In situ and non-cytotoxic cross-linking strategy for 3D printable biomaterials.

3D bioprinting allows the production of patient-specific tissue constructs with desired structural characteristics such as high resolution, controlled swelling degree, and controlled degradation behavior by mostly using hydrogels. Crosslinking of hydrogels is an essential parameter in bioprinting applications, which is beneficial for tuning structural specifications. In this study, gelatin-alginate-whey protein isolate based hydrogels have been used for 3D printing structures in a layer-by-layer fashion. These structures were cross-linked by the Amino Acid (monomer) Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method, which is a unique, non-invasive photosensitive cross-linking technique for protein-based mixtures. In that aim, hydrogel properties (e.g., printability, biocompatibility, rheologic and mechanical behavior) and cross-linking properties (e.g., swelling and degradation behavior) were studied. Results were compared with UV and ionic cross-linking techniques, which are the abundantly used techniques in such studies. The results showed that the ANADOLUCA method can be used for in situ cross-linking under mild conditions for the printing of bio-inks, and the proposed method can be used as an alternative for UV-based and chemical cross-linking techniques.

RuBisCO nano enzyme for mimicking CO2 conversion system in plants.

In this study, carbon dioxide (CO2 ) capture and conversion systems based on the combination of biomimetic systems with nano enzymes have been developed. The effectiveness of the developed system has been investigated toward CO2 conversion. For this aim, nano ribulose bisphosphate carboxylase/oxygenase (RuBisCO) enzyme that plays role in the Calvin cycle in photosynthesis has been synthesized in 93 nm size according to AmiNoAcid (monomer) Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method. Enzymatic activity of synthesized nano RuBisCO enzyme has been spectrophotometrically determined by the formation of 3-phosphoglycerate (3-PGA) at the end of the reaction between CO2 and d-ribulose-1,5 biphosphate with the catalysis of RuBisCO enzyme at 340 nm. The effect of substrate concentration, pH, temperature, and Mg2+ ion concentration on the conversion reaction have investigated comparatively with nano and free RuBisCO enzyme. Besides this, the reusability feature of synthesized nano RuBisCO enzyme in conversion of CO2 reaction is indicated. When all data were evaluated, it has been seen that the nano RuBisCO enzyme is effective on the conversion of CO2 into 3-PGA and can be used for CO2 capture and conversion systems repeatedly without any deformation in its structure.

A new potentiometric platform: Antibody cross-linked graphene oxide potentiometric immunosensor for clenbuterol determination.

An immunosensor is based on the signal measurement obtained upon the reaction of an antibody antigen complex. It plays a significant role in various fields such as environmental analysis, production monitoring, drug detection or screening, veterinary medicine, and animal food. In this study, an antibody crosslinked graphen oxide (GO)-based potentiometric sensor has been developed for fast, simple, and economical detection of clenbuterol. In this context, the photosensitive amino acid bound GO platform is synthesized and used for the preparation of electrode material. Then, polymeric structure is characterized by infrared spectroscopy, and the performance of immunonano platform prepared by potentiometric sensor is evaluated. The effect of pH, response time, selectivity, and sensitivity is investigated. Under the optimized conditions, a simple and rapid method for the determination of clenbuterol from milk sample is established by immuno-potentiometric sensor. The detection limit has found to be 0.87 × 10-9 mmol L-1 for this immuno-potentiometric sensor. This immuno-potentiometric sensor has optimum pH at 7.0, a wide linear response (1.0 × 10-2 to 1.0 × 10-9 mmol L-1 ), rapid response time (2 Min) and 36 weeks operational lifetime.

Proteinous Polymeric Shell Decorated Nanocrystals for the Recognition of Immunoglobulin M.

