Fabrication Of Immunosensors Research Articles

Application of three-dimensional flower-like nanomaterials in the fabrication of sandwich-type electrochemical immunosensors

A novel and ultrasensitive sandwich-type electrochemical immunosensor was developed for the quantitative detection of carcinoembryonic antigen (CEA) in this work.

A label-free electrochemiluminescence immunosensor based on silver nanoparticle hybridized mesoporous carbon for the detection of Aflatoxin B1

A novel electrochemiluminescence (ECL) immunosensor was fabricated for the detection of Aflatoxin B1 (AFB1) by luminol functionalized with silver nanoparticles which were loaded on mesoporous carbon (luminol-AgNPs@MC) as antibody carriers and sensing platform. The immunosensor was prepared by modifying luminol-AgNPs@MC which conjugated AFB1 antibody (anti-AFB1) on glassy carbon electrode. The use of single-step cycle pulse can limit the consecutive reaction between luminol and H2O2 and can be efficient to keep luminol ECL intensity. This proposed method is simple, fast, sensitive, specific, stable and reliable. The results show that the ECL immunosensor has high sensitivity for AFB1 with a linear range from 0.1pg/mL to 50ng/mL and a low detection limit of 50fg/mL. This work reveals that the luminol-AgNPs@MC is excellent sensing platform for the fabrication of simple and sensitive immunosensors. Our study provided a promising technique for the quality inspection of agricultural products.

Copper-doped titanium dioxide nanoparticles as dual-functional labels for fabrication of electrochemical immunosensors

Constructions of versatile electroactive labels are key issues in the development of electrochemical immunosensors. In this study, copper-doped titanium dioxide nanoparticle (Cu@TiO2) was synthesized and used as labels for fabrication of sandwich-type electrochemical immunosensors on glassy carbon electrode (GCE). Due to the presence of copper ions, Cu@TiO2 shows a strong response current when coupled to an electrode. The prepared nanocomposite also shows high electrocatalytic activity towards reduction of hydrogen peroxide (H2O2). The dual functionality of Cu@TiO2 enables the fabrication of immunosensor using different detection modes, that is, square wave voltammetry (SWV) or chronoamperometry (CA). While Cu@TiO2 was used as labels of secondary antibodies (Ab2), carboxyl functionalized graphene oxide (CFGO) was used as electrode materials to immobilize primary antibodies (Ab1). Using human immunoglobulin G (IgG) as a model analyte, the immunosensor shows high sensitivity, acceptable stability and good reproducibility for both detection modes. Under optimal conditions, a linear range from 0.1pg/mL to 100ng/mL with a detection limit of 0.052pg/mL was obtained for SWV analysis. For CA analysis, a wider linear range from 0.01pg/mL to 100ng/mL and a lower detection limit of 0.0043pg/mL were obtained. The proposed metal ion-based enzyme-free and noble metal-free immunosensor may have promising applications in clinical diagnoses and many other fields.

Enzyme modified peptide nanowire as label for the fabrication of electrochemical immunosensor

Ferrocene (Fc) functionalized self-assembled peptide nanowire (Fc-PNW) was synthesized, functionalized and used as label for the fabrication of electrochemical immunosensors to detect substance P (SP). To prepare the label, gold nanoparticles (AuNPs) were immobilized onto the Fc-PNW for the following adsorption of horseradish peroxidase (HRP) and secondary anti-SP antibody (Ab2, Fc-PNW-HRP-Ab2). The role of Fc-PNW in the electrochemical label was not only as supporting matrix for HRP and Ab2, but also as the mediator for HRP to enhance the sensitivity of HRP toward H2O2 detection. The immunosensor was fabricated based on the traditional sandwich protocol with primary anti-SP antibody (Ab1) immobilized onto graphene surface. The catalytic current of HRP toward H2O2 in the presence of mediator Fc was used as signal of the immunosensor. Different parameters, such as the weight ratio of HRP to Ab2 on the Fc-PNW and the antibody–antigen incubation time were optimized to improve the performance of the immunosensor. The resulting immunosensor has high sensitivity, wide linear range (0.01–50ng/mL) and low detection limit (5pg/mL).

