Electroactive Probe Research Articles

Ferrocene-functionalized nanocomposites as signal amplification probes for electrochemical immunoassay of Salmonella typhimurium.

Anelectrochemical immunosensor based on ferrocene (Fc)-functionalized nanocomposites was fabricatedas an efficient electroactive signal probe to amplify electrochemical signals for Salmonella typhimurium detection. The electrochemical signal amplification probe was constructed by encapsulating ferrocene into S. typhimurium-specific antimicrobial peptides Magainin I (MI)-Cu3(PO4)2 organic-inorganic nanocomposites (Fc@MI) through a one-step process. Magnetic beads (MBs) coupled with antibody were used as capture ingredient for target magnetic separation, and Fc@MI nanoparticles were used as signal labels in the immunoassays. The sandwich of MBs-target-Fc@MI assay was performed using a screen-printed carbon electrode as transducer surface. The immunosensor platform presents a low limit of detection (LOD) of 3CFU·mL-1 and a linear range from 10 to 107CFU·mL-1, with good specificity and precision, and was successfully applied for S. typhimurium detection in milk. Graphical abstract One-pot process antimicrobial peptides Magainin I-Cu3(PO4)2 organic-inorganic nanocomposites (Fc@MI) were used as ideal electrochemical signal label, integrating both essential functions of biological recognition and signal amplification. Screen-printed carbon electrode (SPCE) was used as the electrochemical system for Salmonella typhimurium detection.

An electrochemical DNA biosensor fabricated from graphene decorated with graphitic nanospheres

Graphene decorated with graphitic nanospheres functionalized with pyrene butyric acid (PBA) is used for the first time to fabricate a DNA biosensor. The electrode was formed by attaching a DNA probe onto PBA, which had been stacked onto a graphene material decorated with graphene nanospheres (GNSs). The nanomaterial was drop-coated onto a carbon screen-printed electrode (SPE) to create the GNS-PBA modified electrode (GNS-PBA/SPE). A simple method was used to produce GNS by annealing graphene oxide (GO) solution at high temperature. Field emission scanning electron micrographs confirmed the presence of a spherical shape of GNS with a diameter range of 40–80 nm. A stable and uniform PBA-modified GNS (GNS-PBA) was obtained with a facile ultrasonication step. Thus allowing aminated DNA probes of genetically modified (GM) soybean to be attached to the nanomaterials to form the DNA biosensor. The GNS-PBA/SPE exhibited excellent electrical conductivity via cyclic voltammetry (CV) and differential pulse voltammetry (DPV) tests using potassium ferricyanide (K3[Fe(CN)6]) as the electroactive probe. By employing an anthraquinone monosulfonic acid (AQMS) redox intercalator as the DNA hybridization indicator, the biosensor response was evaluated using the DPV electrochemical method. A good linear relationship between AQMS oxidation peak current and target DNA concentrations from 1.0 × 10−16 to 1.0 × 10−8 M with a limit of detection (LOD) of less than 1.0 × 10−16 M was obtained. Selectivity experiments revealed that the voltammetric GM DNA biosensor could discriminate complementary sequences of GM soybean from non-complementary sequences and hence good recoveries were obtained for real GM soybean sample analysis. The main advantage of using GNS is an improvement of the DNA biosensor analytical performance.

Electrochemical immunosensor with Cu(I)/Cu(II)-chitosan-graphene nanocomposite-based signal amplification for the detection of newcastle disease virus

An electrochemical immunoassay for the ultrasensitive detection of Newcastle disease virus (NDV) was developed using graphene and chitosan-conjugated Cu(I)/Cu(II) (Cu(I)/Cu(II)-Chi-Gra) for signal amplification. Graphene (Gra) was used for both the conjugation of an anti-Newcastle disease virus monoclonal antibody (MAb/NDV) and the immobilization of anti-Newcastle disease virus polyclonal antibodies (PAb/NDV). Cu(I)/Cu(II) was selected as an electroactive probe, immobilized on a chitosan-graphene (Chi-Gra) hybrid material, and detected by differential pulse voltammetry (DPV) after a sandwich-type immune response. Because Gra had a large surface area, many antibodies were loaded onto the electrochemical immunosensor to effectively increase the electrical signal. Additionally, the introduction of Gra significantly increased the loading amount of electroactive probes (Cu(I)/Cu(II)), and the electrical signal was further amplified. Cu(I)/Cu(II) and Cu(I)/Cu(II)-Chi-Gra were compared in detail to characterize the signal amplification ability of this platform. The results showed that this immunosensor exhibited excellent analytical performance in the detection of NDV in the concentration range of 100.13 to 105.13 EID50/0.1 mL, and it had a detection limit of 100.68 EID50/0.1 mL, which was calculated based on a signal-to-noise (S/N) ratio of 3. The resulting immunosensor also exhibited high sensitivity, good reproducibility and acceptable stability.