This study demonstrates the preparation of photosensitively orientated and crosslinked proteinous polymeric shell having quantum dot based nanocrystals through Amino acid Decorated and Light Underpinning Conjugation Approach (ANADOLUCA). ANADOLUCA is based on photo-electron transfer method and uses these decorated nanocrystals for specifically and effectively recognition and detection of Immunoglobulin M in the aqueous environment. The conjugation method effectively provides an orientation of affinity pairs on the surface of quantum dots nanocrystals. This photosensitive ruthenium-based amino acid monomer is a synthetic and inexpensive material for the preparation of bioconjugates. The nanocrystals give advantages for using a wide pH and temperature range. The construction and preparation method is applicable to silica materials, superparamagnetic particles, quantum dots, carbon nanotubes, Ag/Au nanoparticles, Au surfaces, and polymeric materials. This prepared proteinous polymeric shell decorated nanocrystals are of great potential in applications in life sciences and can be used in infection case studies or allergy symptoms.

Molecular Imprinting Technology in Quartz Crystal Microbalance (QCM) Sensors.

Molecularly imprinted polymers (MIPs) as artificial antibodies have received considerable scientific attention in the past years in the field of (bio)sensors since they have unique features that distinguish them from natural antibodies such as robustness, multiple binding sites, low cost, facile preparation and high stability under extreme operation conditions (higher pH and temperature values, etc.). On the other hand, the Quartz Crystal Microbalance (QCM) is an analytical tool based on the measurement of small mass changes on the sensor surface. QCM sensors are practical and convenient monitoring tools because of their specificity, sensitivity, high accuracy, stability and reproducibility. QCM devices are highly suitable for converting the recognition process achieved using MIP-based memories into a sensor signal. Therefore, the combination of a QCM and MIPs as synthetic receptors enhances the sensitivity through MIP process-based multiplexed binding sites using size, 3D-shape and chemical function having molecular memories of the prepared sensor system toward the target compound to be detected. This review aims to highlight and summarize the recent progress and studies in the field of (bio)sensor systems based on QCMs combined with molecular imprinting technology.

Molecularly imprinted cryogels for human interferon-alpha purification from human gingival fibroblast culture.

Interferons are important proteins for the immune system because of their antiviral, anti-proliferating and immunomodulatory activities. Therapeutic value of these proteins against certain types of tumors caused interest and investigations aimed to obtain highly purified interferons. Molecular imprinting is an efficient method for purification with high selectivity, specificity and good reproducibility. In this study, we utilized advantages of molecular imprinting technique for the purification of interferon from human gingival fibroblast culture. For this purpose, interferon α-2b imprinted poly(hydroxyethyl methacrylate) cryogel (hIFN-α-MIP) was prepared. Optimum adsorption conditions were determined, and maximum adsorption capacity of hIFN-α-MIP cryogel was found as 254.8 × 10(4) IU/g from aqueous solution. All interferon measurements are expressed as International Unit (IU), which is a unit measurement used to quantify biologically active substances like interferon based on their biological activity or effect. Selectivity experiments were performed using competitive proteins and repeated adsorption-desorption studies showed that the adsorption capacity maintained almost at a constant value after ten cycles. For the purification of interferon from human gingival fibroblast culture, fast protein liquid chromatography was used and the specific activity of the purified interferon α-2b on HeLa cell line was found between the values 3.45 × 10(8) IU/mg and 3.75 × 10(8) IU/mg. The results are promising, and the molecular imprinting technique is effective for the purification of interferon α-2b.

New synthesis method for 4-MAPBA monomer and using for the recognition of IgM and mannose with MIP-based QCM sensors.

