3D nanogap interdigitated electrode array biosensors

Three-dimensional interdigitated electrodes (IDEs) have been investigated as sensing elements for biosensors. Electric field and current density were simulated in the vicinity of these electrodes as a function of the electrode width, gap, and height to determine the optimum geometry. Both the height and the gap between the electrodes were found to have significant effect on the magnitude and distribution of the electric field and current density near the electrode surface, while the width of the electrodes was found to have a smaller effect on field strength and current density. IDEs were fabricated based on these simulations and their performance tested by detecting C-reactive protein (CRP), a stress-related protein and an important biomarker for inflammation, cardiovascular disease risk indicator, and postsurgical recuperation. CRP-specific antibodies were immobilized on the electrode surface and the formation of an immunocomplex (IC) with CRP was monitored. Electrochemical impedance spectroscopy (EIS) was employed as the detection technique. EIS data at various concentrations (1 pg/mL to 10 μg/mL) of CRP spiked in buffer or diluted human serum was collected and fitted into an equivalent electrical circuit model. Change in resistance was found to be the parameter most sensitive to change in CRP concentration. The sensor response was linear from 0.1 ng/mL to 1 μg/mL in both buffer and 5% human serum samples. The CRP samples were validated using a commercially available ELISA for CRP detection. Hence, the viability of IDEs and EIS for the detection of serum biomarkers was established without using labeled or probe molecules.     

Enhancement of Enzymatic IDE Biosensor Response Using Gold Nanoparticles. Example of the Detection of Urea

A new conductometric biosensor based on interdigitated electrodes (IDEs) has been developed for the detection of enzymatic substrates using gold nanoparticles (GNPs), synthesized bellowing the citrate process, with an average diameter of 23 nm and functionalized with urease using layer‐by‐layer technique. A detection limit of 100 µM of urea is obtained when cross‐linked urease is directly immobilized on top of the IDEs (interdigitated distance: 20 µm) whereas a detection limit of 2 µM is obtained when urease functionalized gold nanoparticles are deposited on the top of the IDEs. The use of gold nanoparticles allows the increase of the sensitivity of detection (from 10 µS/mM to 107 µS/mM) due to the decrease of the thickness of probed zone.     

Pb2+ induced DNA conformational switch from hairpin to G-quadruplex: electrochemical detection of Pb2+

Conformational switch from hairpin DNA to G-quadruplex induced by Pb(2+) is studied by electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)(6)](3-/4-) as the redox probe. In the presence of Pb(2+), the G-rich hairpin DNA opens the stem-loop and forms G-quadruplex structure, which gives rise to a sharp increase in the charge-transfer resistance (R(CT)) of the film reflected by the EIS. This structural change is also confirmed by circular dichroism (CD) measurements and UV-Vis spectroscopic analysis and calculated by density functional theory (DFT). On the basis of this, we develop a label-free electrochemical DNA biosensor for Pb(2+) detection. With increasing concentrations of Pb(2+), the differences in the charge-transfer resistance R(CT) before and after the Pb(2+) incubation is linearly dependent on the logarithm of Pb(2+) concentration within a range from 50 μM to 0.5 nM. The biosensor also exhibits good selectivity for Pb(2+) over other metal ions. This is a simple and label-free electrochemical method for Pb(2+) detection making use of the G-quadruplex.     

Label-free oligonucleotide detection method based on a new L-cysteine-dihydroartemisinin complex electroactive indicator

A novel base-mismatched oligonucleotide assay method based on label-free electrochemical biosensor was developed, in which the L-cysteine (Cys)-dihydroartemisinin (DHA) complex was used as a new electroactive indicator. In DNA sensor, Cys-DHA complex was initially formed on electrode surface by cathodic scanning, and target oligonucleotide was conjugated with Cys-terminated DHA indicator through electrostatic interaction under optimal pH. The subsequent sequence assay was responsive to hybridization recognition, which target oligonucleotide was captured by the surface-anchored DNA/Cys-DHA probe. The electrochemical signals of biosensor before and after hybridization were compared basing the measurements of semi-derivative linear scan voltammetry (SDLSV) and electrochemical impedance spectroscopy (EIS). On the basis of signal amplification of electroactive indicator and specific recognition of DNA probe, five target oligonucleotides with different mismatched bases were assayed, and a detection limit reached 0.3 nM. Furthermore, atomic force microscopy (AFM) was used to visually characterize specific recognition spots of biosensor at nanoscale. This study demonstrated a new electroactive molecule-based, biomolecule-involved electroactive indicator and its application in recognition and detection of complementary and base-mismatched oligonucleotide.     

