Electrochemical glucose biosensor by electrostatic binding of PQQ-glucose dehydrogenase onto self-assembled monolayers on gold

Efficient binding of enzymes onto the electrode surface has been prerequisite for the construction of sensitive biosensors and biochips. Here, a simple and robust construction of electrochemical glucose biosensor based on pyrroloquinoline quinone-glucose dehydrogenase was demonstrated. The glucose biosensor was fabricated by binding the enzyme onto the anionic self-assembled monolayers on gold electrode via electrostatic interactions. The resulting glucose biosensor gave rise to twofold higher detection sensitivity than that by covalent conjugation under the same condition. Surface plasmon resonance and atomic force microscopy analyses revealed that electrostatic binding of the enzyme leads to much higher surface density of the enzyme. This approach will find wide applications to the development of robust enzyme-based biosensors and biochips.     

Semi‐specific biosensors for measuring BOD in dairy wastewater

BACKGROUND: Semi‐specific microbial biochemical oxygen demand (BOD) biosensors were constructed using living cells of Escherichia coli R17.1.3 (E. coli) and Raoultella terrigena P74.3 (R. terrigena) immobilized in agarose gel matrix. The research involved comparison with the Pseudomonas fluorescens P75 (P. fluorescens) biosensor that had no specificity in decomposing lactose and milk derivates. The constructed BOD biosensors were calibrated with OECD synthetic wastewater and tested with different wastewater samples. RESULTS: The linear range of the calibration curve was 5–200 mg L^?1 BOD₇ for R. terrigena and P. fluorescens based biosensors and 150 mg L^?1 BOD₇ for the E. coli based biosensor. Service life was 60 days for E. coli and P. fluorescens based biosensors and 40 days for R. terrigena based biosensors. BOD values for dairy industry wastewater obtained with current semi‐specific biosensors considerably overestimate BOD₇, while universal biosensors underestimate BOD₇ obtained by the conventional 7‐day BOD test. CONCLUSION: In spite of extensive overestimation of BOD₇ the semi‐specific biosensors enabled better estimation of BOD in dairy industry wastewater than a reference P. fluorescens biosensor. The best result, in terms of service life, stability, sensitivity and reproducibility was accomplished with semi‐specific E.coli biosensor. Copyright © 2010 Society of Chemical Industry     

Creation of GPCR-based chemical sensors by directed evolution in yeast

G protein-coupled receptors (GPCRs) form a class of biological chemical sensors with an enormous diversity in ligand binding and sensitivity. To explore structural aspects of ligand recognition, we subjected the human UDP-glucose receptor (P2Y14) functionally expressed in the yeast Saccharomyces to directed evolution. We sought to generate new receptor subtypes with ligand-binding properties that would be useful in the development of practical biosensors. Mutagenesis of the entire UDP-glucose receptor gene yielded receptors with increased activity but similar ligand specificities, while random mutagenesis of residues in the immediate vicinity of the ligand-binding pocket yielded mutants with altered ligand specificity. By first sensitizing the P2Y14 receptor and then redirecting ligand specificity, we were able to create mutant receptors suitable for a simple biosensor. Our results demonstrate the feasibility of altering receptor ligand-binding properties via a directed evolution strategy, using standard yeast genetic techniques. The novel receptor mutants can be used to detect chemical ligands in complex mixtures and to discriminate among chemically or stereochemically related compounds. Specifically, we demonstrate how engineered receptors can be applied in a pairwise manner to differentiate among several chemical analytes that would be indistinguishable with a single receptor. These experiments demonstrate the feasibility of a combinatorial approach to detector design based on the principles of olfaction.     

Flow-injection amperometric glucose biosensors based on graphene/Nafion hybrid electrodes

In this research, we demonstrated the fabrication of flow-injection amperometric glucose biosensors based on RGO/Nafion hybrids. The nanohybridization of the reduced graphene oxide (RGO) by Nafion provided the fast electron transfer (ET) for the sensitive amperometric biosensor platforms. The ET rate (k_s) and the charge transfer resistance (R_CT) of GOx-RGO/Nafion hybrids were evaluated to verify the accelerated ET. Moreover, hybrid biosensors revealed a quasi-reversible and surface controlled process, as confirmed by the low peak-to-peak (ΔE_p) and linear relations between I_p and scan rate (ν). Hybrid biosensors showed the fast response time of ∼3 s, the sensitivity of 3.8 μA mM⁻¹ cm⁻², the limit of detection of 170 μM, and the linear detection range of 2–20 mM for the flow-injection amperometric detection of glucose. Furthermore, interference effect of oxidizable species such as ascorbic acid (AA) and uric acid (UA) on the performance of hybrid biosensors was prevented at the operating potential of −0.20 V even under the flow injection mode. Therefore, the fast, sensitive, and stable amperometric responses of hybrid biosensors in the flow injection system make it highly suitable for automatically monitoring glucose.     

