Quantum dot-based electrochemical DNA biosensor using a screen-printed graphite surface with embedded bismuth precursor

This work reports the development of screen-printed quantum dots (QDs)-based DNA biosensors utilizing graphite electrodes with embedded bismuth citrate as a bismuth precursor. The sensor surface serves both as a support for the immobilization of the oligonucleotide and as an ultrasensitive voltammetric QDs transducer relying on bismuth nanoparticles. The utility of this biosensor is demonstrated for the detection of the C634R mutation through hybridization of the biotin-tagged target oligonucleotide with a surface-confined capture complementary probe and subsequent reaction with streptavidin-conjugated PbS QDs. The electrochemical transduction step involved anodic stripping voltammetric determination of the Pb(II) released after acidic dissolution of the QDs. Simultaneously with the electrolytic accumulation of Pb on the sensor surface, the embedded bismuth citrate was converted in situ to bismuth nanoparticles enabling ultra-trace Pb determination. The biosensor showed a linear relationship of the Pb(II) peak current with respect to the logarithm of the target DNA concentrations from 0.1 pmol L^− 1 to 10 nmol L^− 1, and the limit of detection was 0.03 pmol L^− 1. The biosensor exhibited effective discrimination between a single-base mismatched sequence and the fully complementary target DNA. These “green” biosensors are inexpensive, lend themselves to easy mass production, and hold promise for ultrasensitive bioassay formats.     

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H₂O₂ product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL⁻¹ to 1000 ng mL⁻¹, and a low detection limit was 0.02 ng mL⁻¹. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.     

Resonance energy transfer between ZnCdHgSe quantum dots and gold nanorods enhancing photoelectrochemical immunosensing of prostate specific antigen

Gold nanorods (AuNRs) integrated with ZnCdHgSe near-infrared quantum dots (AuNRs-ZnCdHgSe QDs) were successfully synthesized and characterized by transmission electron microscope, X-ray photoelectron spectroscopy, and X-ray diffraction. A glassy carbon electrode was decorated with the aforementioned AuNRs-ZnCdHgSe QDs nanocomposite, which provides a biocompatible interface for the subsequent immobilization of prostate specific antibody (anti-PSA). After being successively treated with glutaraldehyde vapor and bovine serum albumin solution, a photoelectrochemical immunosensing platform based on anti-PSA/AuNRs-ZnCdHgSe QDs/GCE was established. The photocurrent response of ZnCdHgSe QDs was tremendously improved by AuNRs due to the effect of resonance energy transfer which can be deduced from the dependence of the enhanced efficiency on the AuNRs with different length-to-diameter ratios and spectral absorption characteristics. A maximum photocurrent was obtained when the absorption spectrum of AuNRs matched well with the emission spectrum of ZnCdHgSe QDs. A photoelectrochemical immunosensor for prostate specific antigen (PSA) was achieved by monitoring the photocurrent variation. The photocurrent variation before and after being interacted with PSA solution exhibits a good linear relationship with the logarithm of its concentration (logc_PSA) in the range from 1.0 pg mL⁻¹ to 50.0 ng mL⁻¹. The detection limit of this photoelectrochemical immunosensor is able to reach 0.1 pg mL⁻¹ (S/N = 3). Determining PSA in clinical human serum was also demonstrated by using the developed anti-PSA(BSA)/AuNRs-ZnCdHgSe QDs/GCE electrode. The results were comparable with those obtained from an enzyme-linked immunosorbent assay method.     

