Jae Kyoo Lee

Microdroplet fusion mass spectrometry for fast reaction kinetics

Significance Time-resolved mass spectrometry is a powerful approach for identifying reaction intermediates and measuring reaction kinetics, yet one critical limitation is temporal resolution. Here, we describe microdroplet fusion mass spectrometry on timescales as short as microseconds. In our approach, two high-speed streams of liquid microdroplets collide to make fused droplets of 13 ± 6 μm in diameter, where the reactants are mixed in a negligible time. After a short flying time of 50 μs or less during which the reaction proceeds, the fused droplets enter a mass spectrometer for chemical analysis of intermediates and reaction products. This enables observation of early events of various fast chemical reactions in the liquid phase. We investigated the fusion of high-speed liquid droplets as a way to record the kinetics of liquid-phase chemical reactions on the order of microseconds. Two streams of micrometer-size droplets collide with one another. The droplets that fused (13 μm in diameter) at the intersection of the two streams entered the heated capillary inlet of a mass spectrometer. The mass spectrum was recorded as a function of the distance x between the mass spectrometer inlet and the droplet fusion center. Fused droplet trajectories were imaged with a high-speed camera, revealing that the droplet fusion occurred approximately within a 500-μm radius from the droplet fusion center and both the size and the speed of the fused droplets remained relatively constant as they traveled from the droplet fusion center to the mass spectrometer inlet. Evidence is presented that the reaction effectively stops upon entering the heated inlet of the mass spectrometer. Thus, the reaction time was proportional to x and could be measured and manipulated by controlling the distance x. Kinetic studies were carried out in fused water droplets for acid-induced unfolding of cytochrome c and hydrogen–deuterium exchange in bradykinin. The kinetics of the former revealed the slowing of the unfolding rates at the early stage of the reaction within 50 μs. The hydrogen–deuterium exchange revealed the existence of two distinct populations with fast and slow exchange rates. These studies demonstrated the power of this technique to detect reaction intermediates in fused liquid droplets with microsecond temporal resolution.

Acceleration of reaction in charged microdroplets

Using high-resolution mass spectrometry, we have studied the synthesis of isoquinoline in a charged electrospray droplet and the complexation between cytochrome c and maltose in a fused droplet to investigate the feasibility of droplets to drive reactions (both covalent and noncovalent interactions) at a faster rate than that observed in conventional bulk solution. In both the cases we found marked acceleration of reaction, by a factor of a million or more in the former and a factor of a thousand or more in the latter. We believe that carrying out reactions in microdroplets (about 1–15 μm in diameter corresponding to 0·5 pl – 2 nl) is a general method for increasing reaction rates. The mechanism is not presently established but droplet evaporation and droplet confinement of reagents appear to be two important factors among others. In the case of fused water droplets, evaporation has been shown to be almost negligible during the flight time from where droplet fusion occurs and the droplets enter the heated capillary inlet of the mass spectrometer. This suggests that (1) evaporation is not responsible for the acceleration process in aqueous droplet fusion and (2) the droplet–air interface may play a significant role in accelerating the reaction. We argue that this ‘microdroplet chemistry’ could be a remarkable alternative to accelerate slow and difficult reactions, and in conjunction with mass spectrometry, it may provide a new arena to study chemical and biochemical reactions in a confined environment.

Real-Time Dynamics of Ca2+, Caspase-3/7, and Morphological Changes in Retinal Ganglion Cell Apoptosis under Elevated Pressure

Quantitative information on the dynamics of multiple molecular processes in individual live cells under controlled stress is central to the understanding of the cell behavior of interest and the establishment of reliable models. Here, the dynamics of the apoptosis regulator intracellular Ca2+, apoptosis effector caspase-3/7, and morphological changes, as well as temporal correlation between them at the single cell level, are examined in retinal gangling cell line (differentiated RGC-5 cells) undergoing apoptosis at elevated hydrostatic pressure using a custom-designed imaging platform that allows long-term real-time simultaneous imaging of morphological and molecular-level physiological changes in large numbers of live cells (beyond the field-of-view of typical microscopy) under controlled hydrostatic pressure. This examination revealed intracellular Ca2+ elevation with transient single or multiple peaks of less than 0.5 hour duration appearing at the early stages (typically less than 5 hours after the onset of 100 mmHg pressure) followed by gradual caspase-3/7 activation at late stages (typically later than 5 hours). The data reveal a strong temporal correlation between the Ca2+ peak occurrence and morphological changes of neurite retraction and cell body shrinkage. This suggests that Ca2+ elevation, through its impact on ion channel activity and water efflux, is likely responsible for the onset of apoptotic morphological changes. Moreover, the data show a significant cell-to-cell variation in the onset of caspase-3/7 activation, an inevitable consequence of the stochastic nature of the underlying biochemical reactions not captured by conventional assays based on population-averaged cellular responses. This real-time imaging study provides, for the first time, statistically significant data on simultaneous multiple molecular level changes to enable refinements and testing of models of the dynamics of mitochondria-mediated apoptosis. Further, the platform developed and the approach has direct significance to the study of a variety of signaling pathway phenomena.

