Graphenoxide Cross-Linker Based Potentiometric Biosensor Design for Sarcosine Determination.

BACKGROUND: Sarcosine, also known as N-methyl glycine, is a natural amino acid that is an intermediate and by product in glycine synthesis and degradation. Recently found in many peptides, sarcosine has been researched as a newly accepted prostate cancer marker. The increased concentration of sarcosine in blood serum and the urine showed that malignancy of measured prostate cancer cells is active. OBJECTIVE: In this article, we aimed to design a potentiometric biosensor for detection of sarcosine with a low detection limit, high selectivity, short response time, wide linear range, and satisfactory long-term stability. METHODS: In this article, we developed a new Graphene oxide (GFOX) photosensitive cross-linker based potentiometric biosensor based on the AmiNoAcid (monomer) Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method. The functional groups determined using Raman, FT-IR, XPS analyzes, and surface characterization, the morphology of synthesized GFOX photosensitive cross-linker were determined by TEM and AFM studies. Then, the performance of the GFOX based potentiometric biosensor has been evaluated. RESULTS: When the usage of the developed GFOX doped potentiometric biosensor against sarcosine determination, it was found that 10-4 mM sarcosine was determined in 60 seconds in the solution. In addition, the detection limit of the GFOX doped potentiometric biosensor was found to be 9.45x10-7 mM, and the linear potentiometric biosensor was found to be in the concentration range of 10-1 to 10-5 mM. The selectivity studies of the developed potentiometric biosensor were investigated using glycine solutions, and it was determined that GFOX doped potentiometric biosensor was more selective against sarcosine. Besides this, a reusability test using 10-3 mM sarcosine solution showed that reproducible studies were performed without the loss of potential of designed potentiometric biosensor and no loss of sensitivity. CONCLUSION: After applying the framework, we get a new potentiometric biosensor for sarcosine determination. GFOX photosensitive cross-linker was used in designing potentiometric biosensors, and this increased the stability and efficiency of the biosensor. Therefore, the developed potentiometric biosensor for sarcosine determination could be easily used for the early diagnosis of prostate cancer.

Carbonic Anhydrase Carrying Electrospun Nanofibers for Biocatalysis Applications.

BACKGROUND: Enzymes are efficient biocatalysis that catalysis a large number of reactions due to their chemical, regional, or stereo specifities and selectivity. Their usage in bioreactor or biosensor systems has great importance. Carbonic anhydrase enzyme catalyzes the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid. In organisms, the carbonic anhydrase enzyme has crucial roles connected with pH and CO2 homeostasis, respiration, and transport of CO2/bicarbonate, etc. So, immobilization of the enzyme is important in stabilizing the catalyst against thermal and chemical denaturation in bioreactor systems when compared to the free enzyme that is unstable at high temperatures and extreme pH values, as well as in the presence of organic solvents or toxic reagents. Nano-scale composite materials have attracted considerable attention in recent years, and electrospinning based all-nanocomposite materials have a wide range of applications. In this study, electrospun nanofibers were fabricated and used for the supporting media for carbonic anhydrase enzyme immobilization to enhance the enzyme storage and usage facilities. OBJECTIVE: In this article, our motivation is to obtain attractive electrospun support for carbonic anhydrase enzyme immobilization to enhance the enzyme reusability and storage ability in biocatalysis applications. METHODS: In this article, we propose electrospun nanofibers for carbonic anhydrase carrying support for achieving our aforementioned object. In the first part of the study, agar with polyacrylonitrile (PAN) nanofibers was directly fabricated from an agar-PAN mixture solution using the electrospinning method, and fabricated nanofibers were cross-linked via glutaraldehyde (GA). The morphology, chemical structure, and stability of the electrospun nanofibers were characterized. In the second part of the study, the carbonic anhydrase enzyme was immobilized onto fabricated electrospun nanofibers. Then, enzyme activity, the parameters that affect enzyme immobilization such as pH, enzyme amount, immobilization time, etc. and reusability were investigated. RESULTS: When the scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) analysis results are combined in the characterization process of the synthesized electrospun nanofibers, the optimum cross-linking time is found to be 8 hours using 5% glutaraldehyde cross-linking agent. Then, thermal stability measurements showed that the thermal stability of electrospun nanofibers has an excellent characteristic for biomedical applications. The optimum temperature value was found 37°C, pH 8 was determined as an optimum pH, and 100 ppm carbonic anhydrase enzyme concentration was found to be optimum enzyme concentration for the carbonic anhydrase enzyme immobilization. According to the kinetic data, carbonic anhydrase immobilized electrospun nanofibers acted as a biocatalyst in the conversion of the substrate to the product in 83.98%, and immobilized carbonic anhydrase enzyme is reusable up to 9 cycles in biocatalysis applications. CONCLUSION: After applying the framework, we get a new biocatalysis application platform for carbonic anhydrase enzyme. Electrospun nanofibers were chosen as the support material for enzyme immobilization. By using this approach, the carbonic anhydrase enzyme could easily be used in the industrial area by cost-effective advantageous aspects.

