Evaluation of Polytyramine Film and 6-Mercaptohexanol Self-Assembled Monolayers as the Immobilization Layers for a Capacitive DNA Sensor Chip: A Comparison.

The performance of a biosensor is associated with the properties of an immobilization layer on a sensor chip. In this study, gold sensor chips were modified with two different immobilization layers, polytyramine film and 6-mercaptohexanol self-assembled monolayer. The physical, electrochemical and analytical properties of polytyramine film and mercaptohexanol self-assembled monolayer modified gold sensor chips were studied and compared. The study was conducted using atomic force microscopy, cyclic voltammetry and a capacitive DNA-sensor system (CapSenze™ Biosystem). The results obtained by atomic force microscopy and cyclic voltammetry indicate that polytyramine film on the sensor chip surface possesses better insulating properties and provides more spaces for the immobilization of the capture probe than a mercaptohexanol self-assembled monolayer. A capacitive DNA sensor hosting a polytyramine single-stranded DNA-modified sensor chip displayed higher sensitivity and larger signal amplitude than that of a mercaptohexanol single-stranded DNA-modified sensor chip. The linearity responses for polytyramine single-stranded DNA- and mercaptohexanol single-stranded DNA-modified sensor chips were obtained at log concentration ranges, equivalent to 10-12 to 10-8 M and 10-10 to 10-8 M, with detection limits of 4.0 × 10-13 M and 7.0 × 10-11 M of target complementary single-stranded DNA, respectively. Mercaptohexanol single-stranded DNA- and polytyramine single-stranded DNA-modified sensor chips exhibited a notable selectivity at an elevated hybridization temperature of 50 °C, albeit the signal amplitudes due to the hybridization of the target complementary single-stranded DNA were reduced by almost 20% and less than 5%, respectively.

A Sensitive Capacitive Biosensor for Protein a Detection Using Human IgG Immobilized on an Electrode Using Layer-by-Layer Applied Gold Nanoparticles.

A capacitive biosensor for the detection of protein A was developed. Gold electrodes were fabricated by thermal evaporation and patterned by photoresist photolithography. A layer-by-layer (LbL) assembly of thiourea (TU) and HAuCl4 and chemical reduction was utilized to prepare a probe with a different number of layers of TU and gold nanoparticles (AuNPs). The LbL-modified electrodes were used for the immobilization of human IgG. The binding interaction between human IgG and protein A was detected as a decrease in capacitance signal, and that change was used to investigate the correlation between the height of the LbL probe and the sensitivity of the capacitive measurement. The results showed that the initial increase in length of the LbL probe can enhance the amount of immobilized human IgG, leading to a more sensitive assay. However, with thicker LbL layers, a reduction of the sensitivity of the measurement was registered. The performance of the developed system under optimum set-up showed a linearity in response from 1 × 10-16 to 1 × 10-13 M, with the limit detection of 9.1 × 10-17 M, which could be interesting for the detection of trace amounts of protein A from affinity isolation of therapeutic monoclonal antibodies.

Development of an automated flow-based spectrophotometric immunoassay for continuous detection of zearalenone.

Considering the widespread contaminations of food products with mycotoxins, it is important to develop, robust, time- and cost-effective detection methods. We developed and optimized an immunoassay using a continuous flow system for the detection of zearalenone (ZEN). The assay was performed in a flow mode using an automated sequential injection system. Time for an assay cycle was 18 Min. Under optimal conditions, the limit for quantification for ZEN was 0.40 µg L-1 with a linear dependency between concentration and signal amplitude between 0.10 and 10 µg L-1 . The assay proved to be robust and reliable with 13% relative standard deviation between measurements. By dissociating the antigen-antibody complex using a regeneration solution, we showed 50 times reusability of the immobilized antibodies without affecting the antigen-binding properties.

Combined protein A imprinting and cryogelation for production of spherical affinity material.

