Controlled growth of redox polymer network on single enzyme molecule for stable and sensitive enzyme electrode.

The electrochemical applications of enzymes are often hampered by poor enzyme stability and low electron conductivity. In this work, a novel enzyme nanogel based on atom transfer radical polymerization (ATRP) has been developed for highly sensitive detection of glucose based on ferrocene (Fc) embedded in crosslinked polymer network nanogel. Enzyme surfaces are successively modified with Br initiator, and then in situ atom transfer radical polymerization (ATRP) was performed to build up crosslinked polyacrylamide network. The resulting single enzyme nanogel (ATRP-SEG) is uniform in size fairly. ATRP-SEG reveals bi-phasic inactivation, and the half-life of stable ATRP-SEG after 18-day incubation at 50 °C is 47 days, which is 197 times longer than that of free Gox (5.7 h). By introducing a ferrocene (Fc) containing redox polymer, poly(acrylamide-co-vinylferrocene), the half-life of Fc-ATRP-SEG after 18-day incubation at 50 °C is 49 days. Fc-ATRP-SEG is used for preparation of glucose-sensing electrode, and the sensitivity of Fc-ATRP-SEG electrode is 111 µA cm-2 mM-1, which is 366 and 1270 times higher than those of free GOx (0.303 µA cm-2 mM-1) and ATRP-SEG (0.0874 µA cm-2 mM-1), respectively. Fc-ATRP-SEG electrode maintained 90% of initial current density under 4 °C storage condition and repetitive usages every day for 7 days. Even the electrode repeatedly used in continuous harsh condition (250 rpm, room temperature), the current density maintained 96% after 12 h incubation at operational condition.

Phenolic Tyrosinase Substrate with a Formal Potential Lower than That of Phenol to Obtain a Sensitive Electrochemical Immunosensor.

The high and selective catalytic activities of tyrosinase (Tyr) have frequently led to its application in sensitive biosensors. However, in affinity-based biosensors, the use of Tyr as a catalytic label is less common compared to horseradish peroxidase and alkaline phosphatase owing to the fact that phenolic Tyr substrates have yet to be investigated in detail. Herein, four phenolic compounds that have lower formal potentials than phenol were examined for their applicability as Tyr substrates, and three reducing agents were examined as potential strong reducing agents for electrochemical-chemical (EC) redox cycling involving an electrode, a Tyr product, and a reducing agent. The combination of 4-methoxyphenol (MP) and ammonia-borane (AB) allows for (i) a high electrochemical signal level owing to rapid EC redox cycling and (ii) a low electrochemical background level owing to the slow oxidation of AB at a low applied potential and no reaction between MP and AB. When this combination was applied to an electrochemical immunosensor for parathyroid hormone (PTH) detection, a detection limit of 2 pg/mL was obtained. This detection limit is significantly lower than that obtained when a combination of phenol and AB was employed (300 pg/mL). It was also found that the developed immunosensor works well in PTH detection in clinical serum samples. This new phenolic substrate could therefore pave the way for Tyr to be more commonly used as a catalytic label in affinity-based biosensors.

Boosting electrochemical immunosensing performance by employing acetaminophen as a peroxidase substrate.

In horseradish peroxidase (HRP)-based electrochemical immunosensing, an appropriate HRP substrate needs to be chosen to obtain a high electrochemical signal-to-background ratio. This is limited by the unwanted electrochemical reduction of H2O2, oxidation of the substrate, and the slow electrochemical reduction of the product. Herein, we report acetaminophen (AMP) as a new HRP substrate that allows for highly sensitive immunosensing. Electrochemical behavior and immunosensing performance using AMP are compared with those using the most popular HRP substrate, hydroquinone (HQ). To maintain a high electrocatalytic activity even at an electrode modified with an immunosensing layer, an indium tin oxide electrode partially modified with reduced graphene oxide is employed. AMP allows for a higher signal-to-background ratio than HQ, because the formal potential of AMP is 0.28 V higher than that of HQ and the redox reaction of AMP is as reversible as that of HQ, resulting in a lower detection limit in a sandwich-type immunoassay using AMP for thyroid-stimulating hormone detection. The calculated detection limit is ~0.2 pg/mL. The use of AMP as an HRP substrate is especially promising for highly sensitive electrochemical immunoassays.

Diaphorase-Catalyzed Formation of a Formazan Precipitate and Its Electrodissolution for Sensitive Affinity Biosensors.

