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Application of Molecular Tools and Biosensors for Monitoring Water Microbiota
Published in Maulin P. Shah, Wastewater Treatment, 2022
Pyrosequencing is a technique developed in 1996 by Mostafa Ronaghi and Pal Nyren at the Royal Institute of Technology in Stockholm. It is a DNA sequencing method that works on the principle of sequencing by synthesis. This technique is widely used for metagenomics detection of pathogens in various environmental and clinical samples. Pyrosequencing utilizes enzyme coupled reaction and bioluminescence in combination to monitor the release of pyrophosphate after the addition of a nucleotide in real time. This technique can be potentially applied to sequence a large number of reads in a single run. There are mainly four enzymes required for pyrosequencing: apyrase, luciferase, ATP sulfurylase, and Klenow fragment of DNA polymerase 1 (41). Apyrase is an enzyme incorporated into the process of pyrosequencing for degrading the free nucleotides and ATPs. The reaction mixture of pyrosequencing also demands luciferase, adenosine phosphosulfate, and the DNA template annealed to a primer as a starting material. The recognition and addition of a nucleotide to its complementary base in the single-stranded template leads to release of a pyrophosphate molecule (PPi) and the growth of the DNA strand. This released inorganic pyrophosphate is further converted to ATP in the presence of the enzyme ATP sulfurylase by utilizing the adenosine phosphosulfate molecule as a substrate. Finally, this ATP is utilized by the luciferase enzyme to generate a light signal. This generated light is identified and used as evidence for the incorporation of the nucleotide into the growing chain.
Thermistor Probes
Published in John V. Twork, Alexander M. Yacynych, Sensors in Bioprocess Control, 2020
The first reported enzyme thermistor [20] was designed as shown in Figure 6, to contain a thermistor in paraffin oil surrounded by a coil of tubing cemented in place to form a cup with a diameter of 4 mm. The tubing itself had a volume of 180 μL and was filled with approximately 100 mg of glass beads to which enzyme was attached. When substrate was pumped through the tubing a chemical reaction, catalyzed by the enzyme was sufficient to cause a temperature change noted by the thermistor. The first enzyme systems studied by this device were trypsin and apyrase. A similar device was reported at the same time by Canning and Carr [23].
Kinetic study of NTPDase immobilization and its effect of haemocompatibility on polyethylene terephthalate
Published in Journal of Biomaterials Science, Polymer Edition, 2019
Balaji Ramachandran, Vignesh Muthuvijayan
Heparin is one of the widely studied bioactive molecules on various material surfaces to improve haemocompatibility [5]. Despite heparin-mediated inhibition of thrombin formation, researchers have explored the inhibition of early stages of thrombus formation through inhibiting platelet activation. Upon adhesion to the foreign material surfaces, platelets can lead to activation and release of pro-coagulant agonists from dense granules such as thromboxane A2, ADP, serotonin [6]. For platelet-specific improvement of haemocompatibility of material surfaces, many molecules such as nitric oxide, dipyridamole, hirudin had been explored [7,8]. Apyrase/ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) is also one such molecule which can be used for enhancing haemocompatibility of the material surface [9,10]. NTPDase is an important transmembrane protein that phosphohydrolyses ATP to ADP and then to AMP, thereby regulating ADP-dependent platelet activation and adhesion [11,12]. Coating of modified apyrase on the material surface shows improved blood compatibility [13,14]. Clinical studies showed that the platelet adhesion and activation vary between healthy individuals and patients with cardiovascular artery disease [15]. Hence, the coating of the NTPDase should be based on the end user application. This can be only achieved by studying the kinetics of the attached biomolecules.