary pseudo-colour presented by the hybridized Y-DNA can be related to the ratio of the primary pseudo-colours present in its make-up, and thus to the ratio of the three target concentrations. As a proof of concept we detect sequences from the genes of the pathogenic bacterial strains Escherichia coli O157:H7, Vibrio cholera and Salmonella enteric in a semi-quantitative manner across the range 20-167 nM. The assay was relatively quick, with a time from hybridization to completed data interpretation of approximately 4 h.

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.

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.

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.

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.

Voltammetric detection of organohalides by redox catalysis: improved sensitivity by immobilisation within a cubic phase liquid crystal.

Two combined strategies are reported for improving the sensitivity of organohalide detection by redox catalysis. These are, improvement of the second order rate constant (k) for catalytic reduction of the organohalide, and improvement of the rate of substrate diffusion. Values of k are calculated for both alkyl and aryl halides, from slow scan rate cyclic voltammograms in homogeneous solution. It is shown that a Zn(ii) porphyrin exhibits higher catalytic rates than the previously used Co(ii) porphyrin or Co(i) salen. Amperometric and rotating disk electrode studies of electropolymerised films of the Zn(ii) porphyrin, reveal that at optimum thickness, mediator-substrate reaction and substrate diffusion are the rate limiting steps. Hence, immobilisation of the Zn(ii) porphyrin within the more open structure of a cubic phase liquid crystal produces an increase in sensitivity of approx. 10 times, and lowers the limit of detection by one order of magnitude. The optimised sensor responds linearly to seven organohalides in the range 0.1 microM to 1.0 microM with a sensitivity of 6.95 A M(-1) cm(-2). Chronoamperometric experiments with a microdisk electrode show that the rate of charge transport in the cubic phase films (apparent diffusion coefficient, D(E)= 5.65 x 10(-10)+/- 0.11 x 10(-10) cm(2) s(-1)) is faster than in the electropolymerised films (D(E)= 3.64 x 10(-11)+/- 0.02 x 10(-11) cm(2) s(-1)). The variation of D(E) with the concentration of Zn(ii) in the cubic phase suggests that diffusion of charge is predominantly by electron self-exchange, rather than by physical movement.

Electrocatalytic tetracycline oxidation at a mixed-valent ruthenium oxide--ruthenium cyanide-modified glassy carbon electrode and determination of tetracyclines by liquid chromatography with electrochemical detection.

Mixed-valent films of ruthenium oxide-ruthenium cyanide were electrodeposited onto glassy carbon and characterized for the electrocatalytic oxidation of tetracycline. The currents produced by tetracycline were higher than from previously reported electrode modifications or pretreatments. In H(2)SO(4) pH 1.0 + 0.5 M K(2)SO(4), the second-order rate constant for the reaction between tetracycline and the Ru(III/IV) couple of ruthenium oxide was 3 x 10(5) +/- 1 x 10(5) mol(-1) cm(3) s(-1), and the rate of charge diffusion through the films was 4.5 x 10(-7) +/- 3.5 x 10(-7) cm(2) s(-1). Reaction was localized at the film-solution interface. When used as detectors in liquid chromatography (in H(3)PO(4) pH 2.5 + 0.1 M KH(2)PO(4) + 20% CH(3)CN, E = 1.10 V vs SCE), the electrodes gave limits of detection (>3 S/N) of 0.1 ppm for tetracycline and oxytetracycline and 0.5 ppm for doxycycline and chlorotetracycline. These limits were suitable for FDA and Codex Alimentarius guidelines for tetracyclines in food. Recoveries of the four tetracyclines from sea and freshwater shrimp were in the range 73-111%, which was higher or similar to the previously reported recoveries from shrimp.