Signal amplification strategy for sensitive immunoassay of prostate specific antigen (PSA) based on ferrocene incorporated polystyrene spheres

A new kind of signal amplification strategy based on ferrocene (Fc) incorporated polystyrene spheres (PS-Fc) was proposed. The synthesized PS-Fc displayed narrow size distribution and good stability. PS-Fc was applied as label to develop immunosensors for prostate specific antigen (PSA) after the typical sandwich immunoreaction by linking anti-PSA antibody (Ab₂) onto PS-Fc. After the fabrication of the immunosensor, tetrahydrofuran (THF) was dropped to dissolve PS and release the contained Fc for the following stripping voltammetric detection. PS-Fc as a new electrochemical label prevented the leakage of Fc and greatly amplified the immunosensor signal. In addition, the good biocompatibility of PS could maintain the bioactivity of the antibodies. The response current was linear to the logarithm of PSA concentration in the range from 0.01 ng mL⁻¹ to 20 ng mL⁻¹ with a detection limit of 1 pg mL⁻¹. The immunosensor results were validated through the detection of PSA in serum samples with satisfactory results.     

One step electrochemically deposited nanocomposite film of chitosan-carbon nanotubes-gold nanoparticles for carcinoembryonic antigen immunosensor application

A simple and controllable one-step electrodeposition method for the preparation of a chitosan-carbon nanotubes-gold nanoparticles (CS-CNTs-GNPs) nanocomposite film was used to fabricate an immunosensor for detection of carcinoembryonic antigen (CEA). The porous three-dimensional CS-CNTs-GNPs nanocomposite film, which offered a large specific surface area for immobilization of antibodies, exhibited improved conductivity, high stability and good biocompatibility. The morphology of the formed nanocomposite film was investigated by scanning electron microscopy (SEM), and the electrochemical behaviors of the immunosensor were characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Under the optimal conditions, the proposed immunosensor could detect CEA in two linear ranges from 0.1 to 2.0 ng mL⁻¹ and from 2.0 to 200.0 ng mL⁻¹, with a detection limit of 0.04 ng mL⁻¹. The immunosensor based on CS-CNTs-GNPs nanocomposite film as the antibody immobilization matrix could exhibit good sensitivity, stability, and reproducibility for the determination of CEA.     

Multiple signal amplified electrochemiluminescent immunoassay for brombuterol detection using gold nanoparticles and polyamidoamine dendrimers-silver nanoribbon

Electrochemiluminescent (ECL) immunosensor with multiple signal amplification was designed based on gold nanoparticles (AuNPs), polyamidoamine dendrimers (PAMAM) and silver-cysteine hybrid nanoribbon (SNR). Low toxic l-cysteine capped CdSe QDs was chosen as the ECL signal probe. To verify the proposed ultrasensitive ECL immunosensor for β-adrenergic agonists (β-AA), we detected Brombuterol (Brom) as a proof-of-principle analyte. Therein, AuNPs as the substrate can simplify the experiment process, accelerate the electron transfer rate, and carry more coating antigen (Ag-OVA) to enlarge ECL signal. On one hand, SNR on the surface of electrode can avoid the aggregation of AuNPs, and SNR-PAMAM-AuNPs also can be acted as a good accelerator for electron transfer. On the other hand, PAMAM (16 -NH₂) functionalized SNR (SNR-PAMAM) with numerous amino groups could be employed to bond abundant actived QDs to further amplify ECL signal. The new immunosensor can offer a simple, reliable, rapid, and selective detection for Brom, which have a dynamic range of 0.005–700 ng mL⁻¹ with a low detection limit at 1.5 pg mL⁻¹. The proposed biosensor will extend the application of nanomaterials in ECL immunoassays and open a new road for the detection of Brom and other β-AA in the future.     

