Identification of medium- and mechanism-related pitfalls towards improved performance and applicability of electrochemical mercury(II) aptasensors.

The importance of understanding the mercury (II) ion interactions with thymine-rich DNA sequences is the reason for multiple comparative investigations carried out with the use of optical detection techniques directly in the depth of solution. However, the results of such investigations have limited applicability in the interpretation of the Hg2+ binding phenomenon by DNA sequences in thin, interfacial (electrode/solution), self-organized monolayers immobilized on polarizable surfaces, often used for sensing purposes in electrochemical biosensors. Overlooking the careful optimization of the measurement conditions is the source of discrepancies in the interpretation of the registered electrochemical signal. In this study, the chosen effects accompanying the efficiency of surface related recognition of Hg2+ by polyThymine DNA sequences labelled with methylene blue were investigated by voltammetry, QCM and spectro-electrochemical techniques. As was shown, the composition of the biosensing layer and buffers or the analytical procedures have a significant impact on the registered electrochemical readout which translates into signal stability, the biosensor's working parameters or even the mechanism of detection. After elucidation of the above factors, the complete and ready-to-use biosensor-based analytical solution was proposed offering subpicomolar mercury ion determination with high selectivity (also in aqueous real samples), reusability, and high signal stability even after long-term storage. The developed procedures were successfully used during the miniaturization process with self-prepared (PVD) elastic transducers. The obtained sensor, together with the simplicity of its use, low manufacturing cost, and attractive analytical parameters (i.e., LOD < < Hg2+ WHO limit) can present an interesting alternative for on-site mercury ion detection in environmental samples.

Aptamer and Electrochemical Aptasensor towards Selenate Ions (SeO42-).

Selenium is an essential inorganic compound in human and animal nutrition, involved in the proper functioning of the body. As a micronutrient, it actively contributes to the regulation of various metabolic activities, i.e., thyroid hormone, and protection against oxidative stress. However, Se exhibits a narrow concentration window between having a positive effect and exerting a toxic effect. In higher doses, it negatively affects living organisms and causes DNA damage through the formation of free radicals. Increased reactivity of Se anions can also disrupt the integrity and function of DNA-repairing proteins. As the permissible concentration of Se in drinking water is 10 µg/L, it is vital to develop sensitive and robust methods of Se detection in aqueous samples. In this study, for the first time, we proposed a selective aptamer for selenate ion detection, chosen following the SELEX process, and its application in the construction of an electrochemical aptasensor towards SeO42- ions. Measurement conditions such as the used redox marker and pH value of the measurement solution were chosen. The proposed aptasensor is characterized by good selectivity and an LOD of 1 nM. Conditions for biosensor regeneration and storage were also investigated in this research.

Modern Electrochemical Biosensing Based on Nucleic Acids and Carbon Nanomaterials.

To meet the requirements of novel therapies, effective treatments should be supported by diagnostic tools characterized by appropriate analytical and working parameters. These are, in particular, fast and reliable responses that are proportional to analyte concentration, with low detection limits, high selectivity, cost-efficient construction, and portability, allowing for the development of point-of-care devices. Biosensors using nucleic acids as receptors has turned out to be an effective approach for meeting the abovementioned requirements. Careful design of the receptor layers will allow them to obtain DNA biosensors that are dedicated to almost any analyte, including ions, low and high molecular weight compounds, nucleic acids, proteins, and even whole cells. The impulse for the application of carbon nanomaterials in electrochemical DNA biosensors is rooted in the possibility to further influence their analytical parameters and adjust them to the chosen analysis. Such nanomaterials enable the lowering of the detection limit, the extension of the biosensor linear response, or the increase in selectivity. This is possible thanks to their high conductivity, large surface-to-area ratio, ease of chemical modification, and introduction of other nanomaterials, such as nanoparticles, into the carbon structures. This review discusses the recent advances on the design and application of carbon nanomaterials in electrochemical DNA biosensors that are dedicated especially to modern medical diagnostics.

Self-Assembling Graphene Layers for Electrochemical Sensors Printed in a Single Screen-Printing Process.

