Electrophoresis and Biosensor-Based DNA Interaction Analysis of the First Paraben Derivatives of Spermine-Bridged Cyclotriphosphazenes.

Cancer is the uncontrolled growth of abnormal cells via malignant cell division and rapid DNA replication. While DNA damaging molecules can cause cancer, their role as anticancer drugs are very significant. For this purpose, the novel series of paraben substituted spermine bridged(dispirobino) cyclotriphosphazene compounds 2-6 were synthesized for the first time, and their structures were characterized by various spectroscopic techniques. The solid-state structures and geometries of compounds 2-6 were determined using single-crystal X-ray structural analysis. In addition, it was confirmed by TGA that all compounds 1-6 showed high thermal stability. Two methods were used in order to investigate DNA interaction properties of the targeted molecules. While biosensor-based screening test that measures DNA hybridization efficiency on a biochip surface, the agarose gel electrophoresis method examines the effect of compounds on plasmid DNA structure. The results collected from the automated biosensor device and agarose gel electrophoresis have indicated that compounds 1, 5, and 6 showed higher DNA damage than the compounds 2-4. According to the biosensor results, compounds 1, 5, and 6 showed 85%, 69%, and 77% activity, respectively.

Lab-on-a-chip based biosensor for the real-time detection of aflatoxin.

Polymers were synthesized and utilized for aflatoxin detection coupled with a novel lab-on-a-chip biosensor: MiSens and high performance liquid chromatography (HPLC). Non-imprinted polymers (NIPs) were preferred to be designed and used due to the toxic nature of aflatoxin template and also to avoid difficult clean-up protocols. Towards an innovative miniaturized automated system, a novel biochip has been designed that consists of 6 working electrodes (1mm diameter) with shared reference and counter electrodes. The aflatoxin detection has been achieved by a competition immunoassay that has been performed using the new biochips and the automated MiSens electrochemical biosensor device. For the assay, aflatoxin antibody has been captured on the Protein A immobilized electrode. Subsequently the sample and the enzyme-aflatoxin conjugate mixture has been injected to the electrode surfaces. The final injection of the enzyme substrate results in an amperometric signal. The sensor assays for aflatoxin B1 (AFB1) in different matrices were also performed using enzyme link immunosorbent assay (ELISA) and HPLC for confirmation. High recovery was successfully achieved in spiked wheat samples using NIP coupled HPLC and NIP coupled MiSens biosensor [2ppb of aflatoxin was determined as 1.86ppb (93% recovery), 1.73ppb (86.5% recovery), 1.96ppb (98% recovery) and 1.88ppb (94.0% recovery) for immunoaffinity column (IAC)-HPLC, NIP-HPLC, Supel™ Tox SPE Cartridges (SUP)-HPLC and NIP-MiSens, respectively]. Aflatoxin detection in fig samples were also investigated with MiSens biosensor and the results were compared with HPLC method. The new biosensor allows real-time and on-site detection of AFB1 in foods with a rapid, sensitive, fully automated and miniaturized system and expected to have an immense economic impact for food industry.

First paraben substituted cyclotetraphosphazene compounds and DNA interaction analysis with a new automated biosensor.

Cancer, as one of the leading causes of death in the world, is caused by malignant cell division and growth that depends on rapid DNA replication. To develop anti-cancer drugs this feature of cancer could be exploited by utilizing DNA-damaging molecules. To achieve this, the paraben substituted cyclotetraphosphazene compounds have been synthesized for the first time and their effect on DNA (genotoxicity) has been investigated. The conventional genotoxicity testing methods are laborious, take time and are expensive. Biosensor based assays provide an alternative to investigate this drug/compound DNA interactions. Here for the first time, a new, easy and rapid screening method has been used to investigate the DNA damage, which is based on an automated biosensor device that relies on the real-time electrochemical profiling (REP™) technology. Using both the biosensor based screening method and the in vitro biological assay, the compounds 9 and 11 (propyl and benzyl substituted cyclotetraphosphazene compounds, respectively), have resulted in higher DNA damage than the others with 65% and 80% activity reduction, respectively.

Microfluidics and nanoparticles based amperometric biosensor for the detection of cyanobacteria (Planktothrix agardhii NIVA-CYA 116) DNA.

