Label‐Free Impedimetric Immunosensor for Nerve Growth Factor Protein Constructed Using an Automated Dispensing System

We describe the development, validation and optimisation of label‐free immunosensors for Nerve Growth Factor (NGF) protein. Screen‐printed carbon electrodes were electrochemically coated with polyaniline, and onto this antibodies to NGF were immobilised using electrostatic or neutravidin‐biotin interactions. These were constructed manually or using an automated liquid dispensing system. QCM demonstrated deposition of individual layer stages and binding of NGF. Studies revealed clear increases in immunosensor impedance following antigen exposure. Automated methodologies produced sensors with lowered sample‐to‐sample variation. Affinity produced electrodes offered higher sensitivities than those produced utilising electrostatic interactions. NGF between 25–1000 pg mL^?1 could be determined using the optimised sensor.     

On‐Off PVC Membrane Based Potentiometric Immunosensor for Label‐Free Detection of Alpha‐Fetoprotein

A poly(vinylchloride) (PVC) membrane based potentiometric immunosensor for the direct detection of alpha‐fetoprotein (AFP) has been developed. First, Au colloid particle was chemisorbed upon amino groups of o‐phenylenediamine, which were dissolved in plasticized PVC membrane. Then alpha‐fetoprotein antibody (anti‐AFP) was immobilized upon the surface of the Au colloid particle to prepare a potentiometric AFP immunosensor. The Au colloid particle modified PVC membrane was characterized by digital photo and transmission electron microscope (TEM). The immunosensor exhibited fast potentiometric response (≤4 min) and showed specific response to AFP in the range of 4.9 to 158.5 ng/mL with a correlation coefficient of 0.9971 and a detection limit of 1.6 ng/mL. The factors influencing the performance of the immunosensor were also studied in detail. Moreover, the proposed method is economical and efficient as well as potentially attractive for clinical immunoassays.     

Label‐Free and Ultra‐Low Level Detection of Salmonella enterica Serovar Typhimurium Using Electrochemical Impedance Spectroscopy

An immunosensor for rapid and low level detection of the bacterial pathogen Salmonella enterica Serovar Typhimurium was designed and developed based upon label‐free electrochemical impedance spectroscopy and correlated to viable cell counts. The immunosensor was fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode by immobilizing monoclonal antibody (MAb) specific against Salmonella typhimurium cell surface lipopolysaccharide (LPS) onto the surface of the electrode. Use of mass‐fabricated and electroplated PCB electrodes allowed for disposable, highly sensitive, and rapid detection of Salmonella in an aqueous environment. Results demonstrate that in purified solution, Salmonella can be detected as low as 10 CFU in a 100 μL volume and label‐free and rapid manner in fewer than 90 s. The cost effective approach described here can be used for detection of pathogens with relevance for healthcare, food, and environmental applications.     

Label‐Free Bioelectronic Detection of Point Mutation by Using Peptide Nucleic Acid Probes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes with a label‐free protocol is described. The detection of PNA‐DNA and DNA‐DNA hybridizations were accomplished based on the oxidation signal of guanine by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE). It was observed that the oxidation signals of guanine obtained from the PNA and DNA probe modified CPEs were higher than those obtained from the PNA‐DNA and DNA‐DNA hybrid modified CPEs due to the accessible unbound guanine bases. The detection of hybridization between PNA probe and point mutation containing DNA target sequences was clearly observed due to the difference of the oxidation signals of guanine bases, because the point mutation was guanine nearly at the middle of the sequence. The effect of the DNA target concentration on the hybridization signal was also observed. The PNA probe was also challenged with excessive and equal amount of noncomplementary DNA and also mixtures of point mutation and target DNA.     

Label‐Free Amperometric Detection of Albumin with an Oil/Water‐type Flow Cell for Urine Protein Analysis

The 1,2‐dichloroethane (DCE)/water interface, with an anionic surfactant, dinonylnaphthalenesulfonate (DNNS^?), being present in DCE, was utilized for label‐free detection of albumin. An oil/water‐type flow cell was prepared using a porous PTFE tube and dipping the tube in the DCE solution containing DNNS^?. This flow cell provided a well‐defined current response linear to the albumin concentration up to 10 µM with a detection limit of 1.2 µM. The current response is due to the interfacial adsorption of albumin molecules depending on the Galvani potential difference. Possible interference from creatinine in the urine could be avoided by a conventional dialysis treatment.     

