| 1. |
- Ma, Su, et al.
(författare)
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Direct Electron-Transfer Anisotropy of a Site-Specifically Immobilized Cellobiose Dehydrogenase
- 2019
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; , s. 7607-7615
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Tidskriftsartikel (refereegranskat)abstract
- To study the direct electron transfer (DET) of the multicofactor enzyme cellobiose dehydrogenase (CDH) in regard to its orientation on an electrode surface, a recently published, maleimide-based immobilization method was used in combination with site-directed mutagenesis to establish different orientations on an electrode surface. CDH from Myriococcum thermophilum was chosen for this study because its protein structure is resolved and the factors influencing the movement of its mobile cytochrome domain (CYT) are established. Seven CDH variants with a surface-exposed cysteine residue in different spatial positions were generated for site-specific maleimide coupling. Surface plasmon resonance and cyclic voltammetry showed that all CDH variants, but not the wild-type CDH, bound covalently to gold electrodes or glassy carbon electrodes and were catalytically active. For DET, the CYT domain needs to move from the closed-state conformation, where it obtains an electron from the catalytic flavin adenine dinucleotide (FAD) cofactor to the open state where it can donate an electron to the electrode. We therefore hypothesized that the mobility of the CYT domain and its distance to the electrode is central for DET. We found that the uniform spatial orientations of CDH influenced DET as follows: an orientation of the two-domain enzyme on the side, with CYT in proximity to the electrode, resulted in high DET currents. Orientations with a bigger distance between CYT and the electrode, or orientations where CYT could not swing back to the dehydrogenase domain to form the closed enzyme conformation, reduced DET. In the latter case, calcium ions that stabilize the closed conformation of CDH fully recovered DET. The study demonstrates that a mobile CYT domain can compensate unfavorable orientations of the catalytic domain to a great extent and allows CDH as a multicofactor enzyme to transfer electrons even in awkward orientations. The mobile CYT domain reduces the anisotropy of DET, which is also essential for CDH's physiological function as an extracellular, electron-transferring enzyme.
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| 2. |
- Moreira, Beatriz, et al.
(författare)
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Surface Plasmon Resonance for Measuring Exocytosis from Populations of PC12 Cells: Mechanisms of Signal Formation and Assessment of Analytical Capabilities
- 2017
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:5, s. 3069-3077
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Tidskriftsartikel (refereegranskat)abstract
- Because of cell to cell variation, it is difficult to obtain statistically significant data on the frequency of exocytosis events (R-exocytosis, t(-1) m(-2)) with traditional single cell electrophysiological or fluorescence microscopy based methods. Here we take the first steps toward a rapid cost-effective surface plasmon resonance (SPR) based method for measuring the R-exocytosis for populations of PC12 cells. The conditions for culturing confluent monolayers on the sensor slides were optimized, and neurotransmitter exocytosis was evoked by injecting solutions with elevated Exocytosis caused a shift of the resonance angle (Delta theta(r)) that was linearly proportional to R-exocytosis The Delta theta(r) was mainly due to elevated concentration of secretory vesicles close to the cell membrane. The increased vesicle concentration thus acted as a proxy for the Rexocytosis that could not be measured directly. The Delta theta(r) was calibrated for it R-exocytosis using single cell amperometry on parallel cell cultures. The cell populations were large enough for variation in responses between sensor slides to only reflect actual differences in biological condition. The applicability for drug screening is demonstrated by studying the effects of EGTA, reserpine, and prolonged stimulation by K+
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| 3. |
- Tuoriniemi, Jani, 1982, et al.