This article reports findings on screen-printed electrodes employed in microfluidic diagnostic devices. The research described includes developing a series of graphene- and other carbon form-based printing pastes compared to their rheological parameters, such as viscosity in static and shear-thinning conditions, yield stress, and shear rate required for thinning. In addition, the morphology, electrical conductivity, and electrochemical properties of the electrodes, printed with the examined pastes, were investigated. Correlation analysis was performed between all measured parameters for six electrode materials, yielding highly significant (p-value between 0.002 and 0.017) correlations between electron transfer resistance (Ret), redox peak separation, and static viscosity and thinning shear-rate threshold. The observed more electrochemically accessible surface was explained according to the fluid mechanics of heterophase suspensions. Under changing shear stress, the agglomeration enhanced by the graphene nanoplatelets' interparticle affinity led to phase separation. Less viscous pastes were thinned to a lesser degree, allowing non-permanent clusters to de-agglomerate. Thus, the breaking of temporary agglomerates yielded an unblocked electrode surface. Since the mechanism of phase ordering through agglomeration and de-agglomeration is affected by the pastes' rheology and stress during the printing process and requires no further treatment, it can be appropriately labeled as a self-assembling electrode material.

Comparative Evaluation of Different Surface Coatings of Fe3O4-Based Magnetic Nano Sorbent for Applications in the Nucleic Acids Extraction.

Nucleic acid extraction and purification are crucial steps in sample preparation for multiple diagnostic procedures. Routine methodologies of DNA isolation require benchtop equipment (e.g., centrifuges) and labor-intensive steps. Magnetic nanoparticles (MNPs) as solid-phase sorbents could simplify this procedure. A wide range of surface coatings employs various molecular interactions between dsDNA and magnetic nano-sorbents. However, a reliable, comparative evaluation of their performance is complex. In this work, selected Fe3O4 modifications, i.e., polyethyleneimine, gold, silica, and graphene derivatives, were comprehensively evaluated for applications in dsDNA extraction. A family of single batch nanoparticles was compared in terms of morphology (STEM), composition (ICP-MS/MS and elemental analysis), surface coating (UV-Vis, TGA, FTIR), and MNP charge (ζ-potential). ICP-MS/MS was also used to unify MNPs concentration allowing a reliable assessment of individual coatings on DNA extraction. Moreover, studies on adsorption medium (monovalent vs. divalent ions) and extraction buffer composition were carried out. As a result, essential relationships between nanoparticle coatings and DNA adsorption efficiencies have been noticed. Fe3O4@PEI MNPs turned out to be the most efficient nano sorbents. The optimized composition of the extraction buffer (medium containing 0.1 mM EDTA) helped avoid problems with Fe3+ stripping, which improved the validity of the spectroscopic determination of DNA recovery.

Versatile and Easily Designable Polyester-Laser Toner Interfaces for Site-Oriented Adsorption of Antibodies.

Laser toners appear as attractive materials for barriers and easily laminated interphases for Lab-on-a-Foil microfluidics, due to the excellent adhesion to paper and various membranes or foils. This work shows for the first time a comprehensive study on the adsorption of antibodies on toner-covered poly(ethylene terephthalate) (PET@toner) substrates, together with assessment of such platforms in rapid prototyping of disposable microdevices and microarrays for immunodiagnostics. In the framework of presented research, the surface properties and antibody binding capacity of PET substrates with varying levels of toner coverage (0-100%) were characterized in detail. It was proven that polystyrene-acrylate copolymer-based toner offers higher antibody adsorption efficiency compared with unmodified polystyrene and PET as well as faster adsorption kinetics. Comparative studies of the influence of pH on the effectiveness of antibodies immobilization as well as measurements of surface ζ-potential of PET, toner, and polystyrene confirmed the dominant role of hydrophobic interactions in adsorption mechanism. The applicability of PET@toner substrates as removable masks for protection of foil against permanent hydrophilization was also shown. It opens up the possibility of precise tuning of wettability and antibody binding capacity. Therefore, PET@toner foils are presented as useful platforms in the construction of immunoarrays or components of microfluidic systems.

Recent Achievements in Electrochemical and Optical Nucleic Acids Based Detection of Metal Ions.