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.

Albumin Based Nanoparticles for Detection of Pancreatic Cancer Cells.

BACKGROUND: Molecular imaging of cancer cells using effective drug targeting systems are most interested research area in recent years. Albumin protein is a soluble and most abundant protein in circulatory system. It has a ligand-binding function and acts as a transport protein. Researchers are interested in developing albumin based nanostructured specific anti-tumor drugs in cancer therapy. Pancreatic cancer treatment or drug design for targeted pancreatic cancer cell has great importance due to it has a high mortality rate comparing other cancer types. OBJECTIVE: In this article, our goal is to develop new targeting nanoparticles based on the conjugation of albumin and Hyaluronic Acid (HA) for pancreatic cancer cells. METHOD: In this article, we proposed a new technique for conjugation of albumin (BSA) and HA in nano formation. Firstly, cationic BSA is synthesized. Then, BSA-HA conjugation is obtained by interacted cationic BSA with 1000 ppm HA. Secondly, nano BSA-HA particles and nano BSA particles were synthesized according to AmiNoAcid Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method which provides a special cross-linking strategy for biomolecules using ruthenium-based amino acid monomer haptens. After characterization studies, in vitro cytotoxic activity of synthesized nano BSA-HA particles were determined for PANC-1 ATCC® CRL146 cells. RESULTS: According to the data, nano BSA and nano BSA-HA particles synthesized uniquely using special ruthenium-based amino acid decorated cross-linking agent, (MATyr)2-Ru-(MATyr)2.based on ANDOLUCA method. Characterization results showed that there was not any change in protein folding structures during nano formation process. In addition, nano protein particles gained fluorescence feature. When interacting synthesized nano BSA and nano BSA-HA particles with pancreatic cells, it was found that BSA nanoparticles were usually around cells and membranes, but BSA-HA nanoparticles were identified around the cells, in the cytoplasm inside the cell, and next to the cell nucleus. So, nano BSA-HA particles could be used as cancer cell imaging agent for PANC-1 ATCC® CRL146 cells. CONCLUSION: The satisfactory conclusion of this study is that synthesized nano BSA-HA particles are fundamental materials for targeting pancreatic cancer cells due to HA receptors located on pancreatic cancer cells and imaging agents due to fluorescence feature of the BSA-HA nanoparticles.

Reusable nanocopy machine particles for the replication of DNA.

As one of the most important components copying DNA molecules in the PCR system, Taq DNA polymerase has a high processivity, however, lower persistence when compared to other polymerases. Studies for the enhancement of stability of Taq DNA polymerase is of great importance. The present study describes the integration of PCR application of cross-linked Taq DNA polymerase enzyme in a nanochamber using a ruthenium based MATyr-Ru-(bipyr)2)-MATyr monomer hapten prepared by photosensitive microemulsion polymerization technique. The conjugation and cross-linking have achieved using our previously invented Aminoacid (monomer) Decorated and Light Underpining Conjugation Approach (ANADOLUCA) method. Microemulsion polymerization media has prepared by dispersing PVA in deionized water. The nano enzyme could be easily prepared at room temperature, in daylight and under nitrogen atmosphere using ruthenium based photosensitive cross-linking agents. The nano copy machine particles (nano Taq DNA polymerase) are very stable against more acidic or more basic conditions, high temperatures and could be reusable in PCR analysis for many times without any deformation in their structures.

Developing column material for the separation of serum amyloid P and C reactive protein from biological sources.

In this study, we have investigated the isolation of serum amyloid P (SAP) and C-reactive protein (CRP) from rainbow trout. It has recently been found that SAP is deposited in atherosclerotic lesions or neurofibrillary tangles, which are related to aging process and Alzheimer's disease. Given the importance of CRP, the CRP level in blood is becoming recognized as a potential means of monitoring cardiovascular risk. These two proteins, members of the pentraxin family of oligomeric serum proteins, were isolated from rainbow trout using N-methacryloyl-phosphoserine (MA-pSer) immobilized poly (2-hydroxy ethylmethacrylate) (PHEMA) cryogels as a column material in a fast protein liquid chromatography system. The separation process was verified in two steps. First, SAP and CRP proteins were isolated together from serum sample of rainbow trout using MA-pSer/PHEMA cryogel columns. Second, SAP protein was separated chromatographically from CRP protein using the Ca(2+) ion immobilized PHEMA cryogel column. According to the data, a new and effective technique has been developed for the isolation of SAP and CRP proteins from a biological source, rainbow trout. Finally, purified SAP and CRP were loaded using sodium dodecyl sulfate-polyacrylamide gel and western blot analysis to investigate the purity of chromatographically isolated SAP and CRP compared with commertial SAP and CRP.