Cryogels have been demonstrated to be efficient when applied for protein isolation. Owing to their macroporous structure, cryogels can also be used for treating particle-containing material, e.g. cell homogenates. Another challenging development in protein purification technology is the use of molecularly imprinted polymers (MIPs). These MIPs are robust and can be used repeatedly. The paper presents a new technology that combine the formation of cryogel beads concomitantly with making imprints of a protein. Protein A was chosen as the print molecule which was also be the target in the purification step. The present paper describes a new method to produce protein-imprinted cryogel beads. The protein-imprinted material was characterized and the separation properties were evaluated with regard to both the target protein and whole cells with target protein exposed on the cell surface. The maximum protein A adsorption was 18.1 mg/g of wet cryogel beads. The selectivity coefficient of protein A-imprinted cryogel beads for protein A was 5.44 and 12.56 times greater than for the Fc fragment of IgG and protein G, respectively.

Capacitive Biosensors and Molecularly Imprinted Electrodes.

Capacitive biosensors belong to the group of affinity biosensors that operate by registering direct binding between the sensor surface and the target molecule. This type of biosensors measures the changes in dielectric properties and/or thickness of the dielectric layer at the electrolyte/electrode interface. Capacitive biosensors have so far been successfully used for detection of proteins, nucleotides, heavy metals, saccharides, small organic molecules and microbial cells. In recent years, the microcontact imprinting method has been used to create very sensitive and selective biorecognition cavities on surfaces of capacitive electrodes. This chapter summarizes the principle and different applications of capacitive biosensors with an emphasis on microcontact imprinting method with its recent capacitive biosensor applications.

Molecular Imprinting Techniques Used for the Preparation of Biosensors.

Molecular imprinting is the technology of creating artificial recognition sites in polymeric matrices which are complementary to the template in their size, shape and spatial arrangement of the functional groups. Molecularly imprinted polymers (MIPs) and their incorporation with various transducer platforms are among the most promising approaches for detection of several analytes. There are a variety of molecular imprinting techniques used for the preparation of biomimetic sensors including bulk imprinting, surface imprinting (soft lithography, template immobilization, grafting, emulsion polymerization) and epitope imprinting. This chapter presents an overview of all of these techniques with examples from particular publications.

Why Using Molecularly Imprinted Polymers in Connection to Biosensors?

The area of biosensor-oriented research has grown rapidly during recent years.

Imprinting of Microorganisms for Biosensor Applications.

There is a growing need for selective recognition of microorganisms in complex samples due to the rapidly emerging importance of detecting them in various matrices. Most of the conventional methods used to identify microorganisms are time-consuming, laborious and expensive. In recent years, many efforts have been put forth to develop alternative methods for the detection of microorganisms. These methods include use of various components such as silica nanoparticles, microfluidics, liquid crystals, carbon nanotubes which could be integrated with sensor technology in order to detect microorganisms. In many of these publications antibodies were used as recognition elements by means of specific interactions between the target cell and the binding site of the antibody for the purpose of cell recognition and detection. Even though natural antibodies have high selectivity and sensitivity, they have limited stability and tend to denature in conditions outside the physiological range. Among different approaches, biomimetic materials having superior properties have been used in creating artificial systems. Molecular imprinting is a well suited technique serving the purpose to develop highly selective sensing devices. Molecularly imprinted polymers defined as artificial recognition elements are of growing interest for applications in several sectors of life science involving the investigations on detecting molecules of specific interest. These polymers have attractive properties such as high bio-recognition capability, mechanical and chemical stability, easy preparation and low cost which make them superior over natural recognition reagents. This review summarizes the recent advances in the detection and quantification of microorganisms by emphasizing the molecular imprinting technology and its applications in the development of sensor strategies.

Electrochemical Flow-ELISA for Rapid and Sensitive Determination of Microcystin-LR Using Automated Sequential Injection System.

An amperometric immunoanalysis system based on monoclonal antibodies immobilized on Sepharose beads and packed into a micro-immunocolumn was developed for the quantification of microcystin-LR. Microcystin-LR (MCLR) was used as a reference microcystin variant. Inside the immunocolumn, free microcystins and microcystin-horseradish peroxidase (tracer) were sequentially captured by the immobilized antibodies, and the detection was performed electrochemically using Super AquaBlue ELISA substrate 2,2'-azinobis(3-ethylbenzothiazoline-sulfonic acid) (ABTS). The ABTS●+ generated by enzymatic oxidation of ABTS was electrochemically determined at a carbon working electrode by applying a reduction potential set at 0.4 V versus Ag/AgCl reference electrode. The peak current intensity was inversely proportional to the amount of analyte bound to the immunocolumn. The amperometric flow-ELISA system, which was automatically controlled through the CapSenzeTM (Lund, Sweden) computer software, enabled determination of MCLR as low as 0.01 µg/L. The assay time was very short (20 min for one assay cycle). In addition, the electrochemical signals were not significantly affected by possible interferences which could be present in the real samples. Along with the simplicity of automation, this makes the developed method a promising tool for use in water quality assessment.