Electrochemical sensor array for bioprocess monitoring

A silicon-based biosensor chip consisting of an array of amperometric enzyme sensors has been developed for monitoring the concentration of glutamate and glutamine in cell-culture fermentation processes. The glutamate sensor was constructed by immobilising glutamate oxidase, while the glutamine sensor by sequential coupling of glutaminase and glutamate oxidase. The enzymes were immobilised by means of cross-linking with glutaraldehyde on the surface of the patterned platinum thin-film electrodes. The developed biosensors have been electrochemically characterised in solutions with different concentrations of glutamate and glutamine in terms of sensitivity, response time, linear working range and lifetime. A high sensitivity of 96 nA/mM and 100 nA/mM was registered for the glutamate and glutamine sensors, respectively. The preliminary experiments in cell-culture medium have shown a good correlation between the glutamine and glutamate concentrations measured with the biosensor chip and the commercially available biochemistry analyser. The obtained results demonstrate the feasibility of the realised biosensor chip for monitoring the glutamine and glutamate concentrations in fermentation processes.     

Quality Control Certification of RNA Aptamer‐Based Detection

Aptamers are single‐stranded DNA or RNA molecules with a defined tertiary structure for molecular recognition. Numerous RNA aptamers with excellent binding affinity and specificity have been reported; they constitute an attractive reservoir of molecular recognition elements for biosensor development. However, RNA is relatively unstable owing to spontaneous hydrolysis and nuclease degradation. Thus, RNA aptamer‐based biosensors are prone to producing false‐positive signals. Here, we present an RNA aptamer biosensor design strategy that utilises an internal control to distinguish target binding from false‐positive signals. The sequence of a chosen RNA aptamer is expanded so that it can form three consecutive short RNA–DNA duplexes with 1) a quencher‐labelled DNA strand (Q₁DNA), 2) a dual‐fluorophore‐labelled DNA strand (F₁DNAF₂) and 3) another quencher‐labelled DNA strand (Q₂DNA). The addition of a target releases Q₂DNA from the duplex assembly, and produces the expected positive signal from F₂. However, the authenticity of target recognition is validated only if no signal is generated from F₁. We have successfully engineered two fluorescent reporters by using an RNA aptamer that binds thrombin and one that binds theophylline. Both reporters show the expected binding affinity and specificity, and are capable of reporting system malfunction when treated with nucleases and chemical denaturants. This strategy provides a simple and reliable way to ensure high‐quality detection when RNA aptamers are employed as molecular‐recognition elements.     

Development of an electrochemical‐surface plasmon dual biosensor based on carboxylated conducting polymer thin films

A biosensing platform based on the covalent attachment of biomolecules on electropolymerized carboxylated conducting polymers, poly(3‐aminobenzoic acid) and poly(3‐pyrrole carboxylic acid), were developed for the selective simultaneous detection of two biomolecules using electrochemical‐surface plasmon resonance (EC–SPR) spectroscopy. The surface morphology of the developed biosensors was studied by scanning electron microscopy and atomic force microscopy. The EC–SPR dual biosensor was developed for the label‐free, simultaneous, and selective detection of glucose and human immunoglobulin G (IgG). A change in current density was clearly observed after the injection of glucose, whereas a change in SPR reflectivity was clearly observed after the injection of human IgG. The present work demonstrates the potential of this biosensing platform for real sample analysis in the future. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 45641.     

