Biotinylated Horseradish Peroxidase Research Articles

Development of a Progesterone-Receptor-Based Pseudo-immunoassay for Multi-detection of Progestins in Milk and Studying Its Recognition Mechanism.

The residues of progestins in milk are dangerous to consumers, but an immunoassay capable of multi-determining progestins in milk has not been reported thus far. In this study, the ligand binding domain of the human progesterone receptor was expressed and its intermolecular interactions with the commonly used steroid hormones were studied. The docking results showed that the receptor fragment only recognized progestins and did not recognize other steroid hormones. Then, it was used as recognition material to develop a pseudo-direct competitive enzyme-linked immunosorbent assay for multi-determination of five progestins in milk. Because biotinylated horseradish peroxidase was combined with streptavidinated horseradish peroxidase to enhance the signal, the sensitivities for the five progestins (IC50 of 0.029-0.097 ng/mL) were improved 96-143-fold in comparison to the use of the conventional horseradish peroxidase signal system (IC50 of 3.0-12.5 ng/mL). This method showed negligible cross-reactivities to other steroid hormones, consistent with the docking results. This was the first paper developing a progesterone-receptor-based method for detection of progestins, and this method exhibited generally better performance than all of the previously reported immunoassays for progestins.

Determination of niclosamide and its two metabolites in fish by molecularly imprinted microsphere-based pseudo-ELISA

Niclosamide is usually used for the treatment of parasite infections in animals. However, niclosamide and one of its metabolites 2-chloro-4-nitroaniline are mutagenic substances, and their residues in animal-derived foods are potential risks to consumers. As far as we know, there has been no immunoassay or pseudo immunoassay reported to determine niclosamide and its metabolites in animal-derived foods. In this study, a molecularly imprinted microsphere for niclosamide was first synthesized, and a streptavidin-horseradish peroxidase labelled conjugate was also synthesized. The two reagents were used to develop a pseudo enzyme-linked immunosorbent assay on conventional microplates for the determination of niclosamide and its two metabolites (2-chloro-4-nitroaniline and 5-chlorosalicylic acid) in fish. Because biotinylated horseradish peroxidase was used to amplify the signal, the method sensitivities for the three analytes were increased fivefold to 27.5-fold (limits of detection of 0.004–0.03 ng/mL) in comparison with the use of single horseradish peroxidase labelled conjugate (limits of detection of 0.11–0.16 ng/mL). Their recoveries from the standards fortified blank fish samples were in the range of 70.6–95.5%. This is the first study reporting a molecularly imprinted polymer-based pseudo immunoassay for screening of niclosamide and its metabolites in food sample.

Determination of Xylazine and Its Metabolite in Milk by a Molecularly Imprinted Microsphere Based Pseudo Immunoassay

The residues of xylazine and its metabolite in foods of animal origin are dangerous to consumers, but there has been no enzyme-linked immunosorbent assay (ELISA) or ELISA-like method reported to determine them so far. In this study, molecularly imprinted microspheres with xylazine as the template were synthesized, and 2,6-dimethylaniline (the metabolite of xylazine) was used as a hapten to prepare a streptavidinated horseradish peroxidase-labeled conjugate. A direct competitive pseudo-ELISA was developed on a microplate for xylazine and 2,6-dimethylaniline in milk. Because biotinylated horseradish peroxidase was employed to enhance the signal, the limits of detection for xylazine (0.003 ng/mL) and 2,6-dimethylaniline (0.005 ng/mL) were increased by 16.7- and 14-fold, respectively, compared to a horseradish peroxidase-labeled conjugate. Moreover, this material may be reused three times. The analysis of milk samples was consistent with values obtained by liquid chromatography tandem mass spectrometry. This is the first paper reporting a molecularly imprinted polymer-based method for the determination of xylazine and its metabolites in food.

Development of a Magnetic Molecularly Imprinted Microsphere-Based Signal Amplified Semi-Homogeneous Method for Multidetection of Five Progestins in Milk.