Detection of Cocaine Using the Flow Immunosensor

Abstract

A continuous flow immunosensor has been designed for the detection of cocaine in aqueous samples. The continuous flow immunosensor relies on the displacement of fluorophore-labeled antigen from immobilized monoclonal antibody. The sensitivity and accuracy of the flow immunosensor were investigated while varying the parameters of immobilized antibody density, flow rate, amount of antibody-coated Sepharose used in each column, and the saturation of antibody binding sites with fluorophore-labeled antigen. Using a low density of immobilized anti-benzoylecgonine antibody, as little as 5 ng/ml cocaine could be detected. Small amounts of antibody-coated Sepharose could be used repeatedly and the lifetime of the column was proportional to the amount of Sepharose used. Results were obtained in less than a minute and cross-reactivity against various other drugs was negligible.     

The Signal‐Enhanced Label‐Free Immunosensor Based on Assembly of Prussian Blue‐SiO2 Nanocomposite for Amperometric Measurement of Neuron‐Specific Enolase

A signal‐enhanced label‐free electrochemical immunosensor was constructed by the employment of Prussian blue doped silica dioxide (PB‐SiO₂) nanocomposite. At first, PB‐SiO₂ nanocomposite which was produced by using a microemulsion method was used to obtain a nanostructural monolayer on a glassy carbon electrode (GCE) surface. Next amino‐functionalized interface were prepared by self‐assembling 3‐aminopropyltriethoxy silane (APTES) on the PB‐SiO₂ nanoparticle surface. Then chitosan stabled gold nanoparticle (CS‐nanoAu) was subsequently attached, while the entire surface was finally loaded with neuron‐specific enolase antibody (anti‐NSE) via the adsorption of gold nanoparticle. The sensitivity of the proposed immunosensor has greatly improved as the PB‐SiO₂ nanostructural sensing film provides plenty of active sites which might catalyze the reduction of H₂O₂. The immunosensor exhibited good linear behavior in the concentration range from 0.25–5.0 and 5.0–75 ng/mL for the quantitative analysis of neuron‐specific enolase (NSE), a putative serum marker of small‐cell lung carcinoma (SCLC), with a limit of detection of 0.08 ng/mL. The resulting NSE immunosensor showed high sensitivity and long‐term lifetime which can be attributed to the extremely high catalytic activity and biocompatibility of CS‐nanoAu/APTES/PB‐SiO₂ nanostructural multilayers.     

A Renewable Potentiometric Immunosensor Based on Fe3O4 Nanoparticles Immobilized Anti‐IgG

A renewable potentiometric immunosensor for detection of immunoglobulin G (IgG) has been developed by magnetic force attraction of Fe₃O₄ nanoparticles immobilized goat‐anti‐human IgG antibody. For preparing sensitive film of the sensor, cysteine was bonded on the nano‐Fe₃O₄ particles surface. The cysteine functionalized magnetic nanoparticles was attracted on a solid paraffin carbon paste electrode surface to covalently immobilize of anti‐immunoglobulin G (anti‐IgG) by employing a conventional glutaraldehyde‐crosslinking method. The immunosensor showed a specific response to human immunoglobulin G in the range of 0.1–1.2 ng/mL with a detection limit of 0.023 ng/mL. The immunosensor based on the magnetic nanoparticles was made easily by this method. It can be used expediently, renewed easily and low‐cost relatively. The renewable potentiometric immunosensor with better stability and higher sensitivity can be employed extensively in clinical diagnosis, monitoring of disease and environmental studies and etc.     