Label-free amperometric immunobiosensor based on a gold colloid and Prussian blue nanocomposite film modified carbon ionic liquid electrode

A novel experimental methodology based on a Prussian blue (PB) and gold nanoparticles (AuNPs) modified carbon ionic liquid electrode (CILE) was developed for use in a label-free amperometric immunosensor for the sensitive detection of human immunoglobulin G (HIgG) as a model protein. The CILE was fabricated by using the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate as binder. Controllable electrodeposition of PB on the surface of the CILE and coating with 3-aminopropyl triethylene silane (APS) formed a film with high electronic catalytic activity and large surface area for the assembly of AuNPs and further immobilization of HIgG antibody. The electrochemistry of the formed nanocomposite biofilm was investigated by electrochemical techniques including cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The HIgG concentration was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody. The decreased differential pulse voltammetric values were proportional to the HIgG concentration in two ranges, 0.05–1.25 ng mL⁻¹ and 1.25–40 ng mL⁻¹, with a detection limit of 0.001 ng mL⁻¹ (S/N = 3). This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of the AuNPs, ionic liquid, and PB amplified electrochemical detection and would therefore be valuable for clinical immunoassays.     

Development of a sensitive surface plasmon resonance immunosensor for detection of 2,4-dinitrotoluene with a novel oligo (ethylene glycol)-based sensor surface

A surface plasmon resonance (SPR) immunosensor for detection of 2,4-dinitrotoluene (2,4-DNT), which is a signature compound of 2,4,6-trinitrotoluene-related explosives, was developed by using a novel oligo (ethylene glycol) (OEG)-based sensor surface. A rabbit polyclonal antibody against 2,4-DNT (anti-DNPh-KLH-400 antibody) was prepared, and the avidity for 2,4-DNT and recognition capability were investigated by indirect competitive ELISA. The sensor surface was fabricated by immobilizing a 2,4-DNT analog onto an OEG-based self-assembled monolayer formed on a gold surface via an OEG linker. The fabricated surface was characterized by Fourier-transform infrared-refractive absorption spectrometry (FTIR-RAS). The immunosensing of 2,4-DNT is based on the indirect competitive principle, in which the immunoreaction between the anti-DNPh-KLH-400 antibody and 2,4-DNT on the sensor surface was inhibited in the presence of free 2,4-DNT in solution. The limit of detection for the immunosensor, calculated as three times the standard deviation of a blank value, was 20 pg mL⁻¹, and the linear dynamic range was found to be between 1 and 100 ng mL⁻¹. Additionally, the fabricated OEG-based surface effectively prevented non-specific adsorption of proteins, and the specific response to anti-DNPh-KLH-400 antibody was maintained for more than 30 measurement cycles.     

New competitive dendrimer-based and highly selective immunosensor for determination of atrazine in environmental,feed and food samples: The importance of antibody selectivity for discrimination among related triazinic metabolites

A new voltammetric competitive immunosensor selective for atrazine, based on the immobilization of a conjugate atrazine-bovine serum albumine on a nanostructured gold substrate previously functionalized with poliamidoaminic dendrimers, was realized, characterized, and validated in different real samples of environmental and food concern. Response of the sensor was reliable, highly selective and suitable for the detection and quantification of atrazine at trace levels in complex matrices such as territorial waters, corn-cultivated soils, corn-containing poultry and bovine feeds and corn flakes for human use. Selectivity studies were focused on desethylatrazine, the principal metabolite generated by long-term microbiological degradation of atrazine, terbutylazine-2-hydroxy and simazine as potential interferents. The response of the developed immunosensor for atrazine was explored over the 10⁻²–10³ ng mL⁻¹ range. Good sensitivity was proved, as limit of detection and limit of quantitation of 1.2 and 5 ng mL⁻¹, respectively, were estimated for atrazine. RSD values <5% over the entire explored range attested a good precision of the device.     