The residues of progestins in milk are significant risk factors for teenage acne and may cause hormone-dependent cancers in consumers, so the determination of these residues in milk is very important. However, an immunoassay or immunoassay-like method capable of determining multiple progestins in milk has not been reported so far. The present study, for the first time, synthesized a type of magnetic molecularly imprinted microsphere that was capable of simultaneously recognizing five progestins. At the same time, an enzyme labeled conjugate was synthesized by coupling progesterone 3-(o-carboxymethyl)oxime with streptavidinated horseradish peroxidase. The above two reagents were used to develop a semi-homogeneous method for the simultaneous detection of the residues of the five progestins in milk. During the experiments, biotinylated horseradish peroxidase was used to amplify the signal, so the sensitivity to the five drugs (limits of detection 0.04-0.1 pg/mL) was increased 44-75-fold. In addition, the magnetic molecularly imprinted microsphere could be regenerated four times by using simple elution. Through general comparison of its detection spectrum, sensitivity, simplicity, and reusability, the present method exhibited better performance than the previous immunoassays for the detection of progestins, and so it could be used as a routine tool for the screening of progestins residues in milk.

Evaluation of a novel affinity-label reporter protein with SNAP-tag and monomeric streptavidin

Reporter proteins are often used to detect pathogenic bacteria and viruses that are challenging to detect with straightforward assays. However, reporter protein assays are limited by their difficulty in detecting low levels of reporters in large sample sizes and/or in complex matrices. When reporter concentration techniques are employed, enzymatic reporters tend to lose their catalytic activity. In this study, we designed a heterobifunctional reporter composed of SNAP-tag and monomeric streptavidin (SNAP-mSA) that would allow its rapid concentration and easy detection. SNAP protein was used to capture the reporter using functionalized magnetic beads that offered an irreversible binding. Monomeric streptavidin was used to allow labeling the reporter instead of an enzymatic assay for detection. For the proof-of-principle, biotinylated horseradish peroxidase was used as the label for colorimetric detection. Immobilization of the reporter was achievable with a maximum loading of 1.5 μg/μL of magnetic beads for SNAP-mSA. The immobilized reporter was successfully labeled with biotinylated horseradish peroxidase and allowed for a detection limit of 0.2 ng SNAP-mSA. The reporter was able to retain the structure of their active sites after the washing steps and did not lose their binding ability to O6-benzylguanine (BG) and Biotin, respectively. SNAP-mSA was expressed through a T7 phage to determine its ability to detect E. coli BL21. The engineered phage could give a limit of detection of 105−6 CFU of E. coli BL21 in 7 h without an initial enrichment step. These findings suggest that SNAP-mSA can be used to successfully concentrate and label reporter proteins.

Synthesis of magnetic molecularly imprinted microsphere and development of an enhanced semi-homogeneous method for detection of pentachlorophenol in fish

In this study, pentachlorobenzene was used as dummy template to synthesize a type of magnetic molecularly imprinted microsphere for pentachlorophenol. At the same time, pentachloroaniline was coupled to streptavidinated horseradish peroxidase to synthesize an enzyme labeled tracer. The two reagents were used to develop a semi-homogeneous method on conventional microplate for detection of pentachlorophenol in fish samples. Due to the use of biotinylated horseradish peroxidase as signal amplification agent, the method sensitivity was improved for 375 folds in comparison with the use of conventional horseradish peroxidase labeled tracer with a limit of detection of 0.8 fg/g. After comparison, it was found that this method showed better performances than the previous immunoassays and molecularly imprinted polymer based methods for detection of pentachlorophenol. This study for the first time reported a magnetic molecularly imprinted microsphere based semi-homogeneous method for detection of small molecule, and this method could be used for routine determination of pentachlorophenol residue in fish samples.

Impedimetric and amperometric genosensors for the highly sensitive quantification of SARS-CoV-2 nucleic acid using an avidin-functionalized multi-walled carbon nanotubes biocapture platform

