Label-free Electrochemical Impedance Research Articles

A sensitive and label-free electrochemical impedance biosensor for protein detection based on terminal protection of small molecule-linked DNA

In this paper, a label-free electrochemical impedance biosensor has been developed for protein detection based on terminal protection of small molecule linked-DNA. This strategy was demonstrated using folate (FA) and its binding protein folate receptor (FR) as a model case. In the present terminal protection system, with the increase of FR, it could protect more FA linked-DNA from hydrolysis by exonuclease I (Exo I), resulting in an increased electron transfer resistance response. By taking the advantage of the terminal protection assay, this label-free electrochemical biosensor achieved highly sensitive and selective detection of FR with a detection limit of 3pM and a linear range from 10pM to 500nM, which was lower than some previously reported methods. Moreover, the label-free method did not need specific labelling and showed sequence-independent with DNA probes. Thus, it may offer a simple, cost-effective, highly selective and sensitive platform for electrochemical impedance detection of small molecule–protein interactions in clinical diagnostics.

Perylene derivative-bridged Au-graphene nanohybrid for label-free HpDNA biosensor.

Along with the challenges of wet-chemically preparing graphene-based nanohybrids, for example easy aggregation, low-stability in solution environment and insufficient loading amount, here we report the preparation and application of a type of π-conjugated molecule, perylenetetracarboxylic acid di-imide (PDI)-functionalized graphene material with high density of gold nanoparticles (AuNPs). In this nanohybrid, the PDI molecule comprises five-connected benzene rings and positively charged terminals composed of two symmetrical imidazole rings and amine groups, which offers the intrinsic driving force for π-π interactions with graphene and also serves as the active sites for immobilization of AuNPs. Transmission electron microscopy results demonstrated that AuNPs were uniformly dispersed and densely covered the PDI-functionalized graphene compared to the control experiment without PDI. To prove its biological application, the Au-PDI-graphene nanohybrid was chosen as a sensing material for fabricating a label-free electrochemical impedance hairpin DNA (hpDNA) biosensor for detection of human immunodeficiency virus 1 gene. When hpDNA was hybridized, it exhibited a sensitive electrochemical impedance variation on an Au-PDI-graphene modified electrode. This fabricated hpDNA biosensor reveals a wide linear detection range and a relatively low detection limit. Thanks to its high stability and efficient electrochemical impedance sensitivity, this nanohybrid would offer a broad range of possible DNA sequences for specific applications in biodiagnostics and bionanotechnology.

Coiled-coil peptide based sensor for ultra-sensitive thrombin detection

Comb structured gold microelectrode array (CSGMA) functionalized with self-assembled monolayer of thiol terminated coiled-coil peptide (CCP) linked together by the thrombin specific cleavage site (Leu–Val–Pro–Arg–Gly–Ser) has been used to fabricate an ultrasensitive, disposable, electrochemical thrombin biosensor. CCP with thiol at one end provides the ease of CSGMA functionalization and the presence of thrombin specific peptide in the middle of coiled-coil peptide provides site for thrombin capture and detection. CCP/CSGMA electrodes were characterized using label-free electrochemical impedance (EIS) technique and exposed to solutions with different thrombin concentrations for its estimation. Results reveal that CCP/CSGMA electrodes have a limit of detection (LOD) of 10fg/ml (28fM) and are able to detect catalytic activity of thrombin within 30min time frame. CCP/CSGMA electrodes were found to be selective against other IgG anti-bodies such as DO1 and HA. Thus, CCP/CSGMA electrodes provide high specificity toward thrombin detection and mechanistic details of binding and cleavage process.

Effects of the Electrode Size and Modification Protocol on a Label-Free Electrochemical Biosensor

In the present work, the effect of a surface modification protocol along with the electrode size has been investigated for developing an efficient, label-free electrochemical biosensing method for diagnosis of traumatic brain injury (TBI) biomarkers. A microdisk electrode array (MDEA) and a macroelectrode with a comb structure (MECS) were modified with an anti-GFAP (GFAP = glial fibrillary acidic protein) antibody using two protocols for optimum and label-free detection of GFAP, a promising acute-phase TBI biomarker. For the MDEA, an array of six microdisks with a 100 μm diameter and, for the MECS, a 3.2 mm × 5.5 mm electrode 5 μm wide with 10 μm spaced comb fingers were modified using an optimized protocol for dithiobis(succinimidyl propionate) (DSP) self-assembled monolayer formation. Anti-GFAP was covalently bound, and the remaining free DSP groups were blocked using ethanolamine (Ea). Sensors were exposed to solutions with different GFAP concentrations, and a label-free electrochemical impedance spectroscopy (EIS) technique was used to determine the concentration. EIS results confirmed that both types of Ea/anti-GFAP/DSP/Au electrodes modified with an optimized DSP-based protocol can accurately detect GFAP in the range of 1 pg mL(-1) to 100 ng mL(-1) with a detection limit of 1 pg mL(-1). However, the cross-use of the MDEA protocol on the MECS and vice versa resulted in very low sensitivity or poor signal resolution, underscoring the importance of proper matching of the electrode size and type and the surface modification protocol.

