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Monitoring Apoptosis and Anticancer Drug Activity in Single Cells Using Nanosensors
Published in Tuan Vo-Dinh, Nanotechnology in Biology and Medicine, 2017
The ELISA technique utilizes substrates that produce soluble products that can be detected. Ideally, these enzyme substrates should be stable, safe, and inexpensive. Popular enzymes are those that convert a colorless substrate into a colored product, for example, p-nitrophenylphosphate (pNPP), which is converted into the yellow p-nitrophenol by alkaline phosphatase, or DDAO-phosphate, which is converted into DDAO (excitation/emission maxima ∼646/659 nm), a compound with fluorescence characteristics that are distinctly different from those of DDAO-phosphate (∼478/628 nm). After a baseline measurement is acquired, the emitted fluorescence signal produced after excitation by the appropriate laser excitation source can be correlated with the amount of protein or antibody that is present [19]. This sensing scheme employing an ELISA-based format has been successfully applied to measure an important intermediate protein in apoptosis, cytochrome c, in a single living cell [26]. The details of this procedure involve micro-manipulating and inserting optical nanobiosensors with capture antibody into single MCF-7 cells for a brief incubation period. This allows the formation of a primary antibody–cytochrome c complex to form. The optical nanobiosensor is gently micromanipulated and withdrawn from within the living cell and incubated in hybridization solution containing Biotin-labeled antibody, resulting in the formation of a secondary immunocomplex. This immunocomplex is incubated subsequently in a solution containing AP–streptavidin conjugate. This conjugate binds to the biotin-labeled antibody that is part of the secondary immunocomplex and then finally reacted with DDAO-phosphate to produce fluorescent DDAO that is detected using an inverted microscope setup via laser-induced fluorescence. The fluorescence signal generated is recorded and is indicative of the presence of cytochrome c. In this case, ELISA is most appropriate for amplification of the signal to facilitate detection of the target molecule, cytochrome c [26].
Design and development of biomimetic electrospun sulphonated polyether ether ketone nanofibrous scaffold for bone tissue regeneration applications: in vitro and in vivo study
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Rajalakshmi Ekambaram, Sangeetha Dharmalingam
Undifferentiated osteogenic cells show weak alkaline phosphatase (ALP) activity, whereas differentiated osteoblasts feature very high phosphatase activity. The osteogenic differentiation potential of MG63 cell lines was elucidated by using the alkaline phosphatase (ALP) [35], a yellow liquid substrate system (Sigma Life Sciences, USA). In this reaction, ALP catalyzes the hydrolysis of colorless organic phosphate ester substrate, p-nitro phenyl phosphate (pNPP) to a yellow product, p-nitro phenol and phosphate. The scaffolds were washed thrice with PBS at different time intervals during our cell culture study and 400 μl of pNPP liquid was added, incubated for 30 min till the color of the solution became yellow. The reaction was then arrested by the addition of 100 μl of 2 M NaOH solution and the yellow color product formed was aliquoted in 96-well plate and read at 405 nm in microplate assay reader.
Porous PVA/SA/HA hydrogels fabricated by dual-crosslinking method for bone tissue engineering
Published in Journal of Biomaterials Science, Polymer Edition, 2020
Mengjie Xu, Miao Qin, Xiumei Zhang, Xiaoyu Zhang, Jingxuan Li, Yinchun Hu, Weiyi Chen, Di Huang
The ALP activity was determined by using Alkaline Phosphatase Assay Kit (Beyotime, China) to quantify the amount of p-nitrophenol phosphate (pNPP), which was generated by the reaction of alkaline phosphatase. 1 mL cell suspension was seeded on each sample in 24-well plates at a density of 1 × 104 cells/mL. After culturing for 7 and 14 days, the media were removed and the samples were washed with PBS. Then, 200 µL of cell lysis solution (Beyotime, China) was added to each well. The lysates were collected immediately and centrifuged, and the supernatant was collected. 50 µL of the supernatant was added to the wells of a 96-well plate and incubated with 50 µL of the substrate solution (pNPP) at 37 °C for 10 min. Finally, 100 µL of stop solution was added and the OD values were measured with a microplate reader at 450 nm. According to the definition of the ALP Kit instruction, a calibration curve was obtained using the p-nitrophenol solution of known concentrations and the diethanolamine (DEA) enzyme activity unit was calculated.
Generation of bioactive nano-composite scaffold of nanobioglass/silk fibroin/carboxymethyl cellulose for bone tissue engineering
Published in Journal of Biomaterials Science, Polymer Edition, 2018
The early stage osteogenic activity of hMSCs on scaffolds was evaluated by measuring alkaline phosphatase (ALP) activity. The quantitative evaluation of ALP activity was done by using p-nitrophenyl phosphate (pNPP) (SIGMAFASTTM p-nitrophenyl phosphate tablets) as the reaction substrate for alkaline phosphatase. The hMSCs (105 cells/cm2) were cultured on scaffolds in 96 well plates for two weeks. The cell seeded scaffolds were cultured in osteogenic media with media change after 48 hr. The samples were then washed twice with PBS and 50 µl lysis solutions (0.5% TritonX-100) were added to each well. 100 µl of 1 mg/ml p-NPP solution was then added to each wells and incubated for 1.5 hr at 37 °C following the manufacturer protocol. After incubation, absorbance was measured at 405nm.