Quartz crystal microbalance (QCM) sensors coated with molecularly imprinted polymers (MIP) have been developed for the recognition of immunoglobulin M (IgM) and mannose. In this method, methacryloylamidophenylboronic acid (MAPBA) was used as a monomer and mannose was used as a template. For this purpose, initially, QCM electrodes were modified with 2-propene-1-thiol to form mannose-binding regions on the QCM sensor surface. In the second step, the methacryloylamidophenylboronic acid-mannose [MAPBA-mannose], pre-organized monomer system, was prepared using the MAPBA monomer. Then, a molecularly imprinted film was coated on to the QCM electrode surface under UV light using ethylene glycol dimethacrylate (EDMA), and azobisisobutyronitrile (AIBN) as a cross-linking agent and an initiator, respectively. The mannose can be simultaneously bound to MAPBA and fitted into the shape-selective cavities. The binding affinity of the mannose-imprinted sensors was investigated using the Langmuir isotherm. The mannose-imprinted QCM electrodes have shown homogeneous binding sites for mannose (K(a): 3.3 × 10(4) M(-1)) and heterogeneous binding sites for IgM (K(a1): 1.0 × 10(4) M(-1); K(a2): 3.3 × 10(3) M(-1)).

Rapid real-time detection of procalcitonin using a microcontact imprinted surface plasmon resonance biosensor.

Procalcitonin (PCT) is a promising biomarker for identification of the origin and severity of sepsis, which is a deadly body infection. In this work, we report the preparation of a surface plasmon resonance (SPR) biosensor which utilizes a molecular imprinted polymer surface for rapid and reliable detection of PCT. The molecular imprinted surface was prepared using a microcontact imprinting technique, in which PCT molecules were first immobilized onto a glass support and brought into contact with a solution of 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA) on a SPR sensor, then the polymerization process was performed. After removal of the PCT molecules, specific molecular recognition sites were obtained, where PCT molecules can selectively rebind, only at the surface of the polymer matrix. PCT detection studies were carried out using PCT solutions in phosphate buffer and simulated blood plasma (SBP) at different concentrations. The SPR biosensor can detect very low concentrations (9.9 ng mL(-1)) of PCT within approximately 1 h, in both phosphate buffer and SBP. High selectivity of the biosensor against PCT was also demonstrated in the presence of several competitive proteins such as human serum albumin, myoglobin and cytochrome c.

Oriented immobilized anti-hIgG via F(c) fragment-imprinted PHEMA cryogel for IgG purification.

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F(c) fragment-imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti-hIgG for IgG purification from human plasma. Non-imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti-hIgG to compare the adsorption capacities of oriented (MIP/anti-hIgG) and random (NIP/anti-hIgG) cryogel columns. The amount of immobilized anti-hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti-hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti-hIgG column (29.7 mg/g) for the MIP/anti-hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti-hIgG cryogel column. Adsorbed IgG was eluted using 1.0 M NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti-hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification.

Bioconjugated and cross-linked bionanostructures for bifunctional immunohistochemical labeling.

The present study describes the development and use of a new bioconjugate combining targeted quantum dot labeling with an immunoperoxidase method and explores whether these bioconjugates could specifically and effectively label Cu/Zn superoxide dismutase (SOD1). The new bioconjugate is designed for the examination of samples both under fluorescent and bright-field microscopy at the same time. For this purpose chlorobis(2-2'-bipyridyl) methacryloyl tyrosine-ruthenium(II) and bis (2-2'-bipyridyl) methacryloyltyrosine-methacryloyltryptophan-ruthenium (II) photosensitive monomers and photosensitive poly(Bis (2-2'-bipyridyl)) methacryloyltyrosine-methacryloyltryptophan-ruthenium(II) were synthesized and characterized. The anti-SOD1 antibody and horseradish peroxidase (HRD) conjugated quantum dots were prepared by using this polymer. The anti-SOD1 antibody and HRD conjugated quantum dots were used in labeling and imaging of SOD1 in rat liver sections. Quantum dot particles were observed as a bright fluorescence in their specific binding locations inside the hepatocytes. The HRD-diaminobenzidine reaction product was observed as brown-colored particles at the same locations under bright-field microscopy. Structural details of the tissue sections could be examined at the same time. The conjugation protocol is simple; the bioconjugate is applicable for efficient cell labeling and can be adapted for imaging of other targets in different tissues. Also, the prepared nanobioconjugates have mechanic stability and can be used for a long period.