Recently nucleic acids gained considerable attention as selective receptors of metal ions. This is because of the possibility of adjusting their sequences in new aptamers selection, as well as the convenience of elaborating new detection mechanisms. Such a flexibility allows for easy utilization of newly emerging nanomaterials for the development of detection devices. This, in turn, can significantly increase, e.g., analytical signal intensity, both optical and electrochemical, and the same can allow for obtaining exceptionally low detection limits and fast biosensor responses. All these properties, together with low power consumption, make nucleic acids biosensors perfect candidates as detection elements of fully automatic portable microfluidic devices. This review provides current progress in nucleic acids application in monitoring environmentally and clinically important metal ions in the electrochemical or optical manner. In addition, several examples of such biosensor applications in portable microfluidic devices are shown.

From Small Molecules Toward Whole Cells Detection: Application of Electrochemical Aptasensors in Modern Medical Diagnostics.

This paper focuses on the current state of art as well as on future trends in electrochemical aptasensors application in medical diagnostics. The origin of aptamers is presented along with the description of the process known as SELEX. This is followed by the description of the broad spectrum of aptamer-based sensors for the electrochemical detection of various diagnostically relevant analytes, including metal cations, abused drugs, neurotransmitters, cancer, cardiac and coagulation biomarkers, circulating tumor cells, and viruses. We described also possible future perspectives of aptasensors development. This concerns (i) the approaches to lowering the detection limit and improvement of the electrochemical aptasensors selectivity by application of the hybrid aptamer-antibody receptor layers and/or nanomaterials; and (ii) electrochemical aptasensors integration with more advanced microfluidic devices as user-friendly medical instruments for medical diagnostic of the future.

Recent Advancements in Receptor Layer Engineering for Applications in SPR-Based Immunodiagnostics.

The rapid progress in the development of surface plasmon resonance-based immunosensing platforms offers wide application possibilities in medical diagnostics as a label-free alternative to enzyme immunoassays. The early diagnosis of diseases or metabolic changes through the detection of biomarkers in body fluids requires methods characterized by a very good sensitivity and selectivity. In the case of the SPR technique, as well as other surface-sensitive detection strategies, the quality of the transducer-immunoreceptor interphase is crucial for maintaining the analytical reliability of an assay. In this work, an overview of general approaches to the design of functional SPR-immunoassays is presented. It covers both immunosensors, the design of which utilizes well-known and often commercially available substrates, as well as the latest solutions developed in-house. Various approaches employing chemical and passive binding, affinity-based antibody immobilization, and the introduction of nanomaterial-based surfaces are discussed. The essence of their influence on the improvement of the main analytical parameters of a given immunosensor is explained. Particular attention is paid to solutions compatible with the latest trends in the development of label-free immunosensors, such as platforms dedicated to real-time monitoring in a quasi-continuous mode, the use of in situ-generated receptor layers (elimination of the regeneration step), and biosensors using recombinant and labelled protein receptors.

DNA Delivery Systems Based on Peptide-Mimicking Cationic Lipids-The Effect of the Co-Lipid on the Structure and DNA Binding Capacity.

In continuation of previous work, we present a new promising DNA carrier, OO4, a highly effective peptide-mimicking lysine-based cationic lipid. The structural characteristics of the polynucleotide carrier system OO4 mixed with the commonly used co-lipid DOPE and the saturated phospholipid DPPE have been studied in two-dimensional and three-dimensional model systems to understand their influence on the physical-chemical properties. The phase behavior of pure OO4 and its mixtures with DOPE and DPPE was studied at the air-water interface using a Langmuir film balance combined with infrared reflection-absorption spectroscopy. In bulk, the self-assembling structures in the presence and absence of DNA were determined by small-angle and wide-angle X-ray scattering. The amount of adsorbed DNA to cationic lipid bilayers was measured using a quartz crystal microbalance. The choice of the co-lipid has an enormous influence on the structure and capability of binding DNA. DOPE promotes the formation of nonlamellar lipoplexes (cubic and hexagonal structures), whereas DPPE promotes the formation of lamellar lipoplexes. The correlation of the observed structures with the transfection efficiency and serum stability indicates that OO4/DOPE 1:3 lipoplexes with a DNA-containing cubic phase encapsulated in multilamellar structures seem to be most promising.