We report two novel genosensors for the quantification of SARS-CoV-2 nucleic acid using glassy carbon electrodes modified with a biocapture nanoplatform made of multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with avidin (Av) as a support of the biotinylated-DNA probes. One of the genosensors was based on impedimetric transduction offering a non-labelled and non-amplified detection of SARS-CoV-2 nucleic acid through the increment of [Fe(CN)6]3-/4- charge transfer resistance. This biosensor presented an excellent analytical performance, with a linear range of 1.0 × 10−18 M - 1.0 × 10−11 M, a sensitivity of (5.8 ± 0.6) x 102 Ω M−1 (r2 = 0.994), detection and quantification limits of 0.33 aM and 1.0 aM, respectively; and reproducibilities of 5.4% for 1.0 × 10−15 M target using the same MWCNTs-Av-bDNAp nanoplatform, and 6.9% for 1.0 × 10−15 M target using 3 different nanoplatforms. The other genosensor was based on a sandwich hybridization scheme and amperometric transduction using the streptavidin(Strep)-biotinylated horseradish peroxidase (bHRP)/hydrogen peroxide/hydroquinone (HQ) system. This genosensor allowed an extremely sensitive quantification of the SARS-CoV-2 nucleic acid, with a linear range of 1.0 × 10−20 M - 1.0 × 10−17 M, detection limit at zM level, and a reproducibility of 11% for genosensors prepared with the same MWCNTs-Av-bDNAp1 nanoplatform. As a proof-of-concept, and considering the extremely high sensitivity, the genosensor was challenged with highly diluted samples obtained from SARS-CoV-2 RNA PCR amplification.

Production of a Natural Dihydropteroate Synthase and Development of a Signal-Amplified Pseudo-Immunoassay for the Determination of Sulfonamides in Pork.

In this study, a type of magnetic photoaffinity-labeled activity-based protein profiling probe for sulfonamide drugs was first synthesized for the purpose of capturing the natural dihydropteroate synthase of Escherichia coli by using simple incubation and magnetic separation. After characterization of its identity with LC-ESI-MS/MS, this enzyme was used as a recognition reagent to develop a direct competitive pseudo-ELISA for the determination of the residues of 40 sulfonamides in pork. Because of the use of streptavidin-horseradish peroxidase and biotinylated horseradish peroxidase as a signal-amplified system, the limits of detection for the 40 drugs were in the range of 0.001-0.016 ng/mL. Compared to the steps in a conventional assay formation, the operation steps were the same, but the sensitivities increased 32-88-fold. Furthermore, the assay performances were better than the previously reported immunoassays performances for sulfonamides. Therefore, this method could be used as a practical tool for multiscreening the trace levels of sulfonamides residues in food samples.

Electrochemical Impedance Spectroscopy Using Interdigitated Gold–Polypyrrole Electrode Combination

A novel electrochemical impedance spectroscopy (EIS) sensor design, based on a standard interdigitated electrode arrangement in which the working electrode consists of gold and the combined counter and reference electrodes of polypyrrole doped with polystyrene sulfonate (PPy:PSS), is evaluated for biosensing applications. The performance is successfully proved by immobilization of a thiolated biotin as a self‐assembled monolayer (SAM), followed by streptavidin and a biotinylated horseradish peroxidase. It is shown that specific binding of biomolecules takes place only at the gold electrode. The binding activities are not influenced by the addition of small amounts of the nonionic surfactant Pluronic F‐68. The immobilization process is monitored online with EIS showing an excellent repeatability of the EIS signals in comparison with Au–Au electrode configuration even after electrode regeneration.

Avidin and Glucose Oxidase‐non‐covalently Functionalized Multi‐walled Carbon Nanotubes: A New Analytical Tool for Building a Bienzymatic Glucose Biosensor

AbstractWe report an innovative supramolecular architecture for bienzymatic glucose biosensing based on the non‐covalently functionalization of multi‐walled carbon nanotubes (MWCNTs) with two proteins, glucose oxidase (GOx) (to recognize glucose) and avidin (to allow the specific anchoring of biotinylated horseradish peroxidase (b‐HRP)). The optimum functionalization was obtained by sonicating for 10 min 0.50 mg mL−1 MWCNTs in a solution of 2.00 mg mL−1 GOx+1.00 mg mL−1avidin prepared in 50 : 50 v/v ethanol/water. The sensitivity to glucose for glassy carbon electrodes (GCE) modified with MWCNTs‐GOx‐avidin dispersion and b‐HRP (GCE/MWCNTs‐GOx‐avidin/b‐HRP), obtained from amperometric experiments performed at −0.100 V in the presence of 5.0×10−4 M hydroquinone, was (4.8±0.3) μA mM−1 (r2=0.9986) and the detection limit was 1.2 μM. The reproducibility for 5 electrodes using the same MWCNTs/GOx‐avidin dispersion was 4.0 %, while the reproducibility for 3 different dispersions and 9 electrodes was 6.0 %. The GCE/MWCNT‐GOx‐avidin/b‐HRP was successfully used for the quantification of glucose in a pharmaceutical product and milk.

