A highly selective and sensitive cocaine aptasensor based on covalent attachment of the aptamer-functionalized AuNPs onto nanocomposite as the support platform

Based on the conformational changes of the aptamer-functionalized gold nanoparticles (AuNPs) onto MWCNTs/IL/Chit nanocomposite as the support platform, we have developed a sensitive and selective electrochemical aptasensor for the detection of cocaine. The 5′-amine-3′-AuNP terminated aptamer is covalently attached to a MWCNTs/IL/Chit nanocomposite. The interaction of cocaine with the aptamer functionalized AuNP caused the aptamer to be folded and the AuNPs with negative charge at the end of the aptamer came to the near of electrode surface therefore, the electron transfer between ferricyanide (K₃Fe(CN)₆) as redox probe and electrode surface was inhibited. A decreased current of (K₃Fe(CN)₆) was monitored by differential pulse voltammetry technique. In an optimized condition the calibration curve for cocaine concentration was linear up to 11 μM with detection limit (signal-to-noise ratio of 3) of 100 pM. To test the selectivity of the prepared aptasensor sensing platform applicability, some analgesic drugs as the interferes were examined. The potential of the aptasensor was successfully applied for measuring cocaine concentration in human blood serum. Based on our experiments it can be said that the present method is absolutely beneficial in developing other electrochemical aptasensor.     

An Electrochemical Aptamer Biosensor for Bisphenol A on a Carbon Nanofibre-silver Nanoparticle Immobilisation Platform

A nanocomposite platform of silver nanoparticles and carbon nanofibres (AgCNFs) was used to immobilise a bisphenol A specific 63-mer ssDNA aptamer to form a biosensor. The fabrication process of the biosensor was studied with electrochemical impedance spectroscopy and cyclic voltammetry in the presence of [Fe(CN)₆]^3−/4− as redox probe. The biosensor detected bisphenol A in a linear range of 0.1–10 nM, with a limit of detection of 0.39 nM using square wave voltammetry (SWV). The biosensor exhibited good selectivity in the presence of interfering species at 100-fold concentrations and was used to detect BPA in real water sample.     

Electrochemical impedance spectroscopy detection of lysozyme based on electrodeposited gold nanoparticles

A simple, highly sensitive, and label-free electrochemical impedance spectroscopy (EIS) aptasensor based on an anti-lysozyme-aptamer as a molecular recognition element, was developed for the detection of lysozyme. Improvement in sensitivity was achieved by utilizing gold nanoparticles (AuNPs), which were electrodeposited onto the surface of a gold electrode, as a platform for immobilization of the aptamer. To quantify the amount of lysozyme, changes in the interfacial electron transfer resistance (R_et) of the aptasensor were monitored using the redox couple of an [Fe(CN)₆]^3−/4− probe. The R_et increased with lysozyme concentration. The plot of R_et against the logarithm of lysozyme concentration is linear over the range from 0.1 pM to 500 pM with a detection limit of 0.01 pM. The aptasensor also showed good selectivity for lysozyme without being affected by the presence of other proteins.     

Synthesis and Characterization of MWNTs/Au NPs/HS(CH2)6Fc Nanocomposite: Application to Electrochemical Determination of Ascorbic Acid

In this article, a detailed electrochemical study of a novel 6‐ferrocenylhexanethiol (HS(CH₂)₆Fc) self‐assembled multiwalled carbon nanotubes‐Au nanoparticles (MWNTs/Au NPs) composite film was demonstrated. MWNTs/Au NPs were prepared by one‐step in situ synthesis using linear polyethyleneimine (PEI) as bifunctionalizing agent. HS(CH₂)₆Fc, which acted as the redox mediator, was self‐assembled to MWNTs/Au NPs via Au‐S bond. Transmission electron microscopy (TEM), energy‐dispersive X‐ray analysis (EDX), Fourier transformed infrared absorption spectroscopy (FT‐IR), UV‐visible absorption spectroscopy, and cyclic voltammetry were used to characterize the properties of the MWNTs/Au NPs/HS(CH₂)₆Fc nanocomposite. The preparation of the nanocomposite was very simple and effectively prevented the leakage of the HS(CH₂)₆Fc mediator during measurements. The electrooxidation of AA could be catalyzed by Fc/Fc⁺ couple as a mediator and had a higher electrochemical response due to the unique performance of MWNTs/Au NPs. The nanocomposite modified electrode exhibited excellent catalytic efficiency, high sensitivity, good stability, fast response (within 3 s) and low detection limit toward the oxidation of AA at a lower potential.     