Lead and Cadmium Content in Grass Growing Near An Expressway.

The purpose of the study was to evaluate the effect of distance from a road on lead and cadmium content in grass species near an expressway and to assess bioaccumulation of these elements by morphological parts of the plants. The material for the research was the following grass species in their flowering stage: Dactylis glomerata, Arrenatherum elatius, and Alopecurus pratensis. Plant samples were collected along the international E30 road, the ring-road of Siedlce, in May 2015. A 9-km road section was examined with samples collected on both sides, covering a stretch of 700 m, at the following distances from the edge of the road: 1, 5, 10, and 15 m. Five samples of each plant species and at each distance from the road were collected. Lead and cadmium concentration was determined with the AAS method. The largest amounts of Pb were absorbed by A. pratensis L. (3.843 mg kg-1DM), while the lowest by A. elatius L. (2.523 mg kg-1DM). Of the above plants, the highest amount of Cd (0.286 mg kg-1DM) was accumulated by D. glomerata L. Underground parts of the grass species accumulated greater amounts of Pb and Cd than aboveground parts. It indicates that considerable amounts of heavy metals released by expressway vehicles contaminated the soil. The highest content of Pb and Cd was found in the grass growing at a distance of 5 m from the edge of the roadway, and this applies both to underground and aboveground parts.

Isothermal DNA amplification combined with lateral flow dipsticks for detection of biothreat agents.

The recently developed methods of nucleic acids isothermal amplification are promising tools for point-of-care diagnostics and in the field detection of pathogenic microorganisms. However, application of these methods outside a laboratory faces some challenges such as the rapid and sensitive detection of amplified products and the absence of cross-reactivity with genetically related microorganisms. In the presented study we compared three methods of isothermal DNA amplification loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA), for detection of highly dangerous pathogens, such as Bacillus anthracis, Francisella tularensis and Yersinia pestis, and combined them with lateral flow dipsticks for the rapid visualization of amplified products. We observed low specificity of the three methods for B. antharcis, medium for Y. pestis and high for F. tularensis detection. Sensitivity and the detection limit were high and comparable for all the methods. We concluded that the lateral flow dipsticks have been a very useful tool for product detection of the isothermal amplification methods and enable reading the results without the use of any equipment. However, our results showed that the use of isothermal amplification methods is strongly related to the risk of false positive results.

The Effect of Liming and Sewage Sludge Application on Heavy Metal Speciation in Soil.

The aim of this paper is to assess the effect of liming and low doses of municipal sewage sludge (5%, 10%, 15% of the soil mass) on lead, chromium and nickel speciation in soil. The 420-day experiment was carried out in laboratory conditions. In all the samples lead, chromium and nickel concentration was determined with the ICP-AES method, while the content of those metals in different fractions was measured with the seven-step Zeien and Brümmer method, on the 30th and 420th days of the experiment. Sewage sludge doses significantly diversified lead, chromium and nickel amounts in the soil. The highest dose of sludge caused a significant increase, compared to the control, in the content of those metals. In the sludge the dominant forms of metals tested in the experiment were lead and chromium bound to organic matter (F4) as well as nickel bound to amorphous iron oxides (F5). Liming decreased the mobility of the metals in the soil.

Evaluation of biological activity of quantum dots in a microsystem.

The presented work aimed at systematic investigation of biological activity of CdSex S1- x /ZnS and CdSe/ZnS quantum dots (QDs), whose surface was modified with different ligands. For these studies, we used a microfluidic system combined with fluorescence microscopy techniques, which enabled analysis of cells' morphology, viability, and QDs uptake. PDMS and glass-based microfluidic system enabled the precise control of the cell environment, allowed to examine five replications of each tested QDs concentrations (statistically significant number), monitor multiple cellular events, and avoid manual preparation of QDs dilutions. We investigated the influence of the core composition and the type of surface modifiers on QDs toxicity. We also determined whether the examined nanoparticles penetrate into the cells. For all tested nanoparticles, the decrease of cells' viability was observed when increasing nanoparticles concentration. The decrease of live cells' number in microchambers and the accumulation of the nanoparticles around cultured cells were observed. The effect of hydrocarbon chain length of surface modifiers and QDs core composition on the cell viability was confirmed in our tests.

