Electrochemical Immunobiosensor Research Articles

Noninvasive and Continuous Monitoring of On-Chip Stem Cell Osteogenesis Using a Reusable Electrochemical Immunobiosensor.

Noninvasive monitoring of biofabricated tissues during the biomanufacturing process is needed to obtain reproducible, healthy, and functional tissues. Measuring the levels of biomarkers secreted from tissues is a promising strategy to understand the status of tissues during biofabrication. Continuous and real-time information from cultivated tissues enables users to achieve scalable manufacturing. Label-free biosensors are promising candidates for detecting cell secretomes since they can be noninvasive and do not require labor-intensive processes such as cell lysing. Moreover, most conventional monitoring techniques are single-use, conducted at the end of the fabrication process, and, challengingly, are not permissive to in-line and continual detection. To address these challenges, we developed a noninvasive and continual monitoring platform to evaluate the status of cells during the biofabrication process, with a particular focus on monitoring the transient processes that stem cells go through during in vitro differentiation over extended periods. We designed and evaluated a reusable electrochemical immunosensor with the capacity for detecting trace amounts of secreted osteogenic markers, such as osteopontin (OPN). The sensor has a low limit of detection (LOD), high sensitivity, and outstanding selectivity in complex biological media. We used this OPN immunosensor to continuously monitor on-chip osteogenesis of human mesenchymal stem cells (hMSCs) cultured 2D and 3D hydrogel constructs inside a microfluidic bioreactor for more than a month and were able to observe changing levels of OPN secretion during culture. The proposed platform can potentially be adopted for monitoring a variety of biological applications and further developed into a fully automated system for applications in advanced cellular biomanufacturing.

Metal-organic Skeleton (MOF) Composite based Using Electrochemical Immunobiosensor to Detect the Important Tumor Marker of Breast Cancer Glycotype Antigen 15-3

Breast cancer is one of the biggest threats to women's life and health in the world, and the mortality rate has reached the second highest in the world. With the development of metal organic skeleton (MOF) and the advantages of the composite in the field of electrochemistry and biosensing, various metal organic skeleton composites have been widely used in electrochemical biosensors. Thus, electrochemical biosensors have received extensive attention and research in the field of rapid breast cancer detection. This article mainly introduces the detection of sugar-type important tumor marker antigen 15-3, and electrochemical immunobiosensor Excellent performance in breast cancer detection and promising prospects for its application.

A tricarboxylic-ligand decorated neodymium-encapsulated polyoxotungstate with mixed heteroatom fragments

A tricarboxylic-ligand decorated neodymium-encapsulated polyoxotungstate with mixed heteroatom fragments [(CH3)2NH2]2Na7H3[Nd4(HPIII)W8(H2O)12 (H2ptca)2O28][SbIIIW9O33]2·27H2O (1) (H3ptca = 1,2,3-propanetricarboxylic acid) was successfully synthesized. In particular, the distinguished [Nd4(HPIII)W8(H2O)12(H2ptca)2O28][SbIIIW9O33]212− polyanion contains one organic − inorganic hybrid [W4Nd4(H2O)12O10(H2ptca)2]14+ central core, two trivacant Keggin [SbIIIW9O33]9− and one specific [HPIIIW4O20]12− fragments. Furthermore, the composite of 1 and CFMCNs was used to modify GCE to manufacture the 1@CFMCNs-GCE. The 1@CFMCNs-GCE was served as electrochemical immunobiosensor (ECIB) to detect indole-3-acetic acid (IAA), revealing a low detection limit (52.48 pg/mL) and favourable stability as well as reproducibility. This finding not only demonstrates a fascinating 1,2,3-propanetricarboxylic acid decorated neodymium-encapsulated heteropolyoxotungstate with mixed-heteroatom fragments, but also exhibits its promising potential in electrochemical detecting plant growth hormone.

