Affinity Biosensors Research Articles

A Novel Mass-Producible Capacitive Sensor with Fully Symmetric 3D Structure and Microfluidics for Cells Detection.

Affinity biosensors of interdigitated electrodes have been widely used in cell detection. This research presents a mass-producible and disposable three-dimensional (3D) structure capacitive sensor based on the integrated circuit package lead frames for cell concentration detection. The fully symmetric 3D interdigital electrode structure makes the sensor more homogeneous and sensitive. (3-Aminopropyl) triethoxysilane (APTES) and glutaraldehyde are immobilized onto gold-plated electrodes. By overlaying the microfluidic channels on top, the volume of the solution is kept constant to obtain repeatable measured capacitance values. Moreover, using the upgraded reading and writing functions and circular measurement of the E4980A Data Transfer Program, an automatic multigroup test system is developed. It is shown that the cell concentration and capacitance are inversely correlated, and the cell concentration range of 103–106 CFU∙mL−1 is achieved. In addition, the rate of capacitance change matches that of state-of-the-art biosensors reported. A program is developed to find the optimal voltage and frequency for linear fitting between the capacitance change and cell concentration. Future work will employ machine learning-based data analysis to drug resistance sensitivity test of cell lines and cell survival status.

Pushing the limits of electrochemistry toward challenging applications in clinical diagnosis, prognosis, and therapeutic action.

Constant progress in the identification of biomarkers at different molecular levels in samples of different natures, and the need to conduct routine analyses, even in limited-resource settings involving simple and short protocols, are examples of the growing current clinical demands not satisfied by conventional available techniques. In this context, the unique features offered by electrochemical biosensors, including affordability, real-time and reagentless monitoring, simple handling and portability, and versatility, make them especially interesting for adaptation to the increasingly challenging requirements of current clinical and point-of-care (POC) diagnostics. This has allowed the continuous development of strategies with improved performance in the clinical field that were unthinkable just a few years ago. After a brief introduction to the types and characteristics of clinically relevant biomarkers/samples, requirements for their analysis, and currently available methodologies, this review article provides a critical discussion of the most important developments and relevant applications involving electrochemical biosensors reported in the last five years in response to the demands of current diagnostic, prognostic, and therapeutic actions related to high prevalence and high mortality diseases and disorders. Special attention is paid to the rational design of surface chemistry and the use/modification of state-of-the-art nanomaterials to construct electrochemical bioscaffolds with antifouling properties that can be applied to the single or multiplex determination of biomarkers of accepted or emerging clinical relevance in particularly complex clinical samples, such as undiluted liquid biopsies, whole cells, and paraffin-embedded tissues, which have scarcely been explored using conventional techniques or electrochemical biosensing. Key points guiding future development, challenges to be addressed to further push the limits of electrochemical biosensors towards new challenging applications, and their introduction to the market are also discussed.

Layer-by-layer biofunctionalization of nanostructured porous silicon for high-sensitivity and high-selectivity label-free affinity biosensing

Nanostructured materials premise to revolutionize the label-free biosensing of analytes for clinical applications, leveraging the deeper interaction between materials and analytes with comparable size. However, when the characteristic dimension of the materials reduces to the nanoscale, the surface functionalization for the binding of bioreceptors becomes a complex issue that can affect the performance of label-free biosensors. Here we report on an effective and robust route for surface biofunctionalization of nanostructured materials based on the layer-by-layer (LbL) electrostatic nano-assembly of oppositely-charged polyelectrolytes, which are engineered with bioreceptors to enable label-free detection of target analytes. LbL biofunctionalization is demonstrated using nanostructured porous silicon (PSi) interferometers for affinity detection of streptavidin in saliva, through LbL nano-assembly of a bi-layer of positively-charged poly(allylamine hydrochloride) (PAH) and negatively-charged biotinylated poly(methacrylic acid) (b-PMAA). High sensitivity in streptavidin detection is achieved, with high selectivity and stability, down to a detection limit of 600 fM.