Pitfalls and capabilities of various hydrogen donors in evaluation of peroxidase-like activity of gold nanoparticles.

Catalytic nanomaterials, widely used as substitutes of peroxidase, exhibit unique properties, which are unattainable for native enzymes. However, their activity is usually examined by means of substrates developed and methods standardized for horseradish peroxidase (HRP). The aim of the presented work was to determine the scope of usefulness of chromogenic substrates for gold nanoparticle (AuNP) activity studies under conditions which significantly extend beyond the activity range of a native HRP. The applicability of chromogens such as 3,3'5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) beyond the typical range of pH, and for the samples of high concentration of hydrogen peroxide was examined. The conducted research confirmed the usefulness of ABTS and TMB in acidic media (pH 2.5-3.5). At the same time, potential interferences from chloride anion, unobservable for HRP-based assays, were indicated. Moreover, a number of potentially useful hints concerning relations of concentration of substrates and catalyst for aromatic amine oxidation (TMB and OPD) were proposed. By increasing the concentration of chromogens and thanks to assuring the relatively low conversion of the reaction, the stability of TMB and OPD oxidation product was improved even in acidic media. The comparative studies of H2O2 affinity to the surface of AuNPs in the presence of various hydrogen donors underlined the superiority of phenolic compounds over aromatic amines and ABTS in the case of the samples of relatively low H2O2 concentration. This work highlights some improvements in the methods of HRP-like activity characterization of NPs. It provides a critical analysis of the major challenges, which may emerge in a case of bioanalytical assays employing the catalytic nanoparticles as labels.

The effects of soil liming and sewage sludge application on dynamics of copper fractions and total copper concentration.

The paper deals with effects of liming and different doses of municipal sewage sludge (5, 10, and 15 % of soil mass) on copper speciation in soil. In all samples, pH was determined together with total copper concentration, which was measured with the ICP-AES method. Concentration of copper chemical fractions was determined using the seven-step procedure of Zeien and Brümmer. In the soil treated with the highest dose of sludge (15 %), there was, compared to the control, a twofold increase in the concentration of copper and a threefold increase in the concentration of nitrogen. Copper speciation analysis showed that in the municipal sewage sludge the easily soluble and exchangeable fractions (F1 and F2) constituted only a small share of copper with the highest amount of this metal in the organic (F4) and residual (F7) fractions. In the soil, at the beginning of the experiment, the highest share was in the organic fraction (F4), the residual fraction (F7) but also in the fraction where copper is bound to amorphous iron oxides (F5). After 420 days, at the end of the experiment, the highest amount of copper was mainly in the organic fraction (F4) and in the fraction with amorphous iron oxides (F5). Due to mineralization of organic matter in the sewage sludge, copper was released into the soil with the share of the residual fraction (F7) decreasing. In this fraction, there was much more copper in limed soil than in non-limed soil.

Expression of adipocyte biomarkers in a primary cell culture models reflects preweaning adipobiology.

A cohort of genes was selected to characterize the adipogenic phenotype in primary cell cultures from three tissue sources. We compared the quantitative expression of biomarkers in culture relative to their expression in vivo because the mere presence or absence of expression is minimally informative. Although all biomarkers analyzed have biochemical functions in adipocytes, the expression of some of the biomarkers varied enormously in culture relative to their expression in the adult fat tissues in vivo, i.e. inguinal fat for white adipocytes and brite cells, interscapular brown adipose tissue for brown adipocytes, and ear mesenchymal stem cells for white adipocytes from adult mice. We propose that the pattern of expression in vitro does not reflect gene expression in the adult mouse; rather it is predominantly the expression pattern of adipose tissue of the developing mouse between birth and weaning. The variation in gene expression among fat depots in both human and rodent has been an extensively studied phenomenon, and as recently reviewed, it is related to subphenotypes associated with immune function, the inflammatory response, fat depot blood flow, and insulin sensitivity. We suggest that adipose tissue biology in the period from birth to weaning is not just a staging platform for the emergence of adult white fat but that it has properties to serve the unique needs of energy metabolism in the newborn. A case in point is the differentiation of brite cells that occurs during this period followed by their involution immediately following weaning.

