A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses

Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R 2 ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R 2 = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.

Comparison between monopolar and bipolar microsecond range pulsed electric fields in enhancement of apple juice extraction

The effect of monopolar and bipolar shaped pulses in additional yield of apple juice extraction is evaluated. The applied electric field strength, pulsewidth, and number of pulses are assessed for both pulse types, and divergences are analyzed. Variation of electric field strength is ranged from 100 to 1300 V/cm, pulsewidth from 20 to 300 mu s, and the number of pulses from 10 to 200, at a frequency of 200 Hz. Two pulse trains separated by 1 s are applied to apple cubes. Results are plotted against reference untreated samples for all assays. Specific energy consumption is calculated for each experiment as well as qualitative indicators for apple juice of total soluble dry matter and absorbance at 390-nm wavelength. Bipolar pulses demonstrated higher efficiency, and specific energetic consumption has a threshold where higher inputs of energy do not result in higher juice extraction when electric field variation is applied. Total soluble dry matter and absorbance results do not illustrate significant differences between application of monopolar and bipolar pulses, but all values are inside the limits proposed for apple juice intended for human consumption.