Electrochemical aptasensor for the detection of vascular endothelial growth factor (VEGF) based on DNA-templated Ag/Pt bimetallic nanoclusters

In this paper, the DNA-templated Ag/Pt bimetallic nanoclusters were successfully synthesized using an optimized synthetic scheme. The obtained DNA-Ag/Pt NCs have an ultrasmall particle size and excellent distribution. The DNA-Ag/Pt NCs show intrinsic peroxidase-mimicking activity and can effectively catalyze the H₂O₂-mediated oxidation of a substrate, 3,3',5,5'-tetramethylbenzidine (TMB), to produce a blue colored product. Based on this specific property, we employed the aptamer of VEGF to design a label-free electrochemical biosensor for VEGF detection. Under the optimized experimental conditions, a linear range from 6.0 pmol/L to 20 pmol/L was obtained with a detection limit of 4.6 pmol/L. The proposed biosensor demonstrated its high specificity for VEGF and could directly detect the VEGF concentration in human serum samples of breast cancer patients with satisfactory results. This novel electrochemical aptasensor was simple and convenient to use and was cost-effective and label-free in design, and would hold potential applications in medical diagnosis and treatment.     

A sandwich-type DNA biosensor based on electrochemical co-reduction synthesis of graphene-three dimensional nanostructure gold nanocomposite films

A novel electrochemical DNA biosensor based on graphene-three dimensional nanostructure gold nanocomposite modified glassy carbon electrode (G-3D Au/GCE) was fabricated for detection of survivin gene which was correlated with osteosarcoma. The G-3D Au film was prepared with one-step electrochemical coreduction with graphite oxide and HAuCl₄ at cathodic potentials. The active surface area of G-3D Au/GCE was 2.629 cm², which was about 3.8 times compared to that of a Au-coated GCE under the same experimental conditions, and 8.8 times compared to a planar gold electrode with a similar geometric area. The resultant nanocomposites with high conductivity, electrocatalysis and biocompatibility were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). A “sandwich-type” detection strategy was employed in this electrochemical DNA biosensor and the response of this DNA biosensor was measured by CV and amperometric current–time curve detection. Under optimum conditions, there was a good linear relationship between the current signal and the logarithmic function of complementary DNA concentration in a range of 50–5000 fM with a detection limit of 3.4 fM. This new biosensor exhibited a fast amperometric response, high sensitivity and selectivity and has been used in a polymerase chain reaction assay of real-life sample with a satisfactory result.     

Label-free detection of telomerase activity in HeLa cells using electrochemical impedance spectroscopy

A simple assay based on electrochemical impedance spectroscopy (EIS) for detection of telomerase activity is developed, and it is demonstrated that the label-free EIS method is capable of detecting the telomerase activity in HeLa cells with a detection limit of 1000 HeLa cells without using any amplification technique.     

A Novel Electrochemical DNA Biosensor Fabricated with Layer‐by‐Layer Covalent Attachment of Multiwalled Carbon Nanotubes and Gold Nanoparticles

A novel DNA biosensor has been fabricated for the detection of DNA hybridization based on layer‐by‐layer (LBL) covalent assembly of gold nanoparticles (GNPs) and multiwalled carbon nanotubes (MWCNTs). The stepwise LBL assembly process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored by differential pulse voltammetry (DPV) measurement of the intercalated doxorubicin, and the factors influencing the performance of the DNA hybridization was investigated in detail. The signal was linearly changed with target DNA concentration increased from 0.5 to 0.01 nM, and had a detection limit of 7.5 pM (signal/noise ratio of 3). In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

