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Integrated Cantilever-Based Biosensors for the Detection of Chemical and Biological Entities
Published in Tuan Vo-Dinh, Nanotechnology in Biology and Medicine, 2017
Elise A. Corbin, Ashkan YekrangSafakar, Olaoluwa Adeniba, Amit Gupta, Kidong Park, Rashid Bashir
In order to calibrate the device, the cantilever array has been flushed in SSC buffer solution and stabilized at 23°C. Then, a heat pulse of 0.7°C has been applied so that all of the cantilevers bend accordingly. This procedure helps the array to gain the mechanical homogeneity. Subsequently, various concentrations of 12-mer ssDNA BioB2 were injected, and deflection of the cantilever has been observed. The BioB2-sensitized cantilever bends with respect to the BioB2 concentration. The deflection of the cantilever increases with the increase of the concentration. Then, the deflection of cantilevers is restored for the subsequent measurement by injecting the buffer solution. With the cantilever thickness of 450 nm, the sensor showed the minimum detectable concentration of 10 pM, which is sufficient for routine diagnostic tests.
Relative Influence of “Physicochemical Variables” and “Indigenous Bacterial Diversity” on the Efficiency of Bioaugmentation-Mediated In Situ Bioremediation
Published in M.H. Fulekar, Bhawana Pathak, Bioremediation Technology, 2020
For CFU counting and colony hybridization analyses, 0.50 g of collected soil samples were suspended in 5 mL of sterile saline. The suspension was serially diluted and plated on selective medium, i.e., minimal salt medium agar (containing: 4.0 g/L NaH2PO4 × 2 H2O, 4.0 g/L K2HPO4, 0.8 g/L (NH4)2SO4, and 0.8 g/L MgSO4, 1/1000 × trace element solution, and 2 % w/v ultrapure agar) supplemented with 0.5 mM PNP as a sole carbon, energy, and nitrogen source. Utilization of PNP was monitored by observing the decolorization of the yellow color of the PNP on these selective medium plates. All the colonies included within the decolorized regions of the PNP plate were counted. Colony hybridization was performed to further substantiate the results of the CFU counts. Bacterial colonies obtained on the earlier CFU counting on selective plates were screened using a 300 bp DNA fragment that is amplified as a partial amplicon to form a benzenetriol dioxygenase (btd) gene of strain RKJ100. The enzyme encoded by this gene has been shown to be involved in the PNP metabolic pathway of A. protophormiae strain RKJ100 and used to monitor cell survival of strain RKJ100 during earlier in situ biodegradation studies [36]. The colony hybridization process used was as follows: bacterial colonies were transferred onto a HybondN+ nylon membrane (GE HealthCare, Amersham, UK). Membranes were then treated sequentially for 10 min each with cell lysis buffer (10% SDS, 0.5 N NaOH), denaturation buffer (1.5 M NaOH, 0.5 M Tris-Cl; pH 7.4), transfer buffer (1.5 M NaCl), and 2× SSC buffer. Afterwards, membranes were air-dried and UV cross-linked before probing them with the P32-labeled 300-bp fragment of the btd gene. After hybridization, the membrane was exposed to film for 2 hours and hybridization signals were scanned with Phosphor-Imager (Bio-Rad Laboratories, Hercules, CA, USA).
Molecular and eco-physiological characterization of arsenic (As)-transforming Achromobacter sp. KAs 3–5T from As-contaminated groundwater of West Bengal, India
Published in Journal of Environmental Science and Health, Part A, 2018
Balaram Mohapatra, Tulasi Satyanarayana, Pinaki Sar
Molar G+C content of the strain KAs 3–5T was determined using the thermal denaturation method[51] where Escherichia coli K-12 NCIM 2563 was used as an internal standard. DNA–DNA hybridization was carried out between strain KAs 3–5T and its closest neighbours using the SyBr green binding fluorimetry-based method.[52] Hybridization was performed in 2X sodium chloride sodium citrate (SSC) buffer incorporating 0.1X SyBr dye following the real-time PCR program as described by Gonzalez and Seiz-Jimenez.[52] Relative binding ratio (RBR%) and Tm were calculated by the method described by Gonzalez and Seiz-Jimenez.[52] All experiments were conducted in triplicate and the mean was indicated as sample values.
Electrochemical nucleic acid hybridization biosensor based on poly(L-Aspartic acid)-modified electrode for the detection of short oligonucleotide sequences related to hepatitis C virus 1a
Published in Preparative Biochemistry & Biotechnology, 2019
Soner Donmez, Leman Çağdaş, Halit Arslan, Fatma Arslan
All the oligonucleotides were purchased from Ella Biotech Company (Germany) (Table 1), and stock solutions (500 μg mL−1) of the oligonucleotides were prepared by using ultra-pure water and were stored frozen. The stock solutions of the probe were prepared by diluting the solutions with 0.5 M of acetate buffer containing 20 mM NaCl (pH 4.80); and the stock solutions of targets were diluted with 2X Saline Sodium Citrate (SSC) buffer solution (pH 7.0). L-Aspartic acid, N-hydroxysulphosuccinimide (NHS), avidin and N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide (EDC) were obtained from Sigma. All other chemicals used were of an analytical purity grade, and ultrapure water (18 MΩ cm−1) was used in the preparation of solutions.