Strategy for Low Background‐Current Levels in the Electrochemical Biosensors Using Horse‐Radish Peroxidase Labels

This article describes an electrochemical strategy to achieve low background‐current levels in horse‐radish peroxidase (HRP)‐based electrochemical immunosensors. The strategy consists of (i) the use of an HRP substrate/product redox couple whose formal potential is high and (ii) the use of an electrode that shows moderate electrocatalytic activity for the redox couple. The strategy is proved by a model biosensor using a catechol/o‐benzoquinone redox couple and an indium tin oxide (ITO) electrode. The combined effect of high formal potential and moderate electrocatalytic activity allows o‐benzoquinone electroreduction with minimal catechol electrooxidation and H₂O₂ electroreduction. The detection limit for mouse‐IgG is 100 pg/mL.     

Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection

A Fe₂O₃@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe₂O₃@Au nanoparticles were prepared by reducing HAuCl₄ on the surfaces of Fe₂O₃ nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.     

Signal amplification for DNA detection based on the HRP-functionalized Fe3O4 nanoparticles

An electrochemical approach for the sensitive detection of sequence-specific DNA has been developed. Horseradish peroxidase (HRP) assembled on the Fe₃O₄ nanoparticles (NPs) were utilized as signal amplification sources. High-content HRP was adsorbed on the Fe₃O₄ NPs via layer-by-layer (LbL) technique to prepare HRP-functionalized Fe₃O₄ NPs. Signal probe and diluting probe were then immobilized on the HRP-functionalized Fe₃O₄ NPs through the bridge of Au NPs. Thereafter, the resulting DNA-Au-HRP-Fe₃O₄ (DAHF) bioconjugates were successfully anchored to the gold nanofilm (GNF) modified electrode surface for the construction of sandwich-type electrochemical DNA biosensor. The electrochemical behaviors of the prepared biosensor had been investigated by the cyclic voltammetry (CV), chronoamperometry (i-t), and electrochemical impedance spectroscopy (EIS). Under optimal conditions, the proposed strategy could detect the target DNA down to the level of 0.7 fmol with a dynamic range spanning 4 orders of magnitude and exhibited excellent discrimination to two-base mismatched DNA and non-complementary DNA sequences.     

A novel amperometric biosensor based on gold nanoparticles-mesoporous silica composite for biosensing glucose

We report a novel bienzyme biosensor based on the assembly of the glucose oxidase (GOD) and horseradish peroxidase (HRP) onto the gold nanoparticles encapsulated mesoporous silica SBA-15 composite (AuNPs-SBA-15). Electrochemical behavior of the bienzyme bioconjugates biosensor is studied by cyclic voltammetry and electrochemical impedance spectroscopy. The results indicate that the presence of mesoporous AuNPs-SBA-15 greatly enhanced the protein loadings, accelerated interfacial electron transfer of HRP and the electroconducting surface, resulting in the realization of direct electrochemistry of HRP. Owing to the electrocatalytic effect of AuNPs-SBA-15 composite, the biosensor exhibits a sensitive response to H₂O₂ generated from enzymatic reactions. Thus the bienzyme biosensor could be used for the detection of glucose without the addition of any mediator. The detection limit of glucose was 0.5 μM with a linear range from 1 to 48 μM. Supported by the National Natural Science Foundation of China (Grant Nos. 20635020 & 90606016)     

Electrochemical analysis of microRNAs with hybridization chain reaction-based triple signal amplification

Selective and sensitive detection of trace microRNA is important for early diagnosis of diseases due to its expression level related to diseases. Herein, a triple signal amplification strategy is developed for trace microRNA-21 (miRNA-21) detection by combining with target-triggered cyclic strand displacement reaction (TCSDR), hybridization chain reaction (HCR) and enzyme catalytic amplification. Four DNA hairpins (H1, H2, H3, H4) are employed to form an ultralong double-strand DNA (dsDNA) structure, which is initiated by target miRNA-21. As H3 and H4 are labeled with horseradish peroxidase (HRP), numerous HRPs are loaded on the long dsDNA, producing significantly enhanced electrocatalytic signals in the hydrogen peroxide (H₂O₂) and 3,3′,5,5′-tetramethylbenzidine (TMB) reaction strategy. Compared with single signal amplification, the triple signal amplification strategy shows higher electrochemical response, wider dynamic range and lower detection limit for miRNA-21 detection with excellent selectivity, reproducibility and stability. Taking advantage of the triple signal amplification strategy, the proposed electrochemical biosensor can detect miRNA-21 in 10 HeLa cell lysates, suggesting that it is a promising method for fruitful assay in clinical diagnosis.     

Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification

Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo⁻), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.     

Hairpin Assembly Amplified Electrochemical Biosensor for Highly Sensitive and Specific Detection of DNA

In this report, a simple electrochemical biosensor has been developed for highly sensitive and specific detection of DNA based on hairpin assembly amplification. In the presence of target DNA, the biotin‐labelled hairpin H1 is opened by hybridizing with target DNA through complementary sequences. Then the opened hairpin H1 assembles with the hairpin H2 to displace the target DNA, generating H1‐H2 complex. The displaced target DNA could trigger the next cycle of hairpins assembly, resulting in the generation of numerous H1‐H2 complexes. Subsequently, the H1‐H2 complex hybridizes with the capture probe immobilized on the electrode. Finally, the streptavidin alkaline phosphatase (ST‐ALP) binds to biotin in the capture probe‐H1‐H2 complex and catalyzes the substrate α‐naphthol (α‐NP) to produce electrochemical signal. To make a more fascinating hairpin assembly amplification strategy in signal amplification, mismatched base sequences are designed in hairpin H2 to decrease non‐specific binding of the hairpin substrates. The developed biosensor achieves a sensitivity of 20 pM with a linear range from 25 pM to 25 nM, and shows high selectivity toward single‐base mismatch. Thus, the proposed electrochemical biosensor might have the potential for early clinical diagnosis and therapy.     

A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Covalently Immobilized on Electrografted Organic Film on Screen‐Printed Carbon Electrode

A new convenient strategy to fabricate a third‐generation hydrogen peroxide biosensor was described. The screen‐printed carbon electrode (SPCE) was first modified with a layer of 4‐nitrophenyl assembled from the 4‐nitroaniline diazonium salt synthesized in situ in acidic aqueous solution. Next, the nitro groups were converted to amines followed by crosslinking to the horseradish peroxidase (HRP) by glutaraldehyde. The redox chemistry of the active center of the HRP was observed and the HRP‐modified electrode displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediators. H₂O₂ was determined in a linear range from 5.0 μM to 50.0 μM, with a detection limit of 1.0 μM. Furthermore, the biosensor exhibited fast amperometric response, good reproducibility and long‐term stability.     

Sensitive pseudobienzyme electrocatalytic DNA biosensor for mercury(II) ion by using the autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification

Herein, a novel sensitive pseudobienzyme electrocatalytic DNA biosensor was proposed for mercury ion (Hg²⁺) detection by using autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification. Thiol functionalized capture DNA was firstly immobilized on a nano-Au modified glass carbon electrode (GCE). In presence of Hg²⁺, the specific coordination between Hg²⁺ and T could result in the assembly of primer DNA on the electrode, which successfully triggered the HCR to form the hemin/G-quadruplex DNAzyme nanowires with substantial redox probe thionine (Thi). In the electrolyte of PBS containing NADH, the hemin/G-quadruplex nanowires firstly acted as an NADH oxidase to assist the concomitant formation of H₂O₂ in the presence of dissolved O₂. Then, with the redox probe Thi as electron mediator, the hemin/G-quadruplex nanowires acted as an HRP-mimicking DNAzyme that quickly bioelectrocatalyzed the reduction of produced H₂O₂, which finally led to a dramatically amplified electrochemical signal. This method has demonstrated a high sensitivity of Hg²⁺ detection with the dynamic concentration range spanning from 1.0 ng L⁻¹ to 10 mg L⁻¹ Hg²⁺ and a detection limit of 0.5 ng L⁻¹ (2.5 pM) at the 3S_blank level, and it also demonstrated excellent selectivity against other interferential metal ions.     

