Label Free Detection of L-Glutamate Using Microfluidic Based Thermal Biosensor

Abstract

A thermoelectric biosensor for the detection of L-glutamate concentration was developed. The thermoelectric sensor is integrated into a micro-calorimeter which measures the heat produced by biochemical reactions. The device contains a single flow channel that is 120 µm high and 10 mm wide with two fluid inlets and one fluid outlet. An antimony-bismuth (Sb-Bi) thermopile with high common mode rejection ratio is attached to the lower channel wall and measures the dynamic changes in the temperature when L-glutamate undergoes oxidative deamination in the presence of glutamate oxidase (GLOD). The thermopile has a Seebeck coefficient of ~7 µV·(m·K)^-1. The device geometry, together with hydrodynamic focusing, eliminates the need of extensive temperature control. Layer-by-layer assembly is used to immobilize GLOD on the surface of glass coverslips by alternate electrostatic adsorption of polyelectrolyte and GLOD. The impulse injection mode using a 6-port injection valve minimizes sample volume to 5 µL. The sensitivity of the sensor for glutamate is 17.9 nVs·mM^-1 in the linear range of 0-54 mM with an R² value of 0.9873. The lowest detection limit of the sensor for glutamate is 5.3 mM.

Keywords: L-glutamate; L-glutamate oxidase; biosensor; label-free; layer-by-layer self-assembly; thermoelectric.

Figure 1

Thermopile (8 mm × 8 mm) fabricated on 100 µm kapton^® support showing measuring and reference junctions.

Figure 2

Thermoelectric microfluidic L-glutamate sensor. (a) Schematic showing microfluidic device fabrication. (b) Microfluidic device showing hydrodynamic flow and thermopile attached on the lower channel wall of the microfluidic device. The dimensions of the components are glass slide—75 × 25 × 1.2 mm, glass coverslip—75 × 25 × 0.17 mm, and channel—60 × 10 × 0.12 mm. Volume of the microfluidic channel is 72 µL.

Figure 3

Layer-by-layer assembly of glutamate oxidase (GLOD) on a substrate. Electrostatic adsorption and charge resaturation occurs on the substrate to form alternate layers of polyelectrolyte and GLOD films.

Figure 4

Experimental setup (schematic) of the thermoelectric detection system. Sample loop size used in the injection valve is 5 µL.

Figure 5

(a) Activity of the immobilized GLOD layers using Amplex^® Red reagent based assay. Fluorescence was measured for every 5 min using a fluorescence microplate reader using excitation at 530 ± 12.5 nm and the fluorescence detection at 590 ± 17.5 nm. Activity of one layer (polyethyleneimine (PEI)/GLOD), two layers ((PEI/GLOD)₂) and three layers ((PEI/GLOD)₃) of GLOD was studied. (b) Stability of immobilized GLOD film when stored in de-ionized (DI) water, Tris-Buffer (pH 7.5) and air at room temperature. GLOD immobilized coverslips were tested for a period of 8 days. Error bars represent standard error when n = 3.

Figure 6

Thermopile output following the injection of 54 mM L-glutamate sample. Sample size used is 5 µL. Secondary ordinate axis represents the temperature rise (calculated from Seebeck coefficient) detected by the thermopile. The dotted line indicated in red shown in the figure is used to determine the area under the curve (AUC).

Figure 7

Integrated thermopile output plotted as a function of L-glutamate concentration. One layer of GLOD was immobilized. Concentrations of L-glutamate samples range from 0–80.2 mM. Dashed line is the linear fit indicating the linear range from 0–54 mM for immobilized GLOD (PEI/GLOD). The sensor has a lower detection limit of 5.3 mM. Error bars represent the standard error when n=4. Inset: thermopile output at the lower detection limit (5.3 mM).

Figure 8

L-glutamate release from brain tumor cell line CRL 2303 media samples following potassium chloride (KCL) stimulation. The cells were plated in a petri dish at a density of 200,000 cells per mL. After 24 h, the cells were stimulated using 50 mM KCl. The samples of the cell media were collected in regular time intervals after treatment and analyzed using (a) fluorescence spectrophotometer using Amplex red assay, (b) the microfluidic thermoelectric L-glutamate sensor. Error bars represent the standard error when n = 3.