Widening the Scope of “Inherently Chiral” Electrodes: Enantiodiscrimination of Chiral Electroactive Probes with Planar Stereogenicity

The Cover Feature shows the fascinating correlation, through a common fil rouge rationale, between the four stereogenic elements. Successful chiral electroanalysis based on inherently chiral selectors has so far involved only three of them, with planar stereogenicity still missing. Six ferrocene model cases are now discriminated in voltammetry experiments on electrodes modified with inherently chiral films, confirming that the inherent chirality strategy can be effective with all four rigid stereogenic elements. The background image is free from known restrictions under copyright law and has been reproduced in line with public domain considerations (Di Maurice Leloir, 1851–1940, Firma in basso a destra incisione su legno by Jules Huyot Firma in basso a sinistra, http://rylibweb.man.ac.uk/data2/spcoll/dmunro/). More information can be found in the Article by S. Grecchi et al.

Advances on the Use of Graphene as a Label for Electrochemical Biosensors

AbstractThere has been an overwhelming interest in the use of graphene and its derivatives for electrochemical biosensors in the last decade. Although the majority of the works describe the use of these materials as platform for the immobilization of the biorecognition element, there is a significant, often unrecognized, research effort that has shown how graphene materials can also beneficially serve as signaling labels for biosensing. Owing to its intrinsic electroactivity and small size, nano‐graphene oxide, for example, has been used for this purpose, where the working signal arises from the reduction of the oxygen functionalities on the material surface. In other approaches, graphene labels modified with electroactive probes were also used to promote the signal generation. This Minireview will illustrate how the unique electrochemical and structural features make graphene a very promising material for the detection of the biorecognition event. In addition, an overview will be provided, showing the plethora of options offered by graphene and its derivatives as labels for signal generation and enhancement.

Widening the Scope of “Inherently Chiral” Electrodes: Enantiodiscrimination of Chiral Electroactive Probes with Planar Stereogenicity

AbstractA series of planar‐stereogenicity ferrocenes, important as chiral promoters in enantioselective catalysis, is characterized in terms of relationships between structure and electronic properties. The enantiomers of six selected model cases are then successfully discriminated in voltammetry experiments on electrodes modified with electrodeposited inherently chiral oligomer films, in terms of significant potential differences, specular inverting probe or selector configuration. Small substituent changes do not alter the enantiomer peak sequence, but result in significant modulation of peak potential differences, which appear consistent with the availability of chiral/selector matching elements. The present stereogenic plane case, combined with former ones involving stereogenic centers, axes, and/or helices, shows that the inherent chirality strategy in electroanalysis can be effective with all four rigid stereogenic elements.

Effective Enantiodiscrimination in Electroanalysis Based on a New Inherently Chiral 1,1'-binaphthyl Selector Directly Synthesizable in Enantiopure Form.

Enantioselective electroanalysis, which aims to discriminate the enantiomers of electroactive chiral probes in terms of potential difference, is a very attractive goal. To achieve this, its implementation is being studied for various "inherently chiral" selectors, either at the electrode surface or in the medium, yielding outstanding performance. In this context, the new inherently chiral monomer Naph2T4 is introduced, based on a biaromatic atropisomeric core, which is advantageously obtainable in enantiopure form without HPLC separation steps by a synthetic route hinging on enantiopure 2,2’-dibromo-1,1’-binaphthalenes. The antipodes of the new inherently chiral monomer can be easily electrooligomerized, yielding inherently chiral electrode surfaces that perform well in both cyclic voltammetry (CV) enantiodiscrimination tests with pharmaceutically interesting molecules and in magnetoelectrochemistry experiments.