Direct electrochemistry and spectroelectrochemistry of osmium substituted horseradish peroxidase

In this contribution the substitution of the central protoporphyrin IX iron complex of horseradish peroxidase by the respective osmium porphyrin complex is described. The direct electrochemical reduction of the Os containing horseradish peroxidase (OsHRP) was achieved at ITO and modified glassy carbon electrodes and in combination with spectroscopy revealed the three redox couples Os^IIIHRP/Os^IVHRP, Os^IVHRP/Os^VHRP and Os^VHRP/Os^VIHRP. The midpoint potentials differ dependent on the electrode material used with E_1/2 (Os^III/IV) of − 0.4 V (ITO) and − 0.25 V (GC), E_1/2 (Os^IV/^V) of − 0.16 V (ITO) and + 0.10 V (GC), and E_1/2 (Os^V/VI)of + 0.18 V (ITO), respectively. Moreover, with immobilised OsHRP the direct electrocatalytic reduction of hydrogen peroxide and tert-butyl hydroperoxide was observed. In comparison to electrodes modified with native HRP the sensitivity of the OsHRP-electrode for tert-butyl hydroperoxide is higher.     

Probing traces of hydrogen peroxide by use of a biosensor based on mediator-free DNA and horseradish peroxidase immobilized on silver nanoparticles

A new electrochemical biosensor for determination of hydrogen peroxide (H₂O₂) has been developed by immobilizing horseradish peroxidase (HRP) on silver colloids (nanosilver) and use of a DNA-functionalized interface. In the presence of the DNA and the nanosilver the immobilized HRP gives a pair of well-defined redox peaks with an electron-transfer rate constant of 3.27 ± 0.91 s⁻¹ in pH 7.0 PBS. The presence of DNA also provides a biocompatible microenvironment for enzyme molecules, greatly amplifies the amount of HRP molecules immobilized on the electrode surface, and improves the sensitivity of the biosensor. Under optimum conditions the biosensor has electrocatalytic activity in the reduction of hydrogen peroxide with linear dependence on H₂O₂ concentration in the range 1.5 × 10⁻⁶ to 2.0 × 10⁻³ mol L⁻¹; the detection limit is 5.0 × 10⁻⁷ mol L⁻¹ at a signal-to-noise ratio of 3. The value of HRP in the composite membrane was found to be 1.62 mmol L⁻¹. These results suggest that the properties of the complex film, with its bioelectrochemical catalytic activity, could make it useful for development of bioelectronic devices and for investigation of protein electrochemistry at functional interfaces.     

Novel Protocol for Covalent Immobilization of Horseradish Peroxidase on Gold Electrode Surface

A novel protocol for immobilization of horseradish peroxidase (HRP) onto diazonium functionalized screen‐printed gold electrode (SPGE) has been successfully developed. This protocol involved 1) electrochemical reduction of p‐nitrophenyl diazonium salts synthesized in situ in acidic aqueous solution to graft a layer of p‐nitrophenyl on SPGE, 2) electrochemical reduction of the nitro groups to convert to amines, 3) chemical reaction with nitrous acid to transform the amine to diazonium derivative and 4) chemical coupling of the enzyme with the diazonium group to form a covalent diazo bond. The fabricated biosensor showed the direct electrochemistry of HRP and displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediator. The biosensor exhibited fast amperometric response to H₂O₂. The catalytic current increased with increasing H₂O₂ concentration from 5 μM to 30 μM and the detection limit of the biosensor was 2 μM. The biosensor exhibited acceptable sensitivity, good reproducibility and long‐term stability.     

Amplification of bioelectrocatalytic signalling based on silver nanoparticles and DNA-derived horseradish peroxidase biosensors

