Recognition Elements In Biosensors Research Articles

(Invited) F-ALDH, a New Catalyst for Enzyme and Aptamer- Based Electrochemical Biosensors

The selectivity, affinity and catalytic activity are key characteristics which recommend enzymes as components of biosensors, whether for use as main recognition element or for signal amplification. Additionally, the enzyme inhibition by various molecules can be exploited for analytical purposes. In the search for new enzymes with good stability and catalytic properties, a recombinant psychrophilic aldehyde dehydrogenase from the Antarctic Flavobacterium PL002 was recently characterized and shown to have potential in biosensing [1-5].The enzyme is active in a wide temperature range up to 60º C and has high catalytic activity [1,2]. It accepts various aldehydes substrates with Michaelis Menten constants of e.g., Km = 0. 137 ± 0.016 mmol L−1 for benzaldehyde. The enzyme may use both nicotinamide adenin dinucleotide (NAD+) and nicotinamide adenin dinucleotide phosphate (NADP+) as cofactors and has higher affinity for NAD+ ( Km=0.033mmol L−1 and 0.431 mmol L−1 for NAD+ and NADP+, respectively [2]). As for other aldehyde dehydrogenases, the enzyme is inhibited by heavy metals and dithiocarbamate fungicides [2,3].To explore its potential as recognition element in biosensors, enzymatic tests and electrochemical biosensors exploiting the catalytic activity of F-ALDH or its inhibition were first developed [4,5]. In biosensors, the enzyme was immobilized by cross linking in a matrix of bovine serum albumin [5] or by nickel-histidine affinity. Enzymatic inks with a stability of several months at 4º C were obtained by immobilizing the enzyme on magnetic particles functionalized with a complex of nickel-nitrilotriacetic acid (Ni-NTA) followed by mixing with different additives. Practical applications for the specific detection of (i) benzaldehyde in a pharmaceutical ingredient [5] and (ii) acetaldehyde in wines were demonstrated. In wines, acetaldehyde is present in a free form or bound to sulphur dioxide or phenolic compounds. Both free and bound acetaldehyde are relevant for wine quality and its evolution. The F-ALDH enzyme enables testing of both forms in cellar like conditions. Moreover, the detection of free acetaldehyde serves as example for aldehydes detection in vapor phase, opening the way for other similar applications.A separate line of research explored the potential of F-ALDH for the quantitative determination of enzyme’s inhibitors. Spectrophotometry measurements emphasized a 15% reduction of enzyme’s activity in the presence of either 28 ppb thiram or 0.5 ppb mercury chloride. The solution advanced for overcoming the selectivity issues in the detection of thiram is to couple the electrochemical biosensor measuring the F-ALDH inhibition with SERS measurements of the dithiocarbamate fungicide [6].Finally, the F-ALDH enzyme may serve as label in electrochemical aptasensors. The enzyme was attached to an aptamer for lysozyme via Ni-histidine affinity and preserved good catalytic activity for the conversion of acetaldehyde. Preliminary studies were focused on investigating the affinity of the enzyme-labeled aptamer for lysozyme in relation to the linker length.Based on the demonstrated catalytic activity of the enzyme and its preservation upon immobilization on different supports or by linking to an aptamer, various sensing configurations can be envisaged that will exploit the potential of the new enzyme. Acknowledgements: This work was financially supported by the UEFISCDI projects ERANET-M-ENZ4IFACES, contract 166/01.05.2020 (for AV, GNP and CP) and PN-III-P4-ID-PCE-2020-2297 E-MAP, contract 84/9.02.2021 (for RMB and AF). References Necula-Petrareanu G, Lavin P, Vasilescu A, Purcarea C (2018) Antarctic Flavobacterium aldehyde dehydrogenase – a cold adapted catalyst for biosensing. J Biotechnol 280:S18. https://doi. org/10.1016/j.jbiotec.2018.06.054Necula-Petrareanu, G.; Lavin, P.; Paun, V.I.; Gheorghita, G.R.; Vasilescu, A.; Purcarea, C. Highly stable cold-active aldehyde dehydrogenase from the marine Antarctic Flavobacterium sp. PL002. Fermentation. 2022 8(1):7-24 https:// doi.org/10.3390/fermentation8010007Vasilescu A, Titoiu A.M, Purcarea C, Necula Petrareanu G, Method for determining thiram fungicide based on enzymatic inhibition and electrochemical sensor, Romanian patent application, OSIM RO133890A2 /13.08.2018Titoiu AM, Lapauw M, Necula-Petrareanu G, Purcarea C, FanjulBolado P, Marty J-L, Vasilescu A (2018) Carbon nanofiber and Meldola Blue based electrochemical sensor for NADH: application to the detection of benzaldehyde. Electroanalysis 30(11):2676– 2688. https://doi.org/10.1002/elan.201800472Titoiu, A. M., Necula-Petrareanu, G., Visinescu, D., Dinca, V., Bonciu, A., Mihailescu, C. N., ... Vasilescu, A. (2020). Flow injection enzymatic biosensor for aldehydes based on a Meldola Blue-Ni complex electrochemical mediator. Microchimica Acta, 187(10).doi:10.1007/s00604-020-04477-3Ibáñez D, González-García MB, Hernández-Santos D, Fanjul-Bolado P. Detection of dithiocarbamate, chloronicotinyl and organophosphate pesticides by electrochemical activation of SERS features of screen-printed electrodes. Spectrochim Acta A Mol Biomol Spectrosc. 2021 Mar 5;248:119174. doi: 10.1016/j.saa.2020.119174