Use of engineered Escherichia coli cells to detect estrogenicity in everyday consumer products

BACKGROUND: Estrogenic activity has been observed in several industrial and household products, and some evidence suggests that this activity may be linked to increased pathologies in humans and animals. Here, an engineered strain of Escherichia coli is evaluated for its ability to detect estrogenic activity in complex mixtures, including natural dietary supplements, hand and body washes, essential oils, and perfumes. The engineered E. coli biosensor strain expresses the ligand‐binding domain of the human estrogen receptor β (ERβ) as part of a larger allosteric reporter enzyme. The result is a simple bacterial growth assay, where estrogenic activity of a test compound is reflected by increased cell growth on a simple defined medium. RESULTS: While most consumer products did not yield a strong estrogenic response in the assay, a consistent estrogenic effect was observed with several perfumes. This effect is probably due to the presence of the known estrogen, benzophenone‐2, which exhibited an EC₅₀ concentration of 0.44 µmol L^?1 for the sensor strains used. CONCLUSIONS: This simple, bacterial biosensor is capable of rapidly and inexpensively detecting estrogenic activity in complex consumer products, and may eventually yield rough estimates of the equivalent estrogen doses associated with their use. Copyright © 2009 Society of Chemical Industry     

Novel Abscisic Acid Antagonists Identified with Chemical Array Screening

Abscisic acid (ABA) signaling is involved in multiple processes in plants, such as water stress control and seed dormancy. Major regulators of ABA signaling are the PYR/PYL/RCAR family receptor proteins, group A protein phosphatases 2C (PP2Cs), and subclass III of SNF1‐related protein kinase 2 (SnRK2). Novel ABA agonists and antagonists to modulate the functions of these proteins would not only contribute to clarification of the signaling mechanisms but might also be used to improve crop yields. To obtain small molecules that interact with Arabidopsis ABA receptor PYR1, we screened 24 275 compounds from a chemical library at the RIKEN Natural Products Depository by using a chemical array platform. Subsequent SnRK2 and PP2C assays narrowed down the candidates to two molecules. One antagonized ABA in a competitive manner and inhibited the formation of the PYR1‐ABA‐PP2C ternary complex. These compounds might have potential as bioprobes to analyze ABA signaling.     

Analogies Between Digital Radio and Chemical Orthogonality as a Method for Enhanced Analysis of Molecular Recognition Events

Acoustic wave biosensors are a real-time, label-free biosensor technology, which have been exploited for the detection of proteins and cells. One of the conventional biosensor approaches involves the immobilization of a monolayer of antibodies onto the surface of the acoustic wave device for the detection of a specific analyte. The method described within includes at least two immobilizations of two different antibodies onto the surfaces of two separate acoustic wave devices for the detection of several analogous analytes. The chemical specificity of the molecular recognition event is achieved by virtue of the extremely high (nM to pM) binding affinity between the antibody and its antigen. In a standard ELISA (Enzyme-Linked ImmunoSorbent Assay) test, there are multiple steps and the end result is a measure of what is bound so tightly that it does not wash away easily. The fact that this “gold standard” is very much not real time, masks the dance that is the molecular recognition event. X-Ray Crystallographer, Ian Wilson, demonstrated more than a decade ago that antibodies undergo conformational change during a binding event[1, 2]. Further, it is known in the arena of immunochemistry that some antibodies exhibit significant cross-reactivity and this is widely termed antibody promiscuity. A third piece of the puzzle that we will exploit in our system of acoustic wave biosensors is the notion of chemical orthogonality. These three biochemical constructs, the dance, antibody promiscuity and chemical orthogonality will be combined in this paper with the notions of in-phase (I) and quadrature (Q) signals from digital radio to manifest an approach to molecular recognition that allows a level of discrimination and analysis unobtainable without the aggregate. As an example we present experimental data on the detection of TNT, RDX, C4, ammonium nitrate and musk oil from a system of antibody-coated acoustic wave sensors.     

Recent Progress in Strategies for the Creation of Protein‐Based Fluorescent Biosensors

The creation of novel bioanalytical tools for the detection and monitoring of a range of important target substances and biological events in vivo and in vitro is a great challenge in chemical biology and biotechnology. Protein‐based fluorescent biosensors—integrated devices that convert a molecular‐recognition event to a fluorescent signal—have recently emerged as a powerful tool. As the recognition units various proteins that can specifically recognize and bind a variety of molecules of biological significance with high affinity are employed. For the transducer, fluorescent proteins, such as green fluorescent protein (GFP) or synthetic fluorophores, are mostly adopted. Recent progress in protein engineering and organic synthesis allows us to manipulate proteins genetically and/or chemically, and a library of such protein scaffolds has been significantly expanded by genome projects. In this review, we briefly describe the recent progress of protein‐based fluorescent biosensors on the basis of their platform and construction strategy, which are primarily divided into the genetically encoded fluorescent biosensors and chemically constructed biosensors.     