Ultrasensitive Bioaffinity Electrochemical Sensors: Advances and New Perspectives

AbstractThis article reviews recent advances and developments in the field of bioaffinity electrochemical sensors with emphasis on a subclass including immunosensors, aptasensors, genosensors and molecularly imprinted sensosr. Recent insights into novel fabrication methodologies and electrochemical techniques have resulted in the demonstration of biosensors able to detection of wide range of target analyte. It has been shown that due to their high specificity and sensitivity, they represent a great tool in practical applications. Furthermore, at last couple decades nanotechnology has become very popular in the sensor fields due to the high capacity of nanomaterials to immobilize of bioaffinity agents and also their inhibiting effects on denaturation of enzymes and other bioaffinity systems, which they could well have beneficial effects for the performance of these sensors. The main focus of this review is to discuss methods for immobilizing bioreceptors onto a solid support, introduction of nanomaterials for improve sensitivity of biosensor and various electrochemical sensing platforms. The use of biosensor in these applications is most appropriate, because the analysis of trace substances in medical and healthcare applications, environmental science, and food industries is so important. In addition, recent advances in nanomaterial‐mediated bioaffinity sensors used for onset biosensing, point‐of‐care testing and healthcare management are also highlighted.

Fully printed one-step biosensing device using graphene/AuNPs composite

Driven by the growing need of simple, cost efficient and flexible sensing systems, we have designed here a fully printed Reduced Graphene Oxide (rGO) based impedimetric sensor for one step sensing of DNA. The DNA sensor was fabricated by stamping of layered rGO and rGO/gold nanoparticles/single stranded DNA (rGO/AuNPs/ssDNA) composites over PET substrates using wax-printing technique. rGO works as an excellent working electrode, while the AuNPs create a suitable environment for ssDNA immobilization. Counter and reference electrodes were previously screen-printed on the plastic substrate, making thus a compact and highly integrated sensing platform. The change in electron transfer resistance after hybridization with a target ssDNA specific of Coxsackie B3 virus was monitored using electrochemical impedance spectroscopy (EIS), finding a linear response in the range of concentrations 0.01–20 µM. The novel, simple and straightforward one-step printing process for fabrication of a biosensing device developed keeps in mind the growing need of large scale device manufacturing. The successful proof-of-concept for the detection of DNA hybridization can be extended to other affinity biosensors, taking advantage of the integration of the bioreceptor on the sensor surface. Such ready-to-use biosensor would lead to a one-step electrochemical detection.

Electrochemical Impedance Spectroscopy Based Biosensors: Mechanistic Principles, Analytical Examples and Challenges towards Commercialization for Assays of Protein Cancer Biomarkers

AbstractImpedimetric affinity biosensors are, without any doubt, among the most sensitive analytical devices available, offering low limits of detection and wide linear response ranges. There are, however, only a few papers detailing the application of impedimetric biosensors for the analysis of clinically relevant samples with due clinical performance. The fact that these devices have not found their way to any commercial or clinical use to date might be surprising, since an electrochemical assay platform based on portable potentiostats is a success story for monitoring a range of clinical parameters such as ions, haematological indicators and glucose. This review discusses the reasons behind this discrepancy and addresses the barriers to be overcome in order to achieve the point‐of‐care diagnostics using such devices for detection of protein oncomarkers approved by FDA. The final part of the review covers the most recent progress in the area.

SPR biosensor for quantification of fetuin-A as a promising multibiomarker.