基于Pd/COF-LZU1非标记型C-反应蛋白免疫传感器的研制

采用溶剂热法, 通过有机单体合成了一种亚胺键连接的共价有机框架材料(COF-LZU1); 在常温常压条件下, 通过后合成的方法将贵金属钯(Ⅱ)引入到COF材料中, 合成了复合材料Pd/COF-LZU1, 该材料具有优良的催化性能. 利用Pd/COF-LZU1多孔复合材料将C-反应蛋白(CRP)抗体(anti-CRP)固定在玻碳电极表面, 构建了一种非标记型CRP免疫传感器. 当抗体与抗原发生免疫反应时, 形成的免疫复合物会阻碍电化学探针[Fe(CN)₆]^4-/3-的电子传递, 降低其响应电流, 从而实现CRP的快速检测. 采用交流阻抗和差示脉冲伏安法(DPV)考察了免疫传感器的电化学特性, 同时考察了测试底液的pH值、 抗原培育时间和抗体固定浓度等实验条件对传感器性能的影响. 在最优的实验条件下, 采用DPV法对CRP进行检测的线性范围为5~180 ng/mL, 检出限为1.66 ng/mL, 线性相关系数为0.992.     

Amperometric biosensor for hydrogen peroxide based on direct electrocatalysis by hemoglobin immobilized on gold nanoparticles/1,6-diaminohexane modified glassy carbon electrode.

A facile strategy of an amperometric biosensor for hydrogen peroxide based on the direct electrocatalysis of hemoglobin (Hb) immobilized on gold nanoparticles (GNPs)/1,6-diaminohexane (DAH) modified glassy carbon electrode (GCE) has been described. A uniform monolayer film of DAH was initially covalently bound on a GCE surface by virtue of the electrooxidation of one amino group of DAH, and another amino group was modified with GNPs and Hb, successively. The fabrication process was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). The proposed biosensor exhibited an effective and fast catalytic response to the reduction of H2O2 with good reproducibility and stability. A linear relationship existed between the catalytic current and the H2O2 concentration in the range of 1.5x10(-6) to 2.1x10(-3) M with a correlation coefficient of 0.998 (n=24). The detection limit (S/N=3) was 8.8x10(-7) M.     

Fabrication of Pt nanoparticles-decorated CVD diamond electrode for biosensor applications

An electrochemical biosensor was developed using boron-doped diamond (BDD) as an electrode material. To enhance the electrical performance of the electrode, the BDD electrode was decorated with Pt-nanoparticles (Pt-NPs) by electrochemical deposition. Their morphology according to the applied potentials for the synthesis of Pt-NPs was characterized by SEM. To identify the performance of the electrode modified with Pt-NPs, glucose detection was used as a sample sensing process, and the results were compared with those of a gold electrode and a bare BDD electrode. The electrochemical characteristics of the modified electrode were examined by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The BDD electrode with the Pt-NPs showed higher sensitivity and a lower detection limit than the Au electrode and BDD electrode. The proposed biosensor based on the Pt-NPs decorated BDD electrode showed high sensitivity, a low detection limit, fast direct electron transfer and good stability.     

Coupling of a Reagentless Electrochemical DNA Biosensor with Conducting Polymer Film and Nanocomposite as Matrices for the Detection of the HIV DNA Sequences

Abstract

This paper describes a reagentless electrochemical DNA biosensor applied to the detection of human immunodeficiency virus (HIV) sequences based on electrochemical impedance spectroscopy (EIS). The novel DNA biosensor has been elaborated by means of an opposite‐charged adsorption Au‐Ag nanocomposite to a conductive polymer polypyrrole (PPy) modified platinum electrode (Pt) and self‐assembly the mercapto oligonucleotide probes onto the surface of modified electrode via the nanocomposite. The duplex formation was detected by measuring the electrochemical impedance signal of nucleic acids in phosphate buffer solution (PBS). Such response is based on the concomitant conductivity changes of the PPy film and nanocomposite. The reagentless scheme has been characterised using 21‐mer synthetic oligonucleotides as models: parameters affecting the hybridization assay were explored and optimized. The detection limit is 5.0×10^?10 M of target oligonucleotides at 3σ. The potential for development of reagentless DNA hybridization analysis in the clinical diagnosis is being pursued.     