Application of HRP-streptavidin bionanoparticles for potentiometric biotin determination.

Biotin is widely used in infant formula to prevent biotin deficiency of newborn babies and in beauty products as nutritional supplements for coenzymatic functions and having strong nails, shiny hair, and skin over the last few years. There is a need for the development of a fast, simple and reusable assay method to perform biotin determination at very low concentrations. Biotin determination has achieved with a prepared potentiometric biotin sensor that has a very wide concentration range (10-15M-10-7M) and a lower detection limit (0.3 10-15M) with a very good regression coefficient (0.9925). A quick response (7 min), good accuracy (recovery 100.4-103.7%), reproducible, reusable (10 times), and long-term stability (3 months) have been obtained using the prepared potentiometric sensor. The obtained results have proved that the prepared potentiometric sensor can be used for biotin determination in real samples.

Investigation of photosensitively bioconjugated targeted quantum dots for the labeling of Cu/Zn superoxide dismutase in fixed cells and tissue sections.

This study presents the development of targeted and antibody cross-linked QDs and explores whether these bioconjugates could specifically and effectively label Cu/Zn superoxide dismutase (SOD1) on fixed cells and tissues. QD-antibody conjugation was achieved by using our previously invented AmiNoacid (monomer) Decorated and Light Underpining Conjugation Approach (ANADOLUCA) method. In this method, we have used a photosensitive aminoacid monomer having ruthenium complex which is a synthetic and inexpensive material for the preparation of bioconjugates. Its specificity was demonstrated by extracting the active enzyme from rat liver lysate by using the bioconjugate. It provided accurate antibody orientation, high specificity and mechanic stability. The protocol steps for QD-antibody conjugation and specimen preparation were described in detail. The nanobioconjugates were prepared under mild conditions (for example in day light), independent of pH and temperature, without affecting conformation and function of protein. This protocol is simple, inexpensive and can be successfully adapted to detect other targets on different cell types and tissues.

Molecularly imprinted supermacroporous cryogels for cytochrome c recognition.

Molecular imprinting is an attractive biomimetic approach that creates specific recognition sites for the shape and functional group arrangement to template molecules. The purpose of this study is to prepare cytochrome c-imprinted poly(hydroxyethyl methacrylate) (PHEMA)-based supermacroporous cryogel which can be used for the separation of cytochrome c from protein mixtures. N-Methacryloyl-(L)-histidinemethylester (MAH) was used as the metal-coordinating monomer. In the first step, Cu(2+) was complexed with MAH, and the cytochrome c imprinted PHEMA (MIP) cryogel was prepared by free radical cryopolymerization initiated by N,N,N',N'-tetramethylene diamine at -12°C. After polymerization is completed, the template cytochrome c molecules were removed from the MIP cryogel using 0.5 M NaCl solution. The maximum cytochrome c binding amount was 126 mg/g polymer. Selective binding studies were performed in the presence of lysozyme and bovine serum albumin. The relative selectivity coefficients of MIP cryogel for cytochrome c/lysozyme and cytochrome c/bovine serum albumin were 1.7 and 5.2 times greater than those of the non-imprinted PHEMA cryogel, respectively. The selectivity of MIP cryogel for cytochrome c was also confirmed with fast protein liquid chromatography. The MIP cryogel could be used many times with no remarkable decrease in cytochrome c binding capacity.

Protein recognition via ion-coordinated molecularly imprinted supermacroporous cryogels.

