Tracking variations in nicotinamide cofactors extracted from cultured cells using capillary electrophoresis with multiphoton excitation of fluorescence
“…Recently developed nonelectrochemical (fluorescence) assays for NADH displayed detection limits as low as 0.02-0.1 µM; however, the linearity range extended only to below 10 µM. [24][25][26] Perhaps the most attractive feature of the CHIT-AZU/CNT system as the NADH detector is its selectivity and stability. The low detection potential of the CHIT-AZU/CNT films (-0.10 V) eliminated interferences from other redox-active molecules such as ascorbic acid, uric acid, and acetaminophen (Figure 7, inset), which are typically present in biological samples.…”
Section: Resultsmentioning
confidence: 99%
“…However, their linear range has been rather limited (0.01−0.50 μM) and detection potential was high (+0.60 V). Recently developed nonelectrochemical (fluorescence) assays for NADH displayed detection limits as low as 0.02−0.1 μM; however, the linearity range extended only to below 10 μM. − 7 Amperometric response ( E = −0.10 V) of a CHIT-AZU/CNT film electrode to additions of NADH aliquots (5.0 μM−1.0 mM) into a stirred solution of phosphate buffer, pH 7.40. Inset: Current trace recorded at the CHIT-AZU/CNT film ( E = −0.10 V) in a stirred buffer solution (pH 7.40) that was spiked with (a) 0.50 mM NADH, (b) 0.10 mM uric acid, (c) 0.10 mM acetaminophen, and (d) 0.10 mM ascorbic acid.…”
An electrochemical sensing platform was developed based on the integration of redox mediators and carbon nanotubes (CNT) in a polymeric matrix. To demonstrate the concept, a redox mediator Azure dye (AZU) was covalently attached to polysaccharide chains of chitosan (CHIT) and interspersed with CNT to form composite films for the amperometric determination of beta-nicotinamide adenine dinucleotide (NADH). The incorporation of CNT into CHIT-AZU matrix facilitated the AZU-mediated electrooxidation of NADH. In particular, CNT decreased the overpotential for the mediated process by an extra 0.30 V and amplified the NADH current by approximately 35 times (at -0.10 V) while reducing the response time from approximately 70 s for CHIT-AZU to approximately 5 s for CHIT-AZU/CNT films. These effects were discussed in terms of the AZU/CNT synergy, which improved charge propagation through the CHIT-AZU/CNT matrix. The concept of CNT-facilitated redox mediation in polymeric matrixes has a potential to be of general interest for expediting redox processes in electrochemical devices such as sensors, biosensors, and biological fuel cells and reactors.
“…Recently developed nonelectrochemical (fluorescence) assays for NADH displayed detection limits as low as 0.02-0.1 µM; however, the linearity range extended only to below 10 µM. [24][25][26] Perhaps the most attractive feature of the CHIT-AZU/CNT system as the NADH detector is its selectivity and stability. The low detection potential of the CHIT-AZU/CNT films (-0.10 V) eliminated interferences from other redox-active molecules such as ascorbic acid, uric acid, and acetaminophen (Figure 7, inset), which are typically present in biological samples.…”
Section: Resultsmentioning
confidence: 99%
“…However, their linear range has been rather limited (0.01−0.50 μM) and detection potential was high (+0.60 V). Recently developed nonelectrochemical (fluorescence) assays for NADH displayed detection limits as low as 0.02−0.1 μM; however, the linearity range extended only to below 10 μM. − 7 Amperometric response ( E = −0.10 V) of a CHIT-AZU/CNT film electrode to additions of NADH aliquots (5.0 μM−1.0 mM) into a stirred solution of phosphate buffer, pH 7.40. Inset: Current trace recorded at the CHIT-AZU/CNT film ( E = −0.10 V) in a stirred buffer solution (pH 7.40) that was spiked with (a) 0.50 mM NADH, (b) 0.10 mM uric acid, (c) 0.10 mM acetaminophen, and (d) 0.10 mM ascorbic acid.…”
An electrochemical sensing platform was developed based on the integration of redox mediators and carbon nanotubes (CNT) in a polymeric matrix. To demonstrate the concept, a redox mediator Azure dye (AZU) was covalently attached to polysaccharide chains of chitosan (CHIT) and interspersed with CNT to form composite films for the amperometric determination of beta-nicotinamide adenine dinucleotide (NADH). The incorporation of CNT into CHIT-AZU matrix facilitated the AZU-mediated electrooxidation of NADH. In particular, CNT decreased the overpotential for the mediated process by an extra 0.30 V and amplified the NADH current by approximately 35 times (at -0.10 V) while reducing the response time from approximately 70 s for CHIT-AZU to approximately 5 s for CHIT-AZU/CNT films. These effects were discussed in terms of the AZU/CNT synergy, which improved charge propagation through the CHIT-AZU/CNT matrix. The concept of CNT-facilitated redox mediation in polymeric matrixes has a potential to be of general interest for expediting redox processes in electrochemical devices such as sensors, biosensors, and biological fuel cells and reactors.