Antibody-Electroactive Probe Conjugates Based Electrochemical Immunosensors.

Suitable immobilization of a biorecognition element, such as an antigen or antibody, on a transducer surface is essential for development of sensitive and analytically reliable immunosensors. In this review, we report on (1) methods of antibody prefunctionalization using electroactive probes, (2) methods for immobilization of such conjugates on the surfaces of electrodes in electrochemical immunosensor construction and (3) the use of antibody-electroactive probe conjugates as bioreceptors and sensor signal generators. We focus on different strategies of antibody functionalization using the redox active probes ferrocene (Fc), anthraquinone (AQ), thionine (Thi), cobalt(III) bipyridine (Co(bpy)33+), Ru(bpy)32+ and horseradish peroxidase (HRP). In addition, new possibilities for antibody functionalization based on bioconjugation techniques are presented. We discuss strategies of specific, quantitative antigen detection based on (i) a sandwich format and (ii) a direct signal generation scheme. Further, the integration of different nanomaterials in the construction of these immunosensors is presented. Lastly, we report the use of a redox probe strategy in multiplexed analyte detection.

The Fabrication of Multiple Stimuli-Responsive Film Electrode and Logic Gates of Rutin

The glassy carbon (GC) electrode surface modified with carboxylic multi-walled carbon nanotubes (c-MWCNTs) and poly(N-isopropylacrylamide) (PNIPAM) films were used to investigate multiple switching electrochemical behaviors of rutin, and a 4-input/3-output logic-gate system was constructed. The drug molecule rutin acted as electroactive probe, and displayed triple stimuli-responsive switchable cyclic voltammetry (CV) properties on c-MWCNTs/PNIPAM film electrodes. From pH 4.0 to pH 9.0 at 25 °C, the peak currents of rutin were decreasing with the pH value increasing due to the property of rutin itself and the electrostatic interaction between polyacrylamide (PAA) in the film and rutin molecule. When external temperature and Na2SO4 concentration increased, the CV response signals of rutin decreased because the hydrogen bonds between amide group of PNIPAM hydrogel polymer and water molecules were broken gradually, and a collapsed globule structure was formed. According to above results, multiple stimulus-responsive hydrogel thin film sensor was constructed. Then a 4-input/3-output logic-gate system including AND, NAND and INHIBIT gates was fabricated successfully. The construction of logic-gate system based on drug molecule rutin may provide a novel approach and model leading to design the new-style drug sensor, which might be applied to the intelligent medical diagnostics and drug release.

Copper electroplating of 3D printed composite electrodes

The manufacture of electrodes by 3D printing approaches has recently been recognized as a fast, inexpensive and environmentally friendly alternative to traditional preparation techniques based on subtractive manufacturing tools. However, as-prepared 3D printed electrodes typically show a considerable intrinsic kinetic barrier for the electron transfer. In this work we employ fused deposition modelling 3D printing to fabricate electrodes from a polylactic acid/copper composite filament and subject them to the surface functionalization by the copper electroplating aiming at eliminating this kinetic barrier. Cyclic voltammetry employing [Ru(NH3)6]3+/2+ couple as the electroactive probe was employed to inspect electron transfer properties of the manufactured electrodes. We demonstrate that electrodes modified by an optimized electroplating procedure show virtually no kinetic barrier and generate faradaic response with the magnitude comparable to that obtained at conventional metallic and carbon-based electrodes. The obtained faradaic peak separation value (70–75 mV) is superior to all values reported for 3D printed electrodes in the literature.

UV/VIS spectroelectrochemistry with 3D printed electrodes

Recent years have witnessed a boom in applying 3D printing technologies to manufacture customized prototypes in various fields of science. In electrochemistry, fused deposition modelling (FDM) 3D printing employing composite filaments based on thermoplastic materials and conductive allotropes of carbon enabled rapid, routine, inexpensive and operationally safe fabrication of conductive electrodes. Nevertheless, results of cyclovoltammetric measurements reported in the literature indicate that 3D printed electrodes give rise to considerable intrinsic kinetic barriers for electron transfer through the electrode/electrolyte interface. In this work we employ FDM-based 3D printing followed by a simple anodic activation procedure to manufacture electrodes from commercially available composites of polylactic acid (PLA) and carbon nanotubes (CNTs). Employing cyclic voltammetry with ruthenium(III) acetylacetonate as the electroactive probe we demonstrate that the previously reported kinetic barrier is almost completely removed upon the activation process. We apply such devised procedure to manufacture electrodes with optical windows allowing UV/VIS absorption spectroscopic detection of electrogenerated products. We are thus the first to perform a UV/VIS absorption spectroelectrochemical experiment employing 3D printed optically transparent working electrodes.