Label‐free Electrochemical Aptasensor for Carcino‐embryonic Antigen Based on Ternary Nanocomposite of Gold Nanoparticles,Hemin and Graphene

In this report, a label‐free electrochemical aptasensor for carcino‐embryonic antigen (CEA) was successfully developed based on a ternary nanocomposite of gold nanoparticles, hemin and graphene nanosheets (AuNPs‐HGNs). This nanocomposite was prepared by decorating gold nanoparticles on the surface of hemin functionalized graphene nanosheets via a simple wet‐chemical strategy. The aptamer can be assembled on the surface of AuNPs‐HGNs/GCE (glassy carbon electrode) through Au‐S covalent bond to form the sensing interface. Hemin absorbed on the graphene nanosheets not only acts as a protective agent of graphene sheets, but also as an in situ probe base on its excellent redox properties. Gold nanoparticles provide with both numerous binding sites for loading CEA binding aptamer (CBA) and good conductivity to promote the electron transfer. The current changes, which are caused by CEA specifically binding on the modified electrode, are exploited for the label‐free detection of CEA in a very rapid and convenient protocol. Therefore, the method has advantages of high sensitivity, wide linear range (0.0001–10 ng mL^?1), low detection limit (40 fg mL^?1) and attractive specificity. The results illustrate that the proposed label‐free electrochemical aptasensor has a potential application in the biological or clinical target analysis for its simple operation and low cost.     

A Dendrimer Supported Electrochemical Immunosensor for the Detection of Alpha‐feto protein – a Cancer Biomarker

The electrochemical detection of alpha‐feto protein based on novel gold nanoparticles‐ poly(propylene imine) dendrimer platform is reported. The platform was prepared by co‐electrodeposition of gold nanoparticles and generation 3 poly (propylene imine) dendrimer on a glassy carbon electrode. Each modifying step was characterised by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical measurements showed that the platform was stable, conducting and exhibited reversible electrochemistry. Results obtained from the electrochemical impedance spectroscopy interrogation in [Fe(CN)₆^3−/4−] redox probe showed a marked reduction in charge transfer resistance (R_ct) after each modification step. The immunosensor was prepared by immobilisation of a probe anti‐alpha feto protein (AFP) on the platform for 3 hrs at 35 °C followed by blocking the surface with bovine serum albumin to minimise non‐specific binding. The prepared immunosensor was used to detect AFP over a wide concentration range from 0.005 to 500 ng/mL and detection limits of 0.0022 and 0.00185 ng/mL were obtained for SWV and EIS measurements respectively. The immunosensor gave good stability over a period of fourteen days when stored at 4 °C.     

Synthesis and Characterization of Co3O4/Multiwalled Carbon Nanotubes Nanocomposite for Amperometric Sensing of Hydrazine

Co₃O₄ nanoparticles (NPs) were synthesized and decorated on the multi‐walled carbon nanotubes (MWCNTs) through a simple hydrothermal procedure. The deposited Co₃O₄ NPs on the sidewalls of MWCNTs were found to be cubic crystal structure and homogenously dispersed with a narrow particle size distribution centered at around 6 nm. The Co₃O₄/MWCNTs nanocomposite was then utilized for the electrochemical detection of hydrazine, and exhibited a high sensitivity of 34.5 µA mM^?1, a low detection limit of 0.8 µM (S/N=3), a wide linear range of 20 µM to 1.1 mM along with a short response time of less than 5 s.     