Fabrication of a highly sensitive adenosine aptasensor based on covalent attachment of aptamer onto chitosan-carbon nanotubes-ionic liquid nanocomposite

The present study describes the fabrication of a novel electrochemical aptasensor for the label-free determination of adenosine. The immobilization surface is prepared by the modification of a glassy carbon (GC) electrode with a robust nanocomposite containing multiwalled carbon nanotubes, ionic liquid and chitosan(MWCNTs-IL-CHIT). Amine-terminated 12-mer capture probe(ssDNA1) is covalently attached onto the nanocomposite using glutaraldehyde (GA) as the linking agent, a 32-mer adenosine-specific aptamer (ssDNA2) immobilized onto the electrode surface through hybridization with the ssDNA1 and methylene blue (MB) used as the redox probe.The peak current of MB decreased linearly with increasing adenosine concentration due to the formation of aptamer-adenosine complex and displacement of the aptamer from the modified electrode surface.The aptasensor showed alow detection limit of 150pM and high sensitivity of 0.67μAnM−1 at a concentration range of up to 0.4μM.Through the control experiments performed by using some other nucleosides such as guanosine, cytidine and uridine, the excellent specificity of this sensor toward adenosine detection is demonstrated. The potential applicability of the aptasensor is successfully applied for measuring adenosine concentration in blood serum and drug formulation samples.The herein described methodology may hold great promise for fabrication of other aptasensors and immunosensors.

Label-free electrochemiluminescence immunosensor for cardiac troponin I using luminol functionalized gold nanoparticles as a sensing platform

A simple and sensitive label-free electrochemiluminescence (ECL) immunosensor based on the use of luminol functionalized gold nanoparticles (luminol-AuNPs) as antibody carriers and sensing platform is described for detecting the acute myocardial infarction biomarker cTnI. The ECL immunosensor was fabricated by the assembly of luminol-AuNPs conjugated with biotinylated antibodies against cTnI (biotin-anti-cTnI-luminol-AuNPs) with the streptavidin coated AuNPs (SA-AuNPs) modified Au electrode directly by virtue of the biotin-SA system. The fabricated sensing platform exhibited stable and strong ECL intensity and could be used for the recognition of target antigen. In the presence of cTnI, a decrease in the ECL intensity was observed. Direct detection of the ECL signal changes during antigen-antibody immunoreactions can be used for the quantification of cTnI. The ECL response exhibited a quite wide dynamic range from 1000 ng mL(-1) down to 0.1 ng mL(-1). The proposed method has been successfully applied in the detection of cTnI in real plasma samples. This protocol is simple, fast, sensitive, specific, stable and reliable. This work reveals that the luminol-AuNPs are excellent sensing platforms for the fabrication of simple and sensitive immunosensors. Moreover, the proposed strategy may also be extended for the detection of other biomarkers, which is of great application potential in clinical and pharmaceutical analysis.

Application of magnetic core–shell microspheres on reagentless immunosensor based on direct electrochemistry of glucose oxidase for detection of carbohydrate antigen 19-9

This paper reported the production of magnetic core–shell composite microspheres (Fe3O4@SiO2–[email protected]2) which consist of a silica-protected magnetite particle core, an active gold nanoparticle transition layer and an outer mesoporous silica shell with pore channels. During the synthesis of the microspheres, the shape of the nanocomposite was changed from hexagons or quadrangles of Fe3O4 to well-defined spheres of Fe3O4@SiO2–[email protected]2. The synthesized microspheres were characterized by scanning electron microscopy, transmission electron microscopy and X-ray photoelectron spectroscopy. A strategy was proposed for the fabrication of reagentless immunosensors based on direct electrochemistry of glucose oxidase (GOD), which employed multiwall carbon nanotubes and the prepared Fe3O4@SiO2–[email protected]2 as immobilization matrix. The immobilized GOD exhibited direct electrochemistry with a rate constant of 1.8 s−1. The cyclic voltammetric (CV) and differential pulse voltammetric (DPV) peak currents of GOD decreased linearly with the increase of carbohydrate antigen 19-9 (CA19-9) concentrations from 0.01 to 1.11 U mL−1 and 11.11 to 476.11 U mL−1 with a relatively low detection limit of 0.004 U mL−1. Furthermore, this strategy avoided the addition of electron transfer mediator, which simplified the immunoassay procedure and decreased the analytical time.