Enhanced lateral flow assay with double conjugates for the detection of exosomes

Exosomes are promising biological biomarkers for monitoring a number of diseases, especially cancers. Here, we developed a double gold nanoparticles(GNPs) conjugates based lateral flow assay(D-LFA) for rapidly and sensitively detecting and molecular profiling of exosomes. Based on these two GNPs conjugates, the signal amplification can be achieved without any additional operation. The antibody on the 1 st GNPs conjugate could recognize exosomes and form a sandwich format on the test zone. The 2 nd GNPs conjugate was designed to bind to the 1 st GNPs conjugate to realize signal amplification. This biosensor enabled visual and quantitative detection of exosomes by the accumulation of GNPs on the test zone and showed a low detection limit of 1.3×10~3 particles/μL, which has been improved 13-fold compared with the normal lateral flow assay. The D-LFA exhibited good sensitivity and reproducibility and has been successfully used for the detection of exosomes in fetal bovine serum,which proved its potential application in practical diagnostics.     

Target-induced conjunction of split aptamer as new chiral selector for oligopeptide on graphene-mesoporous silica-gold nanoparticle hybrids modified sensing platform

A new electrochemical label-free biosensor based on target-induced conjunction of a split aptamer as new chiral selector for oligopeptide using graphene-mesoporous silica-gold NP hybrids (GSGHs) as magnified sensing platform is firstly reported, which showed high sensitivity and selectivity for the detection of D-vasopressin (D-VP).     

[Ru(bpy)2(dcbpy)NHS] Labeling/Aptamer‐Based Biosensor for the Detection of Lysozyme by Increasing Sensitivity with Gold Nanoparticle Amplification

A novel [Ru(bpy)₂(dcbpy)NHS] labeling/aptamer‐based biosensor combined with gold nanoparticle amplification for the determination of lysozyme with an electrochemiluminescence (ECL) method is presented. In this work, an aptamer, an ECL probe, gold nanoparticle amplification, and competition assay are the main protocols employed in ECL detection. With all the protocols used, an original biosensor coupled with an aptamer and [Ru(bpy)₂(dcbpy)NHS] has been prepared. Its high selectivity and sensitivity are the main advantages over other traditional [Ru(bpy)₃]²⁺ biosensors. The electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) characterization illustrate that this biosensor is fabricated successfully. Finally, the biosensor was applied to a displacement assay in different concentrations of lysozyme solution, and an ultrasensitive ECL signal was obtained. The ECL intensity decreased proportionally to the lysozyme concentration over the range 1.0×10^?13–1.0×10^?8 mol L^?1 with a detection limit of 1.0×10^?13 mol L^?1. This strategy for the aptasensor opens a rapid, selective, and sensitive route for the detection of lysozyme and potentially other proteins.     

Characterisation of antibodies to chloramphenicol, produced in different species by enzyme-linked immunosorbent assay and biosensor technologies

Six polyclonal antisera to chloramphenicol (CAP) were successfully raised in camels, donkeys and goats. As a comparison of sensitivity, IC₅₀ values ranged from 0.3 ng mL⁻¹ to 5.5 ng mL⁻¹ by enzyme-linked immunosorbent assay (ELISA) and from 0.7 ng mL⁻¹ to 1.7 ng mL⁻¹ by biosensor assay. The introduction of bovine milk extract improved the sensitivity of four of the antisera by ELISA and two by biosensor assay; a reduction in sensitivity of the remaining antisera ranged by a factor of 1.1-2.6. Porcine kidney extract reduced the sensitivity of all the antisera by a factor ranging from 1.1 to 7 by ELISA and a factor of 1.5 to 4 by biosensor. A low cross-reactivity with thiamphenicol (TAP) and florfenicol (FF) was displayed by antiserum G2 (1.2% and 18%, respectively) when a homologous ELISA assay format was employed. No cross-reactivity was displayed by any of the antisera when a homologous biosensor assay format was employed. Switching to a heterologous ELISA format prompted three of the antisera to display more significant cross-reactivity with TAP and FF (53% and 82%, respectively, using D1). The heterologous biosensor assay also increased the cross-reactivity of D1 for TAP and FF (56% and 129%, respectively) and of one other antiserum (G1) to a lesser degree. However, unlike the ELISA, the heterologous biosensor assay produced a substantial reduction in sensitivity (by a factor of 6 for D1).     