A label-free electrochemical impedance immunosensor based on AuNPs/PAMAM-MWCNT-Chi nanocomposite modified glassy carbon electrode for detection of Salmonella typhimurium in milk

A sensitive and stable label-free electrochemical impedance immunosensor for the detection of Salmonella typhimurium was developed by immobilising anti-Salmonella antibodies onto the gold nanoparticles and poly(amidoamine)-multiwalled carbon nanotubes-chitosan nanocomposite film modified glassy carbon electrode (AuNPs/PAMAM-MWCNT-Chi/GCE). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to verify the stepwise assembly of the immunosensor. Co-addition of MWCNT, PAMAM and AuNPs greatly enhanced the sensitivity of the immunosensor. The immobilisation of antibodies and the binding of Salmonella cells to the modified electrode increased the electron-transfer resistance (Ret), which was directly measured with EIS using [Fe(CN)6]3−/4− as a redox probe. A linear relationship of Ret and Salmonella concentration was obtained in the Salmonella concentration range of 1.0×103 to 1.0×107CFUmL−1 with a detection limit of 5.0×102CFUmL−1. Additionally, the proposed method was successfully applied to determine S. typhimurium content in milk samples with satisfactory results.

Bacteria Screening, Viability, And Confirmation Assays Using Bacteriophage-Impedimetric/Loop-Mediated Isothermal Amplification Dual-Response Biosensors

Here, we integrate two complementary detection strategies for the identification and quantification of Escherichia coli based on bacteriophage T4 as a natural bioreceptor for living bacteria cells. The first approach involves screening and viability assays, employing bacteriophage as the recognition element in label-free electrochemical impedance spectroscopy. The complementary approach is a confirmation by loop-mediated isothermal amplification (LAMP) to amplify specifically the E. coli Tuf gene after lysis of the bound E. coli cells, followed by detection using linear sweep voltammetry. Bacteriphage T4 was cross-linked, in the presence of 1,4-phenylene diisothiocyanate, on a cysteamine-modified gold electrode. The impedimetric biosensor exhibits specific and reproducible detection with sensitivity over the concentration range of 10(3)-10(9) cfu/mL, while the linear response of the LAMP approach was determined to be 10(2)-10(7) cfu/mL. The limit of detection (LOD) of 8 × 10(2) cfu/mL in less than 15 min and 10(2) cfu/mL within a response time of 40 min were achieved for the impedimetric and LAMP method, respectively. This work provides evidence that integration of the T4-bacteriophage-modified biosensor and LAMP can achieve screening, viability, and confirmation in less than 1 h.

The Development of a Label-Free Electrochemical Impedance Based Pointof-care Technology for Multimarker Detection

With an increasing global population, rising healthcare costs, and greater demand on hospitals and clinicians, a growing need for low cost, rapid, Point of Care Technologies (POCT) exists. The overall goal is to detect or monitor a disease in order to give patients and clinicians fast, accurate, and all-encompassing information regarding the state of the disease. A challenge for many currentpoint-of-care technologies is the difficulty of monitoring several biomarkers simultaneously without the complexity of multiple sensors, labels, or spatially separated transducers. We have previously shown that convoluted signals obtained from protein biomarkers monitored by electrochemical impedance spectroscopy can be “tuned” away from one another by conjugation with gold nanoparticles to allow for the potential simultaneous detection of multiple biomarkers. This method of detection yields a sensitive and specific means of biomarker quantification in human media including tears or blood. In this work, we detail the development of a mathematical model that explores the roles of various factors, such as nanoparticle size and the nature of materials, so that a design space could be created for tuningotherwise convoluted biomarkers. Furthermore, we present assessments as to the validity of this model with preliminary bench-top experiments by taking advantage of gold nanoparticle-antibody conjugates of varying sizes. Gold nanoparticle size changes of 5, 10, and 20 nm demonstrated a 10.0, 4.8, and 1.0 Hz shift in frequency, respectively. Future work includes exploration of different sensor configurations, continuous monitoring, and the prospect for implantable sensors were also discussed as potential future avenues.