DNA‐Directed Attachment of Carbon Nanotubes for Enhanced Label‐Free Electrochemical Detection of DNA Hybridization

Multi‐walled carbon nanotubes (MWNTs) were used as nanowires, which combined DNA molecules to a carbon paste electrode (CPE). The attachment of MWNT on the electrode surface was controlled by a hybridization assay between adenine and thymine containing oligonucleotides. The appearance of guanine oxidation signal after hybridization with target DNA greatly simplified the specific sequence DNA detection mechanism. Combination of sidewall‐ and end‐functionalization of MWNT provided a significant enhancement in the voltammetric signal of guanine oxidation in comparison with the signals obtained from only end‐oxidized MWNT modified CPE and a bare CPE. A control experiment involving adenine containing polynucleotide (poly(A)) instead of adenine probe modified MWNT was performed. The effect of target and noncomplementary DNA concentration on the guanine signal was also monitored. Discrimination against single‐base mismatch and noncomplementary DNA was achieved by surfactant containing washing solution. The promising conductivity of carbon nanotubes, and the creation of a larger surface area for DNA immobilization by sidewall‐ and end‐oxidation of MWNT provided a detection limit down to 10 pg/mL, which is compatible with the demand of the genetic tests.     

Miniaturized Electrochemical Immunosensor for Label‐Free Detection of Growth Hormone

A miniaturized immunosensor was developed in connection with disposable screen‐printed carbon strips (SPCS) for the point‐of‐care detection of growth hormone. The performance of the miniaturized system was studied using growth hormone (GH) as the target protein and compared with a conventional electrochemical analyzer. The detection limit of 25 pg/mL was observed for GH in 20 µL sample volume, which indicated that this versatile platform can be easily adapted for decentralized electrochemical immunosensing of clinically important proteins.     

Label‐Free Electrochemical Imaging of Latent Fingerprints on Metal Surfaces

A label‐free electrochemical method based on scanning electrochemical microscopy (SECM) has been developed to image latent fingerprints with high resolution on five kinds of metal surfaces (platinum, gold, silver, copper and stainless steel), as it could measure the minor conductivity differences of the substrate surface and avoid the interference of the background‐color. The images of sebaceous fingerprints on clean metals were revealed by SECM with ferrocene methanol acting as a redox mediator to detect the topology of the fingerprint deposits in constant‐height feedback mode. Inhibition of electrochemical processes on areas of the surface masked by the insulating fingerprint residues generated a negative image of the fingerprint.     

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label‐Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold‐nanoparticles over graphene screen‐printed electrode (SPE). The antibody was covalently bound to CS via its Fc‐terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG‐immunosensor displayed linear response against the logarithm‐hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low‐cost, high sensitivity and selectivity.     

Phenylboronic Acid‐Gold Nanoparticles for Potentiometric Detection of Saccharides

We propose a concept for the analytical application of gold nanoparticles modified by mercaptophenylboronic acid as a receptor of saccharides in a potentiometric sensor based on a conducting polymer.     

Label‐free DNA Hybridization Based on Coupling of a Heated Carbon Paste Electrode with Magnetic Separations

The technique of electrically heated carbon paste electrodes is applied for label‐free detection of DNA hybridization after magnetic isolation. Coupling of both techniques leads to highly selective and sensitive detection of DNA hybridization. Minimal contributions of nonhybridized DNA are thus coupled to an enhanced signal. Application of elevated temperatures during the accumulation step yields a max. 6‐fold enhancement of guanine oxidation signals compared to room temperature deposition. Use of noncomplementary nucleic acids gives no significant peaks at room or elevated temperatures. The noise level remained unaffected. Variation of the target concentration results in linear calibration plots under hot accumulation conditions.     

A Simple and Renewable Nanoporous Gold‐based Electrochemical Sensor for Bisphenol A Detection

A sensitive and simple electrochemical sensor based on nanoporous gold (NPG) was developed for the detection of bisphenol A (BPA). NPG was prepared by the dealloying method. The NPG modified glassy carbon electrode (GCE) displayed excellent catalytic activity towards the electrooxidation of BPA. The mechanism of the electrooxidation of BPA on NPG/GCE sensor was inferred. The sensor showed a linear range from 0.1 μM to 50 μM with a detection limit of 12.1 nM BPA. Specially, a simple but effective approach was attempted to renew the used sensor. The application of the sensor for real sample analysis was demonstrated.     

Label‐Free Logic Modules and Two‐Layer Cascade Based on Stem‐Loop Probes Containing a G‐Quadruplex Domain

A simple, versatile, and label‐free DNA computing strategy was designed by using toehold‐mediated strand displacement and stem‐loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two‐layer logic cascade were constructed. The probes contain a G‐quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light‐up fluorescent signal of G‐quadruplex/NMM complex was used as the output readout. The inputs are the disease‐specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label‐free and modular strategy might be adapted in multi‐target diagnosis through DNA hybridization and aptamer‐target interaction.     

