Carbon Nanotubes‐Based Amperometric Cholesterol Biosensor Fabricated Through Layer‐by‐Layer Technique

A carbon nanotubes‐based amperometric cholesterol biosensor has been fabricated through layer‐by‐layer (LBL) deposition of a cationic polyelectrolyte (PDDA, poly(diallyldimethylammonium chloride)) and cholesterol oxidase (ChOx) on multi‐walled carbon nanotubes (MWNTs)‐modified gold electrode, followed by electrochemical generation of a nonconducting poly(o‐phenylenediamine) (PPD) film as the protective coating. Electrochemical impedance measurements have shown that PDDA/ChOx multilayer film could be formed uniformly on MWNTs‐modified gold electrode. Due to the strong electrocatalytic properties of MWNTs toward H₂O₂ and the low permeability of PPD film for electroacitve species, such as ascorbic acid, uric acid and acetaminophen, the biosensor has shown high sensitivity and good anti‐interferent ability in the detection of cholesterol. The effect of the pH value of the detection solution on the response of the biosensor was also investigated. A linear range up to 6.0 mM has been observed for the biosensor with a detection limit of 0.2 mM. The apparent Michaelis‐Menten constant and the maximum response current density were calculated to be 7.17 mM and 7.32 μA cm^?2, respectively.     

高灵敏度双介体基胆固醇生物传感器的研制

研制了一种高灵敏度、高准确性、微小取样（－滴血,Ｖ≥２５μＬ）并可快速测量（≤６０ｓ）的实用型夹心式胆固醇生物传感器。它是由双介体、三酶生化氧还体系构成的电流型传感器。其中,第一介体含于敏感电极石墨电导层内,第二介体及三种酶、相试剂含于试剂纤维层中。本传感器可用来直接测量从正常人到异常人的血胆固醇含量（２～１０ｍｍｏｌ／Ｌ）,并将实现商品化。     

Binderless Solution Processed Zn Doped Co3O4 Film on FTO for Rapid and Selective Non‐enzymatic Glucose Detection

A simple solution based deposition process has been used to fabricate Zn doped Co₃O₄ electrode as an electrocatalyst for non‐enzymatic oxidation of glucose. XRD, HRTEM, SEM, EELS, AFM, EIS was used to characterise the electrode. The addition of Zn as dopant on Co₃O₄ resulted in enhanced electrochemical performance of Zn:Co₃O₄ material compared to pristine Co₃O₄ due to increased charge transferability. The as prepared electrode showed fast response (<7 s) time, good sensitivity (193 μA mM⁻¹ cm⁻²) in the linear range of 5 μM–0.62 mM, good selectivity towards glucose at a relatively lower applied potential of +0.52 V in 0.1 M NaOH solution. A detection limit of ∼2 μM was measured for the Zn:Co₃O₄ electrode. The applied fabrication method resulted in good inter and intra electrode reproducibility as was shown by the lower relative standard deviation values (R.S.D). The electrode retained 70 % of initial current response after 30 days. Although the as prepared Zn:Co₃O₄ electrodes did not result in highest reported sensitivity, and lowest limit of detection; the ease of fabrication and scalability of production, good inter and intra electrode reproducibility makes it a potential candidate for commercial application as glucose sensor.     

MOF‐Derived Spinel NiCo2O4 Hollow Nanocages for the Construction of Non‐enzymatic Electrochemical Glucose Sensor

Non‐enzymatic glucose sensor is greatly expected to take over its enzymatic counterpart in the future. In this paper, we reported on a facile strategy to construct a non‐enzymatic glucose sensor by use of NiCo₂O₄ hollow nanocages (NiCo₂O₄ HNCs) as catalyst, which was derived from Co‐based zeolite imidazole frame (ZIF‐67). The NiCo₂O₄ HNCs modified glassy carbon electrode (NiCo₂O₄ HNCs/GCE), the key component of the glucose sensor, showed highly electrochemical catalytic activity towards the oxidation of glucose in alkaline media. As a result, the proposed non‐enzymatic glucose sensor afforded excellent analytical performances assessed with the aid of cyclic voltammetry and amperometry (i–t). A wide linear range spanning from 0.18 μΜ to 5.1 mM was achieved at the NiCo₂O₄ HNCs/GCE with a high sensitivity of 1306 μA mM^?1 cm^?2 and a fast response time of 1 s. The calculated limit of detection (LOD) of the sensor was as low as 27 nM (S/N=3). Furthermore, it was demonstrated that the non‐enzymatic glucose sensor showed considerable anti‐interference ability and excellent stability. The practical application of the sensor was also evaluated by determination of glucose levels in real serum samples.     

