The limits of detection (LOD) for capillary electrophoresis (CE) are constrained by the dimensions of the capillary. For example, the small volume of the capillary limits the total volume of sample that can be injected into the capillary. In addition, the reduced pathlength hinders common optical detection methods such as UV detection. Many different techniques have been developed to improve the LOD for CE. In general these techniques are designed to compress analyte bands within the capillary, thereby increasing the volume of sample that can be injected without loss of CE efficiency. This on-line sample preconcentration, generally referred to as stacking, is based on either the manipulation of differences in the electrophoretic mobility of analytes at the boundary of two buffers with differing resistivities or the partitioning of analytes into a stationary or pseudostationary phase. This article will discuss a number of different techniques, including field-amplified sample stacking, large-volume sample stacking, pH-mediated sample stacking, on-column isotachophoresis, chromatographic preconcentration, sample stacking for micellar electrokinetic chromatography, and sweeping.

The development of a cellulose acetate decoupler for on-column electrochemical detection in microchip capillary electrophoresis is presented. The capillary based laser-etched decoupler is translated to the planar format to isolate the detector circuit from the separation circuit. The decoupler is constructed by aligning a series of 20 30-microm holes through the coverplate of the microchip with the separation channel and casting a thin film of cellulose acetate within the holes. The decoupler shows excellent isolation of the detection circuit for separation currents up to 60 microA, with noise levels at or below 1 pA at a carbon fiber electrode. Detection limits of 25 nM were achieved for dopamine. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.

Severe malaria due to Plasmodium falciparum remains a significant global health threat. DXR, the second enzyme in the MEP pathway, plays an important role to synthesize building blocks for isoprenoids. This enzyme is a promising drug target for malaria due to its essentiality as well as its absence in humans. In this study, we designed and synthesized a series of α,β-unsaturated analogues of fosmidomycin, a natural product that inhibits DXR in P. falciparum. All compounds were evaluated as inhibitors of P. falciparum. The most promising compound, 18a, displays on-target, potent inhibition against the growth of P. falciparum (IC = 13 nM) without significant inhibition of HepG2 cells (IC > 50 μM). 18a was also tested in a luciferase-based Plasmodium berghei mouse model of malaria and showed exceptional in vivo efficacy. Together, the data support MEPicide 18a as a novel, potent, and promising drug candidate for the treatment of malaria.

A new decoupler for on-column electrochemical detection in capillary electrophoresis is presented. The decoupler is constructed by etching a series of holes through the side of the separation capillary with a CO2 laser and then coating the holes with cellulose acetate. The decoupler shows isolation of the detection circuit for separation currents up to 30 microA. Detection limits below 1 nM were achieved for four model compounds, including anions, neutrals, and cations, using the laser-etched decoupler. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.

The ability to measure chemical gradients surrounding single cells provides novel insights into several areas of cell dynamicssparticularly metabolism. Detection of metabolic oxygen consumption can be achieved from a single mammalian cell using a modulated amperometric sensor in a self-referencing mode. To date, however, apart from visual cues, we do not have a reliable and cell-compatible method for determining and stabilizing the position of such probes. In this paper, we report on having successfully measured the increase in the uncompensated resistance of an electrochemical cell upon approach to single, living, biological cells, while simultaneously measuring the metabolic oxygen consumption. This was accomplished by applying an ac and a dc excitation signal to the electrode. The applied ac waveform was a 100-kHz sine wave with an amplitude of 10 mV rms, while the dc voltage applied was -600 mV. The two signals were shown not to interfere with one another. Furthermore, it is shown that the sample-probe distance can be measured for approach to single cells on the order of 10-15-µm diameter and 5-µm height, with 100-nm resolution.Several groups have previously demonstrated that electrochemical microsensors have the required properties for successful single-cell studies. In addition to good spatial resolution, these microsensors exhibit high temporal resolution enabling the investigation of relatively fast processes. For example, manually positioned carbon-fiber sensors have been employed to study the time course of the release of neurotransmitters. 1,2 With the same method, hydrogen peroxide flux and corresponding oxygen concentration changes, from a single human fibroblast in response to mechanical stimulation, have been monitored. 3 Oxygen consumption and photosynthetic production at substructures of cells have been demonstrated with isolated retinal photoreceptors 4 and the alga Spirogyra greveilina. 5 It has also been shown that the modulation of neuronal oxygen flux can be measured using electrochemical microsensors. 5 To measure oxygen flux in the latter experiments, 4,5 the electrode was operated in a modulation format, stepping over a known distance (∆x) usually 10-20 µm, between near and far positions with respect to the cell at ∼0.3 Hz. The procedure, termed self-referencing, has been previously described for both potentiometric and amperometric sensors. 6,7 This technique is particularly useful in measuring small extracellular gradients, such as oxygen consumption, in the presence of a large background concentration. The electrode spends ∼1.5 s at each position, allowing a large number of data points to be averaged, and then the difference in signal between the two positions is recorded. Because this is a relatively slow technique, error due to noise and drift is reduced and small differences in current of tens to hundreds of femtoamperes can be determined. The difference current obtained by an electrode with a step distance ∆x represents the slope of the extracellular diffusion gradient (∆C/∆x) cre...