(författare)
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A new peak recognition algorithm for detection of ultra-small nano-particles by single particle ICP-MS using rapid time resolved data acquisition on a sector-field mass spectrometer
- 2015
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Ingår i: Journal of Analytical Atomic Spectrometry. - : Royal Society of Chemistry (RSC). - 0267-9477 .- 1364-5544. ; 30:8, s. 1723-1729
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Tidskriftsartikel (refereegranskat)abstract
- The applicability of single particle inductively coupled plasma mass spectrometry (spICPMS) is currently limited to particles larger than similar to 10 nm in diameter. In this work, the size detection limit (DLs) was improved by resolving the ion bursts originating from silver or gold nanoparticles (AgNPs or AuNPs) using real time data acquisition with 0.1 ms time resolution. Such acquisition is here called the Fast Acquisition Speed Technique (FAST). The analytical capabilities of the FAST were evaluated on a sector field instrument with high ion transmission efficiency (ITE). An algorithm for distinguishing particle events from dissolved and/or background signals was developed, and it was possible to detect 6.4 nm AuNPs that delivered only 2 ions to the detector. The influence of dwell time was investigated and it was concluded that the minimum DLs is achieved for dwell times close to the duration of particle events similar to 0.2 ms. Attempts to further improve the DLs should therefore be focused on increasing the ITE of the mass spectrometer.
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| 4. |
- Tuoriniemi, Jani, 1982, et al.
(författare)
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Determining Number Concentrations and Diameters of Polystyrene Particles by Measuring the Effective Refractive Index of Colloids Using Surface Plasmon Resonance
- 2016
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 32:41, s. 10632-10640
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Tidskriftsartikel (refereegranskat)abstract
- The capabilities of surface plasmon resonance (SPR) for characterization of colloidal particles were evaluated for 100, 300, and 460 nm nominal diameter polystyrene (PS) latexes. First the accuracy of measuring the effective refractive index (n(eff)) of turbid colloids using SPR was quantified. It was concluded that for submicrometer sized PS particles the accuracy is limited by the reproducibility between replicate injections of samples. An SPR method was developed for obtaining the particle mean diameter (d(part)) and the particle number concentration (c(p)) by fitting the measured n(eff) of polystyrene (PS) colloids diluted in series with theoretical values calculated using the coherent scattering theory (CST). The d(part) and c(p) determined using SPR agreed with reference values obtained from size distributions measured by scanning electron microscopy (SEM), and the mass concentrations stated by the manufacturer. The 100 nm particles adsorbed on the sensing surface, which hampered the analysis. Once the adsorption problem has been overcome, the developed SPR method has potential to become a versatile tool for characterization of colloidal particles. In particular, SPR could form the basis of rapid and accurate methods for measuring the c(p) of submicrometer particles in dispersion.
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| 5. |
- Tuoriniemi, Jani, 1982, et al.
(författare)
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Size dependence of silver nanoparticle removal in a wastewater treatment plant mesocosm measured by FAST single particle ICP-MS
- 2017
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Ingår i: Environmental Science-Nano. - : Royal Society of Chemistry (RSC). - 2051-8153 .- 2051-8161. ; 4:5, s. 1189-1197
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Tidskriftsartikel (refereegranskat)abstract
- The quantities of engineered nanoparticles (NP) released to the environment are often influenced by their fate in waste water treatment plants (WWTP). Here, 40 nm silver NP (AgNP) were spiked into a mesocosm simulating the process used at a major municipal WWTP. The evolution of the mass distributions and number concentrations were followed by fast acquisition speed technique single particle inductively coupled mass spectrometry (FAST spICP-MS) using a high-resolution ICP-MS. It was thus possible to detect smaller Ag containing NP than hitherto possible in similar studies. These small particles (ca. 5-10 nm in corresponding metallic Ag equivalent spherical diameter) were possibly dissolved Ag+ precipitated as Ag2S particles. They were detected immediately upon spiking and were stable with respect to aggregation and thus much less removed by the WWTP process compared to the 40 nm AgNP. The results also suggested that any transformation of the latter AgNP occurred without dissolution. Most of these larger AgNP were probably removed by aggregation with large floc particles and subsequent sedimentation with the suspended particulate matter in the simulated WWTP process. The results have implications for differentiating the fate of nanoparticles as a function of size and demonstrate how spICP-MS can reveal such size-dependent fate dynamics.
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