Highly sensitive lable-free electrochemical aptasensor for thrombin detection with cobalt hexacyanoferrate as the electrochemical probe

A highly sensitive label-free electrochemical aptasensor has been constructed for the electrochemical detection of thrombin (TB), where two layers of cobalt hexacyanoferrate (CoHCF) redox probes sandwiched with carbon nanotubes–Nafion were directly immobilized on the electrode surface by electrodeposition. Through the strong interaction between CN^? (CoHCF) and gold nanoparticles (GNPs), GNPs were assembled on the CoHCF-modified electrode for the immobilization of thiolated thrombin aptamers (TBA). In the presence of target TB, TBA on the electrode surface could catch TB to form TBA–TB complex, which made a barrier for the electron transfer, resulting in a greater decrease in CoHCF redox probe signals. Thus, the proposed aptasensor showed a high sensitivity and a much wider linearity to TB in the range of 1.0 pg/mL?～?1.0 μg/mL with a detection limit of 0.28 pg/mL.     

An amperometric horseradish peroxidase inhibition biosensor for the determination of phenylhydrazine

An amperometric horseradish peroxidase (HRP) inhibition biosensor has been substantially constructed by the help of N,N-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS). The preparation steps and the biosensor response to phenylhydrazine were monitored by electrochemical impedance spectroscopy (EIS), cyclic voltammetry, and chronoamperometry. The proposed biosensor could be applied to determine phenylhydrazine in a 0.10 M phosphate buffer solution containing 1.2 mM hydroquinone and 0.50 mM H(2)O(2) by phenylhydrazine, inhibiting the catalytic activity of the HRP enzyme in the reduction of H(2)O(2). The system was optimized to realize a reliable determination of phenylhydrazine in the range of 2.5 x 10(-7) to 1.1 x 10(-6) M with a detection limit of 8.2 x 10(-8) M and a correlation coefficient of 0.999. The modified electrode displayed good reproducibility, sensitivity and stability for the determination of phenylhydrazine.     

Tunable nanogap devices for ultra-sensitive electrochemical impedance biosensing

A wealth of research has been available discussing nanogap devices for detecting very small quantities of biomolecules by observing their electrical behavior generally performed in dry conditions. We report that a gold nanogapped electrode with tunable gap length for ultra-sensitive detection of streptavidin based on electrochemical impedance technique. The gold nanogap is fabricated using simple monolayer film deposition and in-situ growth of gold nanoparticles in a traditional interdigitated array (IDA) microelectrode. The electrochemical impedance biosensor with a 25-nm nanogap is found to be ultra-sensitive to the specific binding of streptavidin to biotin. The binding of the streptavidin hinder the electron transfer between two electrodes, resulting in a large increase in electron-transfer resistance (R_et) for operating the impedance. A linear relation between the relative R_et and the logarithmic value of streptavidin concentration is observed in the concentration range from 1 pM (picomolar) to 100 nM (nanomolar). The lowest detectable concentration actually measured reaches 1 pM. We believe that such an electrochemical impedance nanogap biosensor provides a useful approach towards biomolecular detection that could be extended to a number of other systems.     

A glucose biosensor based on chitosan-Prussian blue-multiwall carbon nanotubes-hollow PtCo nanochains formed by one-step electrodeposition

In this paper, a simple one-step electrodeposition method is described to fabricate chitosan-Prussian blue-multiwall carbon nanotubes-hollow PtCo nanochains (CS-PB-MWNTs-H-PtCo) film onto the gold electrode surface, then glucose oxidase (GOD) and Nafion were modified onto the film subsequently to fabricate a glucose biosensor. The morphologies and electrochemistry of the composite were investigated by using Fourier transform infrared (FTIR) spectrometry, scanning electron microscopy (SEM) and electrochemical techniques including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. The performances of the biosensor have been investigated by chronoamperometry method under the optimized conditions. This biosensor showed a linear response to glucose range from 1.5 μM to 1.12 mM with a detection limit of 0.47 μM (S/N=3), a high sensitivity of 23.4 μA mM(-1) cm(-2), and a fast response time. The apparent Michaelis-Menten constant (K(M)(app)) was 1.89 mM. In addition, the biosensor also exhibited strong anti-interference ability, excellent stability and good reproducibility.