Band 69, Heft 1

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes expressed in the human body under physiological conditions. AChE is an important part of the cholinergic nerves where it hydrolyses neurotransmitter acetylcholine. Both cholinesterases are sensitive to inhibitors acting as neurotoxic compounds. In analytical applications, the enzymes can serve as a biorecognition element in biosensors as well as simple disposable sensors (dipsticks) and be used for assaying the neurotoxic compounds. In the present review, the mechanism of AChE and BChE inhibition by disparate compounds is explained and methods for assaying the enzymes activity are shown. Optical, electrochemical, and piezoelectric biosensors are described. Attention is also given to the application of sol-gel techniques and quantum dots in the biosensors’ construction. Examples of the biosensors are provided and the pros and cons are discussed.

The functions of metal structures of micro- or nano-dimensions in the sensing mechanisms of amperometric enzyme-based biosensors are considered in the light of the principles of detection of the latter. The applications of metal mono- or bimetallic nanoparticles-modified materials as catalytic electrodes in the fabrication of first-generation and the role which metal nanoparticles play in promoting or enhancing the electron transfer rates in third-generation electrochemical biosensors are reviewed. Some examples of gold NPs functionalised with enzymes via gold-thiol chemistry as a strategy for enzyme immobilisation and spatial orientation when developing amperometric biosensors are also discussed.

Gluconobacter oxydans bacteria exhibit a unique metabolism for quick and incomplete oxidation of a wide range of different compounds (aldoses, ketoses, mono- and poly-alcohols, etc.). Such biotransformation efficiency with simple biomass production led to the industrial applications of these bacteria in the production of several important commodities. Their respiratory activity can also be successfully studied and used in the field of bioelectrochemistry. The main aim of this review is to present various strategies to improve selectivity of assays using intact/treated cells of G. oxydans, to introduce the application of G. oxydans-based biosensors in selective monitoring of analytes during biotransformation processes and to provide information about utilizable sugars in fermentation media or in biological oxygen demand value determination. The final part of the review describes potential application of G. oxydans cells in the generation of electricity from complex fuels within microbial fuel cells by advanced direct electron transfer route between bacterial cells and electrodes.

Microbial cell biosensors, where cells are in direct connection with a transducer enabling quantitative and qualitative detection of an analyte, are very promising analytical tools applied mainly for assays in the environmental field, food industry or biomedicine. Microbial cell biosensors are an excellent alternative to conventional analytical methods due to their specificity, rapid detection and low cost of analysis. Nowadays, nanomaterials are often used in the construction of biosensors to improve their sensitivity and stability. In this review, the combination of microbial and other individual cells with different nanomaterials (carbon nanotubes, graphene, gold nanoparticles, etc.) for the construction of biosensors is described and their applications are provided as well.

Due to the considerable stability of green fluorescent proteins and their capacity to be readily permutated or mutated, they may be exploited in multiple ways to enhance the functionality of in vitro biosensors. Many possibilities, such as the formation of chimeras with other proteins or antibodies, as well as Förster resonance emission transfer performance, may be used for the highly sensitive and specific detection of the target molecules. This review considers the great potential of green fluorescent proteins as the fluorescent probing or recognition biomolecule in various in vitro biosensors applications, as well as obstacles associated with their use.

A fundamental molecular beacon (MB) consists of a special short nucleic acid strand with a fluorophore-quencher pair attached to its ends. It provides a unique framework that is susceptible to conformational transitions between a hairpin (closed) conformation and an extended (open) conformation. These two conformations are readily discernible because of their differing fluorescence emission characteristics. The broad applicability of the robust MB sensing platform has attracted widespread interest, resulting in extensive research studies ranging from theoretical and bioanalytical chemistry to molecular biology and biomedical applications. In this paper, the principles of MB design and the modes and mechanisms of MB operation are reviewed, including MB modifications based on the utilisation of a thymidine-thymidine mismatch in hybridised MB stem, aptamers, peptides and locked nucleic acid strands in an MB loop, as well as plasmonic quenchers, quantum dots and interactions with graphene and graphene oxide. Specific applications of MBs in the analysis of enzymes, DNA mutation, phosphorylation, methylation and ligation, followed by the detection of pathogens and applications in cancer and other disease diagnostics and therapeutics are also reviewed. Molecular beacon-based sensing platforms are expanding rapidly and offer a promising bioanalytical tool for inexpensive and reliable analysis for research and field diagnostics.