Reports on the sensitivity enhancement in interferometric based biosensors by biotin-streptavidin system

Antibody biotinylation is a process of attaching biotin molecules to antibodies by chemically modifying specific functional groups on the antibodies without altering their antigen recognition specificity. Biotin, a small vitamin, forms a strong and specific interaction with the protein streptavidin, resulting in a stable biotin-streptavidin (biotin-STV) complex. This biotin-STV interaction is widely exploited in various biotechnological applications, including biosensors. Biosensors are analytical devices that employ biological recognition elements, such as antibodies, enzymes, or nucleic acids, to detect and quantify target analytes in a sample. Antibodies are commonly used as recognition elements in biosensors due to their high specificity and affinity. In this study, the antibody anti-Bovine Serum Albumin (αBSA) has been biotinylated at different antibody:biotin ratios, and the stability of this labeling over time has been investigated. Furthermore, the sensitivity of the biosensor for detecting the Bovine Serum Albumin (BSA) protein has been compared using the biotinylated antibody and the non-biotinylated form, showing a four-fold improvement in detection. This system was also compared with the Enzyme-Linked ImmunoSorbent Assay (ELISA) technique. The advantages of using biotinylated antibodies in biosensors include increased stability and reproducibility of the biorecognition layer, as well as flexibility in sensor design, as different biotinylated antibodies can be utilized for diverse target analytes without altering the sensor's architecture.

Nucleic Acid Aptamer-Based Biosensors: A Review.

Aptamers, short strands of either DNA, RNA, or peptides, known for their exceptional specificity and high binding affinity to target molecules, are providing significant advancements in the field of health. When seamlessly integrated into biosensor platforms, aptamers give rise to aptasensors, unlocking a new dimension in point-of-care diagnostics with rapid response times and remarkable versatility. As such, this review aims to present an overview of the distinct advantages conferred by aptamers over traditional antibodies as the molecular recognition element in biosensors. Additionally, it delves into the realm of specific aptamers made for the detection of biomarkers associated with infectious diseases, cancer, cardiovascular diseases, and metabolomic and neurological disorders. The review further elucidates the varying binding assays and transducer techniques that support the development of aptasensors. Ultimately, this review discusses the current state of point-of-care diagnostics facilitated by aptasensors and underscores the immense potential of these technologies in advancing the landscape of healthcare delivery.

Aptasensors for full body health checkup

In the last couple of decades, aptamers have been one of the preferred biomolecular recognition elements in biosensors. The platform of aptasensors (aptamer-based biosensors) is demonstrated to solve practical research problems in various areas. Aptamers and biosensors altogether gift a specific, selective, sensitive, affordable, easy to operate, robust, rapid, stable, and low sample volume analytical platform. In diagnostics, numerous review articles are published discussing the detection of various types of non-infectious and infectious diseases using aptasensors, but to the best of our knowledge, no one has reviewed the available information from the perspective of the utility of aptasensors for a full body health checkup. Therefore, to bridge this existing gap in knowledge in terms of the utility of aptasensors for full body health checkup and to provide a new perspective to the aptasensor community, in this review article, we are reviewing a battery of aptasensors, which has been utilized to detect biomarkers that correlate well with an individual's health status. Our motive in this review is to demonstrate the expansion of utility of aptasensors in routine full body checkups. Systematic Evolution of Ligands by Exponential Enrichment (SELEX), design configurations, and various detection methods employed for aptasensors are also discussed. Along with, the existing gap in the field, current challenges, and future of aptamers in diagnostics with concluding remarks are also presented in this review article.