Impedimetric immunoglobulin G immunosensor based on chemically modified graphenes

Immunosensors which display high sensitivity and selectivity are of utmost importance to the biomedical field. Graphene is a material which has immense potential for the fabrication of immunosensors. For the first time, we evaluate the immunosensing capabilities of various graphene surfaces in this work. We propose a simple and label-free electrochemical impedimetric immunosensor for immunoglobulin G (IgG) based on chemically modified graphene (CMG) surfaces such as graphite oxide, graphene oxide, thermally reduced graphene oxide and electrochemically reduced graphene oxide. Disposable electrochemical printed electrodes were first modified with CMG materials before anti-immunoglobulin G (anti-IgG), which is specific to IgG, was immobilized. The principle of detection lies in the changes in impedance spectra of the redox probe after the attachment of IgG to the immobilized anti-IgG. It was found that thermally reduced graphene oxide has the best performance when compared to the other CMG materials. In addition, the optimal concentration of anti-IgG to be deposited onto the modified electrode surface is 10 μg ml(-1) and the linear range of detection of the immunosensor is from 0.3 μg ml(-1) to 7 μg ml(-1). Finally, the fabricated immunosensor also displays selectivity for IgG.

A new method for non-labeling attomolar detection of diseases based on an individual gold nanorod immunosensor

Herein, we present the use of a single gold nanorod sensor for detection of diseases on an antibody-functionalized surface, based on antibody-antigen interaction and the localized surface plasmon resonance (LSPR) λ(max) shifts of the resonant Rayleigh light scattering spectra. By replacing the cetyltrimethylammonium bromide (CTAB), a tightly packed self-assembled monolayer of HS(CH(2))(11)(OCH(2)CH(2))(6)OCH(2)COOH(OEG(6)) has been successfully formed on the gold nanorod surface prior to the LSPR sensing, leading to the successful fabrication of individual gold nanorod immunosensors. Using prostate specific antigen (PSA) as a protein biomarker, the lowest concentration experimentally detected was as low as 111 aM, corresponding to a 2.79 nm LSPR λ(max) shift. These results indicate that the detection platform is very sensitive and outperforms detection limits of commercial tests for PSA so far. Correlatively, its detection limit can be equally compared to the assays based on DNA biobarcodes. This study shows that a gold nanorod has been used as a single nanobiosensor to detect antigens for the first time; and the detection method based on the resonant Rayleigh scattering spectrum of individual gold nanorods enables a simple, label-free detection with ultrahigh sensitivity.

Development of Label-Free Impedimetric hCG-Immunosensor Using Screen-Printed Electrode

Screen-printing (thick-film) technology is well identified as a reliable technique for fabrication of electrodes which can be used as transducer in biosensor, with several advantages including low cost, design flexibility, process automation, good reproducibility and a wide choice of materials. However, the immobilization of antibody molecules is a decisive factor for successful fabrication of immunosensors. Besides, the ability to measure human Chorionic Gonadotropin (hCG) is important in establishing the diagnosis of gestational trophoblastic disease and germ cell tumors. Moreover, Electrochemical Impedance Spectroscopy (EIS) recently has been being chosen as a main detection method because it is label-free, less destructive to the activities of biomolecule and very sensitive with comparable detection limits as optical-based sensor. In this work, a sensitive label-free impedimetric hCG-immunosensor was constructed by using a commercial screen-printing carbon ink electrode (namely Disposable Electrochemical Printed chip) as a basis. The hCG antibody was immobilized via the entrapment technique on the carbon ink electrode of DEP chip using functional molecule, 1-pyrenebutanoic acid, succinimidyl ester. The experimental results exposed that the designed immunosensor is more sensitive than other previously reported immunosensors, in the case of detection limit and linear range for antigen detection. With optimal fabrication parameters, the detection limit for ?-hCG was 33 pg/mL in 10mM phosphate buffer saline (PBS) solution containing 1% bovine serum albumine (BSA). Furthermore, the use of inexpensive DEP chip as a basis for these immunosensors will allow simple instrumentation, disposable and portable at low cost. This work also demonstrates a new approach to develop a sensitive and labelfree impedimetric immunosensor based on screen-printed electrode for applications in clinical diagnosis.

