Simple flow based systems for downscaling of chemical analysis of free fatty acids, glutathione and cell neurons' chemicals

Abstract

In this work, three microfluidic systems have been developed for various chemical analysis, i.e., systems for determination of free fatty acid, glutathione and system for culturing and stimulating of neuron cells for the study of neuropeptide release. A simple flow injection titrimetric system was developed for determination of free fatty acids (FFA) in vegetable oil samples. The method is based on the reaction between free fatty acid and sodium hydroxide which changes the color of phenolphthalein from pink to colorless (pH 10.4 to 8.3). Seventy-five µL of standard/sample diluting in isopropanol solution was injected into the carrier stream and merged with 0.6 mM sodium hydroxide solution containing 1 mM phenolphthalein in the mixing chamber (1.5 mL volume). The decrease in color intensity of the indicator was monitored by a simple home-made colorimeter, employing light emitting diode (LED) as a light source and photodiode (PD) as a light sensor. A linear calibration graph was obtained in a range of 0.1 - 0.9 mM palmitic acid. Relative standard deviation was less than 2%. Thirty three different commercial vegetable oil samples were analyzed, and obtained results were compared with the American Society for Testing and Materials (ASTM) standard method. Good correlation was found (r = 0.9542) between the proposed FI-titrimetric method and the standard ASTM method. A simple and inexpensive method for fabrication of microfluidic platform based on a polydimethylsiloxane (PDMS) was developed. A printed circuit board (PCB) was used for making a master mold for replicating of PDMS microchannel. The master mold was fabricated by a simple photolithographic method, employing a photoresist dry film. The process did not use hazardous chemicals, clean room and expensive instrument. The PDMS microchannel was clamped with polymethylmethacrylate (PMMA) plates, where a light emitting diode (LED) as a light source and a light dependent resistor (LDR) as a light sensor were attached to form a simple optical sensor. The system was successfully employed as a micro flow injection analysis for determination of glutathione in dietary supplement samples. A linear calibration graph in the range of 5 - 60 mg L-1 glutathione was obtained with a detection limit of 0.01 mg L-1. The system provided sample throughput of 48 h-1, with the consumption of microliter amounts of reagent and sample. A poly(dimethylsiloxane) (PDMS) microfluidic device was fabricated for culturing neurons, chemically stimulating them, and collecting the released neuropeptides. The device consists of a straight channel with a main reservoir for cell loading and culture, and two microvalve-controlled stimulation channels that are functionalized with neat 2-[methoxy (polyethylenxy)propyl]trimethoxysilane (OEG) to minimize the peptide absorption into PDMS. The well characterized of peptidergic of Aplysia californica bag cell neurons are cultured within the main reservoir. By temporally controlling the introduction of stimulation solutions to the neurons, the precise time for release to occur can be determined. Bag cell neurons from Aplysia californica can obviously culture within OEG-grafted PDMS microfluidic device, which cover with cell culture dish. Specific secretogogues (KCl and insulin) was used for stimulation of cell neuron. It was found that neuropeptide was released after stimulation for at least 5 min and 20 min by KCl and insulin, respectively. Sample solution exited the device through small tubing, sample was collected, then desalted and concentrated using ZipTipC18 pipette tips. The collected spot sample was detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) that monitoring several known peptides such as acidic peptide (AP) (m/z 2959.7) and -bag cell peptide (BCP) (m/z 1122.7). Furthermore, the microfluidic device was also used to study amino acid release from R3-13 cell neuron. Therefore, the OEG-grafted PDMS microfluidic device with two controllablemicrovalves is an efficient system for investigation of neuronal growth and signaling of cell neuron, which involved minute amounts of chemicals and small numbers of cells. Simple flow based system is a promising tool for downscaling chemical analyses. It provides fast and efficient analysis with low consumption of chemicals ixand less generation of waste. Moreover, it makes possible the analysis of minute amounts of sample which could not be performed in conventional methods.