MWCNTs/Ionic Liquid/Graphene Quantum Dots Nanocomposite Coated with Nickel‐Cobalt Bimetallic Catalyst as a Highly Selective Non‐enzymatic Sensor for Determination of Glucose

In this work, a glassy carbon electrode (GCE) was modified with multiwall carbon nanotubes/ionic liquid/graphene quantum dots (MWCNTs/IL/GQDs) nanocomposite. Then, the nanocomposite was decorated with nickel‐cobalt nanoparticles (Ni?Co NPs), and it was used as a non‐enzymatic glucose sensor. Field emission scanning electron microscopy, X‐ray diffraction spectroscopy, and energy dispersive spectroscopy were employed to prove the electrodeposition of the Ni?Co NPs on the surface of MWCNTs/IL/GQDs/GCE. Also, cyclic voltammetric and amperometric methods were utilized for the investigation of the electrochemical behaviour of the Ni?Co NPs/MWCNTs/IL/GQDs/GCE for glucose oxidation. The novel amperometric sensor displayed two linear ranges from 1.0 to 190.0 μmol L^?1 and 190.0 to 4910 μmol L^?1 with a low detection limit of 0.3 μmol L^?1 as well as fast response time (2 s) and high stability. Also, the sensor showed good selectivity for glucose determination in the presence of ascorbic acid, citric acid, dopamine, uric acid, fructose, and sucrose, as potential interference species. Finally, the performance of the proposed sensor was investigated for the glucose determination in real samples. Ni?Co NPs/MWCNTs/IL/GQDs/GCE showed good sensitivity and excellent selectivity.     

A Novel Amperometric Glucose Biosensor Based on Fe3O4-Chitosan-β-Cyclodextrin/MWCNTs Nanobiocomposite

A novel enzymatic biosensing platform toward glucose is achieved with nanocomposite of magnetic nanoparticles (Fe₃O₄−CS−CD) and multi-walled carbon nanotubes (MWCNTs). The synergistic effect of chitosan, β-cyclodextrin and MWCNTs can facilitate electron transfer between enzyme and electrode based on the promoting results of the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The new biosensors exhibited direct electron transfer (DET) from enzyme to electrode after glucose oxidase (GOx) was immobilized on the modified electrode with the nanocomposite. Consequently, the enzymatic glucose biosensor displayed a considerably wide linear range (40 μM to 1.04 mM) with a high sensitivity of 23.59 μA mM⁻¹cm⁻², low detection limit of 19.30 μM, good selectivity, reproducibility and repeatability for detecting glucose. In addition, the current response still retained at 93.4 % after 25 days. Furthermore, the practical application of glucose biosensor was test in human serum samples with satisfactory accuracy, demonstrating promising and practical potential in biomedical diagnostics.     

Electrochemical Detection of β-Lactoglobulin Allergen Using Titanium Dioxide/Carbon Nanochips/Gold Nanocomposite-based Biosensor

A label-free electrochemical immunosensor was developed for the ultra-sensitive detection of β-lactoglobulin (β-LG). The novel nanocomplex of carbon nanochips, colloidal gold nanoparticles and titanium dioxide nanoparticles TiO₂/CNC/AuC were constructed on conducting polymer, chitosan, and were characterised using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). This nanocomplex interface was studied using cyclic voltammetry (CV) and showed great improvement at the gold electrode surface with enhanced electrochemical performance, sensitivity and selectivity for β-lactoglobulin. Under optimal parameters, the square wave voltammetry (SWV) response curve was determined from 0.01 pg/mL to 500 pg/mL using [Fe(CN)₆]^3−/4−] redox probe. The calibration plot illustrates a linear relationship between log β-LG concentration and SWV current, with the limit of detection determined to be 0.01 pg/mL. This immunosensor displayed high sensitivity, selectivity, reproducibility and stability, and can be utilised for the detection of β-LG in real food samples.     