High-Resolution Live-Cell Imaging and Analysis by Laser Desorption/Ionization Droplet Delivery Mass Spectrometry.

We have developed a new ambient-ionization mass spectrometric technique named laser desorption/ionization droplet delivery mass spectrometry (LDIDD-MS). LDIDD-MS permits high-resolution, high-sensitivity imaging of tissue samples as well as measurements of both single-cell apoptosis and live-cell exocytosis. A pulsed (15 Hz) UV laser beam (266 nm) is focused on a surface covered with target analytes to trigger their desorption and ionization. A spray of liquid droplets is simultaneously directed onto the laser-focused surface region to capture the ionized analytes and deliver them to a mass spectrometer. The approach of rapid and effective capturing of molecules after laser desorption/ionization allows the limit of detection for the amino acid lysine to be as low as 2 amol under ambient ionization conditions. Two-dimensional maps of the desorbed/ionized species are recorded by moving the sample on an XY translational stage. The spatial resolution for imaging with LDIDD-MS was determined to be 2.4 μm for an ink-printed pattern and 3 μm for mouse brain tissue. We applied LDIDD-MS to single-cell analysis of apoptotic HEK cells. Differences were observed in the profiles of fatty acids and lipids between healthy HEK cells and those undergoing apoptosis. We observed upregulation of phosphatidylcholine (PC) with a relatively shorter carbon chain length and downregulation of PC with a relatively longer carbon chain length. We also applied LDIDD-MS for a real-time direct measurements of live-cell exocytosis. The catecholamine dopamine and trace amines (phenethylamine and tyramine) were detected from live PC12 cells without damaging them.

Abiotic production of sugar phosphates and uridine ribonucleoside in aqueous microdroplets

Significance Phosphorylation is essential for life. Phosphorylated molecules play diverse functions in cells, including metabolic (e.g., sugar phosphates), structural (e.g., phospholipids), and instructional (e.g., RNA and DNA). In nature, the phosphorylation of sugars via condensation is thermodynamically and kinetically unfavorable in bulk solution. Thus, a key question arising within prebiotic chemistry concerning the origin of life is, “How was phosphorus incorporated into the biological world?” Here, we show that sugar phosphates and a ribonucleoside form spontaneously in microdroplets, without enzymes or an external energy source. Sugar phosphorylation in microdroplets has a lower entropic cost than in bulk solution. Therefore, thermodynamic obstacles of prebiotic condensation reactions can be circumvented in microdroplets. Phosphorylation is an essential chemical reaction for life. This reaction generates fundamental cell components, including building blocks for RNA and DNA, phospholipids for cell walls, and adenosine triphosphate (ATP) for energy storage. However, phosphorylation reactions are thermodynamically unfavorable in solution. Consequently, a long-standing question in prebiotic chemistry is how abiotic phosphorylation occurs in biological compounds. We find that the phosphorylation of various sugars to form sugar-1-phosphates can proceed spontaneously in aqueous microdroplets containing a simple mixture of sugars and phosphoric acid. The yield for d-ribose-1-phosphate reached over 6% at room temperature, giving a ΔG value of −1.1 kcal/mol, much lower than the +5.4 kcal/mol for the reaction in bulk solution. The temperature dependence of the product yield for the phosphorylation in microdroplets revealed a negative enthalpy change (ΔH = −0.9 kcal/mol) and a negligible change of entropy (ΔS = 0.0007 kcal/mol·K). Thus, the spontaneous phosphorylation reaction in microdroplets occurred by overcoming the entropic hurdle of the reaction encountered in bulk solution. Moreover, uridine, a pyrimidine ribonucleoside, is generated in aqueous microdroplets containing d-ribose, phosphoric acid, and uracil, which suggests the possibility that microdroplets could serve as a prebiotic synthetic pathway for ribonucleosides.