Nanostructured Gold Microelectrodes for Non‐enzymatic Glucose Sensor

A novel, highly stable, selective, and sensitive non‐enzymatic glucose sensor was developed by simple and effective modification procedure. The modification of gold microelectrodes by electrochemically deposited gold nanoparticles resulted in increase of surface area up to 37 %. The nanostructured surfaces of the gold microelectrodes obtained by different modifications were studied by confocal microscopy, atomic force microscopy, and scanning electron microscopy. The gold nanoclusters exhibit great electrocatalytic properties toward glucose with a wide linear range from 0.5 to 50 mM, with a limit of detection 218 μM, and sensitivity of 185.2 mA mM^?1cm^?2. Moreover, the modified microelectrodes display good reproducibility, stability, and selectivity in the presence of poisoning compounds. Due to the small dimensions of gold microelectrodes and a very small volume of the sample, the microelectrodes make a contribution to miniaturisation of the system.     

Glutathione Peroxidase‐Based Amperometric Biosensor for the Detection of S‐Nitrosothiols

A new biosensor is described for the detection of S‐nitrosothiols (RSNOs) based on their decomposition by immobilized glutathione peroxidase (GPx), an enzyme containing selenocysteine residue that catalytically produces nitric oxide (NO) from RSNOs. The enzyme is entrapped at the distal tip of a planar amperometric NO sensor. The new biosensor shows good sensitivity, linearity, reversibility, and response times towards various RSNO species in PBS buffer, pH 7.4 . In most cases, the response time is less than 5 min, and the response is linear up to 6 μM of the tested RSNO species. The lowest detection limit is obtained for S‐nitrosocysteine (CysNO), at approx. 0.2 μM. The biosensor's sensitivity is not affected by the addition of EDTA as a chelating agent; an advantage over other potential catalytic enzymes that contain copper ion centers, such as CuZn‐superoxide dismutase and xanthine oxidase. However, lifetime of the new sensor is limited, with sensitivity decrease of 50% after two days of use. Nonetheless, the new amperometric GPx based RSNO sensor could prove useful for detecting relative RSNO levels in biological samples, including whole blood.     

A Biosilification Fusion Protein for a ‘Self‐immobilising’ Sarcosine Oxidase Amperometric Enzyme Biosensor

Monomeric sarcosine oxidase (mSOx) fusion with the silaffin peptide, R5, designed previously for easy protein production in low resource areas, was used in a biosilification process to form an enzyme layer electrode biosensor. mSOx is a low activity enzyme (10–20 U/mg) requiring high amounts of enzyme to obtain an amperometric biosensor signal, in the clinically useful range <1 mM sarcosine, especially since the K_m is >10 mM. An amperometric biosensor model was fitted to experimental data to investigate dynamic range. mSOx constructs were designed with 6H (6×histidine) and R5 (silaffin) peptide tags and compared with native mSOx. Glutaraldehyde (GA) cross‐linked proteins retained ～5 % activity for mSOx and mSOx‐6H and only 0.5 % for mSOx‐R5. In contrast R5 catalysed biosilification on (3‐mercaptopropyl) trimethoxysilane (MPTMS) and tetramethyl orthosilicate (TMOS) particles created a ‘self‐immobilisation’ matrix retaining 40 % and 76 % activity respectively. The TMOS matrix produced a thick layer (>500 μm) on a glassy carbon electrode with a mediated current due to sarcosine in the clinical range for sarcosinemia (0–1 mM). The mSOx‐R5 fusion protein was also used to catalyse biosilification in the presence of creatinase and creatininase, entrapping all three enzymes. A mediated GC enzyme linked current was obtained with dynamic range available for creatinine determination of 0.1–2 mM for an enzyme layer ～800 nm.     

