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.

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.

Electrochemical detection of manganese ions using aptamer-based layers.

Heavy metals are one of the major pollutants found in drinking water and their abnormal level may pose a threat to human's health and life. Manganese also belongs to heavy metals group, and it is generally used in production of batteries, fertilizers, and ceramics. Even though, Mn is necessary for proper development of central nervous system, its elevated concentration might lead to certain diseases such as epilepsies, cell death in focal cerebral ischemia as well as neurodegenerative diseases such as Huntington and Alzheimer. Hence, it is crucial to elaborate novel methods for manganese ions detection that could be applied for in situ analysis of water samples. Herein, we present the studies on the electrochemical detection of manganese ions using aptamer-modified electrodes. This is the first attempt of application of aptamer strands as receptor layers for electrochemical analysis of manganese ions and for that purpose gold disk electrodes served as transducers, which were further modified with disulfide - based aptamers and 6-mercapto-1-hexanol blocking agent. The electrochemical measurements concerned the choice of the conditions for formation of aptamer receptor layer as well as the type of redox indicator that served as the source of current signal. The studies referred to the definition of aptasensor working parameters including the verification of the possibility of manganese ion detection in cell culture media. It was shown that it was possible to detect Mn2+ ions within 25 nM-1 µM concentration and the proposed aptasensor exhibited high selectivity towards target analyte for which at least 2 - times higher response was recorded than for interfering ions. Moreover, the possibility of Mn2+ detection in real samples was depicted followed by stability and regeneration studies.

A Careful Insight into DDI-Type Receptor Layers on the Way to Improvement of Click-Biology-Based Immunosensors.

Protein-based microarrays are important tools for high-throughput medical diagnostics, offering versatile platforms for multiplex immunodetection. However, challenges arise in protein microarrays due to the heterogeneous nature of proteins and, thus, differences in their immobilization conditions. This article advocates DNA-directed immobilization (DDI) as a solution, emphasizing its rapid and cost-effective fabrication of biosensing platforms. Thiolated single-stranded DNA and its analogues, such as ZNA® and PNA probes, were used to immobilize model proteins (anti-CRP antibodies and SARS-CoV nucleoprotein). The study explores factors influencing DDI-based immunosensor performance, including the purity of protein-DNA conjugates and the stability of their duplexes with DNA and analogues. It also provides insight into backfilling agent type and probe surface density. The research reveals that single-component monolayers lack protection against protein adsorption, while mixing the probes with long-chain ligands may hinder DNA-protein conjugate anchoring. Conventional DNA probes offer slightly higher surface density, while ZNA® probes exhibit better binding efficiency. Despite no enhanced stability in different ionic strength media, the cost-effectiveness of DNA probes led to their preference. The findings contribute to advancing microarray technology, paving the way for new generations of DDI-based multiplex platforms for rapid and robust diagnostics.

Studies on the Aptasensor Miniaturization for Electrochemical Detection of Lead Ions.

Lead poses severe effects on living organisms, and since Pb2+ ions tend to accumulate in different organs, it is crucial to monitor Pb2+ concentration in samples such as water and soil. One of the approaches is the utilization of biosensors combined with aptamer-based layers for the electrochemical detection of lead ions. Herein, we present the studies of applying miniaturized screen-printed transducers as solid surfaces to fabricate aptamer layers. As the research is the direct continuation of our previous studies regarding the use of gold disk electrodes, the working parameters of elaborated aptasensors were defined, including the range of linear response (10-100 nM), selectivity as well as stability, regeneration, and feasibility of application for the analysis of real samples. This was achieved using voltammetric techniques including cyclic and square-wave voltammetry in the presence of methylene blue redox indicator.

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.

Heteroaggregation of virions and microplastics reduces the number of active bacteriophages in aqueous environments.

