Optimized sample buffer for dispersed, high-resolution capillary zone electrophoretic separation of Escherichia coli B.

Capillary zone electrophoresis (CZE) is a powerful tool for high resolution chemical separations. Applying CZE to microbial samples may facilitate a deeper understanding of bacterial physiology and behavior. However, the study of complex microbial samples has been limited by the uncontrolled hetero-aggregation of bacterial cells under an applied electric field. We tested a wide range of sample buffers and buffer additives for the optimization of bacterial CZE separations using a 20 mM Tris-HCl background electrolyte. By modifying the sample buffer, but not the background electrolyte, we retain constant separation conditions, which aids in the comparison of the sample buffer additives. We report optimized methods for automated CZE separation and simultaneous fractionation of Escherichia coli B, which is one of the two most widely used wild-type strains. A modified sample buffer containing neutral salts and the addition of glycerol produced a 20-fold increase in loading capacity and a reduction in peak width/broadening of 86% in comparison to previously reported work. In addition, the glycerol-modified sample buffer appears to reduce the persistent aggregation and adhesion to the capillary walls during electrophoretic separations of complex environmental microbiota.

Comparison of RT-dPCR and RT-qPCR and the effects of freeze-thaw cycle and glycine release buffer for wastewater SARS-CoV-2 analysis.

Public health efforts to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic rely on accurate information on the spread of the disease in the community. Acute and surveillance testing has been primarily used to characterize the extent of the disease. However, obtaining a representative sample of the human population is challenging because of limited testing capacity and incomplete testing compliance. Wastewater-based epidemiology is an agnostic alternative to surveillance testing that provides an average sample from the population served by the treatment facility. We compare the performance of reverse transcription quantitative PCR (RT-qPCR) and reverse transcription digital droplet PCR (RT-dPCR) for analysis of SARS-CoV-2 RNA in a regional wastewater treatment facility in northern Indiana, USA from the earliest stages of the pandemic. 1-L grab samples of wastewater were clarified and concentrated. Nucleic acids were extracted from aliquots and analyzed in parallel using the two methods. Synthetic viral nucleic acids were used for method development and generation of add-in standard-curves. Both methods were highly sensitive in detecting SARS-CoV-2 in wastewater, with detection limits as low as 1 copy per 500 mL wastewater. RT-qPCR and RT-dPCR provided essentially identical coefficients of variation (s/[Formula: see text] = 0.15) for triplicate measurements made on wastewater samples taken on 16 days. We also observed a sevenfold decrease in viral load from a grab sample that was frozen at - 80 °C for 92 days compared to results obtained without freezing. Freezing samples before analysis should be discouraged. Finally, we found that treatment with a glycine release buffer resulted in a fourfold inhibition in RT-qPCR signal; treatment with a glycine release buffer also should be discouraged. Despite their prevalence and convenience in wastewater analysis, glycine release and freezing samples severely and additively (~ tenfold) degraded recovery and detection of SARS-CoV-2.

3-D printed injection system for capillary electrophoresis.

Commercial systems for capillary electrophoresis are designed for the unattended analysis of several samples, and are usually large, complex, and expensive. We report a compact system for manual injection of a single sample in capillary electrophoresis, which is ideal for method development and for student training. The injector consists of two parts that are manufactured by three-dimensional printing (STL and STEP files are included as ESI). One part is immobile and holds an electrode for powering electrophoresis and a gas line for pressurized injection and pumping fluids through the capillary. The second part is removable and is used to hold washing solutions, separation electrolyte, or sample. Conventional machining is used to tap holes to hold the electrode, separation capillary, gas line, and safety interlock. The system is used for either pressure or electrokinetic sample injection, and can be used to pump fluids through the capillary for changing background electrolytes and reconditioning the capillary between runs. We coupled the injection system to our high-dynamic range laser-induced fluorescence detector and evaluated the system by performing capillary zone electrophoresis on solutions of fluorescein. Electrokinetic injection produced a linear response across five orders of magnitude dynamic range (slope of the log-log calibration curve was 1.02), concentration detection limits of 5 pM, and mass detection limits of 1 zmol. Pressure injection produced a linear response across at least four orders of magnitude (slope of the log-log calibration curve was 0.92), concentration detection limits of 2 pM, and mass detection limits of 10 zmol.

Capillary zone electrophoresis separation and collection of spermatozoa for the forensic analysis of sexual assault evidence.