A Strategy for Accessing Nanobody-Based Electrochemical Sensors for Analyte Detection in Complex Media.

Nanobodies are single variable domain antibodies isolated from camelids and are rapidly distinguishing themselves as ideal recognition elements in biosensors due to their comparative stability, ease of production and isolation, and high binding affinities. However, transducing analyte binding by nanobodies in real time is challenging, as most nanobodies do not directly produce an optical or electrical signal upon target recognition. Here, we report a general strategy to fabricate sensitive and selective electrochemical sensors incorporating nanobodies for detecting target analytes in heterogeneous media, such as cell lysate. Graphite felt can be covalently functionalized with recombinant HaloTag-modified nanobodies. Subsequent encapsulation with a thin layer of a hydrogel using a vapor deposition process affords encapsulated electrodes that directly display a decrease in current upon antigen binding, without added redox mediators. Differential pulse voltammetry affords clear and consistent decreases in electrode current across multiple electrode samples for specific antigen concentrations. The change in observed current vs increasing antigen concentration follows Langmuir binding characteristics, as expected. Importantly, selective and repeatable target binding in unpurified cell lysate is only demonstrated by the encapsulated electrode, with an antigen detection limit of ca. 30 pmol, whereas bare electrodes lacking encapsulation produce numerous false positive signals in control experiments.

Phage-Based Sensors in Medicine: A Review

Bacteriophages are interesting entities on the border of biology and chemistry. In nature, they are bacteria parasites, while, after genetic manipulation, they gain new properties, e.g., selectively binding proteins. Owing to this, they may be applied as recognition elements in biosensors. Combining bacteriophages with different transducers can then result in the development of innovative sensor designs that may revolutionize bioanalytics and improve the quality of medical services. Therefore, here, we review the use of bacteriophages, or peptides from bacteriophages, as new sensing elements for the recognition of biomarkers and the construction of the highly effective diagnostics tools.

Long-Term Functional Stability of Functional Nucleic Acid–Gold Nanoparticle Conjugates with Different Secondary Structures

Thiolated functional nucleic acid-gold nanoparticle conjugates (FNA-AuNPs) are the core recognition elements in biosensors. The long-term functional stability (LTFS) is critical for their practical applications and, however, has been overlooked. Here we report on the huge effects of multiple experimental factors on LTFS, including spacer- and buffer-composition, secondary structures of FNAs, and surface blocking. We quantitatively determined these effects by measuring the relative hybridization capacity (RHC, the relative amount of complementary DNA hybridized with the same amount of conjugates) for linear DNA-AuNP or the relative signal change generated by their function (RSC-F) for molecular beacon (MB) and G-quadruplex (G4)-AuNPs. There is a positive relationship between the spacer affinity [oligoadenine (A10) > oligothymine (T10) > oligoethlyene glycol (EG18)] of the linear DNA probes and the LTFS. The LTFS of linear DNA-AuNP in phosphate buffer (PB) was much better than that in Good's buffers such as HEPES, Tris, and MES. The secondary structure of FNAs also strongly impacted the LTFS, showing the substantially decreased LTFS from G4- to linear DNA- to MB-AuNPs, where EG18 spacer was used for all these conjugates. The surface blocking of FNA-AuNPs greatly improved the LTFS. We experimentally determined that the LTFS of FNA-AuNPs was directly related to the dissociation of DNAs caused by the in situ generated H2O2 due to the oxidase activity of AuNP and thereby oxidation of Au-thiol bonds. The oxidase activity of AuNP was favored at high temperature, low pH, high AuNP concentration, high Good's buffer concentration, and high salt concentration, corresponding well with the positive effects of high affinity spacer, PB, and surface blocking on the LTFS of FNA-AuNPs. Our study has implications on both fundamental surface science and practical applications.

Phage Display in the Quest for New Selective Recognition Elements for Biosensors.