New bioanalytical platform based on the use of avidin for the successful exfoliation of multi-walled carbon nanotubes and the robust anchoring of biomolecules. Application for hydrogen peroxide biosensing

We are reporting an innovative building-block for the development of biosensors based on the non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with avidin (MWCNTs-avidin). In this work, at variance with previous reports, avidin has the double role of simultaneously being the exfoliating agent of MWCNTs and the platform for anchoring different biotinylated biomolecules. The optimum dispersion was obtained by sonicating for 5.0 min 0.50 mgmL−1 MWCNTs with 1.00 mgmL−1 avidin solution prepared in 50:50 v/v ethanol/water. As proof-of-concept, we immobilized biotinylated horseradish peroxidase (b-HRP) at glassy carbon electrodes (GCE) modified with MWCNTs-avidin to develop a hydrogen peroxide biosensor using hydroquinone as redox mediator. Surface plasmon resonance, electrochemical impedance spectroscopy, cyclic voltammetry and amperometry demonstrated that, even after the partial denaturation of avidin due to the drastic conditions used to functionalize the MWCNTs, it preserves the biorecognition properties and efficiently interacts with biotinylated horseradish peroxidase (b-HRP). The analytical characteristics of the resulting hydrogen peroxide biosensor are the following: linear range between 1.0 × 10−6 M and 1.4 × 10−5 M, sensitivity of (1.37 ± 0.04) x 105 μAM−1, detection limit of 24 nM and reproducibility of 2.9%. The sensor was challenged with different samples, a mouthwash solution, human blood serum and milk, with very good performance.

A highly sensitive and reliable detection of CA15-3 in patient plasma with electrochemical biosensor labeled with magnetic beads

An early on-time detection of breast cancer can effectively affect the outcome of the treatment. Here, we developed an ultrasensitive, simple and reliable immunosensor to detect the lowest alteration of CA 15-3, the standard biomarker of breast cancer patients. The proposed immunosensor was achieved by modification of gold electrode by streptavidin to immobilize the biotinylated anti-CA 15-3 monoclonal antibody (mAb). Bovine serum albumin was used to prevent nonspecific binding. To improve the sensitivity of modified immunosensor, the sandwich signal enhancer consisting of streptavidin-coated magnetic beads conjugated with biotinylated horseradish peroxidase (HRP) and anti-CA 15-3 biotinylated mAb was applied. The electrochemical measurements were obtained in the presence of hydroquinone as a redox agent and H2O2 as the activating agent of HRP. Under optimized condition and using square wave voltammetry, the lower limit of quantification was obtained as 15 × 10−6 U/mL and the linear CA 15-3 concentration range was 50–15 × 10−6 U/mL. While showing significant stability, the immunosensor displayed an excellent sensitivity and specificity for the detection of CA 15-3 even in the human serum as compared to the enzyme-linked immunosorbent assay (ELISA) as a gold standard method. Based on our findings, the engineered immunosensor is proposed as a robust diagnostic tool for the clinical determination of CA 15-3 and other cancer biomarkers.

Amperometric low potential aptasensor for the fucosylated Golgi protein 73, a marker for hepatocellular carcinoma

The fucosylated Golgi protein 73 (fuc-GP73) has been used as a criterion to distinguish hepatocellular carcinoma (HCC) from other chronic liver diseases. We describe an amperometric aptasensor for ultrasensitive detection of fuc-GP73 that uses a thiolated aptamer against GP73 as the capture probe, and gold nanoparticles (AuNPs) modified with Avidinlens culinaris agglutinin (A-LCA) as the detection probe. The AuNPs on the surface of a gold electrode provide a large surface for immobilization of A-LCA, so that they can be heavily loaded with biotinylated horse radish peroxidase (B-HRP) via avidin-biotin interactions. This results in enhanced analytical sensitivity. Under optimized conditions and a typical working potential as low as 48 mV (vs. SCE), the dynamic response of the electrode covers the 10 pg·mL−1 to 25 ng·mL−1 fuc-GP73 concentation range, with a 7 pg·mL−1 detection limit (for an S/N ratio of 3). The assay is precise, selective and reproducible. It was applied to the determination of fuc-GP73 in serum.