Ag2S quantum dots loaded dendritic mesoporous silica nanospheres as signal amplification labels for ultrasensitive electrochemical immuno-biosensor for Staphylococcus aureus

An electrochemical immuno-biosensor based on Ag 2 S/DMSNs labels was established to detect S. aureus . • An electrochemical immuno-biosensor based on Ag 2 S/DMSNs labels was established to detect S. aureus . • Using DMSNs for Ag 2 S QDs enrichment and signal amplification to improve the detection sensitivity. • A wide linear detection range from 10 to 10 8 CFU mL −1 , with a detection limit of 2 CFU mL −1 . • The immuno-biosensor showed excellent selectivity and stability. • The immuno-biosensor was successfully applied to detect actual samples. Developing novel signal amplification labels in electrochemical immuno-biosensors for ultrasensitive, fast and selective detection of foodborne pathogen is urgent and significant for people's health and life safety. Herein, dendritic mesoporous silica nanospheres (DMSNs) loaded with silver sulfide quantum dots (Ag 2 S QDs) were fabricated and employed as signal amplification labels for highly sensitive electrochemical detection of Staphylococcus aureus ( S. aureus ). DMSNs were treated with 3-aminopropyltrioxysilane to introduce amino group on surfaces to facilitate the immobilization of Ag 2 S QDs. Benefiting from the highly accessible surface areas and large pore channels characteristics given by the unique radial flower-like structure of DMSNs, large amount of Ag 2 S QDs were loaded into the surfaces and pores of the DMSNs to form Ag 2 S/DMSNs. To specifically recognize S. aureus cells, Ag 2 S/DMSNs were modified by anti- S. aureus antibody (Ab) to form Ab-Ag 2 S/DMSNs. When the sandwich-type electrochemical immuno-biosensor operated, Ag(Ⅰ) ions were released from the Ag 2 S/DMSNs label by HNO 3 leaching, and the detection of S. aureus was realized by the differential pulse voltammetry. The electrochemical signal of the prepared immuno-biosensor was substantially magnified when compared to the immuno-biosensor employing Ag 2 S QDs as signal label, since much more Ag 2 S QDs were labeled to each bacterial cell due to the high loading of Ag 2 S QDs on the DMSNs. As a consequence, the developed immuno-biosensor was capable of detecting S. aureus cells down to a limit of 2 colony forming units per mL (CFU mL −1 ) and exhibited a wide linear detection range from 10 CFU mL −1 to 10 8 CFU mL −1 . In addition, the immuno-biosensor showed excellent selectivity and stability for the detection of S. aureus , and was successfully applied to detected S. aureus in milk samples.

Tri-Channel Electrochemical Immunobiosensor for Combined Detections of Multiple Exosome Biomarkers of Lung Cancer.

Current methods for the early diagnosis of cancer can be invasive and costly. In recent years, exosomes have been recognized as potential biomarkers for cancer diagnostics. The common methods for quantitative detection of exosomes, such as nanoparticle tracking analysis (NTA) and flow cytometry, rely on large-scale instruments and complex operation, with results not specific for cancer. Herein, we present a tri-channel electrochemical immunobiosensor for enzyme-free and label-free detecting carcino-embryonic antigen (CEA), neuron-specific enolase (NSE), and cytokeratin 19 fragments (Cyfra21-1) from exosomes for specific early diagnosis of lung cancer. The electrochemical immunobiosensor showed good selectivity and stability. Under optimum experimental conditions, the linear ranges were from 10−3 to 10 ng/mL for CEA, 10−4 to 102 ng/mL for NSE, and 10−3 to 102 ng/mL for Cyfra21-1, and a detection limit down to 10−4 ng/mL was achieved. Furthermore, we performed exosome analysis in three kinds of lung cancer. The results showed a distinct expression level of exosomal markers in different types. These works provide insight into a promising alternative for the quantification of exosomal markers in specific diseases in the following clinical bioassays.

Development of a novel electrochemical immuno-biosensor for circulating biomarkers of the inner ear

Current approaches for diagnosis of hearing or vestibular disorders are mostly based on physical examinations that cannot provide information about the exact location of cellular damage inside the inner ear. Therefore, there is a need for new diagnostic methods capable of identifying the sites of damage through the detection of inner ear blood-circulating biomarkers. Here, we developed the first biosensor platform for rapid detection of otolin-1 and prestin, blood-circulating proteins specifically expressed in the vestibule and cochlea, respectively. The platform was designed on a DNA-based immunoassay that employed conjugated antibodies for target protein recognition, which when bound, altered the DNA-DNA hybridization on the surface, resulting in generation of a concentration-dependent signal. The signal was recorded when the redox moiety brought to the surface by the target enabled a selective electrochemical output directly in whole blood. Signal amplification was acquired by employing high-curvature nanostructured electrodes for sensitive sample analysis at picomolar concentrations with a three-fold quantitative range. The combination of nanostructuring and optimum density of the probes on the surface provided low-picomolar detection limits while utilizing small 10 μL sample volume with a 10-min response time. The proposed immuno-biosensor is highly selective and quantitative and can easily be adapted for rapid detection of any blood-circulating protein using their specific antibodies as recognition elements.