Phages are bacterial viruses that have gained a significant role in biotechnology owing to their widely studied biology and many advantageous characteristics. Perhaps the best-known application of phages is phage display that refers to the expression of foreign peptides or proteins outside the phage virion as a fusion with one of the phage coat proteins. In 2018, one half of the Nobel prize in chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter “for the phage display of peptides and antibodies.” The outstanding technology has evolved and developed considerably since its first description in 1985, and today phage display is commonly used in a wide variety of disciplines, including drug discovery, enzyme optimization, biomolecular interaction studies, as well as biosensor development. A cornerstone of all biosensors, regardless of the sensor platform or transduction scheme used, is a sensitive and selective bioreceptor, or a recognition element, that can provide specific binding to the target analyte. Many environmentally or pharmacologically interesting target analytes might not have naturally appropriate binding partners for biosensor development, but phage display can facilitate the production of novel receptors beyond known biomolecular interactions, or against toxic or nonimmunogenic targets, making the technology a valuable tool in the quest of new recognition elements for biosensor development.

A Low-Cost Aptamer Immobilization Technique for the Selective Detection of Protein Biomarkers Using Electrochemical Impedance Spectroscopy

An aptamer is a single-stranded DNA (or an RNA)-based oligonucleotide used as a synthetic molecular recognition element in biosensors. Its many advantages include high affinity and binding efficiency, chemical stability, resistance to harsh environment without losing its bioactivity, mass producibility and reusability which make aptamers superior to the natural receptors such as antibodies and enzymes. Aptamers have found their use in the selective detection of a broad range analytes including proteins, peptides, amino acids, drugs, metal ions, and even whole cells. One of the most promising uses of aptamers is in the electrochemical detection of biomarkers for point-of-care (POC) diagnostics for diseases. In order to utilize such aptamers as molecular receptors in the electrochemical sensors, they must be securely immobilized to the surface of the working electrode so that the specific binding between the target and the aptamer can be transduced into a measurable electrical signal. Sophisticated surface chemistries such as thiol-gold binding or carbodiimide cross-linker chemistry (i.e. EDC/NHS coupling) are often used to covalently attach aptamers to the electrodes. However, such surface chemistries make the fabrication of the aptamer-based sensors difficult in terms of mass production or microscale aptamer array development. In our approach, a simpler immobilization technique is proposed to functionalize the electrode with aptamers. In this work, we make use of the pi-pi stacking forces between carbon nanotubes (CNTs) and aptamers to immobilize the aptamers onto the electrode surface. The fabrication procedure is illustrated in Figure 1. First, CNT-aptamer liquid suspension is prepared by mixing CNT and aptamers in deionized water and sonicating until the mixture is fully dispersed. A small droplet of the prepared solution is then deposited on the working electrode of the screen-printed carbon electrode (SPCE) and is left to dry a hot plate. The detection principle of the biosensor is illustrated in Figure 2 where the lysozyme-binding aptamers were used. Due to the strong affinity between a CNT and a DNA, aptamers tend to wrap around the CNTs as the hydrophobic DNA bases of the aptamer interact with the sidewall of the CNTs via pi-stacking. Hence, as shown in Figure 2(a), the charge transfer from the redox probe to the electrode is hindered due to the negatively charged phosphate backbone of the oligonucleotide wrapping around the CNT, hence causing electrical insulation. As the SPCEs are exposed to the target protein (lysozyme), the aptamer unbinds itself (partially or entirely) from the CNT due to the conformational changes caused by the specific recognition of lysozyme as illustrated in Figure 2(b). This enhances the charge transfer rate from the redox probe in the solution to the electrode. Figure 3 shows the Nyquist plots obtained from the electrochemical impedance spectroscopy (EIS) measurements before and after incubation with the solution containing 0, 1, 5, and 10 µg/mL of lysozyme. The radius of the semicircle in the plot indicates the charge transfer resistance of the redox probe (ferro/ferricyanide) at the electrode. The results show that the aptamer-lysozyme binding causes a decrease in the charge transfer resistance (Rct) because of the conformational change of the aptamers. Figure 4(a) shows the post-lysozyme exposure Nyquist curves for different lysozyme concentrations which represents that the degree of Rct change correlates with the lysozyme concentration. Figure 4 (b) shows the calibration curve of the EIS-based lysozyme sensor under exposure to various concentrations of 3 different types of proteins. The plot shows that Rct change is proportional to the lysozyme concentration whereas the other two proteins does not cause significant Rct change. We also characterized the selectivity of our lysozyme biosensor against other protein biomarkers such as thrombin and bovine serum albumin (BSA). Figure 5 presents the relative response (charge transfer resistance) for the three types of proteins with concentrations of 1, 5, and 10 µg/mL. The sensor response is most pronounced for the lysozyme detection while the other two proteins yield minimal responses. This high specificity arises from the high affinity of the anti-lysozyme aptamer. An increase in the charge transfer resistance for BSA at 10 µg/mL can be attributed to the non-specific adsorption to the electrode which further increases the charge transfer resistance of the redox probe at the electrode. The proposed aptamer immobilization technique in conjunction with EIS can be an effective method in developing a simple and low-cost biomarker sensor for point-of-care testing. Figure 1