Biodegradation Capability and Enzymatic Variation of Potentially Hazardous Polycyclic Aromatic Hydrocarbons—Anthracene and Pyrene by Anabaena fertilissima

This study encompasses the biodegradation capacity of Anabaena fertilissima to model PAH (Polycyclic Aromatic Hydrocarbon) compounds namely Anthracene (ANT) and Pyrene (PYR) at different LC₅₀ concentrations viz., 5.0 mg/L and 3.0 mg/L, respectively, on growth in terms of Chlorophyll-a and protein. Depletion in chlorophyll-a and protein content was registered with rise in PAHs concentration while the inhibition of nitrogen fixing enzymes such as nitrate reductase and glutamine synthetase activity was also concentration dependent and showed more sensitivity for high molecular weight aromatic compound PYR as compared to ANT. GC/MS analysis explained the degradation of ANT by 46% and PYR by 33%, at 5.0 mg/L and 3.0 mg/L, respectively. Moreover, the common degraded product for ANT was 2, 4-Dimethyl-1-heptene and for PYR it was 2, 3, 4-Trimethylhexane. Results indicate that PYR was more stable and recalcitrant compared to ANT. This study suggests that Anabaena fertilissima could be used for bioremediation of ANT and PYR pollution in surface waters and terrestrial environments.     

Functional phage display of two murine alpha/beta T-cell receptors is strongly dependent on fusion format, mode and periplasmic folding assistance

Phage display has been instrumental for the success of antibody (Ab) technology. The aim of the present study was to explore phage display of soluble T-cell receptors (TCRs). A library platform that supports engineering and selection of improved TCRs to be used as detection reagents for specific antigen presentation will be very useful. In such applications, high, equal and clone independent display levels are a prerequisite for 'fair' selection. Therefore, we explored how different pIII fusion formats and modes affected the display levels of two murine alpha/beta TCRs. Both are derived from T-cell clones associated with the MOPC315 myeloma model. The results show that the design of the pIII fusion particle significantly affects the subsequent display levels. Furthermore, successful display may be obtained both in phagemid and phage versions. Importantly, improvement of poor display can be achieved by over-expressing the periplasmic chaperone FkpA.     

Radiolytic synthesis of ?OH group functionalized fullerene structures and their biosensor application

In this study, radiolytic functionalization of fullerene in methanol/1,2‐dichlorobenzene mixtures and its applications with respect to biosensor support materials were studied. To obtain supports for biosensors for electron transfer, fullerene was functionalized by γ‐irradiation in a methanol/1,2‐dichlorobenzene mixture solution. The hydroxyl group‐modified fullerene, F‐fullerene, was characterized by Fourier‐transform infrared, Raman spectroscopy, MALDI‐TOF mass spectroscopy, and elemental analysis. As a result, the main hydroxyl group was successfully introduced on the surface of fullerene. F‐fullerene was found to disperse well in water by ultrasonication. The results indicated that F‐fullerene is a good candidate for use in biological systems as a biosensor support material. A biosensor based on F‐fullerene was prepared by hand‐casting the mixture of tyrosinase, F‐fullerene, and 2% chitosan solution on an ITO electrode. Furthermore, the prepared biosensor was optimized pH and temperature. The prepared biosensor was then evaluated for its ability to analyze phenolic compounds contained in commercial red wines. The total phenolic concentration was determined to be in the range of 397–895 mg/L. From these results, the electron transfer ability of F‐fullerene was improved on an enzyme biosensor. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Alcohol dehydrogenase amperometric biosensor based on a colloidal gold-carbon nanotubes composite electrode

A novel ethanol biosensor based on the bulk incorporation of alcohol dehydrogenase (ADH) into a colloidal gold (Au_coll)-multiwalled carbon nanotubes (MWCNTs) composite electrode using Teflon as binding material is reported. The composite Au_coll-MWCNTs-Teflon electrode exhibited significantly improved electrooxidation of NADH when compared with other carbon composite electrodes, including those based on carbon nanotubes. Amperometric measurements for NADH at +0.3 V showed significant differences in sensitivity between Au_coll-MWCNTs-Teflon and MWCNTs-Teflon composite electrodes. Incorporation of ADH into the bulk electrode material allowed the construction of a mediatorless ethanol biosensor. Both the enzyme loading and the NAD⁺ concentration in solution were optimized. The ADH-Au_coll-MWCNTs-Teflon biosensor allowed a limit of detection for ethanol of 4.7 μmol l⁻¹, which is remarkably better than those reported for other CNTs-based ADH biosensors. The apparent Michaelis-Menten constant was 4.95 mmol l⁻¹, which is much lower than that reported by immobilization of ADH onto a gold electrode. Both repeatability of the ethanol amperometric measurements, reproducibility with different biosensors, lifetime and storage ability can be, in general, advantageously compared with other ADH-CNTs biosensors. The biosensor was applied for the rapid determination of ethanol in commercial and certified beer samples.     