Horseradish peroxidase (HRP) was immobilized onto a polyion complex membrane containing positively charged silver nanoparticles (nanosilver), double stranded DNA and poly(thionine) to fabricate highly sensitive and selective electrochemical hydrogen peroxide (H₂O₂) biosensor on a glassy carbon electrode. The presence of nanosilver provided a biocompatible microenvironment for enzyme molecules, greatly amplified the surface coverage of HRP on the electrode surface, and most importantly could act as a charge carrier. The process of the biosensor construction was characterized by scanning electron microscopy. Voltammetric and time-based amperometric techniques were employed to characterize the properties of the derived biosensor. Under optimal conditions, the biosensor has an electrocatalytic behavior towards the H₂O₂ reduction, and exhibits a linear range from 1.1 μM to 5.2 mM, with a lower detection limit of 0.2 μM. The apparent Michaelis–Menten constant of the biosensor to H₂O₂ was estimated to be 1.02 mM. Furthermore, the biosensor exhibited high sensitivity, good reproducibility, and acceptable stability. Importantly, the properties of composite film, together with the bioelectrochemical catalytic activity, could make them useful in the development of bioelectronic devices and investigation of protein electrochemistry at functional interface. Correspondence: Yan Liu, College of Chemistry, Chongqing Normal University, Chongqing 400047, P.R. China     

Room temperature ionic liquid doped DNA network immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

A novel electrochemical H₂O₂ biosensor was constructed by embedding horseradish peroxide (HRP) in a 1-butyl-3-methylimidazolium tetrafluoroborate doped DNA network casting on a gold electrode. The HRP entrapped in the composite system displayed good electrocatalytic response to the reduction of H₂O₂. The composite system could provide both a biocompatible microenvironment for enzymes to keep their good bioactivity and an effective pathway of electron transfer between the redox center of enzymes, H₂O₂ and the electrode surface. Voltammetric and time-based amperometric techniques were applied to characterize the properties of the biosensor. The effects of pH and potential on the amperometric response to H₂O₂ were studied. The biosensor can achieve 95% of the steady-state current within 2 s response to H₂O₂. The detection limit of the biosensor was 3.5 μM, and linear range was from 0.01 to 7.4 mM. Moreover, the biosensor exhibited good sensitivity and stability. The film can also be readily used as an immobilization matrix to entrap other enzymes to prepare other similar biosensors. Figure Horseradish peroxidase (HRP) embedded in a 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM·BF ₄ ) doped DNA network can be used to fabricate a HRP sensor for the determination of H₂O₂     

A hydrogen peroxide biosensor based on multiwalled carbon nanotubes-polyvinyl butyral film modified electrode

A novel approach to construct a amperometric biosensor for determination of H₂O₂ is described. Horseradish peroxidase (HRP) as a base enzyme was immobilized into the mixture of multiwalled carbon nanotubes (MWNTs) and polyvinyl butyral (PVB). Taking the classical hydroquinone as mediator, cyclic voltammetry and amperometric measurements were used to study and optimize the performance of the resulting H₂O₂ biosensor. The effect of the concentration of MWNTs, HRP, hydroquinone, solution pH, and the working potential of amperometry on the electrochemical biosensor was systematically studied. The results showed that the fabricated biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with wide linear range from 0.000832 to 0.6 mM, and low detection limit 0.000167 mM (S/N = 3) with fast response time less than 8 s. The apparent Michaelis–Menten constant was determined to be 0.049 mM. Additionally, the biosensor exhibited high sensitivity, rapid response and good long-term stability.     

Electroactive porous films of myoglobin within calcium alginate

In this work, a novel two-step construction strategy for protein assembly films was proposed. The first step was the preparation of porous calcium alginate (CA) films by spraying calcium chloride (CaCl₂) solution over the mixture surface of sodium alginate and polyethylene glycol on various solid substrates. The second step involved the cast of myoglobin (Mb) onto the porous CA films and then formed the electroactive porous Mb-CA films. The nitrogen adsorption desorption isotherm, scanning electron microscope, alternating current impendence and cyclic voltammetry were used to characterize the porous films. Fully hydrated porous CA films had nearly 90 wt% water contents and UV–vis showed that Mb in the porous films retained its near native conformation at medium pH. The stable films modified on glassy carbon electrode demonstrated good electroactivity in protein-free buffer, which was originated from protein heme Fe(III)/Fe(II) redox couples. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k _s) and formal potential (E ^o′) were estimated by fitting the data of square-wave voltammetry with nonlinear regression analysis. It was observed that the formal potential of the Mb Fe(III)/Fe(II) couple in porous CA films shifted linearly between pH 4.0 and 11.0 with a slope of −52.7 mV/pH, suggesting that one proton transfer was coupled to each electron transfer in the electrochemical reaction. The porous Mb-CA films showed the electrocatalytic activity toward dioxygen, hydrogen peroxide, and nitrite with significant decreases in the electrode potential required, and exhibited good operational and storage stability, reproducibility and fast response time for H₂O₂ detection. It is showing the possible future application of the films for biosensors and biocatalysis.     