Catalytic precipitation and subsequent electrochemical oxidation or reduction of a redox-active precipitate has been widely used in electrochemical biosensors. However, such biosensors often do not allow for low detection limits due to a low rate of precipitation, nonspecific precipitation, loose binding of the precipitate to the electrode surface, and insulating behavior of the precipitate within a normal potential window. Here, we report an ultrasensitive electrochemical immunosensor for parathyroid hormone (PTH) detection based on DT-diaphorase (DT-D)-catalyzed formation of an organic precipitate and electrochemical oxidation of the precipitate. In the present study we found that DT-D can be used as a catalytic label in precipitation-based affinity biosensors because DT-D catalyzes fast reduction of 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan precipitate; the MTT reduction does not occur in the absence of DT-D; and a high electrochemical signal is obtained at low potentials during electrodissolution of MTT-formazan precipitate. The immunosensor is fabricated using a silane copolymer-modified ITO electrode surface that is suitable for both efficient and strong adsorption of MTT-formazan precipitate. When the enzymatic MTT-formazan precipitation and subsequent MTT-formazan electrodissolution is applied to a sandwich-type immunosensor, PTH can be detected over a wide range of concentrations with a very low detection limit (∼1 pg/mL) in artificial serum. The measured concentrations of PTH in clinical serum samples showed high similarity with those obtained using a commercial instrument.

Highly sensitive detection of hydrazine by a disposable, Poly(Tannic Acid)-Coated carbon electrode.

We propose an electrochemical sensor based on the enhanced electrocatalytic oxidation exhibited on a functionalized poly(tannic acid) coating to detect hydrazine. Tannic acid, a naturally abundant and low-cost polyphenol, was enzymatically polymerized with horseradish peroxidase and subsequently adsorbed on a disposable screen-printed carbon electrode with a short incubation time (30 min). The fabrication method proved to be reproducible (4.2 % relative standard deviation), with the sensors displaying high sensitivity (7 × 10-3 µA mm-2 µM-1) and selectivity even in the presence of various common interfering agents. The low detection limit (100 nM) and robustness of the sensor demonstrated its suitability for environmental applications. It can be used to quantify hydrazine in tap and river water samples.

Universal method for direct bioconjugation of electrode surfaces by fast enzymatic polymerization.

We report HRP-catalyzed polymerization of Tannic acid (TA) and application of the poly (Tannic acid) (p(TA)) as a versatile platform for covalent immobilization of biomolecules on various electrode surfaces based on electrochemical oxidation of the p(TA) and subsequent oxidative coupling reactions with the biomolecules. We also used this method for capturing cancer cells through a linker molecule, folic acid (FA). Furthermore, we have demonstrated that enhanced electrocatalytic activity of the p(TA)-modified surface could be used for simultaneous electrochemical determination of biologically important electroactive molecules such as ascorbic acid (AA), dopamine (DA), and uric acid (UA). This HRP-catalyzed polymerization of TA and p(TA)-mediated surface modification method can provide a simple and new framework to construct multifunctional platforms for covalent attachment of biomolecules and development of sensitive electrochemical sensing devices.

Washing-Free Displacement Immunosensor for Cortisol in Human Serum Containing Numerous Interfering Species.

Simple and sensitive competitive immunosensors for small molecules are difficult to obtain, especially in serum containing numerous interfering species (ISs) with different concentrations. Herein, we report a washing-free and sensitive (competitive) displacement immunosensor for cortisol in human serum, based on electron mediation of Os(bpy)2Cl2 between an electrode and a redox label [oxygen-insensitive diaphorase (DI)] (i.e., electrochemical-enzymatic redox cycling). The anticortisol IgG-DI conjugate bound to a cortisol-immobilized electrode is displaced by competitive binding of cortisol in serum and diffuses away from the electrode during incubation; therefore, the concentration of the displaced conjugate near the electrode becomes very low, even without washing. Electrochemically interfering ascorbic acid is converted to a redox-inactive species by ascorbate oxidase during incubation. The remaining bound conjugate mainly contributes to electrochemical currents. Compared with ferrocene methanol, Fe(CN)64-, and Ru(NH3)63+, the electrochemical and redox cycling behaviors of Os(bpy)2Cl2 are influenced significantly less by ISs in serum. Comparative studies reveal that washing-free displacement assay shows better cortisol-induced signal change than three other assays. The surface concentration of cortisol immobilized on the electrode is optimized, because the electrochemical signal is highly dependent on the surface concentration. When the washing-free displacement immunosensor is applied for the detection of cortisol in artificial serum, cortisol is measured with a detection limit of ∼30 pM within 12 min. The cortisol concentrations measured in clinical serum samples agree well with those obtained using a commercial instrument. The new immunosensor is highly promising for the simple, sensitive, and rapid point-of-care detection of small molecules.

Immunosensor Employing Stable, Solid 1-Amino-2-naphthyl Phosphate and Ammonia-Borane toward Ultrasensitive and Simple Point-of-Care Testing.