The processing of sexual assault kits (SAKs) relies on the genetic analysis of material extracted from swabs collected from the assault victim. A vital step in producing an identifiable DNA profile of the perpetrator is the effective separation of perpetrator (sperm) and victim (epithelial) DNA that have been isolated from the collected evidence. We report the use of capillary zone electrophoresis for the separation of intact sperm from whole and lysed epithelial cells in SAKs. The separated components are deposited into wells of a microtiter plate using a computer-controlled fraction collector, and quantitative PCR is used to verify the collection of sperm cells by targeted amplification of male DNA. We present results from simulated sexual assault samples that have been aged for up to 18 months, as well as vaginal swabs from authentic forensic kits. Components extracted from the vaginal swabs from the SAK comigrated with an aged semen sample at 6.25 ± 0.25 min. Epithelial cells migrated from 10-12 min, producing baseline resolution of the components. Sperm cells were collected in a microtiter plate for downstream analysis.

Capillary Zone Electrophoresis with Fraction Collection for Separation, Culturing, and Identification of Bacteria from an Environmental Microbiome.

Capillary zone electrophoresis (CZE) can produce high-resolution separations of biological samples, including microbial mixtures. The study of complex populations of microorganisms using CZE is limited because most detectors have limited sensitivity, are destructive, and provide limited information for microbial identification. To address these issues, we developed an integrated capillary zone electrophoresis apparatus to fractionate bacteria from complex mixtures. We deposited fractions onto nutrient agar in a Petri dish for microbial culturing, and we subjected the observed colonies to Sanger sequencing of a phylogenetic marker, the 16S rRNA gene, for microbial identification. We separated and cultured both a single bacteria species, the model Gram-negative organism Escherichia coli, and a complex environmental isolate of primary sewage effluent. Sequence analysis of the 16S rRNA genes from this mixture identified 15 ± 5 distinct bacterial species per run. This approach requires minimal manipulation of microbial populations and combines electrophoretic fractionation of bacterial cells with automated collection for accurate identification of species. This approach should be applicable to microorganisms in general and may enable discrimination of physiologically different cells in complex assemblages, such as in microbiome samples.

Chemical cytometry of thiols using capillary zone electrophoresis-laser induced fluorescence and TMPAB-o-M, an improved fluorogenic reagent.

Low molecular weight thiol compounds play crucial roles in many physiological processes. Most methods for determination of thiol compounds are population-averaged; few methods for quantification of thiol compounds in single cells have been reported. We report an ultrasensitive method for determination of thiol compounds in single cells by use of 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), a fluorogenic probe with useful spectral properties, coupled with capillary zone electrophoresis and laser induced fluorescence detection using a post-column sheath flow cuvette. TMPAB-o-M provides low background, high sensitivity, and excellent reactivity. After optimization of the separation method, we achieved baseline separation of labeled glutathione (GSH), cysteine (Cys), homocysteine, and γ-glutamylcysteine within 11 min, and produced concentration limits of detection from 10 to 20 pM and mass LODs of 65 to 100 zmol. The method was applied for analysis of thiol containing compounds in both cell homogenates and in single HCT-29 and MCF-10A cells. GSH was the main thiol, and Cys was also detected in both cell types. Cells were treated with N-ethylmaleimide, which significantly attenuated thiol levels.

Capillary electrophoresis coupled with automated fraction collection.

A fraction collector based on a drop-on-demand ink-jet printer was developed to interface capillary zone electrophoresis with a 96 well microtiter plate. We first evaluated the performance of the collector by using capillary zone electrophoresis to analyze a 1mM solution of tetramethylrhodamine; a fluorescent microtiter plate reader was then used to detect the analyte and characterize fraction carryover between wells. Relative standard deviation in peak height was 20% and the relative standard deviation in migration time was 1%. The mean and standard deviation of the tetramethylrhodamine peak width was 5 ± 1 s and likely limited by the 4-s period between droplet deposition. We next injected a complex mixture of DNA fragments and used real-time PCR to quantify the product in a CE-SELEX experiment. The reconstructed electrophoretic peak was 27 s in duration. Finally, we repeated the experiment in the presence of a 30-µM thrombin solution under CE-SELEX conditions; fractions were collected and next-generation sequencing was used to characterize the DNA binders. Over 25,000 sequences were identified with close matches to known thrombin binding aptamers.