Enzymes as Sensors.

Over the last few decades the development of new technologies, the fabrication of new materials, and the introduction of nanotechnologies created new trends in a series of advances that produced innovations in biological sensing devices with a wide range of application from health, security, defense, food, and medicine, to the environment. Specificity, low cost, rapidity, sensitivity, and multiplicity are some of the reasons for their growth, and their commercial success is expected to increase in the next future. Biosensors are devices in which the recognition part of the target molecule is accomplished by biological macromolecules such as proteins, enzymes, antibodies, aptamers, etc. These biomolecules are able to bind to the target molecules with high selectivity and specificity. The interaction between the target molecule and the specific biomolecule is reflected as a change of the biomolecule structural features. The extent of this change is strictly related to the biosensor response. Fluorescence spectroscopy, due to its sensitivity, is often used as the principal technique to monitor biological interactions, and thus the biosensor response as well. Both the intrinsic ultraviolet fluorescence of protein, arising from aromatic amino acids (tryptophan, tyrosine, and phenylalanine), and extrinsic fluorescent labels emitting in the visible region of the spectrum together allow for very flexible transduction of the analyte recognition, suitable for many different applications. This chapter focuses special attention on enzymes as practically unmatched recognition elements for biosensors and emphasizes the potential advantages of customized biosensor devices using apo- or holo forms of enzymes also isolated from thermophile sources.

Electrochemical bacterial detection using poly(3-aminophenylboronic acid)-based imprinted polymer

Biosensors can deliver the rapid bacterial detection that is needed in many fields including food safety, clinical diagnostics, biosafety and biosecurity. Whole-cell imprinted polymers have the potential to be applied as recognition elements in biosensors for selective bacterial detection. In this paper, we report on the use of 3-aminophenylboronic acid (3-APBA) for the electrochemical fabrication of a cell-imprinted polymer (CIP). The use of a monomer bearing a boronic acid group, with its ability to specifically interact with cis-diol, allowed the formation of a polymeric network presenting both morphological and chemical recognition abilities. A particularly beneficial feature of the proposed approach is the reversibility of the cis-diol-boronic group complex, which facilitates easy release of the captured bacterial cells and subsequent regeneration of the CIP. Staphylococcus epidermidis was used as the model target bacteria for the CIP and electrochemical impedance spectroscopy (EIS) was explored for the label-free detection of the target bacteria. The modified electrodes showed a linear response over the range of 103–107cfu/mL. A selectivity study also showed that the CIP could discriminate its target from non-target bacteria having similar shape. The CIPs had high affinity and specificity for bacterial detection and provided a switchable interface for easy removal of bacterial cell.

Biosensors based on oxidative enzymes for detection of environmental pollutants

AbstractIn recent years, the continuous and accumulative discharge of toxic and contaminating compounds to the environment makes necessary to propose precise and quick methods for their detection and quantitation. Especially when one considers that the environmental impact of some of these emerging contaminants has not been clearly determined. Enzyme-based biosensors are an interesting alternative when inspecting different pollutants present in the environment in a quick, efficient, automatized, and economic way. Oxidative enzymes such as peroxidases and polyphenol oxidases (laccases and tyrosinases) are versatile and highly functional enzymes used for analyte recognition. Therefore, these enzymes are considered attractive and interesting biomolecules to act as recognition elements in biosensors. In this regard, detection of pollutants such as pesticides, phenols, heavy metals, and pharmaceutical compounds by using oxidative enzymes as recognition elements in biosensors is a versatile field, and it is the focus of the present review.

Development of a wavelength-shifting fluorescent module for the adenosine aptamer using photostable cyanine dyes.