A Novel Hydrogen Peroxide Biosensor Based on the Synergistic Effect of Gold‐Platinum Alloy Nanoparticles/Polyaniline Nanotube/Chitosan Nanocomposite Membrane

Based on the immobilization of horseradish peroxidase (HRP) in chitosan(CS) on a glassy carbon electrode (GCE) modified with the Au‐Pt alloy nanoparticles (NPs) / polyaniline nanotube (nanoPAN) nanocomposite film, a novel hydrogen peroxide biosensor was constructed. The modified processes of GCE were monitored by cyclic voltammetry and electrochemical impedance spectroscopy. Au‐PtNPs/nanoPAN/CS had a better synergistic electrochemical effect than did AuNPs/nanoPAN/CS or PtNPs/nanoPAN/CS. The amperometric response of the biosensor towards H₂O₂ was investigated by successively adding aliquots of H₂O₂ to a continuous stirring phosphate buffer solution under the optimized conditions. Because Au‐PtNPs have unique catalytic properties and good biocompatibility, and especially Au‐PtNPs and nanoPAN have synergistic augmentation for facilitating electron‐transfer, the biosensor displayed a fast response time (<2 s) and broad linear response to H₂O₂ in the range from 1.0 to 2200 μmol L^?1 with a relatively low detection limit of 0.5 μmol L^?1 at 3 times the background noise. Moreover, the biosensor can be applied in practical analysis and exhibited high sensitivity, good reproducibility, and long‐term stability.     

FeS2−AuNPs Nanocomposite as Mimicking Enzyme for Constructing Signal‐off Sandwich‐type Electrochemical Immunosensor Based on Electroactive Nickel Hexacyanoferrate as Matrix

In this work, a novel sandwich‐type electrochemical immunosensor with electroactive nickel hexacyanoferrate nanoparticles (NiHCFNPs) as matrix was constructed for α‐fetoprotein (AFP) detection in a signal‐off manner by using FeS₂?AuNPs nanocomposite catalyzed insoluble precipitation to significantly inhibit the electrochemical signal. Initially, the NiHCFNPs with excellent electrochemical property was modified on the electrodeposited nano‐Au electrode to obtain a strong initial electrochemical signal. Subsequently, another nano‐Au layer was formed for immobilization of capture antibody (Ab₁). In the presence of target AFP, the prepared FeS₂?AuNPs‐Ab₂ bioconjugate could be specifically recognized and immobilized on electrode through the sandwich‐type immunoreaction. The FeS₂ with large specific surface areas were used as scaffolds to load abundant mimicking enzyme AuNPs. With the help of hydrogen peroxide (H₂O₂), FeS₂?AuNPs with peroxidase‐like activity accelerated the 4‐chloro‐1‐naphthol (4‐CN) oxidation with generation of insoluble precipitation on electrode, which would greatly hinder the electron transfer and thus caused the decrease of electrochemical signal for quantitative determination of AFP. This approach achieved a wide dynamic linear range from 0.0001 to 100 ng mL^?1 with an ultralow limit detection of 0.028 pg mL^?1. Especially, the proposed AFP immunosensor can be applied to detect human serum samples with satisfactory results, indicating a potential application in clinical monitoring of tumor biomarkers.     

An Electrochemical Aptasensor for Detection of Thrombin based on Target Protein‐induced Strand Displacement

A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd²⁺ ions which offered sensitive electrochemical signal transduction. The peak current of Cd²⁺ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10^?9–2.3×10^?12 mol/L and the detection limit was 4.3×10^?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.     

Fabrication of Fe(CN)63− Functionalized PDDA/CdTe Layer‐by‐layer Film with Good Electron Transfer Ability

(PDDA/CdTe)_n layer‐by‐layer (LBL) film immobilized with Fe(CN)₆^3? was fabricated on the gold electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to investigate the electrochemical properties of this film. The peak current of the immobilized Fe(CN)₆^3? increased as the number of the bilayers increased and was proportional to the scan rate. Compared with pure (PDDA/CdTe)_n and (PDDA/PSS)_n LBL film, Fe(CN)₆^3? immobilized (PDDA/CdTe)_n LBL film had good electron transfer ability. The immobility of Fe(CN)₆^3? into the film was attributed to its interaction with Cd²⁺ on the surface of CdTe QDs. Fe(CN)₆^3? also can interact with other metal ions, which would make Fe(CN)₆^3? release from the film. The concentrations of metal ions will affect the CV response of Fe(CN)₆^3? immobilized LBL film. It has provided a novel prototype of device or sensor for quantitative detection of metal ions.     