Microdroplet fusion mass spectrometry: accelerated kinetics of acid-induced chlorophyll demetallation

Kinetics of acid-induced chlorophyll demetallation was recorded in microdroplets by fusing a stream of microdroplets containing 40 μM chlorophyll a or b dissolved in methanol with a stream of aqueous microdroplets containing 35 mM hydrochloric acid (pH = 1·46). The kinetics of the demetallation of chlorophyll in the fused microdroplets (14 ± 6 μm diameter; 84 ± 18 m s−1 velocity) was recorded by controlling the traveling distance of the fused microdroplets between the fusion region and the inlet of a mass spectrometer. The rate of acid-induced chlorophyll demetallation was about 960 ± 120 times faster in the charged microdroplets compared with that reported in bulk solution. If no voltage was applied to the sprayed microdroplets, then the acceleration factor was about 580 ± 90, suggesting that the applied voltage is not a major factor determining the acceleration. Chlorophyll a was more rapidly demetallated than chlorophyll b by a factor of ~26 in bulk solution and ~5 in charged microdroplets. The demetallation kinetics was second order in the H+ concentration, but the acceleration factor of microdroplets compared with bulk solution appeared to be unchanged in going from pH = 1·3 to 7·0. The water:methanol ratio of the fused microdroplets was varied from 7:3 to 3:7 causing an increase in the reaction rate of chlorophyll a demetallation by 20%. This observation demonstrates that the solvent composition, which has different evaporation rates, does not significantly affect the acceleration. We believe that a major portion of the acceleration can be attributed to confinement effects involving surface reactions rather than either to evaporation of solvents or to the introduction of charges to the microdroplets.

The topographical guidance of neurons cultured on holographic photo-responsive polymer

Neuronal cells to respond to submicron-scale groove structure. On the grooved structure of particular dimension, it has been reported that neuronal cells grew perpendicular to the groove direction. We used holographic photo-responsive polymer to form a submicron-scale surface relief grating structure. A sinusoidal groove pattern is built up by holographic interference of 488 nm Ar ion laser beams. The primary hippocampal neurons cultured on the surface of the polymer film grew extending their neurites in a perpendicular orientation to the groove direction. This suggests that laser holography can be used to control the neurites orientation and growth. The holographic grating and photo-responsive polymer will raise the possibility of controlling neural network formation between living cells by light.

Neural Prosthesis in the Wake of Nanotechnology : Controlled Growth of Neurons Using Surface Nanostructures

Neural prosthesis has been successfully applied to patients with motional or sensory disabilities for clinical purpose. To enhance the performance of the neural prosthetic device, the electrodes for the biosignal recording or electrical stimulation should be located in closer proximity to target neurons than they are now. Instead of revising the prior implanting surgery to improve the electrical contact of neurons, we propose a technique that can bring the neurons closer to the electrode sites. A new method is investigated that can control the direction of neural cell growth using surface nanostructures. We successfully guide the neurons to the position of the microelectrodes by providing a surface topographical cue presented by the surface nanostructure on a photoresponsive polymer material. Because the surface structure formed by laser holography is reversible and repeatable, the geometrical positioning of the neurons to microelectrodes can be adjusted by applying laser treatment during the surgery for the purpose of improving the performance of neural prosthetic device.

Photo-Triggering of the Membrane Gates in Photo-Responsive Polymer for Drug Release

The use of light stimulus for triggering drug is a promising method for accurate drug delivery. A new approach using azopolymer membrane and laser holography was investigated for developing light-triggering drug delivery system. Polymeric drug delivery system was prepared by covering azopolymer membrane on a drug agent. Holographic laser interference generated surface relief grating pattern on the azopolymer surface. The widths and depths of gates on the polymer membrane were easily modified by adjusting incident angle and irradiation time. Ar laser made the polymeric membrane permeable to the drug agent and release it in a solution. This result indicated that the azopolymer and laser holography would provide a strong foundation for the light-triggering drug delivery system

Spatial Patterning of Fibroblast Cells with Fabricating Holographic Patterning on the Photoresponsive Polymer

Microtopographic feature was obtained by laser holographic fabrication on the photoresponsive polymer. Surface feature was localized using photomask for developing two dimensional cellular pattering. Fibroblast cells were cultured and proliferated only on the patterned substrate. Obtained cellular pattering suggests that the laser fabricating with photoresponsive polymer would be applied to regenerating new tissue and developing biomedical device of living cells