DNA-based aptamers are commonly used recognition elements in biosensors for a range of target molecules. Here, the development of a wavelength-shifting optical module for a DNA-based adenosine-binding aptamer is described. It applies the combination of two photostable cyanine-styryl dyes as covalent modifications. This energy-transfer pair is postsynthetically attached to oligonucleotides via a copper(I)-catalyzed azide–alkyne cycloaddition by two structurally different approaches: 1) as nucleotide modifications at the 2′-position of uridines and 2) as nucleotide substitutions using (S)-amino-1,2-propanediol as acyclic linker between the phosphodiester bridges. Both dyes exhibit a remarkable photostability. A library of DNA aptamers consisting of different combinations of the two dyes in diagonal orientations were evaluated by their emission color contrast as readout. Further optimization led to aptasensors with improved fluorescent readout as compared with previously reported aptasensors. This approach described is synthetically facile using simple propargylated phosphoramidites as DNA building blocks. As such, this approach could be applied for other dyes and other chemical/biological applications.

Scaffolded biosensors with designed DNA nanostructures

In addition to its fundamental function as a genetic code carrier, the utilization of DNA in various material applications has been actively explored over the past several decades. DNA is intrinsically an excellent type of self-assembly nanomaterial owing to its predictable base-pairing, high chemical stability and the convenience it possesses for synthesis and modification. Because of these unparalleled properties, DNA is widely used as excellent recognition elements in biosensors and as unique building blocks in nanodevices. A critical challenge in surface-based DNA biosensors lies in the reduced accessibility of target molecules to the DNA probes arranged on heterogeneous surfaces, especially when compared to probe–target recognition in homogeneous solutions. To improve the recognition abilities of these heterogeneous surface-confined DNA probes, much effort has been devoted to controlling the surface chemistry, conformation and packing density of the probe molecules, as well as the size and geometry of the surface. In this review, we aim to summarize the recent progress on the improvement of the probe–target recognition properties by introducing DNA nanostructure scaffolds. A range of new strategies have proven to provide a significantly enhanced range in the spatial positioning and the accessibility of the probes to the surface over previously reported linear structures. We will also describe the applications of DNA nanostructure scaffold-based biosensors.

Sensors based on cytochrome P450 and CYP mimicking systems

Cytochrome P450 enzymes (CYPs) act on more than 90 percent of all drugs currently on the market. The catalytic cycle requires electron supply to the heme iron in the presence of oxygen. Electrochemistry allows to characterise the reaction mechanism of these redox enzymes by observing the electron transfer in real time. According to the number of publications on protein electrochemistry CYP has the third position after glucose oxidase and cytochrome c. CYP based enzyme electrodes for the quantification of drugs, metabolites or pesticides have been developed using different iso-enzymes. A crucial step in the sensor development is the efficiency of coupling the biocatalytic systems with the electrode is.In the 1970s the direct electron transfer of heme and heme peptides called microperoxidases (MPs) was used as model of oxidoreductases. They exhibit a broad substrate spectrum including hydroxylation of selected aromatic substrates, demethylation and epoxidation by means of hydrogen peroxide. It overlaps with that of P450 making heme and MPs to alternate recognition elements in biosensors for the detection of typical CYP substrates. In these enzyme electrodes the signal is generated by the conversion of all substrates thus representing in complex media an overall parameter. By combining the biocatalytic substrate conversion with selective binding to a molecularly imprinted polymer layer the specificity has been improved.Here we discuss different approaches of biosensors based on CYP, microperoxidases and catalytically active MIPs and discuss their potential as recognition elements in biosensors. The performance of these sensors and their further development are discussed.

Reverse Virtual Screening on Persistent Organic Pollutants 4,4’-DDE and CB-153

Persistent organic pollutants(POPs) cause various diseases in both human and wildlife and have become a new global environmental problem.The toxic effects of POPs can be induced through their binding to specific proteins in the body.In the present work,a reverse virtual screening method based on molecular docking was used to search for potential protein targets for two POPs,4,4-dichlorodiphenyldichloroethane(4,4-DDD) and 2,2,4,4,5,5-hexachlorobiphenyl(CB-153),from a protein structure database.Targets ranked in the top 5% were chosen for analysis using experimental information.All known targets appeared in the top 5%of targets.This study not only increases understanding of the toxic mechanism of POPs,but may also aid the design of novel recognition elements in biosensors.