The use of aptamers in biosensing has attracted considerable attention as an alternative to antibodies because of their unique properties such as long-term stability, cost-effectiveness and adjustability to various applications. Among cancers, the early diagnosis of prostate cancer (PCa) is one of the greatest concerns for ageing men worldwide. One of the most commonly used biomarkers for PCa is prostate-specific antigen (PSA), which can be found in elevated levels in patients with cancer. This review presents the gradual transition of research from antibody-based to aptamerbased biosensors, specifically for PSA. A brief description on aptamer-based biosensing for other PCa biomarkers is also presented. Special attention is given to electrochemical methods as analytical techniques for the development of simple, sensitive and cost-effective biosensors. The review also focuses on the different surface chemistries exploited for fabrication and their applications in clinical samples. The use of aptamers represents a promising tool for the development of point-ofcare biosensors for the early detection of prostate cancer. In view of the unmatched upper hand of aptamers, future prospects are also discussed, not only in the point-of-care format but also in other novel applications.

Glycans are chains of carbohydrates attached to proteins (glycoproteins and proteoglycans) or lipids (glycolipids). Glycosylation is a post-translational modification and glycans have a wide range of functions in the human body including involvement in oncological diseases. Change in a glycan structure can not only indicate the presence of a pathological process but, more importantly, in some cases also its stage. Thus, a glycan analysis has the potential to be an effective and reliable tool in cancer diagnostics. Lectins are proteins responsible for natural biorecognition of glycans; even carbohydrate moieties still attached to proteins or whole cells can be recognised by lectins, which makes them an ideal candidate for designing label-free biosensors for glycan analysis. This review seeks to summarise evidence that the glycoprofiling of biomarkers by lectin-based biosensors can be of significant help in detecting prostate cancer.

Early diagnosis of diseases with minimal cost and time-consumption has become achievable due to recent advances in the development of biosensors. These devices use biorecognition elements for the selective interaction with an analyte and the signal read-out is obtained via different types of transducers. The operational characteristics of biosensors have been reported as improving substantially when a diverse range of nanomaterials is employed. This review presents the construction of electrochemical biosensors based on graphene, atomically thin 2D carbon crystals, a nanomaterial currently the subject of intensive studies. Here, the most attractive directions for graphene applications in biosensor preparation are discussed, including novel detection and amplification schemes exploiting graphene’s unique electrochemical, physical and chemical properties. There is probably a very bright future for graphene-based biosensors, but much further work is required to fulfill the high expectations.

The aim of this work was to demonstrate that various types of nanostructures provide different gains in terms of sensitivity or detection limit albeit providing the same gain in terms of increased area. Commercial screen printed electrodes (SPEs) were functionalized with 100 μg of bismuth oxide nanoparticles (Bi 2 O 3 NPs), 13.5 μg of gold nanoparticles (Au NPs), and 4.8 μg of multi-wall carbon nanotubes (MWCNTs) to sense hydrogen peroxide (H 2 O 2 ). The amount of nanomaterials to deposit was calculated using specific surface area (SSA) in order to equalize the additional electroactive surface area. Cyclic voltammetry (CV) experiments revealed oxidation peaks of Bi 2 O 3 NPs, Au NPs, and MWCNTs based electrodes at (790 ± 1) mV, (386 ± 1) mV, and (589 ± 1) mV, respectively, and sensitivities evaluated by chronoamperometry (CA) were (74 ± 12) μA mM −1 cm −2 , (129 ± 15) μA mM −1 cm −2 , and (54 ± 2) μA mM −1 cm −2 , respectively. Electrodes functionalized with Au NPs showed better sensing performance and lower redox potential (oxidative peak position) compared with the other two types of nanostructured SPEs. Interestingly, the average size of the tested Au NPs was 4 nm, under the limit of 10 nm where the quantum effects are dominant. The limit of detection (LOD) was (11.1 ± 2.8) μM, (8.0 ± 2.4) μM, and (3.4 ± 0.1) μM for Bi 2 O 3 NPs, Au NPs, and for MWCNTs based electrodes, respectively.