A rapid and sensitive method to determine 8-oxoguanine (8oxoG) and 8-hydroxydeoxyguanosine (8OHdG), biomarkers for oxidative DNA damage, in cerebral cortex microdialysate samples using capillary electrophoresis with electrochemical detection was developed. Samples were concentrated on-column using pH-mediated stacking for anions. On-column anodic detection was performed with a carbon fiber working electrode and laser-etched decoupler. The method is linear over the expected extracellular concentration range for 8oxoG and 8-OHdG during induced ischemia-reperfusion, with RSD values ≤ 5 % and limits of detection of 0.5 nM for both analytes. Basal concentrations of 8oxoG in rat cerebral cortex microdialysate were determined to be 3.2 ± 0.6 nM. Actual 8oxoG concentration in the brain was estimated to be 5.5 ± 1.3 nM based on probe calibration by in vivo delivery. 8OHdG was not detected under basal conditions in the rat cerebral cortex extracellular fluid. These results were confirmed by LC with tandem mass spectrometry.

With the rising prevalence of multidrug-resistance, there is an urgent need to develop novel antibiotics. Many putative antibiotics demonstrate promising in vitro potency but fail in vivo due to poor drug-like qualities (e.g. serum half-life, oral absorption, solubility, toxicity). These drug-like properties can be modified through the addition of chemical protecting groups, creating “prodrugs” that are activated prior to target inhibition. Lipophilic prodrugging techniques, including the attachment of a pivaloyloxymethyl group, have garnered attention for their ability to increase cellular permeability by masking charged residues and the relative ease of the chemical prodrugging process. Unfortunately, pivaloyloxymethyl prodrugs are rapidly activated by human sera, rendering any membrane permeability qualities absent during clinical treatment. Identification of the bacterial prodrug activation pathway(s) will allow for the development of host-stable and microbe-targeted prodrug therapies. Here, we use two zoonotic staphylococcal species, S. schleiferi and S. pseudintermedius, to establish the mechanism of carboxy ester prodrug activation. Using a forward genetic screen, we identify a conserved locus in both species encoding the enzyme hydroxyacylglutathione hydrolase (GloB), whose loss-of-function confers resistance to carboxy ester prodrugs. We enzymatically characterize GloB and demonstrate that it is a functional glyoxalase II enzyme, which has the capacity to activate carboxy ester prodrugs. As GloB homologs are both widespread and diverse in sequence, our findings suggest that GloB may be a useful mechanism for developing species-or genus-level prodrug targeting strategies.

The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years with numerous lead compounds identified including several tRNA synthetase inhibitors. In this study, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (CpMetRS) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day five. Parasites shed by each recrudescent calf had different amino acid altering CpMetRS mutations, coding either an aspartate 243 to glutamate (D243E) or a threonine 246 to isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant expressing parasites respectively had 2093 EC50s of 613- or 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 by >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.