The objective of this study is to explore the effects of microplastics on the viability of the bacteriophages in an aqueous environment. Bacteriophages (phages), that is, viruses of bacteria, are essential in homeostasis. It is estimated that phages cause up to 40% of the death of all bacteria daily. Any factor affecting phage activity is vital for the whole food chain and the ecology of numerous niches. We hypothesize that the number of active phages decreases due to the virions' adsorption on microplastic particles or by the released leachables from additives used in the production of plastic, for example, stabilizers, plasticizers, colorants, and reinforcements. We exposed three diverse phages, namely, T4 (tailed), MS2 (icosahedral), and M13 (filamentous), to 1 mg/mL suspension of 12 industrial-grade plastics [acrylonitrile butadiene styrene, high-impact polystyrene, poly-ε-caproamide, polycarbonate, polyethylene, polyethylene terephthalate, poly(methyl methacrylate), polypropylene, polystyrene, polytetrafluoroethylene, polyurethane, and polyvinyl chloride] shredded to obtain microparticles of radius ranging from 2 to 50 µm. The effect of leachables was measured upon exposure of phages not to particles themselves but to the buffer preincubated with microplastics. A double-overlay plaque counting method was used to assess phage titers. We employed a classical linear regression model to verify which physicochemical parameters (65 variables were tested) govern the decrease of phage titers. The key finding is that adsorption mechanisms result in up to complete scavenging of virions, whereas leachables deactivate up to 50% of phages. This study reveals microplastic pollution's plausible and unforeseen ecotoxicological effect causing phage deactivation. Moreover, phage transmission through adsorption can alter the balance of the food chain in the new environment. The effect depends mainly on the zeta potentials of the polymers and the phage type.

Studies on the application of single-stranded DNA and PNA probes for electrochemical detection of miRNA 141.

The abnormal concentration of microRNAs (miRNAs) can be associated with occurrence of various diseases including cancer, cardiovascular and neurodegenerative, hence they can be considered as potential biomarkers. An attractive approach could be the application of electrochemical methods, particularly where hybridization event between single-stranded deoxyribonucleic acid (ssDNA) or peptide-nucleic acid (PNA) with miRNA strand happens. Recently, the use of various nanomaterials such as gold nanoparticles, graphene oxide, quantum dots as well as catalyzed hairpin assembly or hybridization chain reaction were proposed to further enhance the performance of elaborated sensors. Herein, we present the studies on selection of receptor layer composition for detection of miRNA 141. The possibility of formation of receptor layer and further duplex monolayer between ssDNA or PNA with miRNA was analyzed by atomic force microscopy (AFM) technique. The interaction of ssDNA and PNA probes with miRNA was further verified using surface plasmon resonance (SPR) and quartz - crystal microbalance (QCM) techniques. On the basis of impedance spectroscopy it was shown that the use of unlabelled ssDNA as receptor layer provided 0.1 pM detection limit. This shows that proposed biosensor that is simple in preparation and use is an attractive alternative to other recently presented approaches.

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.

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.

Detection of SARS-CoV-2 Using Reverse Transcription Helicase Dependent Amplification and Reverse Transcription Loop-Mediated Amplification Combined with Lateral Flow Assay.

Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods-reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)-combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10-20 min, whereas for RT-LAMP, the LOD was 30-300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results.

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 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.

Molecular diagnostic of toxigenic Corynebacterium diphtheriae strain by DNA sensor potentially suitable for electrochemical point-of-care diagnostic.

The presented study is focused on the development of electrochemical genosensor for detection of tox gene fragment of toxigenic Corynebacterium diphtheriae strain. Together with our previous studies it fulfils the whole procedure for fast and accurate diagnostic of diphtheria at its early stage of infection with the use of electrochemical methods. The developed DNA sensor potentially can be used in more sophisticated portable device. After the electrochemical stem-loop probe structure optimization the conditions for real asymmetric PCR (aPCR) product detection were selected. As was shown it was crucial to optimize the magnesium and organic solvent concentrations in detection buffer. Under optimal conditions it was possible to selectively detect as low as 20.8 nM of complementary stand in 5 min or 0.5 nM in 30 min with sensitivity of 12.81 and 0.24 1⋅µM-1 respectively. The unspecific biosensor response was elucidated with the use of new electrode blocking agent, diethyldithiocarbamate. Its application in electrochemical genosensors lead to significant higher current values and the biosensor response even in conditions with magnesium ion depletion. The developed biosensor selectivity was examined using samples containing genetic material originated from a number of non-target bacterial species which potentially can be present in the human upper respiratory tract.