Studies on potential use of tin(IV) porphyrin in a role of proteins' label.

We present an electrochemical and optical characterization of 5,10,15,20-tetraphenylporphyrin tin(IV) dichloride (Sn-tpp) in terms of its potential use as a hybrid proteins' label. Our research comprised Sn-tpp and Sn-tpp in the presence of model proteins selected as to mimic a receptor or surface blocking agents: bovine serum albumin, ovalbumin, and immunoglobulin G. In the course of the study, we determined optimal conditions for analysis by means of differential pulse voltammetry, ultraviolet-visible spectrophotometry, and spectrofluorimetry. In electrochemical detection, the influence of the working electrode, solvent, and supporting electrolyte was examined. Displacements of the received signals along the potential axis (a shift of the potential) and changes in signal intensities due to the addition of proteins were observed and analyzed. Simultaneously, the suitability of Sn-tpp as a label in optical detection mode was assessed by using spectroscopic techniques. The obtained results prove Sn-tpp to be applicable in dual and triple detection systems. Such an approach will improve the reliability of the analysis and, at the same time, will allow for widening the range of the linear response with some overlapping ranges of concentrations.

Electroanalysis of pM-levels of urokinase plasminogen activator in serum by phosphorothioated RNA aptamer.

Protein biomarkers of cancer allow a dramatic improvement in cancer diagnostics as compared to the traditional histological characterisation of tumours by enabling a non-invasive analysis of cancer development and treatment. Here, an electrochemical label-free assay for urokinase plasminogen activator (uPA), a universal biomarker of several cancers, has been developed based on the recently selected uPA-specific fluorinated RNA aptamer, tethered to a gold electrode via a phosphorothioated dA tag, and soluble redox indicators. The binding properties of the uPA-aptamer couple and interference from the non-specific adsorption of bovine serum albumin (BSA) were modulated by the electrode surface charge. A nM uPA electroanalysis at positively charged surfaces, complicated by the competitive adsorption of BSA, was tuned to the pM uPA analysis at negative surface charges of the electrode, being improved in the presence of negatively charged BSA. The aptamer affinity for uPA displayed via the binding/dissociation constant relationship correspondingly increased, ca. three orders of magnitude, from 0.441 to 367. Under optimal conditions, the aptasensor allowed 10(-12)-10(-9) M uPA analysis, also in serum, being practically useful for clinical applications. The proposed strategy for optimization of the electrochemical protein sensing is of particular importance for the assessment and optimization of in vivo protein ligand binding by surface-tethered aptamers.

Peroxidase-like activity of gold nanoparticles stabilized by hyperbranched polyglycidol derivatives over a wide pH range.

The aim of this work was to carry out comparative studies on the peroxidase-like activity of gold nanoparticles (AuNPs) stabilized with low molecular weight hyperbranched polyglycidol (HBPG-OH) and its derivative modified with maleic acid residues (HBPG-COOH). The influence of the stabilizer to gold precursor ratio on the size and morphology of nanoparticles obtained was checked, and prepared nanoparticles were characterized by means of transmission electron microscopy and UV-Vis spectroscopy. The results indicated the divergent effect of increasing the concentration of stabilizers (HBPG-OH or HBPG-COOH) on the size of the nanostructures obtained. The gold nanoparticles obtained were characterized as having intrinsic peroxidase-like activity and the mechanism of catalysis in acidic and alkaline mediums was consistent with the standard Michaelis-Menten kinetics, revealing a strong affinity of AuNPs with 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 3, 3', 5, 5'-tetramethylbenzidine (TMB), and significantly lower affinity towards phenol. By comparing the kinetic parameters, a negligible effect of polymeric ligand charge on activity against various types of substrates (anionic or cationic) was indicated. The superiority of steric stabilization via the application of tested low-weight hyperbranched polymers over typical stabilizers in preventing salt-induced aggregation and maintaining high catalytic activity in time was proved. The applied hyperbranched stabilizers provide a good tool for manufacturing gold-based nanozymes, which are highly stable and active over a wide pH range.