Molecular imprinting is a method for making selective binding sites in synthetic polymers using a molecular template. The aim of this study is to prepare lysozyme-imprinted supermacroporous cryogels which can be used for the purification of lysozyme (Lyz) from egg white. N-Methacryloyl-(L)-histidinemethylester (MAH) was chosen as the metal-coordinating monomer. In the first step, Cu2+ was complexed with MAH and the lysozyme-imprinted poly(HEMA-MAH) [Lyz-MIP] cryogel were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) in an ice bath. After that, the template (i.e., lysozyme) was removed using 0.05 M phosphate buffer containing 1M NaCl (pH 8.0). The maximum lysozyme adsorption capacity was 22.9 mg/g polymer. The relative selectivity coefficients of Lyz-MIP cryogel for lysozyme/bovine serum albumin and lysozyme/cytochrome c were 4.6 and 3.2 times greater than non-imprinted poly(HEMA-MAH) (NIP) cryogel, respectively. Purification of lysozyme from egg white was also monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 94% with recovery about 86%. The Lyz-MIP cryogel could be used many times without decreasing the adsorption capacity significantly.

Preconcentration of phosphate ion onto ion-imprinted polymer.

In this study, selective separation and preconcentration of phosphate ions on the phosphate-imprinted chitosan-succinate beads have investigated. Chitosan-succinate, phosphate, epichlorohydrin were used as the complexing monomer, template and crosslinking agent, respectively. In the first step, chitosan was modified with succinic anhydrides and complex formation occurred between carboxylic acid functional groups and iron(III) ions. Secondly, Fe(III)-chitosan-succinate particles were reacted with phosphate ions. Afterwards, particles were crosslinked with epichlorohydrin and the template (phosphate ions) was removed using 1M KOH solution. Selective cavity for the phosphate ion was obtained in the phosphate-imprinted metal-chelate polymer. These phosphate-imprinted metal-chelate polymer was used in the adsorption-desorption process. The adsorption process was fast and equilibrium was reached around 30 min. The adsorption behaviour of this system was described approximately by the Langmuir equation. Percent extraction, distribution ratio and selectivity coefficients of phosphate and other ions using non-imprinted and phosphate-imprinted polymer were also determined and comparison of these data was reported.

Removal of mercury species with dithiocarbamate-anchored polymer/organosmectite composites.

Mercury is one of the most toxic heavy metals found in solid and liquid waste disposed by chloro-alkali, paint, paper/pulp, battery, pharmaceutical, oil refinery and mining companies. Any form of mercury introduced to nature through any means is converted into a more toxic form such as methylmercury chloride (as produced by aquatic organisms) which usually accumulates in the tissue of fish and birds. The primary aim of this study was to investigate performance of dithiocarbamate-anchored polymer/organosmectite composites as sorbents for removal of mercury from aqueous solution. The modified smectite nanocomposites then were reacted with carbondisulfide to incorporate dithiocarbamate functional groups into the nanolayer of the organoclay. These dithiocarbamate-anchored composites were used for the removal of mercury species [Hg(II), CH(3)Hg(I) and C(6)H(5)Hg(I)]. Mercury adsorption was found to be dependent on the solution pH, mercury concentration and the type of mercury species to be adsorbed. The maximum adsorption capacities were equal to 157.3 mg g(-1) (782.5 micromol g(-1)) for Hg(II); 214.6 mg g(-1) (993.9 micromol g(-1)) for CH(3)Hg(I); 90.3 mg g(-1) (325 micromol g(-1)) for C(6)H(5)Hg(I). The competitive adsorption capacities (i.e. adsorption capacities based on solutions containing all three mercuric ions) are 7.7 mg g(-l) (38.3 micromol g(-1)), 9.2 mg g(-l) (42.6 micromol g(-1)) and 12.7 mg g(-1) (45.7 micromol g(-1)) for Hg(II), CH(3)Hg(I) and C(6)H(5)Hg(I), respectively, at 10 ppm initial concentration. The adsorption capacities on molar basis were in order of C(6)H(5)Hg(I)>CH(3)Hg(I)>Hg(II).

Nano-hemoglobin film based sextet state biomemory device by cross-linked photosensitive hapten monomer.