Glucose oxidase as a blocking agent-based signal amplification strategy for the fabrication of label-free amperometric immunosensors

An effective electrochemical signal amplification strategy based on enzyme membrane modification and redox probe immobilization was proposed to construct an amperometric immunosensor. L-cysteine@ferrocene functionalized chitosan, which possessed not only efficient redox-activity but also excellent film-forming ability, was coated on the bare glass carbon electrode. Moreover, the thiol groups (-SH) in the ferrocenyl compound were used for gold nanoparticles immobilization via the strong bonding interaction, which could further be utilized for the immobilization of antibody biomolecules with well-retained bioactivities. Finally, glucose oxidase (GOD) as the enzyme membrane was employed to block the possible remaining active sites and avoid the nonspecific adsorption. With the excellent electrocatalytic properties of GOD towards glucose, the amplification of antigen-antibody interaction and the enhanced sensitivity could be achieved. Under the optimal conditions, the linear range of the proposed immunosensor for the determination of carcinoembryonic antigen (CEA) was from 0.05 to 100 ng/mL with a detection limit of 0.02 ng/mL (S/N = 3). Moreover, the immunosensor exhibited good selectivity, stability and reproducibility, which provided a promising potential for clinical immunoassay.

Low Density Lipoprotein Detection Based on Antibody Immobilized Self-Assembled Monolayer: Investigations of Kinetic and Thermodynamic Properties

Human plasma low density lipoprotein (LDL) immunosensor based on surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) was fabricated by immobilizing antiapolipoprotein B (AAB) onto self-assembled monolayer (SAM) of 4-aminothiophenol (ATP). The AAB/ATP/Au immunosensor can detect LDL up to 0.252 microM (84 mg/dL) and 0.360 microM (120 mg/dL) with QCM and SPR, respectively. The SPR and QCM measurements were further utilized to study the reaction kinetics of the AAB-LDL interaction. The adsorption process involved was explored using Langmuir adsorption isotherm and Freundlich adsorption models. The thermodynamic parameters such as change in Gibb's free energy (DeltaG(ads)), change in enthalpy (DeltaH(ads)), and change in entropy (DeltaS(ads)) determined at 283, 298, and 308 K revealed that the AAB-LDL interaction is endothermic in nature and is governed by entropy. Kinetic, thermodynamic, and sticking probability studies disclosed that desorption of the water molecules from the active sites of AAB and LDL plays a key role in the interaction process and increase in temperature favors binding of LDL with the AAB/ATP/Au immunosensor. Thus, the studies were utilized to unravel the most important subprocess involved in the adsorption of LDL onto AAB-modified ATP/Au surface that may help in the fabrication of LDL immunosensors with better efficiency.

Fabrication, characterization, and application of potentiometric immunosensor based on biocompatible and controllable three-dimensional porous chitosan membranes

