A Graphene-Based Enzymatic Biosensor Using a Common-Gate Field-Effect Transistor for L-Lactic Acid Detection in Blood Plasma Samples

Abstract

Lactate is an important organic molecule that is produced in excess during anaerobic metabolism when oxygen is absent in the human organism. The concentration of this substance in the body can be related to several medical conditions, such as hemorrhage, respiratory failure, and ischemia. Herein, we describe a graphene-based lactate biosensor to detect the concentrations of L-lactic acid in different fluids (buffer solution and plasma). The active surface (graphene) of the device was functionalized with lactate dehydrogenase enzyme using different substances (Nafion, chitosan, and glutaraldehyde) to guarantee stability and increase selectivity. The devices presented linear responses for the concentration ranges tested in the different fluids. An interference study was performed using ascorbic acid, uric acid, and glucose, and there was a minimum variation in the Dirac point voltage during detection of lactate in any of the samples. The stability of the devices was verified at up to 50 days while kept in a dry box at room temperature, and device operation was stable until 12 days. This study demonstrated graphene performance to monitor L-lactic acid production in human samples, indicating that this material can be implemented in more simple and low-cost devices, such as flexible sensors, for point-of-care applications.

Keywords: biosensor; field-effect transistor; graphene; lactate; lactate dehydrogenase.

Figure 1

Microchannels integrated with the electrode array (eight pairs) of the common-gate graphene-based field-effect transistor (GFET) sensor to detect L-lactic acid in human samples: (a) top view and (b) cross-sectional A–A view.

Figure 2

Immobilization matrix to support the enzyme (lactate dehydrogenase) on the surface of the active layer (graphene) of the GFET biosensor.

Figure 3

Schematic of the study performed to detect the concentration of lactate in the samples that were injected in the microchannels over the immobilization matrix while measuring the transfer characteristics of the graphene-based device.

Figure 4

Characterization of common-gate graphene-based field-effect transistors. (a) Raman spectra (532 nm wavelength; G peak: 1537 cm⁻¹ and 2D peak: 2670 cm⁻¹) of the chemical vapor deposition (CVD)-grown graphene layers that was transferred using polydimethylsiloxane (PDMS) as supportive layer. (b) Normalized average transfer curves (n = 16) using 1× phosphate-buffered saline (PBS) (pH 7.4) as a buffer solution. (c) Comparison between the Dirac point voltage values of the common-gate GFET (mean = 1 ± 0.03 V) and the individual gate GFET (mean = 1.03 ± 0.06 V).

Figure 5

Experiments using the common-gate GFET device to detect L-lactic acid on buffer solution: (a) electrochemical reaction for the lactate assay; (b) the mean Dirac point (V_Dirac) (n = 3) concentration of L-lactic acid (0.25 to 10 mM); and (c) the linear fit of the measured concentrations with a Pearson correlation coefficient of 0.9480 (R² = 0.8735).

Figure 6

Dirac point voltage values of GFET devices: (a) Using Nafion (NA), chitosan (CHI), and glutaraldehyde (GA) to immobilize and stabilize the LDH enzyme over the graphene layer; (b) Dirac point voltage values and the failure percentage measured during the stabilization experiment to evaluate the device for up to 50 days after immobilization of the enzyme. A concentration of 5 mM of L-lactic acid in buffer solution was injected into each channel.

Figure 7

Detection of different concentrations of L-lactic acid in human samples. (a) Dirac point voltage values (n = 3) for blood plasma samples treated with L-lactic acid (0 to 7.5 mM). (b) Comparison between the shift of Dirac point voltage by concentration of lactate (Pearson coefficient correlations: 0.9911).

Figure 8

Measurement of the Dirac point voltages (n = 3) of PBS, plasma sample containing 7.5 mM of L-lactic acid (LA), and after injection of each interference biochemical (uric acid (UA), ascorbic acid (AA), and glucose (GLU)).