Laser‐induced Graphene‐based Non‐enzymatic Sensor for Detection of Hydrogen Peroxide

In this study, a laser‐induced graphene (LIG) loaded platinum nanoparticles (PtNPs) was prepared for precise, rapid and non‐enzymatic electrochemical detection of hydrogen peroxide (H₂O₂). The commercial PI films were used as the substrate of LIG. In order to improve the electrochemical performance of LIG, a layer of PtNPs catalyst was fabricated through a magnetron sputtering process on the surface of LIG (PtLIG). Under optimized conditions, a linear relationship between H₂O₂ reduction current and H₂O₂ concentration was recorded, the correlation coefficient R² is 0.9919 with the detection limit of 0.1 μM (S/N=3) and the sensitivity of 248.4 μA mM^?1cm^?2. Moreover, the PtLIG exhibits excellent selectivity, reproducibility and repeatability. Because of these remarkable advantages, we believe that PtLIG will provide a wider range of applications in biosensors and bioelectronic devices.     

Template‐Directed Copying of RNA by Non‐enzymatic Ligation

The non‐enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA‐based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template‐directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′‐amino group at the 3′‐end of each oligonucleotide, in combination with an N‐alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.     

Facile Fabrication of a Graphene‐based Electrochemical Biosensor for Glucose Detection

A simple and effective glucose biosensor based on immobilization of glucose oxidase (GOD) in graphene (GR)/Nafion film was constructed. The results indicated that the immobilized GOD can maintain its native structure and bioactivity, and the GR/Nafion film provides a favorable microenvironment for GOD immobilization and promotes the direct electron transfer between the electrode substrate and the redox center of GOD. The electrode reaction of the immobilized GOD shows a reversible and surface‐controlled process with the large electron transfer rate constant (k_s) of 3.42±0.08 s^?1. Based on the oxygen consumption during the oxidation process of glucose catalyzed by the immobilized GOD, the as‐prepared GOD/GR/Nafion/GCE electrode exhibits a linear range from 0.5 to 14 mmol·L^?1 with a detection limit of 0.03 mmol·L^?1. Moreover, it displays a good reproducibility and long‐term stability.     

Design of an Interference‐Free Cholesterol Amperometric Biosensor Based on the Electrosynthesis of Polymeric Films of Diaminonaphthalene Isomers

Different cholesterol amperometric biosensors were developed based on entrapment of cholesterol esterase and/or cholesterol oxidase in polymer films of diaminonaphthalene isomers, electrochemically synthesised from aqueous solutions of the monomers and enzymes in phosphate buffer at neutral pH. These conditions permit the growth of films with extraordinary selective properties which allow the preparation of interference‐free biosensors for application in biological media without the response being affected by the presence of either endogenous species (ascorbic and uric acid) or exogenous species like 4‐acetamidophenol. These selective properties were evaluated for the different monolayer and bilayer configurations proposed in function of the film permeation factor. All the steps involved in the preparation of the biosensors and determination of free or total cholesterol were carried out in a flow system. A comparative study was made of the analytical properties of each of the configurations developed and their application to the flow‐injection determination of cholesterol in a synthetic serum.     

Electrochemical Study of Chitosan‐SiO2‐MWNT Composite Electrodes for the Fabrication of Cholesterol Biosensors

We report a novel composite electrode made of chitosan‐SiO₂‐multiwall carbon nanotube (CHIT‐SiO₂‐MWNT) composite coated on the indium‐tin oxide (ITO) glass substrate. Cholesterol oxidase (ChOx) was covalently immobilized on the CHIT‐SiO₂‐MWNT/ITO electrode that resulted in a ChOx/CHIT‐SiO₂‐MWNT/ITO cholesterolactive bioelectrode. The CHIT‐SiO₂‐MWNT/ITO and ChOx/CHIT‐SiO₂‐MWNT/ITO electrodes were characterized with Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The influence of various parameters was investigated, including the applied potential, pH of the medium, and the concentration of the enzyme on the performance of the biosensor. The cholesterol bioelectrode exhibited a sensitivity of 3.4 nA/ mgdL^?1 with a response time of five seconds. The biosensor using ChOx/CHIT‐SiO₂‐MWNT/ITO as the working electrode retained its original response after being stored for six months. The biosensor using ChOx/CHIT‐SiO₂‐MWNT/ITO as the working electrode showed a linear current response to the cholesterol concentration in the range of 50–650 mg/dL.     