Preparation of a novel hydrophobic affinity cryogel for adsorption of lipase and its utilization as a chromatographic adsorbent for fast protein liquid chromatography.

In this study, we have prepared a hydrophobic cryogel for the chromatographic separation of lipase from its aqueous solutions including single protein and protein mixture and also Yarrowia lipolytica cell extract. N-methacryloyl-(l)-phenylalanine methyl ester was used as a monomer to provide the hydrophobic character to the prepared cryogels. The highest adsorption capacity was observed at pH 5.0 at 0.5 mL min(-1) flow rate. The chromatographic separation of lipase was achieved from a binary mixture of lipase:bovine serum albumin (BSA) and lipase:lysozyme, and was also achieved from triple-mixture of lipase:lysozyme:BSA by using fast protein liquid chromatography. Finally, lipase purification was performed from Yarrowia lipolytica cell extract used as a natural source. These studies have shown that the hydrophobic cryogel has good chromatographic performance for the separation and purification of lipase not only from aqueous solution, but also from cell extract as a natural source of lipase.

Separation and purification of hyaluronic acid by embedded glucuronic acid imprinted polymers into cryogel.

Hyaluronic acid (HA) has been used in many applications such as pharmaceutical, clinical and cosmetics, so its separation and purification is very important. In this study, firstly d-glucuronic acid imprinted polymers (MIPs) have been synthesized for the separation of HA which has glucuronic acid part in its structure. MIP particles have characterized by elemental analysis, Fourier Transform Infrared Spectroscopy (FT-IR) and swelling tests. Then, synthesized MIP particles have embedded into polyacrylamide based cryogel. Cryogel has prepared by free radical cryogelation process initiated by N,N,N',N'-Tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) as redox initiators. This cryogel material was characterized by FT-IR, swelling tests, scanning electron microscopy (SEM) and surface adsorption analyze including pore size analyzer (BET) method. The adsorption of HA has investigated by spectrophotometric method using MIPs embedded into cryogel columns (GAIPEC) and the maximum HA adsorption capacity was found to be 318mgg(-1). The selectivity of GAIPEC column has estimated using N-acetylglucose amine as interfering agent since this molecule is a part of HA and the results have shown that GAIPEC has been nearly 35 times selective for HA than N-acetylglucose amine. The optimum chromatographic conditions for separation of HA were investigated. pH 7.0 buffer solution for elution and 0.1M of NaCl solution as desorption agent were used at 0.5mLmin(-1) flow rate. Also, recovery of GAIPEC was investigated and the results have shown that GAIPEC could be used many times without decreasing its adsorption capacity significantly. Here in, combining selectivity of MIP particles and mechanical properties of cryogel, a rigid and stable material was prepared for the separation and purification of HA. To point out this, HA has been isolated from fish eye and fermentation of Streptococcus equi RSKK 679 cell culture. After that, it has characterized and Fast Protein Liquid Chromatography (FPLC) applications have been investigated.

Novel nanoimaging approach: antibodious polymeric nanolabel for intracellular alpha-fetoprotein targeted monitoring.

This study describes preparation and use of novel labeled and antibodious polymeric nanolabels (anti-alpha fetoprotein cross-linked nanolabels) as an immunogenic and semisynthetic nanolabel with potential prognostic and therapeutic roles for hepatoma cancer. Specificity, uptake, and binding efficiencies of the nanolabel have been examined in a human hepatosarcoma cell line HepG2, a human colorectal cell line DLD-1, and a mouse myoblast cell line C2. Labeling of the cells has been performed by treating live and fixed cells with varying concentrations of the nanolabels and then, the cells have been examined under a fluorescence microscope. In addition, all cell lines have also been labeled using FITC-conjugated nanotrastuzumab to compare the results obtained with those of the binding of the FITC-nanoanti-alpha fetoprotein nanolabels. Results show that FITC-conjugated anti-alpha fetoprotein cross-linked nanolabels have been taken up by both live and fixed cells and have efficiently and specifically labeled HepG2 cells at a quite low concentration. Taken all together, the results indicate that the novel targeted nanoimaging tools and technique demonstrated their ability to detect the distribution of the nanolabels as probes in hepatoma cells.