Enzyme-free sandwich-type electrochemical immunosensor for highly sensitive prostate specific antigen based on conjugation of quantum dots and antibody on surface of modified glassy carbon electrode with core–shell magnetic metal-organic frameworks

The early diagnosis of cancer increases the chance of its treatment. In this study, an enzyme-free sandwich type of immunosensors is presented for prostate specific antigen (PSA), as prostate cancer biomarker. In this regard, the quantum dots (QDs) and magnetic metal-organic frameworks (MOFs) were used for labeling the secondary anybody and modifying the electrode surface, respectively. Herein, a bare glassy carbon electrodes is modified with iron magnetic nanoparticles with a core shell MOFs (magnetic framework Fe3O4@ TMU-10) and the primary antibodies (Ab1) are immobilized on the modified electrode. Moreover, the second antibody is successfully conjugated to a nickel-cadmium quantum dots as an electroactive non-enzymatic probe. The prepared immunosensor shows a linear range between 1 pg mL−1- 100 ng mL−1 and the detection limit of 0.45 pg mL−1 with acceptable selectivity, reproducibility and stability. The proposed sensor is also applied for determination of PSA in human serum samples and the results is confirmed by a reference method, which indicates its ability for clinical monitoring of the tumor marker.

3D printed polylactic acid/carbon black electrodes with nearly ideal electrochemical behaviour

In recent years electrochemistry has joined the revolution in 3D printing. Not only electrochemical cells but also conductive electrodes may be now conveniently and inexpensively manufactured directly in electrochemical laboratories. However, 3D printed electrodes typically suffer from considerable intrinsic kinetic barrier that manifests itself as increased separation of faradaic peaks in cyclic voltammograms. In this work we employ fused deposition modelling 3D printing to manufacture electrodes from a conductive polylactic acid (PLA)/carbon black composite filament. We further activate electrodes by a simple electrochemical anodic procedure. Cyclic voltammograms employing Ru(acac)3 as the electroactive probe show the faradaic peak separation values between 80 and 85 mV which is superior to any value reported for a 3D printed PLA-based electrode so far. Furthermore, comparison of experimentally obtained faradaic peak current values with those calculated theoretically shows that the relative effective surface area of electrodes approaches unity. Our work clearly demonstrates that 3D printed electrodes may reach characteristics well comparable to those obtained at conventional metallic or carbon electrodes.

Electrochemical biosensor based on pyruvate oxidase immobilized AuNRs@Cu2O-NDs as electroactive probes loaded poly (diallyldimethylammonium chloride) functionalized graphene for the detection of phosphate

A novel and sensitive electrochemical biosensor for the detection of phosphate was constructed using AuNRs@Cu2O-NDs loaded poly (diallyldimethylammonium chloride) functionalized graphene immobilized pyruvate oxidase (PyO). In this study, PDDA-Gr with good conductive ability was firstly used to increase the active site and serve as supporting substrate of biosensor. Subsequently, the AuNRs@Cu2O-NDs composite material was prepared to enhanced capability for PyO immobilization and achieved rapid electron transfer between hydrogen peroxide and Cu2O-NDs to amplify the electrical signal. Additionally, AuNRs@Cu2O-NDs loaded PDDA-Gr had good biocompatibility and yielded superior current response. The surface concentration (ΓT) of electroactive PyO on the surface of AuNRs@Cu2O-NDs/PDDA-Gr/AuE is estimated as 7.65 × 10−10 mol cm-2. Under the optimal conditions, the proposed biosensor showed a wide linearity range from 0.01 to 80 μM and a low detection limit of 0.4 nM by differential pulse voltammetry. Through a series of assessments, the biosensor exhibited satisfactory anti-interference, good stability, excellent reproducibility and successfully applied to the monitoring of phosphate content in food.