Using flowerlike polymer–copper nanostructure composite and novel organic–inorganic hybrid material to construct an amperometric biosensor for hydrogen peroxide

A new type of amperometric hydrogen peroxide biosensor was fabricated by entrapping horseradish peroxidase (HRP) in the organic–inorganic hybrid material composed of zirconia–chitosan sol–gel and Au nanoparticles (ZrO₂–CS–AuNPs). The sensitivity of the biosensor was enhanced by a flowerlike polymer–copper nanostructure composite (pPA–FCu) which was prepared from co-electrodeposition of CuSO₄ solution and 2,6-pyridinediamine solution. Several techniques, including UV–vis absorption spectroscopy, scanning electron microscopy, cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy were employed to characterize the assembly process and performance of the biosensor. The results showed that this pPA–FCu nanostructure not only had excellent redox electrochemical activity, but also had good catalytic efficiency for hydrogen peroxide. Also the ZrO₂–CS–AuNPs had good film forming ability, high stability and good retention of bioactivity of the immobilized enzyme. The resulting biosensors showed a linear range from 7.80 × 10^?7 to 3.7 × 10^?3 mol L^?1, with a detection limit of 3.2 × 10^?7 mol L^?1 (S/N = 3) under optimized experimental conditions. The apparent Michaelis–Menten constant was determined to be 0.32 mM, showing good affinity. In addition, the biosensor which exhibits good analytical performance, acceptable stability and good selectivity, has potential for practical applications.     

Electrochemical characteristics of a platinum electrode modified with a matrix of polyvinyl butyral and colloidal Ag containing immobilized horseradish peroxidase

A new hydrogen peroxide biosensor was constructed, which consisted of a platinum electrode modified by a matrix of polyvinyl butyral (PVB) and nanometer-sized Ag colloid containing immobilized horseradish peroxidase (HRP), and using Co(bpy)₃³⁺ as mediator in the hydrogen peroxide solution. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The modified process was characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The HRP immobilized on colloidal Ag was stable and retained its biological activity. The sensor displays excellent electrocatalytic response to the reduction of H₂O₂. Analytical parameters such as pH and temperature were also studied. Linear calibration for H₂O₂ was obtained in the range of 1×10^–5 to 1×10^–2 M under optimized conditions. The sensor was highly sensitive to H₂O₂, with a detection limit of 2×10^–6 M, and the sensor achieved 95% of steady-state current within 10 s. The sensor exhibited high sensitivity, selectivity and stability.     

Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry

A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO₂NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy)₃]^2+/3+ redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy)₃]^2+/3+ FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10⁻¹⁵ to 1 × 10⁻⁸ mol L⁻¹. The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL⁻¹ with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy)₃]^2+/3+ interaction with ssDNA before and after hybridization.     

Novel poly (neutral red) nanowires as a sensitive electrochemical biosensing platform for hydrogen peroxide determination

Poly (neutral red) nanowires (PNRNWs) have been synthesized for the first time by the method of cyclic voltammetric electrodeposition using porous anodic aluminum oxide (AAO) template and were examined by scanning electron microscopy (SEM) and transmission electron microscope (TEM). Moreover, horseradish peroxidase (HRP) was encapsulated in situ in PNRNWs (denoted as PNRNWs–HRP) by electrochemical copolymerization for potential biosensor applications. The PNRNWs showed excellent efficiency of electron transfer between the HRP and the glassy carbon (GC) electrode for the reduction of H₂O₂ and the PNRNWs–HRP modified GC electrode showed to be excellent amperometric sensors for H₂O₂ at −0.1 V with a linear response range of 1 μM to 8 mM with a correlation coefficient of 0.996. The detection limit (S/N = 3) and the response time were determined to be 1 μM and <5 s and the high sensitivity is up to 318 μA mM⁻¹ cm⁻².