“…Traditional biochemical methods for the analysis of redox states of specific metabolites, i.e., chromatography, mass spectrometry, enzymatic cycling assays 9,10 , capillary electrophoresis 11 , and isotope-labeling techniques 2,12 , are invasive and do not capture the transient subcellular redox changes associated with metabolic activation or dysfunction in intact individual cells. Furthermore, these methods all suffer from potential sample oxidization or degradation during processing, potentially leading to spurious measurements of the redox-active species.…”
Cellular oxidation-reduction reactions are mainly regulated by pyridine
nucleotides (NADPH/NADP+ and NADH/NAD+), thiols and
reactive oxygen species (ROS), and play central roles in cell metabolism,
cellular signaling and cell fate decisions. A comprehensive evaluation or
multiplex analysis of redox landscapes and dynamics in intact living cells is
important for interrogating cell functions in both healthy and diseased states;
however, until recently, this goal has been limited owing to the lack of a
complete set of redox sensors. We recently reported a series of highly
responsive, genetically encoded fluorescent sensors for NADPH, significantly
strengthening the existing toolset of genetically encoded sensors for thiols,
H2O2, and NADH redox states. By combining sensors with
unique spectral properties and specific sub-cellular targeting domains, our
approach allows for simultaneous imaging of up to four different sensors. In
this protocol, we first describe strategies for multiplex fluorescence imaging
of these sensors in single cells, and we demonstrate how to apply these sensors
to study changes in redox landscapes during the cell cycle, following macrophage
activation, and in living zebrafish. This approach can be adapted to different
genetically encoded fluorescent sensors using various analytical platforms, such
as fluorescence microscopy, high-content imaging systems, flow cytometry, and
microplate readers. Typically, the preparation of cells or zebrafish expressing
different sensors is anticipated to take 2–3 d, and microscopy imaging or
flow cytometry analysis can be performed in 5–60 min.
“…As important cofactors closely related to energy metabolism, nicotinamide nucleotides are also separated by LC [91][92][93][94][99][100] and CE [98,101] When the electric field is applied, the cations in the mobile layer are moved towards the negative electrode (cathode) and drag the water molecules around them together. Thus, we can observe the electroosmotic flow towards the cathode [145].…”
Section: Chromatography With Mass Spectrometry Detection (Lc-ms) and mentioning
confidence: 99%
“…The extraction method we adapted breaks cells on the surface of the culture flask/dish without digesting them. The advantage of this manipulation is to minimize the pipetting process and lower the possibility of sample loss or introduction of interferences [98]. However, it also brings two issues in the extraction.…”
who started the separation project and Carrie Fisk for her input. I have learned a lot from everyone and their encouragement is irreplaceable. Thanks to Dr. Jim Jung-Ching Lin and Alissa van Winkle for helping us to establish the cell culture facility in Geng lab. Thanks to the Center for Biocatalysis and Bioprocessing for awarding me the graduate fellowship from 2006 to 2009. I wish to thank my aunt, Guoqing Lan, who lost her life to colon cancer in 2007, for encouraging me to go further with cancer diagnosis research. May she rest in peace. Finally, I would like to thank my grandfather, Menglin Li, my father, Xikong Li, and my mother, Yingping Wu, who are living in China, half an earth away from their single child, me. I owe them a lot for not being around for them. They may never be able to read this dissertation but they must see that their love to me has crystallized in this work. I am deeply grateful to my beloved husband, Dr. Jianquan Yang. His unconditional support and endless love have been carrying me through all the happiness and difficulties in life. We have come this far and will go farther only because we are together. v TABLE OF CONTENTS LIST OF TABLES .
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