Detection of Human Interleukine‐2 Gene Using a Label‐Free Electrochemical DNA Hybridization Biosensor on the Basis of a Non‐Inosine Substituted Probe

The human interleukine‐2 gene (hIL‐2) is detected with a label‐free DNA hybridization biosensor using a non‐inosine substituted probe. The sensor relies on the immobilization of a 20‐mer antisense single strand oligonucleotide (chIL‐2) related to the human interleukine‐2 gene on the pencil graphite electrode (PGE) as a probe. The guanine oxidation signal was monitored using anodic differential pulse voltammetry (ADPV). The electrochemical pretreatment of the polished PGE at 1.80 V for 5 min is suggested. Then, 5 min immobilization at 0.50 V was found as the optimum condition for immobilization of the probe. The electrochemical detection of hybridization between chIL‐2 and hIL‐2 as a target was accomplished. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Diagnostic performance of the biosensor is described and the detection limit is found 36 pg/μL.     

Sensitive Detection of Small Molecule–Protein Interactions on a Metal–Insulator–Metal Label‐Free Biosensing Platform

The need to develop label‐free biosensing devices that enable rapid analyses of interactions between small molecules/peptides and proteins for post‐genomic studies has increased significantly. We report a simple metal–insulator–metal (MIM) geometry for fabricating a highly sensitive detection platform for biosensing. MIM substrates consisting of an Au–PMMA–Ag nanolayer were extensively studied using both theoretical and experimental approaches. By monitoring reflectivity changes at the normal incidence angle, we observed molecular interactions as the thickness of the biolayer increased on the substrate surface. These interactions included the adsorption of various proteins (Mw=6–150 kD) and interactions between small molecules (Mw≤2 kD) and the immobilized proteins. The interaction of designed monosaccharide‐modified designed peptides with various lectins was also clearly detected. These interactions could not be detected by the conventional Au‐only substrate. Thus, the MIM approach affords a powerful label‐free biosensing device that will aid our understanding of protein interactions and recognition.     

A Facile Electrochemical Immunosensor with Mesoporous Alumina for Detection of Carcinoembryonic Antigen

A label‐free electrochemical immunosensor for the sensitive determination of carcinoembryonic antigen (CEA) was fabricated by immobilizing anti‐CEA onto mesoporous alumina (meso‐Al₂O₃) dispersed in chitosan (0.5 %wt) by the cross‐linking method using glutaraldehyde. Due to its plenty of active sites, meso‐Al₂O₃ showed high catalysis towards hydroquinone. With the electrocatalytic ability of meso‐Al₂O₃ for the reduction of hydroquinone, the current signal of the antigen‐antibody reaction was amplified and the enhanced sensitivity was achieved. The current decreased linearly with CEA concentration in the range of 0.04 to 10 ng/mL (26 pg/mL, S/N=3). The immunosensor had good selectivity and wonderful stability. Furthermore it was applied to the analysis of CEA in serum sample with satisfactory results.     

An Electrochemical Immunosensor for C‐Reactive Protein Based on Multi‐Walled Carbon Nanotube‐Modified Electrodes

C‐reactive protein (CRP) is a nonspecific biomarker of inflammation and infection that can be used as a predictive risk marker of cardiovascular disease in asymptomatic individuals or as a prognostic marker of recurrent ischemia and death among patients with coronary heart disease or stroke. We developed a sensitive, disposable, and easy to operate sensor based on heterogeneous sandwich immunoassay for high sensitivity CRP (hs‐CRP) measurement. We used screen‐printed electrodes modified with multi‐walled carbon nanotubes and protein A to ensure the oriented immobilization of anti‐CRP antibodies. CRP was quantitatively measured down to a concentration of 0.5 ng mL^?1, enabling high dilution of the samples before measurement and consequently reducing interferences present in the serum. The sensor developed is suitable for point of care detection of hs‐CRP at clinically relevant concentrations for the diagnosis of both conventional and low‐grade inflammations.