Myelin basic protein immunosensor for multiple sclerosis detection based upon label-free electrochemical impedance spectroscopy

A novel highly sensitive impedimetric Myelin Basic Protein (MBP) immunosensor for the determination of a Multiple Sclerosis (MS) autoantibody, Anti-Myelin Basic Protein (Anti-MBP) was developed by immobilization of MBP on Gelatin and Gelatin–Titanium Dioxide (TiO2) modified platinium electrode. Cyclic voltammetric (CV) and Electrochemical Impedance Spectroscopic (EIS) methods were employed in determination of the electrode responses and applicability. Gelatin-MBP and gelatin–TiO2-MBP electrodes were prepared by chemical immobilization of the substrates onto the platinium electrodes. The formal potentials of MBP confined on gelatin-MBP and gelatin–TiO2-MBP surfaces are estimated to be 195 and 205mV, respectively. Thus, a little more reversible electron transfer reaction occurs on the gelatin–TiO2-MBP immunosensor surface. The peak separations of MBP (150mV and 110mVs−1 at 100mVs−1) and the asymmetric anodic and cathodic peak currents indicate that the electron transfer between Anti-MBP and gelatin-MBP/gelatin–TiO2-MBP immunosensor is quasireversible. Control samples containing a nonspecific human immunoglobulin G (hIgG) antibody were also studied, and calibration curves were obtained by subtraction of the responses for specific and nonspecific antibody-based sensors. Gelatin-MBP and gelatin–TiO2-MBP immunosensors have detection limit of 0.1528ngml−1 and 0.1495ngml−1 respectively. This immunosensor exhibits high sensitivity and low response times (58s for gelatin-MBP and 46s for gelatin–TiO2-MBP immunosensor). The developed label-free impedimetric immunosensors also provide a simple and sensitive detection method for the specific determination of Anti-MBP in human cerebrospinal fluid (CSF) and serum samples.

Detection and discrimination of alpha-fetoprotein with a label-free electrochemical impedance spectroscopy biosensor array based on lectin functionalized carbon nanotubes

A label-free electrochemical impedance spectroscopy (EIS) biosensor for the sensitive determination and discrimination of alpha-fetoprotein (AFP) was developed by employing wheat-germ agglutinin (WGA) lectin as molecular recognition element. The EIS biosensor was fabricated by adsorbing carboxyl-functionalized single-wall carbon nanotubes (SWNTs) onto a screen-printed carbon electrode (SPCE) and subsequently covalently coupling WGA onto the surface of the SWNTs-modified electrode. Upon binding of AFP to the biosensor, the electron transfer resistance was increased and the increase in the electron transfer resistance was linearly proportional to the logarithm of the concentration of AFP in the range from 1 to 100ng/L with a detection limit of 0.1ng/L. It was found that the employment of SWNTs as immobilization platform could reduce the background and enhance the EIS response. Moreover, the lectin-based biosensor array fabricated with different lectins was used to evaluate the glycan expression of AFP N-glycan and discriminate AFP between healthy and cancer patients serum samples. This work demonstrates that the employment of carbon nanotubes as immobilization platform and lectin as molecular recognition element in biosensor array is a promising approach for the determination and discrimination of glycoproteins for cancer diagnosis. The strategy proposed in this work could further be used for high-throughput, label-free profiling of the glycan expression of cancer-related glycoproteins and to develop methods for cancer diagnosis in the early stages.

A label-free impedimetric DNA sensing chip integrated with AC electroosmotic stirring

AC electroosmosis (ACEO) flow and label-free electrochemical impedance spectroscopy are employed to increase the hybridization rate and specifically detect target DNA (tDNA) concentrations. A low-ionic-strength solution, 6.1μS/cm 1mM Tris (pH 9.3), was used to produce ACEO and proved the feasibility of hybridization. Adequate voltage parameters for the simultaneous ACEO driving and DNA hybridization in the 1mM Tris solution were 1.5 Vpp and 200Hz. Moreover, an electrode set with a 1:4 ring width-to-disk diameter ratio exhibited a larger ACEO velocity above the disk electrode surface to improve collecting efficiency. The ACEO-integrated DNA sensing chips could reach 90% saturation hybridization within 117s. The linear range and detection limit of the sensors was 10aM–10pM and 10aM, respectively. The label-free impedimetric DNA sensing chips with integrated ACEO stirring can perform rapid hybridization and highly-sensitive detections to specifically measure tDNA concentrations.