A novel three-dimensional porous chitosan membrane material was prepared as a matrix to encapsulate hepatitis B surface antibody (HBsAb) for fabrication of immunosensors. The porous chitosan matrix was prepared by electrodepositing a designer nanocomposite solution of chitosan-encapsulated silica nanoparticle hybrid film on an ITO electrode, and then removing the silica nanoparticles with HF solution. Using HBsAb as a model, the potentiometric immunosensor was constructed by linking HBsAb molecules to the three-dimensional porous chitosan film using glutaraldehyde as a cross-linker. Scanning electron microscopy was used to investigate the surface morphology of the three-dimensional porous chitosan films. Cyclic voltammograms and electrochemical impedance spectroscopy were used to probe the interfacial properties of the immunosensor. Results showed that the fabricated immunosensor with three-dimensional porous structure possessed high surface area, good mechanical stability, and good hydrophilicity, which provided a biocompatible microenvironment for maintaining the bioactivity of the immobilized protein and increased the protein loading. Therefore, the present immunosensor exhibits a wide linear range from 6.85 to 708 ng mL −1 with a low detection limit of 3.89 ng mL −1 for the detection of hepatitis B surface antigen (HBsAg). This work implied that the biocompatible and controllable three-dimensional porous chitosan membrane possessed potential applications for biosensing.

Scalable fabrication of immunosensors based on carbon nanotube polymer composites

In this work we present the fabrication and characterization of immunosensors basedon polystyrene (PS)–multiwalled carbon nanotube (MWCNT) composites. Theelectrochemical properties of the sensors have been investigated and show that the surfacearea is increased upon addition of the MWCNT–PS layer. Furthermore, a plasmaactivation process is used to partially remove the PS and expose the MWCNTs. Thisresults in a huge increase in the electrochemical area and opens up the possibility ofbinding biomolecules to the MWCNT wall. The MWCNTs have been functionalizedcovalently with a model antibody (rabbit IgG). The biosensors have been tested usingamperometric techniques and show detection limits comparable to standard techniquessuch as ELISA.

A novel capacitive immunosensor for transferrin detection based on ultrathin alumina sol–gel-derived films and gold nanoparticles

A novel capacitive immunosensor was successfully developed for the direct detection of transferrin based on an ultrathin γ-alumina sol–gel-derived film and gold nanoparticles. The thin film was formed and air-dried by dripping Al 2O 3 sol with a microliter syringe on a gold electrode modified with a self-assembled mercaptoacetic acid (MAA) monolayer. After defects in the film were blocked with a long-chain alkylthiol, gold nanoparticles were deposited on the surface of the modified film via a potential step from +1.1 to 0.0 V (versus Ag|AgCl|KCl) for 15 s in a 0.5 M H 2SO 4 solution containing 0.1 mM HAuCl 4. Finally, the antibody was immobilized on the gold nanoparticles under the optimized experimental conditions. The capacitive sensor prepared by the present method can provide high sensitivity because of the ultrathin inorganic film with high permittivity and good biocompatibility of gold nanoparticles. Compared with a capacitive immunoassay based on antibody-embedded ultrathin γ-alumina sol–gel-derived films, the novel immunosensor presented a lower detection limit of 0.05 ng/ml and a wider linear response range of 1–75 ng/ml for transferrin detection. The prepared procedure of the novel immunosensor also provided a new approach to fabrication of capacitive immunosensors.

Micro- and nanoimmunosensors: technology and applications

Since the technique of surface plasmon resonance (SPR) [1] was first applied to the biosensing of an antibody-antigen interaction, the research and technological development of immunosensors have experienced an exponential growth. This technique showed the enormous potential for the direct, real-time and label-free detection of an immunoreaction. The success of the biosensor technology can be deduced from the increasing number of commercially available instruments. This reflects the growing acceptance of the methodology and the increased availability of the instrumentation. But there is still a long way to go to replace completely conventional immunoassays by biosensor technology in many fields and, especially, in clinical diagnostics [2]. Besides the excellent results obtained with the existing immunosensor technologies (mainly related to SPR sensors) [2], we still need biosensors able to detect, in a direct way, very low levels (picomolar to femtomolar) of a great number of chemical and biochemical substances in the areas of environmental monitoring, industrial and food processes, health care, biomedical technology, clinical analysis, etc. Ideally, we must achieve the fabrication of immunosensors with the following characteristics: very high sensitivity and selectivity, broad dynamic range, immunity to matrix effects, capable of multianalyte determination at the same time, with short analysis time, reversible, stable, simple to operate, robust, cheap and of small size [3]. In order to achieve all or most of the aforementioned characteristics, the application of the recent progress in microand nanotechnologies promises to revolutionise the applications of immunosensors [4]. Indeed, the application of such technologies is allowing the miniaturisation of the biosensor devices, the development of microfluidic platforms and the increase in the sensitivity of the immunoassays performed. We are assisting in the appearance of a cascade of new immunosensor devices at the microand nanoscale which could be easily integrated in portable “lab-on-a-chip” platforms to perform “point-of-care” analysis and which in the future could even work inside the human body as “nano-sentinels”.