Prostate cancer (PCa) diagnostics can be effectively addressed using sensor-based approaches. Proper selection of biomarkers to be included in biosensors for accurate detection becomes the need of the hour. Such biosensor and biochip technologies enable fast and efficient determination of proteins and provide a remarkable insight into the changes in the protein structure, such as aberrant glycosylation, which can increase the performance, sensitivity and specificity of clinic assays. However, for a thorough comprehension of such complex protein modifications, it is crucial to understand their biospecific interactions. Surface plasmon resonance (SPR), one of the most rapidly developing techniques for measuring real-time quantitative binding affinities and kinetics of the interactions of antigens and antibodies, was chosen as an appropriate tool for this purpose. Herein, experiments on the interactions of antibodies specific against different epitopes of free and complexed prostate-specific antigen (PSA), a prominent PCa biomarker, are presented with two main aims: (i) to continue as lectin glycoprofiling studies and; (ii) to be used in microfluidic immunoassay-based platforms for point-of-care devices. Various PSA-specific antibodies were covalently immobilized on the biochip surface via amine coupling, and free or complexed PSA was injected into the dual-flow channels of the SPR device. Kinetic parameters and affinity constants of these interactions, as well as cross-reactivities of the used antibodies were determined. The sandwich assay for PSA determination was developed employing both primary and secondary anti-PSA antibodies. Sensitivity of the assay was 3.63 nM −1 , the detection limit was 0.27 nM and the SPR biosensor response towards free PSA was linear up to 25 nM. All these findings are essential for proper design of a selective, sensitive, and highly reliable biosensor for PCa diagnosis as a lab-on-chip device.

The catalytic oxidation of a synthetic water-soluble analogue of vitamin E (α-tocopherol, Trolox) by tyrosinase enzyme in the presence of molecular oxygen was studied using electrochemical techniques. This specific enzymatic reaction was exploited for the preparation of a biosensor based on the amperometric reduction of the electroactive product (α-tocoquinone) formed. An electroactive surface of the transducers used was covered with a thin conductive layer of Nafion containing tyrosinase. Significant progress in sensitivity towards polyphenolic compounds such as Trolox was achieved at CPE with carbon nanotubes immobilised on its surface (CPE/CNTs) as electric transducers. The biosensor so developed can be used for the direct determination of total phenolic content (TPC). This important nutrition value can be expressed as the mass equivalent of Trolox, i.e. Trolox equivalent antioxidant capacity (TEAC), which could be used as an alternative to the evaluations currently used based on spectrophotometric methods such as total radical-trapping antioxidant parameter (TRAP), ferric reducing-antioxidant power (FRAP) or 1,1-diphenyl-2-picrylhydrazyl spectrometric assay (DPPH). The effects of the enzyme amount in the Nafion layer (3.0 μg), the influence of the nanoparticles present, the optimal pH value suitable for enzymatic activity (7.0), and the kinetics of enzymatic and electrochemical reactions were studied using cyclic voltammetry (CV). The determination of optimal conditions for amperometry in batch configuration (working potential, speed of stirring, volume of sample, calibration curve, etc.) was not a target of this electrochemical study.

A biosensor for putrescine containing a sensing layer with an optical oxygen probe based on ruthenium complex and the enzyme diamine oxidase from pea is described. The diamine oxidase was pre-immobilised on broken micro-beads modified with a ferrofluid. The pre-immobilised enzyme and ruthenium complex were both incorporated into the UV-cured inorganic-organic hybrid polymer ORMOCER ® and deposited on a lens to form a sensitive layer of 210 μm in thickness. The sensitivity to the putrescine concentration determined under air saturation was between 3.50 μs L mmol −1 and 4.50 μs L mmol −1 in a hundred experiments conducted intermittently over a one year period. With the oxygen concentration increasing from 10 % to 100 % of DO (dissolved oxygen), the biosensor sensitivity decreased from 6.87 μs L mmol −1 to 0.70 μs L mmol −1 and its dynamic range increased from 0.10 mmol L −1 to 1.75 mmol L −1 . To estimate the behaviour of the putrescine sensor in parametric space, a mathematical model of the reaction-transport processes inside the sensing layer was developed. The model revealed the qualitative relations between the sensor analytical features, the characteristics of the sensitive layer and concentrations of substrates. The results of the mathematical modelling may serve as guidelines in the design of optodes for specific applications.