Graphene Quantum Dots‐based Electrochemical Biosensor for Catecholamine Neurotransmitters Detection

An useful electrochemical sensing approach was developed for epinephrine (EP) detection based on graphene quantum dots (GQDs) and laccase modified glassy carbon electrodes (GC). The miniature GC biosensor was designed and constructed via the immobilization of laccase in an electroactive layer of the electrode coated with carbon nanoparticles. This sensing arrangement utilized the catalytic oxidation of EP to epinephrine quinone. The detection process was based on the oxidation of catecholamine in the presence of the enzyme – laccase. With the optimized conditions, the analytical performance demonstrated a high degree of sensitivity −2.9 μA mM⁻¹ cm⁻², selectivity in a broad linear range (1–120×10⁻⁶ M) with detection limit of 83 nM. Moreover, the method was successfully applied for EP determination in labeled pharmacological samples.     

Detection of Human Interleukine‐2 Gene Using a Label‐Free Electrochemical DNA Hybridization Biosensor on the Basis of a Non‐Inosine Substituted Probe

The human interleukine‐2 gene (hIL‐2) is detected with a label‐free DNA hybridization biosensor using a non‐inosine substituted probe. The sensor relies on the immobilization of a 20‐mer antisense single strand oligonucleotide (chIL‐2) related to the human interleukine‐2 gene on the pencil graphite electrode (PGE) as a probe. The guanine oxidation signal was monitored using anodic differential pulse voltammetry (ADPV). The electrochemical pretreatment of the polished PGE at 1.80 V for 5 min is suggested. Then, 5 min immobilization at 0.50 V was found as the optimum condition for immobilization of the probe. The electrochemical detection of hybridization between chIL‐2 and hIL‐2 as a target was accomplished. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Diagnostic performance of the biosensor is described and the detection limit is found 36 pg/μL.     

Electrochemical Signal Enhancer Fabricated Using Lysine‐rich Peptide for Ultrasensitive Electrochemical DNA Biosensor Analysis

Here we present a novel design of electrochemical signal enhancer to increase the detection sensitivity of electrochemical DNA biosensors. The key element of this enhancer is a lysine‐rich peptide (LRP). Its C‐terminal is conjugated with a planer molecule, being able to intercalate into the base pairs of probe‐target duplexes. The lysine residues of LRP are covalently linked with electrochemical signal indicators, acting as an assembly of electrochemical signal indicators. Experimental results proved the feasibility of the novel design. We have examined the effects of the numbers of lysine residues and the hybridization conditions on the detection sensitivity. The optimization procedures have led to significant sensitivity enhancement, and the LOD (limit of detection) has been determined to be 1.4 amol. This enhancer demonstrates advantages of easy operation, simple instrumentation, and high exemption from environmental influence.     

Amperometric Biosensor for Detection of Phenolic Compounds Based on Tyrosinase,N‐Acetyl‐L‐cysteine‐capped Gold Nanoparticles and Chitosan Nanocomposite

A novel biosensor was fabricated based on the immobilization of tyrosinase and N ‐acetyl‐L ‐cysteine‐capped gold nanoparticles onto the surface of the glassy carbon electrode via the film forming by chitosan. The NAC‐AuNPs (N ‐acetyl‐L ‐cysteine‐capped gold nanoparticles) with the average size of 3.4 nm had much higher specific surface area and good biocompatibility, which were favorable for increasing the immobilization amount of enzyme, retaining the catalytic activity of enzyme and facilitating the fast electron transfer. The prepared biosensor exhibited suitable amperometric responses at −0.2 V for phenolic compounds vs. saturated calomel electrode. The parameters of influencing on the working electrode such as pH , temperature, working potential were investigated. Under optimum conditions, the biosensor was applied to detect catechol with a linear range of 1.0 × 10⁻⁷ to 6.0 × 10⁻⁵ mol•L⁻¹ , and the detection limit of 5.0 × 10⁻⁸ mol•L⁻¹ (S /N =3). The stability and selectivity of the proposed biosensor were also evaluated.     