Three-dimensional macroporous gold electrodes superior to conventional gold disk electrodes in the construction of an electrochemical immunobiosensor for Staphylococcus aureus detection.

Herein, a three-dimensional macroporous gold (3DMG) electrode is demonstrated to be a better choice than a conventional gold disk electrode in the construction of an electrochemical immunobiosensor for Staphylococcus aureus (S. aureus) detection. The 3DMG electrode was prepared on a gold disk electrode by one-step electrodeposition using hydrogen bubbles as dynamic templates. The 3DMG electrode has a high electrochemically active surface area with pore sizes ranging from 20 to 50 μm, and these unique features are conducive to the immobilization of primary antibodies and the capture of S. aureus. Secondary antibodies (Ab2) and alkaline phosphatase (ALP) were immobilized on mesoporous silica nanospheres (MSNs), and the resulting ALP-MSNs-Ab2 composites were utilized as signal tags to construct a sandwich-type electrochemical immunobiosensor. S. aureus was measured based on alkaline phosphatase-catalyzed silver deposition and differential pulse voltammetric detection. The linear range is from 5 to 109 CFU mL-1, and the detection limit is 2 CFU mL-1 for S. aureus detection. Due to the signal amplification of the 3DMG electrode, the sensitivity of the immunobiosensor constructed on the 3DMG electrode is 9 times that of an immunobiosensor constructed on a gold disc electrode. The proposed biosensor was successfully applied for detecting S. aureus in milk samples.

Silica nanoparticles-assisted electrochemical biosensor for the rapid, sensitive and specific detection of Escherichia coli

To face the emerging issues raised by bacterial infections in biomedical, food and environmental contexts, the combination of nanotechnologies and biosensors characteristics is increasingly seen as a promising tool to develop rapid, highly sensitive and specific devices for reliable bacterial detection and quantification. We present here the design of an electrochemical immuno-biosensor, integrating non cytotoxic silica nanoparticles (NPs), in a two-step spin coating process. Unlike biosensors that integrate a unique antibody-based layer without NPs, this new tool allows, through cyclic voltammetry measurements, the continuous and non-saturated detection of E. coli bacteria in only 5 min. and can reach up to 103 CFU/mL in 30 min. It offers great promises for industrial purposes as electrode functionalization is rather simple and its architecture can be tuned on demand to target a variety of micro-organisms, and the highlighted indicators (variations of current intensity, intensity ratios and/or difference of potentials) can easily be implemented in a screen displayed packaging.

An electrochemical immunobiosensor for ultrasensitive detection of Escherichia coli O157:H7 using CdS quantum dots-encapsulated metal-organic frameworks as signal-amplifying tags

We report here cadmium sulfide quantum dots (CdS QDs)-encapsulated metal-organic frameworks as signal-amplifying tags for ultrasensitive electrochemical detection of Escherichia coli O157:H7 (E. coli O157:H7). CdS QDs were encapsulated in zeolitic imidazolate framework-8 (ZIF-8) to form CdS@ZIF-8 muti-core-shell particles by in situ growth of ZIF-8 in the presence of CdS QDs. To specifically recognize E. coli O157:H7 cells, CdS@ZIF-8 particles were coated with polyethyleneimine to introduce amino groups on their surfaces, followed by surface modification of anti-E. coli O157:H7 antibody. A sandwich-type electrochemical immunobiosensor for the detection of E. coli O157:H7 was fabricated using CdS@ZIF-8 particles as signal tags. Cd(II) ions were released from CdS@ZIF-8 tags by HCl leaching, enabling the detection of E. coli O157:H7 by differential pulse voltammetry. Under the optimized conditions, the linear range of the biosensor is from 10 to 108 colony forming units (CFU) per mL for E. coli O157:H7 detection, with the detection limit of 3 CFU mL−1 (S/N = 3). The sensitivity of the biosensor for E. coli O157:H7 detection using CdS@ZIF-8 particles as signal tags is 16 times that of a biosensor using CdS QDs as signal tags, because the number of CdS QDs labeled to each bacterial cell increases greatly resulting from a great number of CdS QDs encapsulated in each CdS@ZIF-8 label. This method was successfully used to detect E. coli O157:H7 in milk samples.

