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Abstract

The toxicity detection using real testing on human or animal is no longer an appropriate technique. Development of new technical methods for detecting toxic materials is highly demanded. A variety of non-animal methods are available for toxicity detection consists of in-vitro analysis of living cells and tissue cultures, microarray technology and computerized modeling. These methods can provide more reliable, faster and cheaper results. Various cells such as bacterial, mammalian and plant cells have been for the application in pharmacology, toxicology and environmental measurements. In this paper, we present a novel microfluidic-based whole cell biosensor to enable detection of environmental toxic substances through dynamics of oscillatory growth behavior of a whole plant cell in a high-throughput platform. Pollen tube as the fastest tip growing plant cells was used to demonstrate the performance of this plant-cell biosensor due to its extremely sensitivity to the external signals and environmental chemicals. The microfluidic platform was designed from the previously enhanced microfluidic device devised for chemical treatment of tip growing cells. The pollen grains were injected into the chip, conducted through the main chamber towards the growth microchannels and trapped at the entrance of microchannels. The individual pollen tubes grow along the microchannels and were subjected to chemical treatment. The growth of pollen tubes was observed under microscope before and after introducing the toxic medium (Aluminium). While for higher Al concentrations, the change of growth rate and the burst of pollen tube was useful to detect the Al concentration, for lower concentration of Al the dynamic behavior of pollen tube was exploited. The oscillatory dynamic growth includes both fundamental and higher modes of oscillation frequencies associated with growth. The effect of stress from toxic substance on variation of the fundamental and higher modes of dynamic oscillation of growth rate was used to detect the concentration of Al in the media. The experimental results show that for 20μM Al concentration and below, the drop of growth rate was not simply detected, though the variation or disruption of higher mode oscillation frequency in 20μM was simply detectable. The advanced waveform techniques of Fast Fourier transform (FFT) and window filtering techniques were used to identify the primary and secondary peak oscillation frequencies. For a sample pollen tube tested under 20μM Al concentration, the results of FFT analysis. When the cell is subjected to toxic environment, the behavior of the pollen tube was altered at entire range of dynamic growth and the power associated with change of all frequencies was used as a criterion of dynamic response in order to quantify the cell behavior under different concentration of toxic Al. The total power of its corresponding Fourier Transform integrated across all of its frequency components was calculated before and after treatment with Al toxic, each, in time series of 100s. The degree of toxicity (τ) was then defined as the ratio of spectral power of normal growth of pollen tube and spectral power of tube after treatment with Al toxic.