Label-free electrochemical impedance biosensor for sequence-specific recognition of double-stranded DNA

A simple and label-free DNA electrochemical impedance biosensor has been developed for the sequence-specific recognition of double-stranded DNA (ds-DNA). The probe triplex-forming oligonucleotide (TFO) is immobilized on the gold electrode through Au–S interaction. In the presence of the target ds-DNA, the probe TFO interacted with the bases in the major groove of the ds-DNA to form a rigid triplex structure via Hoogsteen hydrogen bonds, leading to an increase of the electron-transfer resistance. It is found that the change of impedance has a linear relationship with the concentration of the target in the range of 0.1–40 nmol L−1, and the detection limit is as low as 0.04 nmol L−1 (S/N = 3).

Label-free electrochemical impedance genosensor based on 1-aminopyrene/graphene hybrids

In this work, we proposed a novel simple protocol for preparing 1-aminopyrene/graphene (ApG) hybrids for fabricating label-free electrochemical impedance genosensor. Graphene, with the structure of a single-atom-thick sheet of sp(2)-bonded carbon atoms, was anchored to 1-aminopyrene (1-Ap) with the pyrenyl group viaπ-stacking interaction. The morphology, conductivity, and interaction of ApG hybrids were characterized by transmission electron microscopy (TEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), UV-visible (UV-vis) and fluorescence spectra. The amino-substituted oligonucleotide probe was conjugated to 1-Ap by the cross-linker glutaraldehyde. The DNA hybridization reaction of oligonucleotide probe with target DNA was monitored by EIS. Under optimum conditions, the proposed biosensor exhibited high sensitivity and a low detection limit for detecting the complementary oligonucleotide. The target oligonucleotide could be quantified in a wide range of 1.0 × 10(-12) to 1.0 × 10(-8) M with good linearity (R = 0.9900) and low detection limit of 4.5 × 10(-13) M (S/N = 3).

The development of a multimarker, label-free electrochemical impedance based point-ofcare technology

The development of a multimarker, label-free electrochemical impedance based point-ofcare technology

One-pot synthesis of carbon-decorated FePt nanoparticles and their application for label-free electrochemical impedance sensing of DNA hybridization

Highly monodispersive FePt nanoparticles (NPs) assembled on carbon nanotubes (CNTs) or graphene oxide (GO) surfaces to form carbon-decorated FePt nanocomposites (FePt–CNTs and FePt–GO) were successfully obtained via a one-pot modified polyol protocol. Using the as-prepared nanocomposites, a novel, ultra-high-resolution electrochemical impedance DNA biosensor for the enhanced detection of the PML–RARA fusion gene in acute promyelocytic leukemia was obtained. The detection limit is up to 10−13 mol L−1 for FePt–CNT-modified electrodes and 10−14 mol L−1 for FePt–GO-modified electrodes, respectively. This work proposed a simple polyol strategy to synthesize carbon-decorated FePt nanocomposites and opened up a new direction in the application of FePt for biosensing.

Label-free electrochemical impedance spectroscopy biosensor for direct detection of cancer cells based on the interaction between carbohydrate and lectin

A novel label-free electrochemical impedance spectroscopy (EIS) biosensor for direct cancer cell detection based on the interaction between carbohydrate and lectin has been developed with good sensitivity and selectivity. In the present work, concanavalin A (Con A), a mannose specific lectin, was immobilized on a gold disk electrode to fabricate the Con A sensor. This sensor was incubated with the cancer cell sample, and the binding of cancer cells with Con A resulted in a change of charge transfer resistance (Rct). EIS measurement was employed to measure the impedance change which reveals the concentration of cancer cells. This method has been successfully applied in human liver cancer cell Bel-7404 for direct and sensitive detection with a detection limit of 234cells/mL. This method could be extended to carry out multi-component diagnosis applications, thus providing enormous potential for applications of cancer monitoring and therapy.

Carboxymethyl chitosan-functionalized graphene for label-free electrochemical cytosensing

Here we demonstrate the fabrication of an effective cytosensor using carboxymethyl chitosan-functionalized graphene (CMC-G) prepared through chemical reduction of graphene oxide. The CMC-G hybrid was further characterized with UV–vis spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis, transmission electron microscopy and atomic force microscopy. Layer-by-layer assembly of CMC-G with polyethyleneimine and folic acid enabled the fabrication of a label-free electrochemical impedance spectroscopy cytosensor with high stability and biocompatibility. The proposed cytosensor exhibited good electrochemical behavior and cell-capture ability for HL-60 cells, and showed a wide linear range and low detection limit for quantification.

Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays

We demonstrate the feasibility of a label-free electrochemical method to detect the kinetics of phosphorylation and dephosphorylation of surface-attached peptides catalyzed by kinase and phosphatase, respectively. The peptides with a sequence specific to c-Src tyrosine kinase and protein tyrosine phosphatase 1B (PTP1B) were first validated with ELISA-based protein tyrosine kinase assay and then functionalized on vertically aligned carbon nanofiber (VACNF) nanoelectrode arrays (NEAs). Real-time electrochemical impedance spectroscopy (REIS) measurements showed reversible impedance changes upon the addition of c-Src kinase and PTP1B phosphatase. Only a small and unreliable impedance variation was observed during the peptide phosphorylation, but a large and fast impedance decrease was observed during the peptide dephosphorylation at different PTP1B concentrations. The REIS data of dephosphorylation displayed a well-defined exponential decay following the Michaelis–Menten heterogeneous enzymatic model with a specific constant, kcat/Km, of (2.1±0.1)×107M−1s−1. Consistent values of the specific constant was measured at PTP1B concentration varying from 1.2 to 2.4nM with the corresponding electrochemical signal decay constant varying from 38.5 to 19.1s. This electrochemical method can be potentially used as a label-free method for profiling enzyme activities in fast reactions.

A Highly Sensitive Electrochemical Impedance Spectroscopy Immunosensor for Determination of 1-Pyrenebutyric Acid Based on the Bifunctionality of Nafion/Gold Nanoparticles Composite Electrode

A simple, highly sensitive and label-free electrochemical impedance spectroscopy (EIS) immunosensor was developed using Nafion and gold nanoparticles (nano-Au/Nafion) composites for the determination of 1-pyrenebutyric acid (PBA). Under the optimal conditions, the amount of immobilized antibody was significantly improved on the nano-Au/Nafion electrode due to the synergistic effect and biocompatibility of Nafion film and gold nanoparticles composites. The results showed that the sensitivity and stability of nano-Au/Nafion composite electrode for PBA detection were much better than those of nano-Au modified glassy carbon electrode (nano-Au/GCE). The plot of increased electron transfer resistances (Rets) against the logarithm of PBA concentration is linear over the range from 0.1 to 150 ng·mL−1 with the detection limit of 0.03 ng·mL−1. The selectivity and accuracy of the proposed EIS immunosensor were evaluated with satisfactory results.

Label-free aptamer-based electrochemical impedance biosensor for 17β-estradiol

A novel aptamer-based label-free electrochemical impedance spectroscopy biosensor for 17β-estradiol has been fabricated. The aptamers were firstly immobilized on the gold electrode through Au-S interaction; the aptamer probe was then bound with the addition of 17β-estradiol to form the estradiol/aptamer complex on the electrode surface. This leads to a significantly larger interfacial electron transfer resistance than that without the addition of 17β-estradiol. The change in the resistance had a linear relationship with 17β-estradiol concentration in the range of 1.0 × 10(-8) to 1.0 × 10(-11) mol L(-1), with a detection limit of 2.0 × 10(-12) mol L(-1). The biosensor showed high selectivity to 17β-estradiol and good stability. The designed biosensor has been applied to detect 17β-estradiol in human urine with satisfactory results.

Green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance DNA hybridization biosensing

Sensitive electrochemical impedance assay of DNA hybridization by using a novel graphene sheets platform was achieved. The graphene sheets were firstly functionalized with 3,4,9,10-perylene tetracarboxylic acid (PTCA). PTCA molecules separated graphene sheets efficiently and introduced more negatively-charged –COOH sites, both of which were beneficial to the decoration of graphene with gold nanoparticles. Then amine-terminated ionic liquid (NH 2-IL) was applied to the reduction of HAuCl 4 to gold nanoparticles. The green-synthesized gold nanoparticles, with the mean diameter of 3 nm, dispersed uniformly on graphene sheets and its outer layer was positively charged imidazole termini. Due to the presence of large graphene sheets and NH 2-IL protected gold nanoparticles, DNA probes could be immobilized via electrostatic interaction and adsorption effect. Electrochemical impedance value increased after DNA probes immobilization and hybridization, which was adopted as the signal for label-free DNA hybridization detection. Unlike previously anchoring DNA to gold nanoparticles, this label-free method was simple and noninvasive. The conserved sequence of the pol gene of human immunodeficiency virus 1 was satisfactorily detected via this strategy.