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.

The application of antibody-aptamer hybrid biosensors in clinical diagnostics and environmental analysis.

The growing number of various diseases and the increase of environmental contamination are the causes for the development of novel methods for their detection. The possibility of the application of affinity-based biosensors for such purposes seems particularly promising as they provide high selectivity and low detection limits. Recently, the usage of hybrid antibody-aptamer sandwich constructs was shown to be more advantageous in terms of working parameters in comparison to aptamer-based and immune-based biosensors. This review is focused on the usage of hybrid antibody-aptamer receptor layers for the determination of clinically and environmentally important target molecules. In this work, antibodies and aptamer molecules are characterized and the methods of their immobilization as well as analytical signal generation are shown. This is followed by the critical presentation of examples of hybrid sandwich biosensors that have been elaborated in the past 12 years.

The influence of a swab type on the results of point-of-care tests.

Most point-of-care tests (POCT) use swabs for sampling and/or for applying a sample on the test. A variety of swabs differing in tip materials is commercially available. Different tip materials have different chemical and physical characteristics which might influence the specimen collection and release. We investigated properties of various types of swabs used in clinical diagnostics with focusing on two kinds of analytes, DNA and proteins, which are most often used targets in POCT. As the model samples we used diphtheria toxoid NIBSC 69/017 for investigating recovery of protein analytes such as antigens and bacterial strains of Escherichia coli ATCC 25922, diphtheria toxin-producing Corynebacterium diphtheriae NCTC 10648, and the clinical isolate nontoxigenic C. diphtheriae 5820/15 for investigating the recovery of nucleic acids. We investigated four types of swabs most commonly used in clinical diagnostics in terms of absorption capacity and efficiency of release of nucleic acids and proteins. Volume uptake was measured in milligrams. For DNA release various washing out buffers were used and the amount of released DNA was measured spectrophotometrically. The amount of protein released from the swabs were examined using the Lowry assay. We observed statistically significant differences (p < 0.05) in the mean weights of absorbed liquid, in the DNA recovery and protein recovery by the four variety of swab examined. However, the efficiency of DNA and protein release was not correlated to the absorbed volume of a sample, but rather to the properties of swabs. The swab composition and structure can have a significant impact on the collection and release efficiency of a sample. Therefore, validation of POCT in relation to the used swabs for sampling is really important. The use of inappropriate swabs could lead to false negative or misleading analysis results.

Reduced nonspecific protein adsorption by application of diethyldithiocarbamate in receptor layer of diphtheria toxoid electrochemical immunosensor.

The immunoassay technology is of particular importance for both the environmental industry and clinical analysis. Biosensors, with the sensing layer based on antibodies or their fragments, offer high selectivity and short detection times. However, analytical devices where the electrochemical signal corresponds to changes in the interfacial region (sensing layer/electrode surface) are very susceptible to any nonspecific adsorption. Unfortunately, proteins (including antibodies) belong to the molecules showing high non-specific interactions with solid substrates. Herein, we propose diethyldithiocarbamate as a new antifouling and highly conductive agent. The investigations were conducted to evaluate its interaction with chosen proteins and the mechanism of its co-adsorption with biotinylated thiol (an anchor point for immune-sensing elements). The developed receptor layer is characterised by reduced nonspecific protein adsorption and high conductivity with the same preserved specificity of the antibodies (immobilised by the streptavidin/biotin bioaffinity technique). This allowed for selective detection of the diphtheria toxoid, an inactive toxin secreted by virulent strains of Corynebacterium diphtheria, at the level of 5 â‹… 10-6 µg⋅ml-1 (1 â‹… 10-6 Lf⋅ml-1) and in the real-life sample.

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.