Impact of Molecular Surface Charge on Biosensing by Electrochemical Impedance Spectroscopy

The effect of the molecular surface charge on sensing sensitivity using the electrochemical impedance spectroscopy (EIS) technique in the presence of [Fe(CN)6]3−/[Fe(CN)6]4− redox couple was investigated using Avidin- biotinylated Horseradish Peroxidase as model proteins. Reducing the positive charge of the probe molecule (Avidin) by chemical modification increased the change in the real part of the system’s impedance, which improved the sensitivity of the sensor. The system’s detection limit was within the range of few nM, where at low analyte concentrations – under 5nM – the signal becomes very noisy. The work presented herein shows that the nature and amount of charge of the molecules involved – both analyte and the binding protein, have major effect on the EIS signal, which should be taken into consideration throughout the construction and use of EIS biosensor. The results of the impedimetric measurements were characterized and compared to results obtained from the widely- established ELISA biochemical method for detection of biorecognition events. A physical model explaining the results obtained is proposed.

Coil-like Enzymatic Biohybrid Structures Fabricated by Rational Design: Controlling Size and Enzyme Activity over Sequential Nanoparticle Bioconjugation and Filtration Steps.

Well-defined enzymatic biohybrid structures (BHS) composed of avidin, biotinylated poly(propyleneimine) glycodendrimers, and biotinylated horseradish peroxidase were fabricated by a sequential polyassociation reaction to adopt directed enzyme prodrug therapy to protein-glycopolymer BHS for potential biomedical applications. To tailor and gain fundamental insight into pivotal properties such as size and molar mass of these BHS, the dependence on the fabrication sequence was probed and thoroughly investigated by several complementary methods (e.g., UV/vis, DLS, cryoTEM, AF4-LS). Subsequent purification by hollow fiber filtration allowed us to obtain highly pure and well-defined BHS. Overall, by rational design and control of preparation parameters, e.g., fabrication sequence, ligand-receptor stoichiometry, and degree of biotinylation, well-defined BHS with stable and even strongly enhanced enzymatic activities can be achieved. Open coil-like structures of BHS with few branches are available by the sequential bioconjugation approach between synthetic and biological macromolecules possessing similar size dimensions.

Biotin determination in food supplements by an electrochemical magneto biosensor

An electrochemical magneto biosensor for the rapid determination of biotin in food samples is reported. The affinity reaction was performed on streptavidin-modified magnetic microbeads as a solid support in a direct competitive format. The biotinylated horseradish peroxidase enzyme (biotin-HRP) competes with free biotin in the sample for the binding sites of streptavidin on the magnetic microbeads. The modified magnetic beads were then easily captured by a magneto graphite–epoxy composite electrode and the electrochemical signal was based on the enzymatic activity of the HRP enzyme under the addition of H2O2 as the substrate and o-phenilendiamine as cosubstrate. The response was electrochemically detected by square wave voltammetry. The limit of detection was 8.4×10−8molL−−1 of biotin (20μgL−−1) with a dynamic range from 0.94 to 2.4×10−7molL−−1. Biotin-fortified commercial dietary supplement and infant formula samples were evaluated obtaining good performances in the results. Total time of analysis was 40min per 20 assays.

Sortase A-Catalyzed Site-Specific Coimmobilization on Microparticles via Streptavidin

A microparticle surface was designed by the unique method incorporating streptavidin-biotin affinity and sortase A (SrtA)-catalyzed transpeptidation. Leucine-proline-glutamate-threonine-glycine-tagged streptavidin (Stav-LPETG)was immobilized on the surface using streptavidin-biotin affinity, and GGGGG-tagged red fluorescent protein (Gly5-RFP) was conjugated with SrtA. Biotinylated fluorescein isothiocyanate (biotin-FITC) was then bound to residual biotin-binding sites in Stav-LPETG. The resulting particles had RFP and FITC immobilized on the surface via Stav-LPETG, and RFP- and FITC-associated fluorescence was observed using fluorescence microscopy. Finally, GGG-tagged glucose oxidase and biotinylated horseradish peroxidase were immobilized on the microparticle surface, resulting in a functional particle capable of detecting glucose. This particle can be repeatedly used and is more sensitive in detecting glucose than particles prepared using chemical modification. Our method provides a simple strategy for site-specific coimmobilization on molecular surfaces and expands the use of protein hybrid devices.