Electrodeposited Biotinylated Polypyrrole as an Immobilization Method for Impedimetric Immunosensors

The potentialities of an electrodeposited biotinylated polypyrrole film as an immobilization matrix for the fabrication of impedimetric immunosensors are described. Biotinylated antibody (anti-human IgG), used as a model system, was attached to free biotin groups on the electrogenerated polypyrrole film using avidin as a coupling reagent. The resulting recognition interface consisted of a highly oriented monolayer immobilized onto the polymer surface. Cyclic voltammetry was used to characterize the polymer film. Additionally, scanning electron microscopy and atomic force microscopy were used to investigate the morphology of the immobilized material. This immobilization method allows the obtention of a highly reproducible and stable device. The resulting immunosensor has a linear dynamic range of 10-80 ng.ml/sup -1/ of antigen and a detection limit of 10 pg.ml/sup -1/. Furthermore, this immunosensor exhibited minor loss in response after two regeneration steps.

Electrical Characterization of a Thiol SAM on Gold as a First Step for the Fabrication of Immunosensors based on a Quartz Crystal Microbalance

In order to develop a robust biosensor based on quartz crystal microbalance technique for antigen detection, a control of the steps of the surface functionalization has been performed by impedance spectroscopy. The gold electrode is functionalized with the self-assembled monolayer technique. The high insulating properties of the acidic thiol monolayer has been characterized with cyclic voltammetry and impedance spectroscopy. The modified surface is activated with N-hydroxysuccinimide(NHS) and 1-(3-(dimethylamino)propyl)-3-ethylcarbodimide hydrochloride(EDC) cross-linker for antibody coupling. The non-specific sites are blocked with bovin serum albumine molecules. Different concentrations of antigen can be detected with a good reversibility in real time with the quartz crystal microbalance.

Macroporous silicon based capacitive affinity sensor—fabrication and electrochemical studies

We report the fabrication and characterization of capacitive immunosensors based on electrolyte-insulator-porous silicon (EIS) structures. The sensor structure, gold∣silicon∣porous silicon∣SiO 2∣aminosilane∣glutaraldehyde∣antibody∣phosphate-buffered-saline (PBS)∣platinum, is fabricated with a low thermal budget process and is found to be five times more sensitive than an immunosensor on polished silicon with identical die area. Macroporous silicon was prepared by electrochemical etching of polished 〈1 0 0〉 oriented p-type silicon wafers and characterized using SEM, optical microscopy, cyclic voltammetry and impedance spectroscopy. Columnar macroporous structures of different column densities and column sizes were used as the sensor substrates. The capacitive immunosensors were fabricated by anodic oxidation of these porous silicon substrates to form oxide followed by covalent immobilization of antibody (mouse IgG). Antibody-analyte (goat anti-mouse IgG) interactions were monitored via fluorescence microscopy and by change in the measured capacitance. Sensor response was dependent on both the porous silicon column structure and oxide quality.

Impedimetric immunosensor using avidin–biotin for antibody immobilization

The potentialities of an electrodeposited biotinylated polypyrrole film as an immobilisation matrix for the fabrication of impedimetric immunosensors are described. Biotinylated antibody (anti-human IgG), used as a model system, was attached to free biotin groups on the electrogenerated polypyrrole film using avidin as a coupling reagent. This immobilization method allows to obtain a highly reproducible and stable device. The resulting immunosensor has a linear dynamic range of 10–80 ng ml −1 of antigen and a detection limit of 10 pg ml −1. Furthermore, this immunosensor exhibited minor loss in response after two regeneration steps.