Microcontact Imprinted Plasmonic Nanosensors: Powerful Tools in the Detection of Salmonella paratyphi.

Identification of pathogenic microorganisms by traditional methods is slow and cumbersome. Therefore, the focus today is on developing new and quicker analytical methods. In this study, a Surface Plasmon Resonance (SPR) sensor with a microcontact imprinted sensor chip was developed for detecting Salmonella paratyphi. For this purpose, the stamps of the target microorganism were prepared and then, microcontact S. paratyphi-imprinted SPR chips were prepared with the functional monomer N-methacryloyl-L-histidine methyl ester (MAH). Characterization studies of the SPR chips were carried out with ellipsometry and scanning electron microscopy (SEM). The real-time Salmonella paratyphi detection was performed within the range of 2.5 × 106-15 × 106 CFU/mL. Selectivity of the prepared sensors was examined by using competing bacterial strains such as Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The imprinting efficiency of the prepared sensor system was determined by evaluating the responses of the SPR chips prepared with both molecularly imprinted polymers (MIPs) and non-imprinted polymers (NIPs). Real sample experiments were performed with apple juice. The recognition of Salmonella paratyphi was achieved using these SPR sensor with a detection limit of 1.4 × 106 CFU/mL. In conclusion, SPR sensor has the potential to serve as an excellent candidate for monitoring Salmonella paratyphi in food supplies or contaminated water and clearly makes it possible to develop rapid and appropriate control strategies.

Cryogelation of chitosan using noble-metal ions: in situ formation of nanoparticles.

One of the purposes of the project was to develop the method of preparation of 3D macroporous hydrogel with a structure of interconnected pores by the use of noncovalent interactions. The combination of chitosan and noble-metal complexes was investigated as cross-linking agents for the preparation of ionic cryogels (ICs). Furthermore, the treatment of the ICs containing gold complex by glutaraldehyde results in spontaneous formation of gold nanoparticles (AuNPs) and chemical cross-linking of the cryogel. The characterization of prepared macroporous materials was carried out by the use of FTIR, SEM, TEM techniques, and texture analyzer. A new strategy for control of size distribution of AuNPs was suggested. The size distribution of obtained AuNPs and their population inside of walls of cryogels was estimated. A method for quantifying unreacted chloroauric acid in the presence of acetic acid was proposed. The possibility of use of prepared cryogels with immobilized AuNPs as a catalytic flow through reactor is shown.

Recovery of struvite via coagulation and flocculation using natural compounds.

One of the major setbacks of struvite recovery processes is the difficulty in harvesting struvite crystals. This study evaluates the use of different coagulants to improve precipitation of struvite (MgNH4PO4.6H20) crystals. Chitosan and poly(diallyldimethyl ammonium chloride) (Poly-DADMAC) as a coagulant-flocculent and alginate and bentonite as a coagulant aid have been examined in jar tests. Also, a continuous three-phase process, i.e., struvite crystallization, coagulation/flocculation and precipitation process, was set up for real wastewater. Addition of chitosan as the coagulant and bentonite as the coagulant aid was significantly more efficient in forming struvite flocs in comparison to Poly-DADMAC alone or with coagulant aid, which did not show any positive effect. The calculated average settling velocity of struvite with chitosan-bentonite addition in synthetic and in real wastewater increased by approximately 5.3 and 2.8 folds, respectively, compared with that of no coagulant/flocculent addition. Phosphorus recovery of over 70% was achieved by the continuous process. Findings in this study clearly confirmed the possibility of using chitosan and bentonite as an efficient coagulant-flocculent to enhance the recovery of struvite crystals.

Microcultivation of anaerobic bacteria single cells entrapped in alginate microbeads.