Electrochemical aptasensor based on one-step synthesis of Cu2O@aptamer nanospheres for sensitive thrombin detection

Cu2O@aptamer nanospheres were prepared via a facile one-step synthesis using aptamer as template. The precursor CuSO4 was reduced in the presence of aptamer to form Cu2O@aptamer nanospheres. In comparison with the pristine Cu2O immobilized with aptamer, the resultant Cu2O@aptamer nanospheres exhibit high sensitivity for detecting thrombin. The results showed that the developed electrochemical biosensor based on Cu2O@aptamer nanospheres has a detection limit of 0.01ngmL−1 (0.33pM) within the linear range from 0.1 to 50ngmL−1 toward the thrombin detection. Moreover, excellent selectivity toward interfering proteins, such as IgG, IgE, and BSA, was achieved. The fabricated electrochemical immunobiosensor provides a promising alternative for determining other biological samples.

Development of an electrochemical immunosensor for the detection of HbA1c in serum

An electrochemical immuno-biosensor for detecting glycosylated haemoglobin (HbA1c) is reported based on glassy carbon (GC) electrodes with a mixed layer of an oligo(phenylethynylene) molecular wire (MW) and an oligo(ethylene glycol) (OEG). The mixed layer is formed from in situ-generated aryl diazonium cations. To the distal end of the MW, a redox probe 1,1'-di(aminomethyl)ferrocene (FDMA) was attached followed by the covalent attachment of an epitope N-glycosylated pentapeptide (GPP), an analogon to HbA1c, to which an anti-HbA1c monocolonal antibody IgG can selectively bind. HbA1c was detected by a competitive inhibition assay based on the competition for binding to anti-HbA1c IgG antibodies between the analyte in solution, HbA1c, and the surface bound epitope GPP. Exposure of the GPP modified sensing interface to the mixture of anti-HbA1c IgG antibody and HbA1c results in the attenuation of ferrocene electrochemistry due to free antibody binding to the interface. Higher concentrations of analyte led to higher Faradaic currents as less anti-HbA1c IgG is available to bind to the electrode surface. It was observed that there is a good linear relationship between the relative Faradaic current of FDMA and the concentration of HbA1c from 4.5% to 15.1% of total haemoglobin in serum without the need for washing or rinsing steps.

An Electrochemical Immunobiosensor for Direct Detection of Veterinary Drug Residues in Undiluted Complex Matrices

AbstractAn electrochemical immunobiosensor is developed that allows the detection of small molecules, such as drugs, in undiluted complex samples, with no washing or rinsing steps via a displacement assay. This is achieved using an interface comprised of a mixed layer of oligo(phenylethynylene) molecular wire (MW), to allow electrochemical communication, and oligo(ethylene glycol) (OEG) to control the interaction of proteins and electroactive interferences with the electrode surface. The mixed layer is formed from in situ‐generated aryl diazonium cations. To the distal end of the MW, a redox probe 1,1′‐di(aminomethyl)ferrocene (FDMA) is attached followed by an epitope (the structural feature the antibody selectively recognizes) to which an antibody would bind. Association or dissociation of the antibody with the sensing interface causes a modulation of the ferrocene electrochemistry. Antibody complexed electrodes are exposed to samples containing spiked enrofloxacin (unbound target analyte), in milk and environmental water and interrogated using square wave voltammetry (SWV). The lowest detected concentration of free enrofloxacin was 10 pg mL−1 in phosphate buffer, 50 mM, pH 7. For free enrofloxacin detection in undiluted complex matrices, by adding disodium EDTA (50 mM), the recovery obtained was 94.1 % in skim milk and 88.5 % in stream water, respectively as compared to clean phosphate buffer. The immunobiosensor response time was 10–15 minutes. The sensor performance in milk was shown to be superior to a standard method based on Liquid Chromatography Mass Spectroscopy (LC‐MS/MS).