DYRK1A-mediated Hyperphosphorylation of Tau

Most individuals with Down syndrome show early onset of Alzheimer disease (AD), resulting from the extra copy of chromosome 21. Located on this chromosome is a gene that encodes the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). One of the pathological hallmarks in AD is the presence of neurofibrillary tangles (NFTs), which are insoluble deposits that consist of abnormally hyperphosphorylated Tau. Previously it was reported that Tau at the Thr-212 residue was phosphorylated by Dyrk1A in vitro. To determine the physiological significance of this phosphorylation, an analysis was made of the amount of phospho-Thr-212-Tau (pT212) in the brains of transgenic mice that overexpress the human DYRK1A protein (DYRK1A TG mice) that we recently generated. A significant increase in the amount of pT212 was found in the brains of DYRK1A transgenic mice when compared with age-matched littermate controls. We further examined whether Dyrk1A phosphorylates other Tau residues that are implicated in NFTs. We found that Dyrk1A also phosphorylates Tau at Ser-202 and Ser-404 in vitro. Phosphorylation by Dyrk1A strongly inhibited the ability of Tau to promote microtubule assembly. Following this, using mammalian cells and DYRK1A TG mouse brains, it was demonstrated that the amounts of phospho-Ser-202-Tau and phospho-Ser-404-Tau are enhanced when DYRK1A amounts are high. These results provide the first in vivo evidence for a physiological role of DYRK1A in the hyperphosphorylation of Tau and suggest that the extra copy of the DYRK1A gene contributes to the early onset of AD.

Intracellular labeling of single cortical astrocytes in vivo

Glial cells have traditionally been considered to play supportive roles in the central nervous system. As recent experimental evidence suggests glial cells’ participation in neural information processing, there has been a need to monitor the physiology of glial cells in vivo in the matured brain. Concurrently, identification and classification of the recorded glial cells is essential as there are at least several different kinds of glial cells. Past studies have achieved in vivo intracellular electrophysiological recording of glial cells using sharp glass microelectrodes, however, morphological recovery and identification of the recorded cells have hardly been done, due to technical difficulties. We demonstrate that use of large fragment biotinylated dextran amine (BDA) is an effective way to label a single glial cell recorded with a sharp microelectrode in vivo. Furthermore, the tracer signal amplification was achieved by a combination of avidin biotinylated horseradish peroxidase macromolecular complex (ABC) and tyramide-based methods, making multiple immunohistochemistry feasible. Using the method described in this study, we have successfully recorded and labeled cortical glial cells including astrocytes, oligodendrocytes, and microglia.

Evaluation of the analytical performances of avidin-modified carbon sensors based on a mediated horseradish peroxidase enzyme label and their application to the amperometric detection of nucleic acids

In this study, neutravidin-coated screen-printed carbon sensors were fully characterized and further used for the amperometric detection of specific DNA sequences of human cytomegalovirus (HCMV DNA). For this purpose, we took advantage of an earlier established relationship between the amount of HRP affinity immobilized on the surface of the electrode and the steady-state current recorded in the presence of H2O2 as substrate and the single electron donor [OsIII(bpy)2pyCl]2+ as cosubstrate. After incubating a saturating concentration of biotinylated horseradish peroxidase (Bio-HRP) onto the neutravidin-modified sensors, a surface concentration of active HRP of 3.6pmolcm−2 was calculated from the measurement of the electrocatalytic plateau current value. This result indicates that monolayers of neutravidin were adsorbed on the screen-printed carbon sensors. These neutravidin-covered platforms were then used to immobilize biotinylated nucleic acid targets. After hybridization with a complementary digoxigenin-labeled detection probe, the extent of hybrids formed was determined with an anti-digoxigenin HRP conjugate. The biosensor assay was applied to the detection of a synthetic oligonucleotide target, and then to the determination of an amplified viral DNA sequence. Monolayers of HRP-labeled oligonucleotide hybrids were immobilized onto the sensing surface whereas one third of the surface was covered with HCMV DNA hybrids. On the other hand, detection limits of 200pM and 1nM were obtained for the short oligonucleotide and the longer DNA targets, respectively. Finally, we demonstrated that the sensitivity of the electrochemical assay could be significantly improved by using high concentrations of the reduced form of the mediator [OsII(bpy)2pyCl]+, thus allowing one to detect as low as 30pM of amplified HCMV DNA fragment.