Some of the cyanobacteria produce protease inhibitor oligopeptides such as cyanopeptolins and cause drinking water contamination; hence, their detection has great importance to monitor the well-being of water sources that is used for human consumption. In the current study, a fast and sensitive nucleic acid biosensor assay has been described where cyanopeptolin coding region of one of the cyanobacteria (Planktothrix agardhii NIVA-CYA 116) genome has been used as target for monitoring of the fresh water resources. A biochip that has two sets of Au electrode arrays, each consist of shared reference/counter electrodes and 3 working electrodes has been used for the assay. The biochip has been integrated to a microfluidics system and all steps of the assay have been performed during the reagent flow to achieve fast and sensitive DNA detection. On-line hybridization of the target on to the capture probe immobilized surface resulted in a very short assay duration with respect to the conventional static assays. The binding of the avidin and enzyme modified Au nanoparticles to the biotinylated detection probe and the subsequent injection of the substrate enabled a real-time amperometric measurement with a detection limit of 6×10(-12) M target DNA (calibration curve r(2)=0.98). The developed assay enables fast and sensitive detection of cyanopeptolin producing cyanobacteria from freshwater samples and hence shows a promising technology for toxic microorganism detection from environmental samples.

Fast and sensitive detection of mycotoxins in wheat using microfluidics based Real-time Electrochemical Profiling.

The objective of the study has been the development of a new sensing platform, called Real-time Electrochemical Profiling (REP) that relies on real-time electrochemical immunoassay detection. The proposed REP platform consists of new electrode arrays that are easy to fabricate, has a small imprint allowing microfluidic system integration, enables multiplexed amperometric measurements and performs well in terms of electrochemical immunoassay detection as shown through the deoxynivalenol detection assays. The deoxynivalenol detection has been conducted according to an optimised REP assay protocol using deoxynivalenol standards at varying concentrations and a standard curve was obtained (y=-20.33ln(x)+124.06; R(2)=0.97) with a limit of detection of 6.25 ng/ml. As both ELISA and REP detection methods use horse radish peroxidase as the label and 3.3',5.5'-Tetramethylbenzidine as the substrate, the performance of the REP platform as an ELISA reader has also been investigated and a perfect correlation between the deoxynivalenol concentration and the current response was obtained (y=-14.56ln(x)+101.02; R(2)=0.99). The calibration curves of both assays have been compared to conventional ELISA tests for confirmation. After assay optimisation using toxin spiked buffer, the deoxynivalenol detection assay has also been performed to detect toxins in wheat grain.

Design and characterisation of a thin-film electrode array with shared reference/counter electrodes for electrochemical detection.

In the current study, a novel electrode array and integrated microfluidics have been designed and characterised in order to create a sensor chip which is not only easy, rapid and cheaper to produce but also have a smaller imprint and good electrochemical sensing properties. The current study includes the assessment of the effects of an Au quasi-reference electrode and the use of shared reference/counter electrodes for the array, in order to obtain a small array that can be produced using a fine metal mask. In the study, it is found that when Au is used as the quasi-reference electrode, the arrays with shared reference and counter electrodes result in faster electron transfer kinetics and prevent the potential change with respect to scan rate, and hence is advantageous with respect to conventional electrodes. In addition, the resulting novel electrode array has been shown to result in higher current density (10.52 µA/cm(2); HRP detection assay) and measured diffusion coefficient (14.40×10(-12) cm(2)/s; calculated from the data of cyclic voltammetry with 1mM potassium ferricyanide) with respect to conventional electrodes tested in the study. Using the new electrode arrays, the detection limits obtained from horse radish peroxidase (HRP) and bisphenol A assays were 12.5 ng/ml (2.84×10(-10) M ) and 10 ng/ml (44×10(-9) M), respectively. Performing the HRP detection assay in a flow injection system using array integrated microfluidics provided 25 times lower detection limit (11.36×10(-12) M), although Ti has been used as electrode material instead of Au. In short, incorporation of this new electrode array to lab-on-a-chip or MEMs (micro-electro mechanic systems) technologies may pave the way for easy to use automated biosensing devices that could be used for a variety of applications from diagnostics to environmental monitoring, and studies will continue to move forward in this direction.

Highly efficient method towards in situ immobilization of invertase using cryogelation.

A novel method was developed for the immobilization of Saccharomyces cerevisiae invertase within supermacroporous polyacrylamide cryogel and was used to produce invert sugar. First, the cross-linking of invertase with soluble polyglutaraldehyde (PGA) was carried out prior to immobilization in order to increase the bulkiness of invertase and thus preventing the leakage of the cross-linked enzyme after immobilization by entrapment. And then, in situ immobilization of PGA cross-linked invertase within cryogel synthesis was achieved by free radical polymerization in semi-frozen state. The method resulted in 100 % immobilization and 74 % activity yields. The immobilized invertase retained all the initial activity for 30 days and 30 batch reactions. Immobilization had no effect on optimum temperature and it was 60 °C for both free and immobilized enzyme. However, optimum pH was affected upon immobilization. Optimum pH values for free and immobilized enzyme were 4.5 and 5.0, respectively. The immobilized enzyme was more stable than the free enzyme at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes and microorganisms.