Design of a New Non‐enzymatic Sensor Based on a Substituted A2BO4+δ Perovskite for the Voltammetric Detection of Glucose

Instant determination of glucose levels is necessary to monitor the treatment of diabetes. The next generation of electrochemical sensors aims to eliminate the use of enzymes because of their lack of stability and the complex procedure to immobilize them on the electrode. In this paper Pr_1.92Ba_0.08Ni0.₉₅Zn_0.05O_4+δ perovskite, a A₂BO_4+δ type, was tested, for the first time for non enzymatic detection of glucose. It was synthesized by a sol‐gel method. The obtained crystallized powder was structurally characterized by XRD, morphologically characterized by SEM and EDX and electrochemically characterized. A monoclinic crystallographic system was formed. The presence of Pr₂O₃ during synthesis and calcination is in agreement with the formation of defects in the crystalline network and the disproportionation of Ni^III sites into Ni^II and Ni^IV, due to the substitution of Pr by Ba. The oxido‐reduction of Ni^II sites is observed by cyclic voltammetry. The electrocatalytic oxidation of glucose through the electrooxidized Ni^II site was observed on a gold electrode, at 481 mV. The analytical performance of this glucose sensor is good in comparison to previously published ABO₃ perovskite modified electrodes, in terms of dynamic range (1.5 μM–7000 μM) and detection limit (0.5 μM). Its application to human serum shows that there is no interference for glucose detection.     

Fabrication of Miniaturised Screen‐printed Glucose Biosensors,Using a Water‐based Ink,and the Evaluation of their Electrochemical Behaviour

This paper describes a simple, convenient approach to the fabrication of microband electrodes and microband biosensors based on screen printing technology. These devices were printed in a three‐electrode configuration on one strip; a silver/silver chloride electrode and carbon counter electrode served as reference and counter electrodes respectively. The working electrodes were fabricated by screen‐printing a water‐based carbon ink containing cobalt phthalocyanine for hydrogen peroxide detection. These were converted into a glucose microband biosensor by the addition of glucose oxidase into the carbon ink. In this paper, we discuss the fabrication and application of glucose microband electrodes for the determination of glucose in cell media. The dimensions (100–400 microns) of the microband electrodes result in radial diffusion, which results in steady state responses in the absence of stirring. The microband biosensors were investigated in cell media containing different concentrations of glucose using chronoamperometry. The device shows linearity for glucose determination in the range 0.5 mM to 2.5 mM in cell media. The screen‐printed microband biosensor design holds promise as a generic platform for future applications in cell toxicity studies.     

Amperometric Biosensor for Choline Based on Gold Screen‐Printed Electrode Modified with Electrochemically‐Deposited Silica Biocomposite

A mediator‐free choline biosensor was developed using the electrochemically assisted sol‐gel deposition on gold screen‐printed electrodes. The addition of 12 mM of cationic surfactant CTAB in silica sol allowed enhancing the stability of the sensor. The modified electrode demonstrated catalytic activity and stable amperometric response to choline for over 3 weeks of exploitation with the sensitivity of 6 µA mM^?1 and LOD of 6 µM. The interference of ascorbic acid was reduced by pretreating the analyzed solution with MnO₂ powder. The application of the sensor with the purpose of identifying choline in the baby milk demonstrated satisfactory metrological characteristics.     

Mathematical Analysis of Reaction–Diffusion Equations Modeling the Michaelis–Menten Kinetics in a Micro-Disk Biosensor

In this study, we have investigated the mathematical model of an immobilized enzyme system that follows the Michaelis–Menten (MM) kinetics for a micro-disk biosensor. The film reaction model under steady state conditions is transformed into a couple differential equations which are based on dimensionless concentration of hydrogen peroxide with enzyme reaction (H) and substrate (S) within the biosensor. The model is based on a reaction–diffusion equation which contains highly non-linear terms related to MM kinetics of the enzymatic reaction. Further, to calculate the effect of variations in parameters on the dimensionless concentration of substrate and hydrogen peroxide, we have strengthened the computational ability of neural network (NN) architecture by using a backpropagated Levenberg–Marquardt training (LMT) algorithm. NNs–LMT algorithm is a supervised machine learning for which the initial data set is generated by using MATLAB built in function known as “pdex4”. Furthermore, the data set is validated by the processing of the NNs–LMT algorithm to find the approximate solutions for different scenarios and cases of mathematical model of micro-disk biosensors. Absolute errors, curve fitting, error histograms, regression and complexity analysis further validate the accuracy and robustness of the technique.