The importance of interfacial design for the sensitivity of a label-free electrochemical immuno-biosensor for small organic molecules

An immuno-biosensing interface comprising a mixed layer of an oligo(ethylene glycol) (OEG) component, and an oligo(phenylethynylene) molecular wire (MW) is described. The OEG controls the interaction of proteins and electroactive interferences with the surface and the MW allows electrochemical communication to the underlying glassy carbon electrode. The layers are formed from in situ generated-aryl diazonium cations. To the distal end of the MW, a redox probe 1,1′-di(aminomethyl)ferrocene is attached followed by the surface bound epitope (the structural feature the antibody selectively recognizes) to which an antibody would bind. Association or disassociation of the antibody with the sensing interface causes a modulation of the ferrocene electrochemistry. X-ray photoelectron spectroscopy, cyclic voltammetry, and square wave voltammetry have been used to characterize the step-wise fabrication of the sensing interface. The influence of the molar ratio of the MW and OEG deposited onto the sensor interface was explored relative to the final sensor sensitivity. Five combinations of MW/OEG 1:0, 1:20, 1:50, 1:75 and 1:100 were tested on sensor sensitivity detection for a model analyte (biotin) free in solution, via a displacement assay. The ratio of 1:50 was found to give the highest sensitivity. At this ratio, good reproducibility (RSD 6.8%) and repeatability (RSD 9.6%) was achieved. This immuno-biosensor provides an intervention free immuno-biosensing platform for agriculture and biomedical samples.

A Highly Sensitive Enzyme-Amplified Immunosensor Based on a Nanoporous Niobium Oxide (Nb2O5) Electrode

We report on the development of an enzyme-amplified sandwich-type immunosensor based on a thin gold film sputtered on an anodic nanoporous niobium oxide (Au@Nb2O5) electrode. The electrocatalytic activity of enzymatically amplified electroactive species and a stable electrode consisting of Au@Nb2O5 were used to obtain a powerful signal amplification of the electrochemical immunobiosensor. The method using this electrochemical biosensor based on an Au@Nb2O5 electrode provides a much better performance than those based on conventional bulk gold or niobium oxide electrodes. Our novel approach does not require any time-consuming cleaning steps to yield reproducible electrochemical signals. In addition, the strong adhesion of gold films on the niobium oxide electrodes offers a very stable substrate during electrochemical biosensing. Cyclic voltammetry measurements indicate that non-specific binding of proteins to the modified Au@Nb2O5 surface is sufficiently low to be ignored in the case of our novel system. Finally, we demonstrated the ability of the biosensor based on an Au@Nb2O5 offering the enhanced performance with a high resolution and sensitivity. Therefore, it is expected that the biosensor based on an Au@Nb2O5 has great potential for highly efficient biological devices.

Electrochemical Immuno-Biosensor for the Rapid Determination of Nuclear Matrix Protein 22 (NMP22) antigen in Urine Samples by Co(III) Phthlocyanine/Fe 3O 4/Au Collide Coimmobilized Electrode

A novel immunosensor is proposed for the rapid separation-free determination of nuclear matrix protein 22 (NMP22) antigen in human urine. The immunosensor is prepared by co-immobilizing HRP-NMP22 antibody, Co(III) phthalocyanine (CoPc), on a Fe 3O 4/Au colloid -modified gold electrode (HRP-Ab-NMP22/Fe 3O 4/Au/CoPc CME) using the self-assembly technique in which CoPc was as the electron transfer medium. The modified electrode shows an excellent electrocatalytic activity for NMP22 antigen in phosphate buffer solution (pH 7.0). After the immunosensor is incubated with NMP22 antigen solution at 37 °C for 20 min, the access of activity center of the HRP to electrode is partly inhibited, which leads to a linear decrease in the catalytic efficiency of HRP in the reduction of immobilized CoPc by H 2O 2 at –50 mV at NMP22 antigen concentration of 1.2–200 ng ml –1. The detection limit was 0.5 ng ml –1. The immunosensor was successfully utilized for the determination of NMP22 antigen from the urine samples of bladder cancer patients, which shows good repeatability, stability, and anti-interference. This method decreases the detection cost and shortens the analytical time, thus would be valuable in clinical immunoassay for the identification of bladder cancer.

Electroactive Silica Nanoparticles for Biological Labeling

Making biosense: Electroactive poly(guanine)-functionalized silica nanoparticles have been synthesized and used as biological labels (see scheme). An electrochemical immunobiosensor based on such labels was developed, which utilizes a mediator-generated catalytic reaction. This immunobiosensor is very sensitive for IgG detection (to a limit of 0.2 ng mL−1 or 1.3 pM), which was attributed to signal amplification from the poly(guanine)-functionalized silica nanoparticles and from the catalytic oxidation of guanine.