Early diagnosis of ongoing malignant disease is crucial to improve survival rate and life quality of the patients and requires sensitive detection of specific biomarkers e.g. prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), etc. In spite of current technological advances, malignant diseases are still identified in rather late stages, which have detrimental effect on the prognosis and treatment of the disease. Here, we present a biosensor able to detect fetuin-A, a potential multibiomarker. The biosensing platform is based on polymer brush combining antifouling monomer units of N-(2-hydroxypropyl)methacrylamide (HPMA) and carboxybetaine methacrylamide (CBMAA), statistically copolymerized by surfaceinitiated atom transfer radical polymerization. The copolymer poly(HPMA-co-CBMAA) exhibits excellent non-fouling properties in the most relevant biological media (i.e. blood plasma) as well as antithrombogenic surface properties by preventing the adhesion of blood components (i.e. leukocytes; platelets; and erythrocytes). Moreover, the polymer brush can be easily functionalized with biorecognition elements maintaining high resistance to blood fouling and the binding capacity can be regulated by tuning the ratio between CBMAA and HPMA units. The superior antifouling properties of the copolymer even after biofunctionalization were exploited to fabricate a new plasmonic biosensor for the analysis of fetuin-A in real clinical blood plasma samples. The assay used in this work can be explored as labelfree affinity biosensor for diagnostics of different biomarkers in real clinical plasma samples and to shift the early biomarker detection toward novel biosensor technologies allowing point of care analysis.

Fabrication of a novel DNA affinity biosensor based on hybridisation induced current by electrostatic repulsion of silicotungstic acid as a redox indicator

We report on a novel DNA affinity biosensor which utilises the capture of a neutral charged single stranded (ss) morpholino DNA on a gold electrode to trigger an electrostatic repulsion of negatively charged silicotungstate anions and, in turn, enabled detection of the hybridisation of complementary base pairs. The repulsion of the anions, as a redox indicator, is reflected by a decrease in its electrochemical response with increasing target ss-DNA concentration. A theoretical framework for DNA detection by the affinity biosensor is proposed and verified by electrochemical measurements in the presence of the target ss-DNA by either dc cyclic voltammetry or Fourier transformed alternating current voltammetry (FTACV). The optimised conditions for the capture of the target ss-DNA and the electrochemical detection include 1 μM thiolated neutral morpholino oligo-nucleotide probe, hybridisation time of 10 min, 0.25 mM [α-SiW12O40]4-, and 25 mM phosphate buffer. In addition, the use of the 5th harmonic component of the FTACV gave the most sensitive response for the detection of the target ss-DNA. Under these conditions, the DNA affinity biosensor, based on FTACV detection, achieved a minimum detectable concentration of 0.1 pM ss-DNA and a linear concentration range of 0.1–1000 pM. The biosensor also successfully distinguished between some matched and mismatched base pairs.

Biosensing based on electrochemical impedance spectroscopy: Influence of the often-ignored molecular charge

When measurement of electrochemical impedance is used as an analytical method for affinity biosensors, its increase is in the most cases considered as a proof of specific binding. The increase of the mass in the protein layer is expected to be the cause. However, we encountered severe inconsistencies for these assumptions. In our experiments during the development of an immunosensor, proteins with diverse charges were immobilised and captured on the electrode surface. The measured values of impedance were predominantly affected by the charge of these proteins - negatively charged proteins caused (an expected) increase of the impedance values. Contrary, immobilization/capture of positively charged proteins and other compounds caused (a surprising) relative decrease of the impedance. The obvious and logical origin of this behaviour – a repulsive or attractive ionic interactions between protein layers and negatively charged redox probe [Fe(CN)6]4−/3− (commonly used in electrochemistry) – is usually neglected throughout the literature although some fragmental information could be traced. Besides, we want to emphasize here the importance of the additional characterization of the studied system using for example gel electrophoresis, since many unexpected and overlooked factors can influence the electrochemical response upon analyte binding.