General Preparation of Heme Protein Functional Fe3O4@Au‐Nps Magnetic Nanocomposite for Sensitive Detection of Hydrogen Peroxide

Stable magnetic nanocomposite of gold nanoparticles (Au‐NPs) decorating Fe₃O₄ core was successfully synthesized by the linker of Boc‐L‐cysteine. Transmission electron microscope (TEM), energy dispersive X‐ray spectroscopy (EDX) and cyclic voltammograms (CV) were performed to characterize the as‐prepared Fe₃O₄@Au‐Nps. The results indicated that Au‐Nps dispersed homogeneously around Fe₃O₄ with the ratio of Au to Fe₃O₄ nanoparticles as 5–10/1 and the apparent electrochemical area as 0.121 cm². After self‐assembly of hemoglobin (Hb) on Fe₃O₄@Au‐Nps by electrostatic interaction, a hydrogen peroxide biosensor was developed. The Fe₃O₄@Au‐Nps/Hb modified GCE exhibited fast direct electron transfer between heme center and electrode surface with the heterogeneous electron transfer rate constant (Ks ) of 3.35 s⁻¹. Importantly, it showed excellent electrocatalytic activity towards hydrogen peroxide reduction with low detection limit of 0.133 μM (S /D =3) and high sensitivity of 0.163 μA μM⁻¹, respectively. At the concentration evaluated, the interfering species of glucose, dopamine, uric acid and ascorbic acid did not affect the determination of hydrogen peroxide. These results demonstrated that the introduction of Au‐Nps on Fe₃O₄ not only stabilized the immobilized enzyme but also provided large surface area, fast electron transfer and excellent biocompatibility. This facile nanoassembly protocol can be extended to immobilize various enzymes, proteins and biomolecules to develop robust biosensors.     

A Photoelectrochemical Biosensor Fabricated using Hierarchically Structured Gold Nanoparticle and MoS2 on Tannic Acid Templated Mesoporous TiO2

In a tannic acid assisted synthesis of mesoporous TiO₂, tannic acid was used as a cost effective and non‐toxic template for pore formation. Meanwhile, a gold nanoparticles (Au NPs) deposited TiO₂ nanocomposite was coated on an indium tin oxide electrode for the fabrication of a photoelectrochemical (PEC) biosensing system. Upon the formation of anatase structure, the electrode was coated with MoS₂ for effective visible light absorption. The mesoporous structure led to an enhanced surface area by improving Au NPs and glucose oxidase adsorption. Incorporation of Au NPs led to an enhanced photonic efficiency due to the generation of Schottky barriers. The obtained nanocomposite was used for the light‐driven, real‐time, and selective PEC glucose sensing. Under visible light irradiation, the enzyme immobilized electrodes yielded significant photocurrent improvement owing to the releasing electron donor H₂O₂. The obtained PEC biosensor demonstrated acceptable reproducibility and stability with a sensitivity of 4.42 μA mM^?1 cm^?2 and a low detection limit of 1.2 μM glucose. Also, the linear measurement range was found to be 0.004–1.75 mM glucose. The results indicated that the proposed production method of mesoporous TiO₂ will pave the way for a green chemistry based porous material production, along with the extension of the implementation of semiconductors in PEC biosensing systems.     

One Step Fabrication of Au Nanoparticles‐Ni‐Al Layered Double Hydroxide Composite Film for the Determination of L‐Cysteine

This paper reports the fabrication of Au nanoparticles (Au NPs)‐Ni‐Al layerd double hydroxide (LDH) composite film by one step electrochemical deposition on the surface of a glass carbon electrode from the mixture solution containing HAuCl₄ and nitrate salts of Ni²⁺ and Al³⁺. Improved conductivity was obtained by Au NPs codeposited on LDH film. The synergic effect of LDHs and Au NPs dramatically improves the performance of L ‐cysteine electro‐oxidation, displaying low oxidation peak potential (0.16 V) and high current response. Thus the electrode was used to sense L ‐cysteine, showing good sensitivity and selectivity.     