Surface plasmon resonance aided electrochemical immunosensor for CK-MB determination in undiluted serum samples

This article presents a simple chronoamperometric immunosensor for the quantitative assessment of creatine kinase MB (CK-MB) in 50 μL undiluted serum samples. The immunosensor consists of gold working and counter electrodes patterned onto a glass chip by thin-film photolithography and an external Ag|AgCl reference electrode. The detection limit (DL) of the chronoamperometric method is 13 ng mL⁻¹ (DL = 2×RMSD/S, where RMSD is the residual mean standard deviation of the measured points around a calibration curve with a slope of S). In spiked serum samples, the response was linear up to 300 ng mL⁻¹ of CK-MB. A surface plasmon resonance (SPR) system with simultaneous electrochemical detection (EC-SPR) aided the development of the sandwich immunoassay. Real-time monitoring of the SPR signal was used to optimize the capture antibody immobilization, CK-MB and detection antibody binding, as well as to minimize the nonspecific adsorption of serum proteins to the sensor surface. The detection antibody has been labeled with alkaline phosphatase (ALP) enzyme for sensitive electrochemical detection. ALP catalyzes the hydrolysis of ascorbic acid phosphate and generates ascorbic acid, which is measured chronoamperometrically. The electrochemical immunoassay for CK-MB was less sensitive to nonspecific adsorption related interferences, had a better detection limit, and required a lower volume of sample than the SPR method.     

An electrochemical immunosensor based on enzyme-encapsulated liposomes and biocatalytic metal deposition

A novel electrochemical immunosensor based on double signal amplification of enzyme-encapsulated liposomes and biocatalytic metal deposition was developed for the detection of human prostate specific antigen (PSA). Alkaline phosphatase (ALP)-encapsulated and detection antibody-functionalized liposomes were first prepared and used as the detection reagent. In the sandwich immunoassay, the model analyte PSA was first captured by anti-PSA capture antibody immobilized on the electrode and then sandwiched with the functionalized liposomes. The bound liposomes were then lysed with surfactant to release the encapsulated ALP, which served as secondary signal amplification means. ALP on the electrode surface initiated the hydrolysis of ascorbic acid 2-phosphate (AA-p) to produce ascorbic acid. The latter, in turn, reduced silver ions on the electrode surface, leading to deposition of the metal silver on the electrode surface. Linear sweep voltammetry (LSV) was chosen to detect the amount of the deposited silver. The results showed that the anodic stripping peak current was linearly dependent on the PSA concentration in the range of 0.01-100 ng mL⁻¹, and a detection limit as low as 0.007 ng mL⁻¹ can be obtained. Since the cut-off value of human PSA is 4 ng mL⁻¹, the proposed electrochemical immunosensor would be expected to gain widespread applications for the detection of PSA in clinical diagnosis.     

Electrochemical sensors for the simultaneous determination of zinc,cadmium and lead using a Nafion/ionic liquid/graphene composite modified screen-printed carbon electrode

A simple, low cost, and highly sensitive electrochemical sensor, based on a Nafion/ionic liquid/graphene composite modified screen-printed carbon electrode (N/IL/G/SPCE) was developed to determine zinc (Zn(II)), cadmium (Cd(II)), and lead (Pb(II)) simultaneously. This disposable electrode shows excellent conductivity and fast electron transfer kinetics. By in situ plating with a bismuth film (BiF), the developed electrode exhibited well-defined and separate peaks for Zn(II), Cd(II), and Pb(II) by square wave anodic stripping voltammetry (SWASV). Analytical characteristics of the BiF/N/IL/G/SPCE were explored with calibration curves which were found to be linear for Zn(II), Cd(II), and Pb(II) concentrations over the range from 0.1 to 100.0 ng L⁻¹. With an accumulation period of 120 s detection limits of 0.09 ng mL⁻¹, 0.06 ng L⁻¹ and 0.08 ng L⁻¹ were obtained for Zn(II), Cd(II) and Pb(II), respectively using the BiF/N/IL/G/SPCE sensor, calculated as 3σ value of the blank. In addition, the developed electrode displayed a good repeatability and reproducibility. The interference from other common ions associated with Zn(II), Cd(II) and Pb(II) detection could be effectively avoided. Finally, the proposed analytical procedure was applied to detect the trace metal ions in drinking water samples with satisfactory results which demonstrates the suitability of the BiF/N/IL/G/SPCE to detect heavy metals in water samples and the results agreed well with those obtained by inductively coupled plasma mass spectrometry.     

Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL⁻¹ with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics.     

Organic-inorganic matrix for electrochemical immunoassay: Detection of human IgG based on ZnO/chitosan composite

A new strategy to construct amperometric immunosensor for human IgG assay based on ZnO/chitosan composite as sensing platform has been described. This material, which combined the advantages of inorganic species, ZnO and organic polymer, chitosan, can maintain biological activity well. A sequential sandwich immunoassay format was performed on the ZnO/chitosan composite supported by glass carbon electrode (GCE) using goat-anti-human IgG antibody (IgG Ab) and human IgG as a model system. Amperometry was used to determine the amount of horse-radish peroxidase (HRP) fixed on the sensor surface, which was related to the content of the desired human IgG. Assay conditions that were optimized included the amount of labeled antibody, the incubation time and temperature, the pH of the substrate solution, etc. Using hydroquinone as a mediator, amperometric detection at −150 mV (versus SCE) resulted in a detection range 2.5-500 ng mL⁻¹, with a detection limit of 1.2 ng mL⁻¹. The simple manipulations of the construction of ZnO/chitosan composite, as well as low-cost and broad linear range, are the main features of the proposed immunosensing method.     

Preparation of TiO2 nanosheet-carbon nanotube composite as immobilization platform for both primary and secondary antibodies in electrochemical immunoassay

TiO₂ nanosheets (TNSs) were synthesized and deposited on multi-wall carbon nanotubes (MWCNTs) to form a nano-composite through a hydrothermal method, followed by the characterization with various spectroscopic and microscopic techniques. The TNS-MWCNT composite was then applied as not only an electrode scaffold to immobilize primary antibody, but also as a carrier to load secondary antibody and horseradish peroxidase (HRP). In both cases, bis(sulfosuccinimidyl) suberate sodium salt acted as an amino cross-linker to covalently bind the biomolecules on TNS-MWCNT composite through their surface primary amino groups. After the sandwich-type immunoreaction, HPR was quantitatively captured on the electrode surface via the binding between secondary antibody and antigen, and electrochemical response of the immunosensor was then amplified by a H₂O₂ mediated HRP catalytic reaction. Using α-Fetoprotein as a model analyte, a linear range between 0.005 and 320 ng mL⁻¹ with a detection limit of 2.0 pg mL⁻¹ was achieved by differential pulse voltammetry. The improved immunosensor performance could be attributed to the biocompatibility and high specific surface area of TNS, and excellent electrical conductivity of MWCNTs, which accelerated the electron transfer at the electrode surface.     

A streptavidin functionalized graphene oxide/Au nanoparticles composite for the construction of sensitive chemiluminescent immunosensor

In this work, a novel streptavidin functionalized graphene oxide/Au nanoparticles (streptavidin/GO/AuNPs) composite is prepared and for the first time used to construct sensitive chemiluminescent immunosensor for the detection of tumor marker. The streptavidin/GO/AuNPs composite and the immunosensor are characterized using scanning electron microscopy, static water contact angle measurement and electrochemical impedance spectroscopy. The biofunctionalized composite has large reactive surface area and excellent biocompatibility, thus the capture antibody can be efficiently immobilized on its surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as a model, the proposed chemiluminescent immunosensor shows a wide linear range from 0.001 to 0.1 ng mL⁻¹ with an extremely low detection limit down to 0.61 pg mL⁻¹. The resulting AFP immunosensor shows high detection sensitivity, fast assay speed, acceptable detection and fabrication reproducibility, good specificity and stability. The assay results of serum samples with the proposed method are in an acceptable agreement with the reference values. This work provides a promising biofunctionalized nanostructure for sensitive biosensing applications.     