Biocatalysed precipitation of an insoluble product accumulated on the enzyme-modified electrode surface was applied as the amplification path for low concentration sensing of hydrogen peroxide and glucose. Sensitive electrochemical and quartz-crystal microbalance (QCM) biosensors based on biocatalytic precipitation were developed. A horseradish peroxidase (HRP) monolayer-modified electrode was used to sense H 2 O 2 via the oxidation of 4-chloro-1-naphthol (4CN) forming insoluble benzo-4-chlorocyclohexadienone. Additionally, the bienzyme system employed glucose oxidase (GOx) linked to HRP/4CN. The amount of the precipitate assembled on the sensing surface corresponded to the concentration of analytes and to the length of the incubation interval. The precipitated deposits were followed as a change of impedance using cyclic voltammetry (CV), mass change was determined continuously using a microgravimetric quartz-crystal microbalance, and optical microscopy enabled the visualisation of the precipitate. Regeneration of the enzyme-modified electrode was performed using cathodic reduction of the insoluble product. Thus, a simple biosensor for multiple analyses with low detection limits and of low cost can be developed.

Various types of carbon nanoparticles were directly mixed with microbial cells of Gluconobacter oxydans within a 3-D bionanocomposite in order to prepare a highly sensitive ethanol biosensor with a short response time. From all carbonaceous nanomaterials tested, single- or multi-walled carbon nanotubes provided the highest sensitivity of detection (117-121 μA cm −2 mM −1 ), but from a practical point of view, Ketjen black 300 and 600 provide very low detection limit (2-6 μM) and high sensitivity for the ethanol analysis (84-88 μA cm −2 mM −1 ) with a short response time (14-33 s). Moreover, the price of Ketjen black is a few orders of magnitude lower compared to that of carbon nanotubes. Finally, the study showed that the morphology of nanoparticles rather than their surface modification is the key element in achieving high sensitivity of ethanol detection.

The present study demonstrates the influences of chlorides, fluorides and bromides of potassium and sodium on the growth and Hg 2+ -induced bioluminescence of bioreporter Escherichia coli ARL1. In a Luria-Bertani medium (LB), cell growth was inhibited by concentrations of sodium and potassium fluorides above 0.2 mol L −1 . The addition of NaCl increased cell tolerance to the toxic effects of fluorides and bromides. Lag periods of 10 h and more were observed for cultivations in LB without NaCl and with halides (NaCl, KCl, NaBr, KBr, NaF and KF) at concentrations lower than 0.06 mol L −1 . In a phosphate buffer (PB), the bioluminescence of E. coli ARL1, induced with HgCl 2 , was increased by the addition of NaCl, KCl, NaBr, KBr, NaF and KF (concentration of 0-0.25 mol L −1 ). In a saline phosphate buffer (PBS), the maxima of induced bioluminescence declined to 50 %, in the case of NaF (0.12 mol L −1 ), and to zero for KF. An addition of tryptone to the induction medium increased induced light emission ten-fold. Concentrated artificial sea water (ASW) (70-100 % ASW) inhibited bioluminescence induction. The new detection assay with E. coli ARL1 made possible the detection of 0.57 μg L −1 of HgCl 2 in double-diluted artificial sea water (25 % ASW).

Nanoparticles as a progressively developing branch offer a tool for studying the interaction of carbon quantum dots (CQDs) with DNA. In this study, fluorescent CQDs were synthesized using citric acid covered with polyethylene glycol (PEG) as the source of carbon precursors. Furthermore, interactions between CQDs and DNA (double-stranded DNA and single-stranded DNA) were investigated by spectral methods, gel electrophoresis, and electrochemical analysis. Primarily, the fluorescent behavior of CQDs in the presence of DNA was monitored and major differences in the interaction of CQDs with tested single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) were observed at different amounts of CQDs (μg mL −1 : 25, 50, 100, 250, 500). It was found that the interaction of ssDNA with CQDs had no significant influence on the CQDs fluorescence intensity measured at the excitation wavelengths of 280 nm, 350 nm, and 400 nm. However, in the presence of dsDNA, the fluorescence intensity of CQDs was significantly increased. Our results provide basic understanding of the interaction between CQDs and DNA. Such fabricated CQDs-DNA might be of great benefit for the emerging nanomaterials based biosensing methods.