Boronate-based fluorescent carbon dot for rapid and selectively bacterial sensing by luminescence off/on system

Boronic acid, which can bind chemo-selectively and reversibly to diols, could be used for the early detection of bacteria through its affinity-binding reaction with diol groups on the bacterial cell wall. Herein, we describe the use of a diol-modified fluorescent probe (DYE) conjugated to a nanosensor consisting of phenylboronic acid-functionalized fluorescent carbon dot (FCD) to allow quenching via the Förster resonance energy transfer (FRET) process. Phenylboronic acid is well-known for its preferential affinity for diol-containing molecules through cyclic ester bond formation. Therefore, in the presence of glucose-containing bacteria, the DYE in the cyclic ester form will be released from the FCD and replaced by the bacterial cell forming a new cyclic boronate ester bond with the nanoparticle, inducing recovery of the fluorescence. Quantitatively, the system’s detection performance at various bacterial concentrations (101–107 CFU/mL) reached ∼100% after 60 min, indicating that the high binding affinity of the diol moeity on the peptidoglycan (sugar)-rich bacteria was enough to displace the DYE from the boronic acid-functionalized FCD platform. Our facile and tunable fluorescence switch-on system was tested for its ability to detect bacteria in water from a contaminated river. Incredibly, the system was most successful in detecting bacteria in the contaminated river water, thus proving it to be a less expensive and more robust affinity biosensor for the detection of contaminating pathogens in various chemoselective ligand-based environments.

Cysteine functionalized bio-nanomaterial for the affinity sensing of Pb(II) as an indicator of environmental damage

This work aims at the development of an electrochemical affinity biosensor for Pb(II) quantification using a platform that combines glassy carbon electrodes (GCE) and an aqueous dispersion of single-walled carbon nanotubes (SWCNT) covalently modified with cysteine residues (Cys). The biosensing protocol includes the accumulation of Pb(II) at the electrode surface through the affinity interaction promoted by Cys residues at open circuit potential, followed by the reduction of the accumulated Pb(II) at ‐0.900 V and the transduction step performed by linear sweep-adsorptive stripping voltammetry (LSAdSV) in a 0.020 M acetate buffer solution pH 5.00. There is a linear relationship between Pb(II) oxidation peak current and Pb(II) concentration. The dynamic linear range extends from 5.0 to 125.0 μg·L−1, exhibiting a sensitivity of 0.061 μAμg−1L and a detection limit of 0.69 μg·L−1. In addition, the selectivity of the biosensor was evaluated in the presence of high concentrations of possible interferents such as Cu(II), Cd(II), Ni(II), Hg(II), Rh(II), Ru(II), Zn(II), Ir(IV), Co(II) and As(III) demonstrating a high discrimination of Pb(II) in complex samples. The sensor was challenged with tap and rain water samples enriched with Pb(II), demonstrating outstanding properties in terms of recovery percentages showing an excellent agreement with ICP- MS.

Shining Light on Molecular Mechanism for Odor-selectivity of CNT-immobilized Olfactory Receptor

Olfactory receptor (OR)-based bioelectronic nose is a new type of bio-affinity sensor applied for detecting numerous odorant molecules. In order to elucidate the effect of the adsorption of nanomaterial carriers on the receptor structure and its selectivity to odors, we used a systematic computation-scheme to study two OR models immobilized onto carbon nanotube. Our result indicates that there is a multistep OR-adsorption process driven by hydrophobic interaction. Many allosteric communication pathways exist between the absorbed residues and the pocket ones, leading to a significant shrinkage of the pocket. Consequently, the size-selectivity of the receptor to the odors is changed to some extent. But, the odor size and its hydrophobicity, rather than specific functional groups of the odor, still play a determinant role in binding OR, at least for the 132 odors under study. Regardless of the limitation for the odor size in initial recognition, the different-size odors could induce significant changes in the pocket conformation so that it could better match the pocket space, indicating the importance of the ligand-fit binding. Due to the CNT-induced shrinkage of the pocket, the CNT immobilization could increase the binding affinity through enhancing van der Waals interaction, in particular for the large odors.

Advances in the design of nanomaterial-based electrochemical affinity and enzymatic biosensors for metabolic biomarkers: A review.