Development of an immunosensor for the detection of testosterone in bovine urine

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H₂O₂) substrate system, at +100 mV. The EC₅₀ values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL⁻¹ and 960 pg mL⁻¹, respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL⁻¹ to 40 ng mL⁻¹; while that in urine ranged from 0.03 ng mL⁻¹ to 1.6 ng mL⁻¹. The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL⁻¹ and 1.8 pg mL⁻¹. Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV < 13%, n = 3) and repeatability (CV < 9%, n = 3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 °C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.     

A novel antibody–antigen based impedimetric immunosensor for low level detection of HER2 in serum samples of breast cancer patients via modification of a gold nanoparticles decorated multiwall carbon nanotube-ionic liquid electrode

A highly sensitive impedimetric immunosensor based on a gold nanoparticles/multiwall carbon nanotube-ionic liquid electrode (AuNPs/MW-CILE) was developed for the determination of human epidermal growth factor receptor 2 (HER2). Gold nanoparticles were used to enhance the extent of immobilization and to retain the immunoactivity of the antibody Herceptin on the electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed for characterization of various layers coated onto the AuNPs/MW-CILE. The impedance measurements at different steps were based on the charge transfer kinetics of the [Fe(CN)₆]^3−/4− redox pair. The immobilization of antibody and the corresponding antigen–antibody interaction at the electrode surface altered the interfacial electron transfer. The interactions of antibody with various concentrations of antigen were also monitored via the change of impedance response. The results showed that the charge transfer resistance increases linearly with increasing concentrations of HER2 antigen. The linear range and limit of detection were found as 10–110 ng mL⁻¹ and 7.4 ng mL⁻¹, respectively. The sensitivity and specificity of the immunosensor were validated. The results showed that the prepared immunosensor is a useful tool for screening of trace amounts of HER2 in serum samples of breast cancer patients.     

An electrochemical biosensor for α-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles

A novel and effective electrochemical immunosensor for the rapid determination of α-fetoprotein (AFP) based on carbon paste electrode (CPE) consisting of room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF₆) and graphite. The surface of the CPE was modified with gold nanoparticles for the immobilization of the α-fetoprotein antibody (anti-AFP). By sandwiching the antigen between anti-AFP on the CPE modified with gold nanoparticles and the secondary antibody, polyclonal anti-human-AFP labeled with horseradish peroxidase (HRP-labeled anti-AFP), the immunoassay was established. The concentration of AFP was determined based on differential pulse voltammetry (DPV) signal, which was generated in the reaction between O-aminophenol (OAP) and H₂O₂ catalyzed by HRP labeled on the sandwich immunosensor. AFP concentration could be measured in a linear range of 0.50-80.00 ng mL⁻¹ with a detection limit of 0.25 ng mL⁻¹. The immunosensor exhibited high sensitivity and good stability, and would be valuable for clinical assay of AFP.     

Hot electron induced cathodic electrochemiluminescence at AuSb alloy electrode for fabricating immunosensor with self-assembled monolayers

Thin antimony oxide covered AuSb alloy electrode was firstly found to be an excellent cold cathode for generating hot electrons during cathodic pulse polarization. Owing to the injection of hot electrons and the subsequent generation of hydrated electrons, fluorescein iso-thiocyanate (FITC) that cannot be excited in common ECL was cathodically excited at the alloy electrode. Self-assembled thiol monolayers were formed on the electrode surface due to the presence of Au in the alloy, to which strepavidin was covalently bound, and then biotinylated antibody was immobilized through the strepavidin-biotin interaction. As a simple model, an immunosensor for the detection of human IgG (hIgG) using FITC as labeling agent was fabricated. ECL signals were responsive to the amount of hIgG bounded to the immunosensor. The ECL intensity was linearly changed with the logarithm of hIgG concentration in the range of 1.0-1000 ng mL⁻¹, and the detection limit was ca. 0.3 ng mL⁻¹ (S/N = 3). The proposed immunosensor showed a broad linear range (three magnitudes), good reproducibility and stability, which is promising in detecting FITC-based labels in various types of bioaffinity assays.