Biosensors for ultrasensitive point-of-care testing require dried reagents with long-term stability and a high signal-to-background ratio. Although ortho-substituted diaromatic dihydroxy and aminohydroxy compounds undergo fast redox reactions, they are not used as electrochemical signaling species because they are readily oxidized and polymerized by dissolved oxygen. In this report, stable, solid 1-amino-2-naphthyl phosphate (1A2N-P) and ammonia-borane (H3N-BH3) are respectively employed as a substrate for alkaline phosphatase (ALP) and a reductant for electrochemical-chemical (EC) redox cycling. ALP converts 1A2N-P to 1-amino-2-naphthol (1A2N), which is then employed in EC redox cycling using H3N-BH3. The oxidation and polymerization of 1A2N by dissolved oxygen is significantly prevented in the presence of H3N-BH3. The electrochemical measurement is performed without modification of indium-tin oxide (ITO) electrodes with electrocatalytic materials. For comparison, nine aromatic dihydroxy and aminohydroxy compounds, including 1A2N, are evaluated to achieve fast EC redox cycling, and four strong reductants, including H3N-BH3, are evaluated to achieve a low background level. The combination of 1A2N and H3N-BH3 allows the achievement of a very high signal-to-background ratio. When the newly developed combination is applied to the detection of creatine kinase-MB (CK-MB), the detection limit for CK-MB is ∼80 fg/mL, indicating that the combination allows ultrasensitive detection. The concentrations of CK-MB in clinical serum samples, determined using the developed system, are in good agreement with the concentrations obtained using a commercial instrument. Thus, the use of stable, solid 1A2N-P and H3N-BH3 along with bare ITO electrodes is highly promising for ultrasensitive and simple point-of-care testing.

Facile degradation of benzenediazonium-grafted thick layers on the electrode surface enabling electrochemical biosensor application.

We found that the formylbenzenediazonium (FBD)-grafted organic layers can be degraded by treating with an aqueous solution of salt and Tween. An electrochemical immunoassay was carried out on the degraded surface, which allowed highly sensitive detection of antigen mouse IgG.

An electrochemically reduced graphene oxide-based electrochemical immunosensing platform for ultrasensitive antigen detection.

We present an electrochemically reduced graphene oxide (ERGO)-based electrochemical immunosensing platform for the ultrasensitive detection of an antigen by the sandwich enzyme-linked immunosorbent assay (ELISA) protocol. Graphene oxide (GO) sheets were initially deposited on the amine-terminated benzenediazonium-modified indiun tin oxide (ITO) surfaces through both electrostatic and π-π interactions between the modified surfaces and GO. This deposition was followed by the electrochemical reduction of graphene oxide (GO) for preparing ERGO-modified ITO surfaces. These surfaces were then coated with an N-acryloxysuccinimide-activated amphiphilic polymer, poly(BMA-r-PEGMA-r-NAS), through π-π stacking interactions between the benzene ring tethered to the polymer and ERGO. After covalent immobilization of a primary antibody on the polymer-modified surfaces, sandwich ELISA was carried out for the detection of an antigen by use of a horseradish peroxidase (HRP)-labeled secondary antibody. Under the optimized experimental conditions, the developed electrochemical immunosensor exhibited a linear response over a wide range of antigen concentrations with a very low limit of detection (ca. 100 fg/mL, which corresponds to ca. 700 aM). The high sensitivity of the electrochemical immunosensor may be attributed not only to the enhanced electrocatalytic activity owing to ERGO but also to the minimized background current owing to the reduced nonspecific binding of proteins.

Reusable bio-functionalized surfaces based on electrochemical desorption of benzenediazonium-grafted organic layers.

We describe an electrochemical oxidative desorption of benzenediazonium-grafted organic layers and immobilized proteins on the layers from indium-tin-oxide electrode surfaces.

Aldehyde-functionalized benzenediazonium cation for multiprobe immobilization on microelectrode array surfaces.

We report in situ generation of aldehyde-functionalized benzenediazonium cation (ABD) and its use as a suitable linker molecule for fast and selective immobilization of biomolecules on indium-tin-oxide (ITO) electrode surfaces. We prepared ABD through a new reaction procedure, a simultaneous diazotation of the amine group and deprotection of the aldehyde group from an aniline derivative, 2-(4-aminophenyl)-1,3-dithiane, which was revealed on the ITO electrode surfaces through the electrodeposition of the reaction product and the characterization of the resulting surfaces with cyclic voltammetry, X-ray photoelectron spectroscopy, and protein immobilization. We also showed that successive electrodeposition of ABD and probe molecules on individually addressable microarray electrode surfaces can provide a useful platform for efficient detection of multianalyte. The usage of ABD has been demonstrated by the patterning of three different probe molecules on a single substrate and the simultaneous detection of two target molecules.

Use of 1,3-dithiane combined with aryldiazonium cation for immobilization of biomolecules based on electrochemical addressing.

We report the use of 1,3-dithiane combined with aryldiazonium cation for the immobilization of biomolecules based on electrochemical addressing.