This review (with 340 refs) focuses on methods for specific and sensitive detection of metabolites for diagnostic purposes, with particular emphasis on electrochemical nanomaterial-based sensors. It also covers novel candidate metabolites as potential biomarkers for diseases such as neurodegenerative diseases, autism spectrum disorder and hepatitis. Following an introduction into the field of metabolic biomarkers, a first major section classifies electrochemical biosensors according to the bioreceptor type (enzymatic, immuno, apta and peptide based sensors). A next section covers applications of nanomaterials in electrochemical biosensing (with subsections on the classification of nanomaterials, electrochemical approaches for signal generation and amplification using nanomaterials, and on nanomaterials as tags). A next large sections treats candidate metabolic biomarkers for diagnosis of diseases (in the context with metabolomics), with subsections on biomarkers for neurodegenerative diseases, autism spectrum disorder and hepatitis. The Conclusion addresses current challenges and future perspectives. Graphical abstract This review focuses on the recent developments in electrochemical biosensors based on theuse of nanomaterials for the detection of metabolic biomarkers. It covers the critical metabolites for some diseases such as neurodegenerative diseases, autism spectrum disorder and hepatitis.

Simultaneous lancet-free monitoring of alcohol and glucose from low-volumes of perspired human sweat

A lancet-free, label-free biosensor for simultaneous detection of sweat glucose and alcohol was demonstrated using zinc oxide thin films integrated into a nanoporous flexible electrode system. Sensing was achieved from perspired human sweat at low volumes (1–3 μL), comparable to ambient conditions without external stimulation. Zinc oxide thin film electrodes were surface functionalized with alcohol oxidase enzyme and with glucose oxidase enzyme towards developing an affinity biosensor specific to the physiological relevant range of alcohol comprising of 0–2 drinks (0–50 mg/dl) and physiologically relevant range of glucose ranging from hypo- to hyper-glycaemia (50–130 mg/dl) in perspired human sweat. Sensing was achieved by measuring impedance changes associated with alcohol and glucose binding onto the sensor interface using electrochemical impedance spectroscopy with a dynamic range from 0.01–200 mg/dl and a limit of detection of 0.01 mg/dl for alcohol in human sweat. Sensor calibration in synthetic sweat containing interferents (25–200 mg/dl) and comparison using regression and Bland-Altman analysis of sweat sensor performance was done with BACtrack®. Combinatorial detection of glucose and ethanol in perspired human sweat and comparison of sweat sensor performance with Accu-Chek® blood glucose monitoring system that we expect would be relevant for pre-diabetics and diabetics for monitoring their glucose levels and alcohol consumption.

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments

This paper describes two different electrochemical affinity biosensing approaches for the simple, fast and bisulfite and PCR-free quantification of 5-methylated cytosines (5-mC) in DNA using the anti-5-mC antibody as biorecognition element. One of the biosensing approaches used the anti-5-mC as capture bioreceptor and a sandwich type immunoassay, while the other one involved the use of a specific DNA probe and the anti-5-mC as a detector bioreceptor of the captured methylated DNA. Both strategies, named for simplicity in the text as immunosensor and DNA sensor, respectively, were implemented on the surface of magnetic microparticles and the transduction was accomplished by amperometry at screen-printed carbon electrodes by means of the hydrogen peroxide/hydroquinone system. The resulting amperometric biosensors demonstrated reproducibility throughout the entire protocol, sensitive determination with no need for using amplification strategies, and competitiveness with the conventional enzyme-linked immunosorbent assay methodology and the few electrochemical biosensors reported so far in terms of simplicity, sensitivity and assay time. The DNA sensor exhibited higher sensitivity and allowed the detection of the gene-specific methylations conversely to the immunosensor, which detected global DNA methylation. In addition, the DNA sensor demonstrated successful applicability for 1 h-analysis of specific methylation in two relevant tumor suppressor genes in spiked biological fluids and in genomic DNA extracted from human glioblastoma cells.

Integrated Affinity Biosensing Platforms on Screen-Printed Electrodes Electrografted with Diazonium Salts.