Influence of intermolecular interactions in the redox kinetics performance of surface confined probes by Square Wave Voltammetry

Analysis of the Square Wave Voltammetry (SWV) responses of surface confined electroactive probes under finite kinetics conditions, by considering the influence of intermolecular interactions on the current, is presented. This non-ideality gives rise to very significant deviations in the most relevant features of the current potential response as compared with predictions of ideal models. The theoretical model does not require that the dependence of the rate constant with the potential (i. e., Butler-Volmer or Marcus-Hush kinetic formalisms) is explicitly established a priori. Both location and height of the Quasi-Reversible Maximum and symmetry of the SWV current-potential curves are strongly affected by the interaction parameters G=aOO+aRR−2aOR and S=aRR−aOO (with aij being the interaction coefficient for species i and j). Quantitative methods for obtaining kinetic and interaction parameters of the redox probes are presented, based on the measurement of the peak currents and potential under quasi-reversible conditions where the current exhibits two asymmetric peaks. A discussion is done concerning the deviations found in the estimation of the rate constants with SWV when intermolecular interactions are not considered. The above methods have been applied to the characterization of mixed ferrocenylundecanethiol/decanethiol monolayers on gold electrodes at an ethanolic medium for which the SWV is clearly non-ideal.

Ultrasensitive peptide-based electrochemical detection of protein kinase activity amplified by RAFT polymerization

Since the oversecretion of protein kinases is indicative of multiple human diseases, the screening of their activities is quite important to clinical diagnosis and targeted therapy. In this work, an ultrasensitive peptide-based electrochemical biosensor was presented for the detection of protein kinase activity by using the reversible addition-fragmentation chain transfer (RAFT) polymerization technique as a signal amplification strategy. First, the substrate peptides were tethered to a gold electrode surface via the thiol terminals. After the phosphorylation of substrate peptides by protein kinases, the carboxyl group-containing dithiobenzoates were labeled to the phosphorylated sites via the robust phosphate-Zr4+-carboxylate linkages. Finally, the RAFT polymerization was initiated using ferrocenylmethyl methacrylates (FcMMAs) and dithiobenzoates as the monomers and the RAFT agents, respectively. The grafting of ferrocenyl polymer chains efficiently recruits a great number of electro-active Fc probes to each phosphorylated site, leading to a drastic amplification of the electrochemical signal. With PKA (protein kinase A) as the target, the detection limit of the peptide-based biosensor can be as low as 1.05 mU mL−1. Moreover, it can selectively differentiate the target from other interferents and is applicable for the screening of potential inhibitors as well as the detection of protein kinase activity in complex cell lysates. Therefore, the peptide-based biosensor shows great promise as a universal tool for protein kinase activity detection and inhibitor screening.

A distinguished cancer-screening package containing a DNA sensor and an aptasensor for early and certain detection of acute lymphoblastic leukemia

A disposable package of biosensors was developed along with the corresponding guidelines for early detection of the acute lymphoblastic leukemia cancer. This proposed cancer-screening package included a DNA sensor and an aptasensor as two main types of biosensors. The biosensors were used simultaneously. This combination of sensors can detect not only the presence of mutant genes but also the biomarkers of cancer. At current work, the combination of sensors were used to detect the presence of BCR-ABL1 as a mutant gene and CEA as a biomarkers of cancer, such a capability makes the package liable for early and certain detection of acute lymphoblastic leukemia.To construct both the DNA sensor and the aptasensor, a nanocomposite consisting of electrosynthesis carbon quantum dots and biosynthesized gold nanoparticles was applied. The construction of these biosensors was characterized using four different electrochemical methods including DPV (Differential Pulse Voltammetry), EIS (Electrochemical Impedance Spectroscopy), CV (Cyclic Voltammetry) and chronoamperometry.The peak current of a catechol solution that was used as an electroactive probe on the biosensor was linearly related to the logarithm of the concentrations of the target DNA and the target antigen in the range of 10 pM to 100 μM and 1 pg mL−1 to 0.001 g mL−1 with the detection limits of 1.5 pM and 0.26 pg mL−1 respectively, which are quite good results.