Contribution of the cashew gum (Anacardium occidentale L.) for development of layer-by-layer films with potential application in nanobiomedical devices

The search for bioactive molecules to be employed as recognition elements in biosensors has stimulated researchers to pore over the rich Brazilian biodiversity. In this sense, we introduce the use of natural cashew gum (Anacardium occidentale L.) as an active biomaterial to be used in the form of layer-by-layer films, in conjunction with phthalocyanines, which were tested as electrochemical sensors for dopamine detection. We investigated the effects of chemical composition of cashew gum from two different regions of Brazil (Piauí and Ceará states) on the physico-chemical characteristics of these nanostructures. The morphology of the nanostructures containing cashew gum was studied by atomic force microscopy which indicates that smooth films punctuated by globular features were formed that showed low roughness values. The results indicate that, independent of the origin, cashew gum stands out as an excellent film forming material with potential application in nanobiomedical devices as electrochemical sensors.

Aptamers for pharmaceuticals and their application in environmental analytics

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications.

Can peroxygenase and microperoxidase substitute cytochrome P450 in biosensors

Aromatic peroxygenase (APO) from the basidiomycetous mushroom Agrocybe aegerita (AaeAPO) and microperoxidases (MPs) obtained from cytochrome c exhibit a broad substrate spectrum including hydroxylation of selected aromatic substrates, demethylation and epoxidation by means of hydrogen peroxide. It overlaps with that of cytochrome P450 (P450), making MPs and APOs to alternate recognition elements in biosensors for the detection of typical P450 substrates. Here, we discuss recently developed approaches using microperoxidases and peroxygenases in view of their potential to supplement P450 enzymes as recognition elements in biosensors for aromatic compounds. Starting as early as the 1970s, the direct electron transfer between electrodes and the heme group of heme peptides called microperoxidases has been used as a model of oxidoreductases. These MP-modified electrodes are used as hydrogen peroxide detectors based on the catalytic current generated by electrically contacted microperoxidase molecules. A similar catalytic reaction has been obtained for the electrode-immobilised heme protein AaeAPO. However, up to now, no MP-based sensors for substrates have been described. In this review, we present biosensors which indicate 4-nitrophenol, aniline, naphthalene and p-aminophenol based on the peroxide-dependent substrate conversion by electrode-immobilised MP and AaeAPO. In these enzyme electrodes, the signal is generated by the conversion of all substrates, thus representing in complex media an overall parameter. The performance of these sensors and their further development are discussed in comparison with P450-based electrodes.

Preparation of Core‐shell Magnetic Molecularly Imprinted Polymer Nanoparticles for Recognition of Bovine Hemoglobin

Imprinting nanoparticles: Core-shell bovine hemoglobin (BHb) imprinted magnetic nanoparticles (MNPs) with a mean diameter of 210 nm have been synthesized for the first time. The imprinted magnetic nanoparticles could easily reach the adsorption equilibrium and magnetic separation under an external magnetic field, thus avoiding problems related to the bulk polymer. In this work, the core-shell bovine hemoglobin (BHb) imprinted magnetic nanoparticles (MNPs) with a mean diameter of 210 nm were synthesized for the first time. In this protocol, the initial step involved co-precipitation of Fe(2+) and Fe(3+) in an ammonia solution. Silica was then coated on the Fe(3)O(4) nanoparticles using a sol-gel method to obtain silica shell magnetic nanoparticles. Subsequently, 3-aminophenylboronic acid (APBA), which is the functional and cross-linking monomer, and poly(APBA) thin films were coated onto the silica-modified Fe(3)O(4) surface through oxidation with ammonium persulfate in an aqueous solution in the presence or absence of protein. The morphology, adsorption, and recognition properties of the magnetic molecularly imprinted nanomaterial were investigated by transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and vibrating sample magnetometer (VSM). Rebinding experiments were carried out to establish the equilibrium time and to determine the specific binding capacity and selective recognition. The protein adsorption results showed that poly(APBA) MIPs-coated magnetic nanoparticles have high adsorption capacity for template protein BHb and comparatively low non-specific adsorption. The imprinted magnetic nanoparticles could easily reach the adsorption equilibrium and magnetic separation under an external magnetic field, thus avoiding problems related to the bulk polymer. We believe that the imprinted polymer-coated magnetic nanoparticles can be one of the most promising candidates for various applications, which include chemical and biochemical separation, cell sorting, recognition elements in biosensors, and drug delivery.