Preparation and modification of carbon nanotubes electrodes by cold plasmas processes toward the preparation of amperometric biosensors

An electrochemical transducer based on vertically aligned carbon nanotubes (CNT) was prepared as a platform for biosensor development. Prior to enzyme immobilization, the CNT were treated using a microwave plasma system (CO₂ and N₂/H₂) in order to functionalize the CNT surface with oxygenated and aminated groups. The morphological aspect of the electrode surface was examined by SEM and its chemical structure was also elucidated by XPS analysis. It was found out that microwave plasma system (CO₂ and N₂/H₂) not only functionalizes the CNT but also permits to avoid the collapse phenomena retaining thus the alignment structure of the electrode surface. The electrochemical properties of the resulting new material based on CNT were carried out by cyclic voltammetry and were found suitable to develop high sensitive enzyme (HRP) biosensors operating on direct electron transfer process.     

Engineering of the Escherichia coli Im7 immunity protein as a loop display scaffold

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.     

Anthrax Protective Antigen Retargeted with Single-Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells

The nontoxic, anthrax protective antigen/lethal factor N-terminal domain (PA/LF_N) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LF_N to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single-chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer-relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA−scFv fusions enable cell-specific delivery of diphtheria toxin catalytic domain and Ras/Rap1-specific endopeptidase to pancreatic cancer cells. Importantly, mPA−scFv fusion-based treatments display potent cell-specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.     

A biosensor platform based on amine functionalized conjugated benzenediamine-benzodithiophene polymer for testosterone analysis

A novel benzenediamine-benzodithiophene polymer is synthesized for use in biosensor fabrication for the detection of testosterone. The sensory platform is constructed via drop coating on a screen-printed carbon electrode, using poly(benzenediamine-Bis[(2-ethylhexyl)oxy]benzodithiophene) (pBDBT) as the polymer layer. Testosterone antibodies are immobilized on the polymer-coated electrode surface via glutaraldehyde, which binds to the surface through the amino functional groups on the polymer backbone. The changes in the surface features due to testosterone binding are investigated via electrochemical techniques such as differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectrometry as well as contact angle measurements. Surface morphology of the modified electrodes is characterized by atomic force microscopy. The linear range and limit of detection of the sensor are calculated. Impact of possible interfering compounds is investigated. Furthermore, the sensory platform is utilized for testosterone analysis in synthetic biological fluids.     

A sensitive electrochemical aptasensor based on water soluble CdSe quantum dots (QDs) for thrombin determination

A novel aptamer biosensor with easy operation and good sensitivity, specificity, stability and reproducibility was developed by immobilizing the aptamer on water soluble CdSe quantum dots (QDs) modified on the top of the glassy carbon electrode (GCE). Methylene blue (MB) was intercalated into the aptamer sequence and used as an electrochemical marker. CdSe QDs improved the electrochemical signal because of their larger surface area and ion centers of CdSe QDs may also had a major role on amplifying the signal. The higher ion concentration caused more combination of aptamer which caused larger signal. The thrombin was detected by differential pulse voltammetry (DPV) quantitatively. Under optimal conditions, the two linear ranges were obtained from 3 to 13 μg mL⁻¹ and from 14 to 31 μg mL⁻¹, respectively. The detection limit was 0.08 μg mL⁻¹ at 3σ. The constructed biosensor had better responses compared with that in the absence of the CdSe QDs immobilizing. The control experiment was also carried out by using BSA, casein and IgG in the absence of thrombin. The results showed that the aptasensor had good specificity, stability and reproducibility to the thrombin. Moreover, the aptasensor could be used for detection of real sample with consistent results in comparison with those obtained by fluorescence method which could provide a promising platform for fabrication of aptamer based biosensors.