A Sensitive Signal-off Electrochemical Aptasensor for Thrombin Detection using PB−Au@MoS2 Nanomaterial as Both Platform and Signal Reporter

Human serum is one of the effective samples for point-of-care testing (POCT). Sensitive and quick determination of thrombin content in human serum samples is important. An electrochemical aptasensor based on Prussian blue and Au nanoparticles loaded MoS₂ nanoflowers (PB−Au@MoS₂) hybrid was constructed. By using PB−Au@MoS₂ as both a substrate and a signal reporter, this aptasensor could demonstrate excellent performance for thrombin detection with a detection linear range from 0.01 pM to 30 nM and detection limit down to 1 fM. This work may provide a strategy to establish effective and sensitive sensing devices for thrombin in clinical diagnosis.     

Self‐assembly of p‐Aminothiophenol on Gold Surface: Application for Impedimetric and Potentiometric Sensing of Cobalt (II) Ions – A Comparative Study

Cobalt, despite an essential biological element, imposes threat to humans when exposed to high concentration or even to low concentration for long term which demands the development of highly sensitive and selective analytical methods for its trace analysis. In the present work, self‐assembly of p‐aminothiophenol (p‐ATP) on gold surface (Au?ATP SAM) was carried out and for the first time, applied as a platform for impedimetric and potentiometric sensing of Co²⁺. Au?ATP SAM was characterized using electrochemical techniques: cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), in the presence of two redox probes: [Fe(CN)₆]^3?/4? and [Ru(NH₃)₆]^2+/3+ to evaluate associated passivating behaviour. Au?ATP SAM completely blocked [Fe(CN)₆]^3?/4? as compared to [Ru(NH₃)₆]^2+/3+ which may be attributed to inner‐sphere and outer‐sphere ET mechanisms, respectively. Au?ATP SAM was found to exhibit excellent sensitivity towards Co²⁺ in a wider concentration range from 1.0×10^?12 M to 1.0×10^?5 M (r²=0.963) at pH 5.5 with a detection limit of 6.0×10^?13 M and superior selectivity. Further, carbon paste electrode (CPE) was prepared by incorporating p‐ATP bound gold nanoparticles and explored for potentiometric sensing of Co²⁺ which exhibited Nernstian slope of 29.2±0.2 mV/dec in linear concentration range of 1.0×10^?6 M–1.0×10^?1 M (r²=0.971) with a detection limit of 8.0×10^?7 M. The proposed sensors were successfully applied for estimation of Co²⁺ content in water samples.     

Lithium Ferrocyanide Catholyte for High-Energy and Low-cost Aqueous Redox Flow Batteries**

Aqueous redox flow batteries (ARFBs) are a promising technology for grid-scale energy storage, however, their commercial success relies on redox-active materials (RAM) with high electron storage capacity and cost competitiveness. Herein, a redox-active material lithium ferrocyanide (Li₄[Fe(CN)₆]) is designed. Li⁺ ions not only greatly boost the solubility of [Fe(CN)₆]⁴⁻ to 2.32 M at room temperature due to weak intermolecular interactions, but also improves the electrochemical performance of [Fe(CN)₆]^4−/3−. By coupling with Zn, ZIRFBs were built, and the capacity of the batteries was as high as 61.64 Ah L⁻¹ (pH-neutral) and 56.28 Ah L⁻¹ (alkaline) at a [Fe(CN)₆]⁴⁻ concentration of 2.30 M and 2.10 M. These represent unprecedentedly high [Fe(CN)₆]⁴⁻ concentrations and battery energy densities reported to date. Moreover, benefiting from the low cost of Li₄[Fe(CN)₆], the overall chemical cost of alkaline ZIRFB is as low as $11 per kWh, which is one-twentieth that of the state-of-the-art VFB ($211.54 per kWh). This work breaks through the limitations of traditional electrolyte composition optimization and will strongly promote the development of economical [Fe(CN)₆]^4−/3−-based RFBs in the future.