In this study, a biomemory device, consisting of hemoglobin (Hb) cross-linked by MACys-Ru(bipyr)2-MACys) photosensitive monomer cross-linkers, which have memory effect through both Ru3+/2+ in hapten monomer and Fe3+/2+ in redox active center of Hb through multi-charge transfer mechanism, has been improved. Cyclic voltammetry (CV) has been used to determine the redox property of the Hb cross-linked MACys-Ru(bipyr)2-MACys) hapten. Three memory functions, writing, reading and erasing of the fabricated biomemory device, have been accomplished by chronoamperometry (CA) and open-circuit potential amperometry (OCPA). The reliability and repeatability of the biodevice consisting of the p(Hb-co-MACys-Ru(bipyr)2-MACys) sextet state bio-memory layer have been analysed. The Hb film based biodevice on gold electrodes has shown ≥ 2 months the retention time and switched until 106 times continuous cycling without degradation in efficiency. Other hand, the topography of p(Hb-co-MACys-Ru(bipyr)2-MACys) layer on the gold surface has investigated by scanning electron microscopy (SEM) and EDX data.

Ferritin based bionanocages as novel biomemory device concept.

Ferritin is an iron cage having protein, capable of extracting metal ions in their cages and a consequence of the electron transfer of metal ions in their cage by reduction and oxidation processes, electrochemical information storage devices can be designed. In this work, ferritin based protein biomemory substrate has been synthesized by using Amino Acid (monomer) Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method, which utilizes photosensitive electron transfer based microemulsion co-polymerization as nanobead form of ferritin. Protein substrate contains metal ions such as silver and copper or metal ion pairs namely, silver-copper (Janus bionanocage) and co-polymeric shell of the photosensitive crosslinker protein. The redox behavior of bionanocages differentiates electrochemical "writing" and "erase" states depending on these metal ions (silver or copper) or metal ion pairs. The bionanocages based biomemory substrates have been immobilized using graphene modified glassy carbon electrodes and the memory functions of ferritin based bionanocages have been confirmed by chronoamperometry (CA) and open circuit potential amperometry (OCPA). The stability and durability of multi-state memory devices represent promising properties for future bioelectronic information technologies.

3D Micropatterned All-Flexible Microfluidic Platform for Microwave-Assisted Flow Organic Synthesis.

A large-area, all-flexible, microwaveable polydimethoxysilane microfluidic reactor was fabricated by using a 3D printing system. The sacrificial microchannels were printed on polydimethoxysilane substrates by a direct ink writing method using water-soluble Pluronic F-127 ink and then encapsulated between polydimethoxysilane layers. The structure of micron-sized channels was analyzed by optical and electron microscopy techniques. The fabricated flexible microfluidic reactors were utilized for the acetylation of different amines under microwave irradiation to obtain acetamides in shorter reaction times and good yields by flow organic synthesis.

Ion-imprinted beads for molecular recognition based mercury removal from human serum.

The aim of this study is to prepare ion-imprinted polymers which can be used for the selective removal of mercury ions [Hg(2+)] from human serum. N-Methacryloyl-(L)-cysteine (MAC) was chosen as the complexing monomer. In the first step, Hg(2+) was complexed with MAC and the Hg(2+)-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-cysteine) (MIP) beads were synthesized by suspension polymerization. After that, the template ions (i.e., Hg(2+)) were removed using thiourea (0.5%, v/v) in 0.05 M HCl. The specific surface area of the MIP beads was found to be 59.04 m(2)/g with a size range of 63-140 micro m in diameter and the swelling ratio was 91.5%. According to the elemental analysis results, the MIP beads contained 87.0 micro mol MAC/g polymer. The maximum adsorption capacity was 0.45 mg Hg(2+)/g beads. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. The relative selectivity coefficients of MIP beads for Hg(2+)/Cd(2+), Hg(2+)/Zn(2+) were 14.7 and 21.5 times greater than the non-imprinted (NIP) matrix, respectively. The MIP beads could be used many times without decreasing in their adsorption capacities significantly.