Adequate selection of the electrode surface and the strategies for its modification to enable subsequent immobilization of biomolecules and/or nanomaterials integration play a major role in the performance of electrochemical affinity biosensors. Because of the simplicity, rapidity and versatility, electrografting using diazonium salt reduction is among the most currently used functionalization methods to provide the attachment of an organic layer to a conductive substrate. This particular chemistry has demonstrated to be a powerful tool to covalently immobilize in a stable and reproducible way a wide range of biomolecules or nanomaterials onto different electrode surfaces. Considering the great progress and interesting features arisen in the last years, this paper outlines the potential of diazonium chemistry to prepare single or multianalyte electrochemical affinity biosensors on screen-printed electrodes (SPEs) and points out the existing challenges and future directions in this field.

Encyclopedia of Interfacial Chemistry: Surface Science and Electrochemistry

There is an increasing interest in electrochemical impedance spectroscopy as the method for the electrical characterization of biointerfaces and for the transducing method for label-free biosensors. In the field of affinity biosensors, this technique allows the direct detection of the complex formation through the variation of the capacitance or of the charge transfer resistance at the biofunctionalized electrode/biological liquid interface.This article presents the physical principles of electrochemical impedance and the performances of detection of different biotargets.

Enhanced Electron Transfer Mediated by Conjugated Polyelectrolyte and Its Application to Washing-Free DNA Detection.

Direct electron transfer between a redox label and an electrode requires a short working distance (<1-2 nm), and in general an affinity biosensor based on direct electron transfer requires a finely smoothed Au electrode to support efficient target binding. Here we report that direct electron transfer over a longer working distance is possible between (i) an anionic π-conjugated polyelectrolyte (CPE) label having many redox-active sites and (ii) a readily prepared, thin polymeric monolayer-modified indium-tin oxide electrode. In addition, the long CPE label (∼18 nm for 10 kDa) can approach the electrode within the working distance after sandwich-type target-specific binding, and fast CPE-mediated oxidation of ammonia borane along the entire CPE backbone affords high signal amplification.

Affinity Biosensors for Detection of Mycotoxins in Food.

This chapter reviews recent achievements in methods of detection of mycotoxins in food. Special focus is on the biosensor technology that utilizes antibodies and nucleic acid aptamers as receptors. Development of biosensors is based on the immobilization of antibodies or aptamers onto various conventional supports like gold layer, but also on nanomaterials such as graphene oxide, carbon nanotubes, and quantum dots that provide an effective platform for achieving high sensitivity of detection using various physical methods, including electrochemical, mass sensitive, and optical. The biosensors developed so far demonstrate high sensitivity typically in subnanomolar limit of detection. Several biosensors have been validated in real samples. The sensitivity of biosensors is similar and, in some cases, even better than traditional analytical methods such as ELISA or chromatography. We believe that future trends will be focused on improving biosensor properties toward practical application in food industry.

Reversible Self-Assembled Monolayers (rSAMs): Adaptable Surfaces for Enhanced Multivalent Interactions and Ultrasensitive Virus Detection.

We report on the design of pH-switchable monolayers allowing a reversible and ordered introduction of affinity reagents on sensor surfaces. The principal layer building blocks consist of α-(4-amidinophenoxy)alkanes decorated at the ω-position with affinity ligands. These spontaneously self-assemble on top of carboxylic acid terminated SAMs to form reversible homo or mixed monolayers (rSAMs) that are tunable with respect to the nature of the head group, layer order and stability while featuring pH responsiveness and the dynamic nature of noncovalent build assemblies. We show that this results in a range of unique biosensor features. As a first example a sialic acid rSAM featuring strong lectin affinity is here used to sense hemagglutinin and influenza virus (H5N1) at the pM and fM level by in situ ellipsometry in a fully reversible fashion. We believe that the rSAM concept will find widespread use in surface chemistry and overall for boosting sensitivity in affinity biosensors.