Au-polythionine nanocomposites: a novel mediator for bisphenol A dual-signal assay based on imprinted electrochemical sensor

In this work, a novel electrochemical sensor was developed by the modification of poly(p-aminobenzene sulfonic acid) (pABSA), Au-polythionine (Au-pTH) nanowires, and molecularly imprinted polymer (MIP) on glassy carbon electrode surface for bisphenol A (BPA) detection. The results of characterizations including scanning electron microscope, transmission electron microscopy, Fourier transform infrared spectra, and X-ray diffraction showed the successful synthesis of helical structural Au-pTH nanowires, which acted as an electro-active probe for BPA detection. Cyclic voltammetry results illustrated that the modified pABSA, Au-pTH, and MIP endowed the sensor with good electrocatalytic activity, the second current signal, and recognition ability, respectively. Since the imprinted cavities provided electron transfer channels for thionine (TH) redox, the peak current of TH can be found in a blank electrolyte. The added BPA molecules can be rebound in imprinted cavities, which are oxidized and then display its current. The rebound BPA molecules in turn blocked the electron transfer channels for TH redox, resulting in the decrease of TH current. A double signal defined, as the sum of the changes of TH current (∆iTH|) and BPA current (|∆iBPA|) (|∆iTH|+|∆iBPA|), was employed as the detected signal for BPA sensitive detection, which was linearly proportional to the logarithm of concentration of BPA ranging from 8.0 × 10-8 to 1.0 × 10-4mol/L with a limit of detection of 3.8 × 10-8mol/L (S/N = 3) in a weakly acidic solution. Moreover, the natural recognition ability of MIP enabled the sensor to selectively detect BPA from its analogues. The proposed dual-signal strategy-based sensor provided a feasible tool for rapid, sensitive, and selective determination of BPA. Graphical abstract.

Label-free poly(ADP-ribose) polymerase-1 activity assay based on perpendicular orientated mesoporous silica films

Since the expression of poly(ADP-ribose) polymerase-1 (PARP-1) was significantly up-regulated in several primary malignancies, it was important to construct a sensitive and rapid PARP-1 assay for early diagnosis of cancer. When PARP-1 was activated by active dsDNA, it generates a number of negatively charged poly(ADP-ribose) (PAR) polymers, which can be readily detected. Here, we developed a label-free electrochemical (EC) biosensor for PARP-1 activity based on the electrostatic attraction between negatively charged PAR and positively charged amino-functioned mesoporous silica films (NH2-MSFs). Negatively charged PAR was absorbed on the NH2-MSFs surface and suppressed the electrochemical signal as the PAR prevented the electroactive probe reaching to the ITO electrode. The electrochemical signal for PAR linearly decreased from 0.01 U to 1.2 U with a detection limit of 0.005 U, which is comparable to the PARP-1 biosensors reported before. The strategy has been used to evaluate PARP-1 inhibitors and to detect PARP-1 activity in human serum and real cancer cells lysate with satisfactory results, indicating that it was a promising candidate for point-of-care diagnostics.

Probe and analogue: Double roles of thionine for aloe-emodin selective and sensitive ratiometric detection

Molecular imprinted polymer (MIP) has been employed for electrochemical sensor preparation with good selectivity. However, to restrict the non-specific adsorption, the conductivity of MIP is usually unsatisfying, which limits its sensitivity for template detection. To achieve both of selectivity and sensitivity, a MIP electrochemical sensor was designed and fabricated by the electropolymerization of pyrrole in the presence of aloe-emodin (AE, template molecule) on carbon nanoparticles modified electrode in this work. Thionine (TH), being added in solution in advance, played double roles for AE recognition and detection. As an analogue of AE, TH was adsorbed into imprinted cavities and then was replaced by AE, indicating the good selectivity of the sensor. This replacement of TH by AE was also demonstrated by the result of computational simulation. As an electro-active probe, the peak current of TH decreased and peak current of AE increased based on the replacement of TH by AE. A current ratiometric signal defined as |ΔiAE|/|ΔiTH| was used for AE sensitive detection. Thus, the replacements of TH by AE demonstrated that the sensor possessed high sensitivity and good selectivity. A linear range of 1.0 × 10−7 -1.0 × 10-4 mol/L for AE detection was obtained with a limit of detection of 7.5 × 10-8 mol/L. And the sensor has been applied to analyze AE in real samples with satisfactory results. The simple, smart, and effective strategy presented in this work can be further used to prepare electrochemical sensors for other compounds selective recognition and sensitive detection.