Alginate microbeads, produced by emulsion/internal gelation, were studied for the entrapment and microcultivation of microbial cells with biotechnological potential. An anaerobic consortium which was selected for its capacity to degrade complex carbohydrates, and a pure culture of cellulose degrading bacteria were used for entrapment studies. Optimization of conditions for the formation of spherical alginate microbeads in sizes between 20 and 80 µm were examined. The best conditions were achieved by combining rapeseed methyl ester as oil phase and stirring at 100 rpm using a rotation impeller. Calcium alginate microbeads produced under these conditions were shown to present morphological stability, with large pores in the internal matrix that favours microcolony development. Finally, single cells were observed inside the beads after the entrapment procedure and microcolony formation was confirmed after cultivation in cellobiose.

Recent Advances in Optical Sensing for the Detection of Microbial Contaminants.

Microbial contaminants are responsible for several infectious diseases, and they have been introduced as important potential food- and water-borne risk factors. They become a global burden due to their health and safety threats. In addition, their tendency to undergo mutations that result in antimicrobial resistance makes them difficult to treat. In this respect, rapid and reliable detection of microbial contaminants carries great significance, and this research area is explored as a rich subject within a dynamic state. Optical sensing serving as analytical devices enables simple usage, low-cost, rapid, and sensitive detection with the advantage of their miniaturization. From the point of view of microbial contaminants, on-site detection plays a crucial role, and portable, easy-applicable, and effective point-of-care (POC) devices offer high specificity and sensitivity. They serve as advanced on-site detection tools and are pioneers in next-generation sensing platforms. In this review, recent trends and advances in optical sensing to detect microbial contaminants were mainly discussed. The most innovative and popular optical sensing approaches were highlighted, and different optical sensing methodologies were explained by emphasizing their advantages and limitations. Consequently, the challenges and future perspectives were considered.

Caloramator boliviensis sp. nov., a thermophilic, ethanol-producing bacterium isolated from a hot spring.

A novel moderately thermophilic, anaerobic, ethanol-producing bacterial strain, 45B(T), was isolated from a mixed sediment water sample collected from a hot spring at Potosi, Bolivia. The cells were straight to slightly curved rods approximately 2.5 µm long and 0.5 µm wide. The strain was Gram-stain-variable, spore-forming and monotrichously flagellated. Growth of the strain was observed at 45-65 °C and pH 5.5-8.0, with optima of 60 °C and pH 6.5. The substrates utilized by strain 45B(T) were xylose, cellobiose, glucose, arabinose, sucrose, lactose, maltose, fructose, galactose, mannose, glycerol, xylan, carboxymethylcellulose and yeast extract. The main fermentation product from xylose and cellobiose was ethanol (0.70 and 0.45 g ethanol per gram of consumed sugar, respectively). Acetate, lactate, propionate, carbon dioxide and hydrogen were also produced in minor quantities. 1,3-Propanediol was produced when glycerol-containing medium was supplemented with yeast extract. The major cellular fatty acids were anteiso-C(15:0), C(16:0), iso-C(16:0), C(15:1), iso-C(14:0), C(13:0) and C(14:0). The polar lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an aminoglycolipid and 15 other unidentified lipids were predominant. The DNA G+C content of strain 45B(T) was 32.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence similarity revealed that strain 45B(T) is located within the Gram-type positive Bacillus-Clostridium branch of the phylogenetic tree. On the basis of morphological and physiological properties and phylogenetic analysis, strain 45B(T) represents a novel species, for which the name Caloramator boliviensis sp. nov. is proposed; the type strain is 45B(T) (=DSM 22065(T)=CCUG 57396(T)).

Composite cryogel with immobilized concanavalin A for affinity chromatography of glycoproteins.

Composite cryogels containing porous adsorbent particles were prepared under cryogelation conditions. The composites with immobilized concanavalin A (Con A) were used for capturing glycoproteins. Adsorbent particles were introduced into the structure in order to improve the capacity and to facilitate the handling of the particles. The monolithic composite cryogels were produced from suspensions of polyvinyl alcohol particles and porous adsorbent particles and cross-linked under acidic conditions at sub-zero temperature. The cryogels were epoxy activated and Con A was immobilized as an affinity ligand. Binding and elution of horseradish peroxidase (HRP) was studied in batch experiment and in a chromatographic setup. Increasing adsorbent concentration in composite cryogels will increase ligand density, which therefore enhances the amount of bound HRP from 0.98 till 2.9 (milligram enzyme per milliliter of gel) in the chromatographic system. The material was evaluated in 10 cycles for binding and elution of HRP.

