Browsing by Author "Urban, P. L."

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Fusion of microlitre water-in-oil droplets for simple, fast and green chemical assays
(2015-01-01)
Chiu, S. -H.
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Urban, P. L.
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應用化學系
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應用化學系分子科學碩博班
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Department of Applied Chemistry
A simple format for microscale chemical assays is proposed. It does not require the use of test tubes, microchips or microtiter plates. Microlitre-range (ca. 0.7-5.0 mu L) aqueous droplets are generated by a commercial micropipette in a non-polar matrix inside a Petri dish. When two droplets are pipetted nearby, they spontaneously coalesce within seconds, priming a chemical reaction. Detection of the reaction product is accomplished by colorimetry, spectrophotometry, or fluorimetry using simple light-emitting diode (LED) arrays as the sources of monochromatic light, while chemiluminescence detection of the analytes present in single droplets is conducted in the dark. A smartphone camera is used as the detector. The limits of detection obtained for the developed in-droplet assays are estimated to be: 1.4 nmol (potassium permanganate by colorimetry), 1.4 pmol (fluorescein by fluorimetry), and 580 fmol (sodium hypochlorite by chemiluminescence detection). The format has successfully been used to monitor the progress of chemical and biochemical reactions over time with sub-second resolution. A semi-quantitative analysis of ascorbic acid using Tillman\'s reagent is presented. A few tens of individual droplets can be scanned in parallel. Rapid switching of the LED light sources with different wavelengths enables a spectral analysis of multiple droplets. Very little solid waste is produced. The assay matrix is readily recycled, thus the volume of liquid waste produced each time is also very small (typically, 1-10 mu L per analysis). Various water-immiscible translucent liquids can be used as the reaction matrix: including silicone oil, 1-octanol as well as soybean cooking oil.
Liquid-phase and gas-phase investigation of biomolecules in a single experiment
(2013)
Huang, H. -Y.
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Lo, T. -J.
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Chen, Y. -C.
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Hu, J. -B.
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Urban, P. L.
Interrogating three-dimensional structures of biomolecules is instrumental to the fields of biochemistry and biophysics. Mass spectrometry is a technique that can provide important data on the molecular weight of protein molecules in the gas phase. However, the relationship between the structure of biomolecules in the native water-rich environment and the artificial gas-phase environment cannot always be established with high certainty. Here we communicate initial results obtained using a simple analytical approach which allows one to perform measurements of structural parameters of biomolecules in liquid phase and gas phase within a single experiment. Single-component samples are injected as plugs to a microscale capillary filled with aqueous medium and driven hydrodynamically. As they traverse the capillary, they undergo zone broadening. The extent of this broadening is measured at two points along the capillary by UV absorption detection, and used to compute hydrodynamic radii according to the Taylor dispersion analysis theory. Further downstream, the dispersed zones leave the capillary through the sharpened outlet aligned to the orifice of a mass spectrometer. This multiple detection strategy allows one to confront protein conformation in the liquid phase (deduced from the hydrodynamic size) and the gas phase (deduced from mass spectral patterns).
Mass spectrometry-guided refinement of chemical energy buffers
(2016-06-01)
Chen, T. -R.
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Urban, P. L.
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應用化學系
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應用化學系分子科學碩博班
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Department of Applied Chemistry
Biocatalytic reactions often require supplying chemical energy and phosphate groups in the form of adenosine triphosphate (ATP). Auxiliary enzymes can be used to convert a reaction by-product-adenosine diphosphate (ADP)-back to ATP. By employing real-time mass spectrometry (RTMS), one can gain an insight into inter-conversions of reactants in multi-enzyme reaction systems and optimize the reaction conditions. In this study, temporal traces of ions corresponding to adenosine monophosphate (AMP), ADP and ATP provided vital information that could be used to adjust activities of the \'\'buffering enzymes\'. Using the RTMS results as a feedback, we also characterized a bienzymatic energy buffer that enables the recovery of ATP in the cases where it is directly hydrolysed to AMP in the main enzymatic reaction. The significance of careful selection of enzyme activities-guided by RTMS-is exemplified in the synthesis of glucose-6-phosphate by hexokinase in the presence of a buffering enzyme, pyruvate kinase. Relative activities of the two enzymes, present in the reaction mixture, influence biosynthetic reaction yields. This observation supports the conclusion that optimization of chemical energy recycling procedures is critical for the biosynthetic reaction economy.