A novel, sensitive and selective electrochemical hybridisation biosensor was developed for the detection of the hepatitis B virus (HBV) using a manganese(II) complex as electrochemical indicator and a DNA probe-modified carbon paste electrode as the biosensor (DNA/CPE). The results showed that this complex could be accumulated electrochemically the immobilised dsDNA layer rather than in the single-stranded DNA (ssDNA) layer. On the basis of this, the manganese complex was used as an electrochemical hybridisation indicator for the detection of oligonucleotides related to HBV. The hybridisation event was evaluated on the basis of the difference between the reduction signals of the manganese(II) complex with the probe DNA prior to and post hybridisation with a target sequence using a differential pulse mode. Several factors affecting the immobilisation and hybridisation of oligonucleotides as well as the indicator’s accumulation were investigated. Experiments with a noncomplementary and mismatch sequences demonstrated the good selectivity of the biosensor. Using this approach, the HBV target oligonucleotide’s sequence could be quantified over arange from 0.22 ng L −1 to 5.40 ng L −1 , with a linear correlation coefficient of 0.9994 and the limit of detection of 0.07 ng L −1 .

The thickness shear mode acoustic method was used to study the binding of thrombin to DNA aptamers immobilised on the gold surface covered by DNA tetrahedrons. The binding of thrombin to conventional aptamers sensitive to fibrinogen (FBT) and heparin (HPT) exosites as well as to HPT in a loop configuration (HPTloop) made it possible to determine the constant of dissociation (K D ) and the limit of detection (LOD). The sensing system composed of a HPTloop was characterised by K D = (66.7 ± 22.7) nM, which was almost twice as low as that of FBT and HPT. For this biosensor, a lower LOD of 5.2 nM compared with 17 nM for conventional HPT aptamers was also obtained. Less sensitive sensors based on FBT aptamers revealed an LOD of 30 nM which is in agreement with the lower affinity of these aptamers to thrombin in comparison with that of HPT. The surface concentration of DNA tetrahedrons was determined by the electrochemical method using [Ru(NH 3 ) 6 ] 3+ as a redox probe. These experiments confirmed that the “step by step” method of forming the sensing layer, consisting first in chemisorption of DNA tetrahedrons onto a gold surface and then in hybridisation of the aptamer-supporting part with complementary oligos at the top of the tetrahedron, is preferable. In addition, atomic force microscopy was applied to analyse the topography of the gold layers modified stepwise by DNA tetrahedrons, DNA aptamers and thrombin. The height profiles of the layers were in reasonable agreement with the dimensions of the adsorbed molecules. The results indicate that DNA tetrahedrons represent an efficient platform for immobilisation of aptamers.

Albumin, as an antifouling agent preventing non-specific adsorption, is widely applied in biosensor construction and in the design of nanocarriers for theranostic applications. However, albumin is not completely inert and it interacts with many endogenous and exogenous compounds. The present work investigates the interactions of bovine serum albumin (BSA) with phenol red dye (PR), a common component of cell-growth media. PR was found to bind to BSA, with the affinity constant KA = (2.5 ± 0.8) × 10 5 L mol −1 , and that it quenches the intrinsic BSA fluorescence through the F¨orster non-radiative resonance energy transfer (FRET) via static modes, with critical FRET distance of R0 = 2.65 nm. Synchronous fluorescence spectroscopy measurements indicated a conformational transition of BSA in the presence of PR to a conformation with tyrosine residues surrounded by a more hydrophobic environment. Thermodynamic parameters, ΔH°, ΔG° and ΔS°, determined using fluorescence and isothermal titration microcalorimetry, indicated that entropic contribution played a major role at ambient temperature. It was also found that common metal ions increased KA but Ca 2+ and Fe 3+ decreased it.

The response of low-density graphite electrodes hosting Trametes hirsuta laccase in a direct electron transfer regime is presented for real-time analysis of O 2 concentrations. The use of contrasting immobilisation methods developed for biocathodes affords good reproducibility and reliability of the amperometric biosensor, which shows a limit of detection below 1 μM and a sensitivity slightly higher than 60 nA cm −2 M −1 .

The analysis of glycan isomers is an important task in glycomics. This study reveals for the first time that two oligosaccharide pentamers can be distinguished electrochemically. 3α,6α-mannopentaose (MPO) and verbascose (VBS) were modified using Os(VI)-temed and the carbohydrate adducts measured directly in the reaction mixture using cyclic voltammetric (CV) and square wave voltammetric (SWV) stripping at the hanging mercury drop electrode. Some CV and SWV responses of MPO were found to differ from those of VBS at full electrode coverage, requiring μM concentrations at short accumulation time-intervals. Similar results were obtained at partial electrode coverage at nM concentrations. These results afford an opportunity for the development of a new simple and inexpensive glycan analysis.