Porous protein-based scaffolds prepared through freezing as potential scaffolds for tissue engineering.

Successful tissue engineering with the aid of a polymer scaffold offers the possibility to produce a larger construct and to mould the shape after the defect. We investigated the use of cryogelation to form protein-based scaffolds through different types of formation mechanisms; enzymatic crosslinking, chemical crosslinking, and non-covalent interactions. Casein was found to best suited for enzymatic crosslinking, gelatin for chemical crosslinking, and ovalbumin for non-covalent interactions. Fibroblasts and myoblasts were used to evaluate the cryogels for tissue engineering purposes. The stability of the cryogels over time in culture differed depending on formation mechanism. Casein cryogels showed best potential to be used in skeletal tissue engineering, whereas gelatin cryogels would be more suitable for compliable soft tissues even though it also seemed to support a myogenic phenotype. Ovalbumin cryogels would be better suited for elastic tissues with faster regeneration properties due to its faster degradation time. Overall, the cryogelation technique offers a fast, cheap and reproducible way of creating porous scaffolds from proteins without the use of toxic compounds.

Struvite-based fertilizer and its physical and chemical properties.

This study describes a method to formulate struvite fine powder into pellets that are easy to spread on agricultural land. To evaluate the quality of produced pellets, some chemical and physical properties commonly measured for fertilizers were tested. The findings indicated that the salt index and heavy metal content ofstruvite pellets were significantly lower than those of commercial NPK fertilizers. In addition, the percentage of nutrient released from struvite pellets after 105 days was in the range of 9.6-23.2, 8.4-26.7 and 11.3-32.6% for nitrogen, phosphorous and magnesium, respectively, which is considerably lower than that of commercial NPK fertilizer. Among different formulations between struvite crystals and binders, starch and bentonite were the most efficient in agglomerating struvite powder, leading to an increase in the crush strength to over the recommended limit of >2.5 kgf for fertilizer hardness.

Preparation of Staphylococcal Protein A Imprinted Supermacroporous Cryogel Beads.

Protein A is the most commonly used ligand in IgG purification due to its specific binding to the Fc receptor of most immunoglobulins, making it commercially important. Molecular imprinting is a method based on the selective recognition of various molecules. Molecular imprinted polymers are materials that are easy to prepare, durable, cheap and have molecular recognition capability. Cryogels are prepared by radical polymerization in a partially frozen environment. The unique structure of cryogels combined with osmotic, chemical and mechanical stability make them attractive chromatography matrices for a variety of biological compounds/specimens (plasmids, pathogens, cells). In this protocol, protein A imprinted supermacroporous poly(2-hydroxyethyl methacrylate) cryogels were prepared in spherical form for protein A purification. The characterization of the prepared cryogels were made by swelling test, scanning electron microscopy (SEM), Fourier transform infrared spectrophotometer (FTIR), and Brunauer-Emmett-Teller (BET) surface area analysis. After characterization, optimum conditions for protein A adsorption were determined in the batch system. The maximum protein A adsorption capacity was determined after optimization of the imprinted cryogels. Protein A relative selectivity coefficients of imprinted cryogels were examined for both Fc and protein G. Protein A was isolated from the bacterial cell wall using fast performance liquid chromatography (FPLC). The separated protein A was determined by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). In the last stage, the reusability of the cryogel was examined.

Upgrading of bio-separation and bioanalysis using synthetic polymers: Molecularly imprinted polymers (MIPs), cryogels, stimuli-responsive polymers.

Bio-separation plays a crucial role in many areas. Different polymers are suitable for bio-separation and are useful for applications in applications in both science and technology. Besides biopolymers, there are a broad spectrum of synthetic polymers with tailor-made properties. The synthetic polymers are characterized by their charges, solubility, hydrophilicity/hydrophobicity, sensitivity to environmental conditions and stability. Furthermore, ongoing developments are of great interest on biodegradable polymers for the treatment of diseases. Smart polymers have gained great attention due to their unique characteristics especially emphasizing simultaneously changing their chemical and physical property upon exposure to changes in environmental conditions. In this review, methodologies applied in bio-separation using synthetic polymers are discussed and efficient candidates are focused for the construction of synthetic polymers.