-lactate is a metabolite generated during anaerobic glucose metabolism. Its production involves an increase in the proton concentration inside the cells. l
-lactate levels in blood range from 0.5 to 1.5 mM in normal people at rest, and can increase to 12.0 mM during exercise [1
]. Under extreme conditions of excessive exercise, lactate levels can become as high as 25 mM. In this case, the cellular proton buffering may be exceeded. Hence, the cell pH decreases, and this may result in cell lactic acidosis, which disrupts the performance of the muscles [2
]. Therefore, knowledge of personal lactic threshold is very important to perform any physical activity, and this is the reason why sport medicine requires the monitoring of l
-lactic levels in order to evaluate the so-called lactic threshold [3
], which indicates the physical training level of an athlete. Lactate levels in plasma depend on the balance between factors that produce lactate and lactate clearance factors [4
]. Any imbalance between the production and clearance of lactate may lead to the lactic acidosis. The increase of lactate levels in blood may be due to the presence of very diverse pathologies, such as haemorrhagic shock or pulmonary embolism [4
], cardiogenic [5
] or endotoxic shocks [6
], respiratory failure [7
], liver diseases [8
], and others. High levels of lactate are also the main cause of acidification in the microenvironment of cancer cells and tumors [9
], due to the imbalance between the dynamics of the production and consumption of lactate, characteristic for these types of cells and tissues [10
]. Thus, the importance of determining lactate level in medical care is evident because the blood lactate level is a valuable indicator of both fitness and clinical states.
Lactic acid fermentation is an important process in the food industry. Lactate levels may indicate the stability, freshness, and quality of many fermented dairy products such as milk, yogurt, and creams, as well as raw meat, fruits, and vegetables [11
]. In wine production, the malolactic fermentation carried out by bacteria present in the grapes and the must is very important. This fermentation takes place after the alcoholic fermentation, and converts the l
-malic acid to l
-lactic reducing the wine acidity, and leading to an improvement of the taste and flavor of the final product. The control of this secondary fermentation is very important in the quality of wines, and requires a continuous monitoring of the levels of lactic and malic acids present in the young wine [11
The examples described above show the great interest that exists in monitoring lactate concentration for clinical diagnosis, intensive care, in the food industry, and in food control, among others. To achieve these goals, there is a great demand for devices capable of determining lactate in a simple and direct way with rapid response, high specificity, low cost, and minimal or no sample preparation. In this regard, a variety of methods have been described, using techniques such as amperometry [12
], potentiometry [13
], high-performance liquid chromatography [14
], fluorometry [15
], chemiluminescence [16
], microwave sensing [17
], holography [18
], and magnetic resonance spectroscopy [19
]. Because of their simplicity, portability, and easy integration in different devices, amperometric biosensors have reached the most significant development—in particular when reagent-less biosensors based on disposable electrodes are employed.
Different biosensors for lactate monitoring have been reported based on immobilized lactate oxidase [20
]. Bienzyme systems such as lactate oxidase/lactate dehydrogenase (LOx/LDH) have also been described [24
]. In these configurations, the enzyme was immobilized in a polymeric matrix inside the electrode material. Recently, in order to improve the biosensor response, lactate biosensors using nanoparticles have been described [25
]. In general, these devices showed linearity in the range of μM to mM lactate concentration.
l-lactate oxidase and lactate dehydrogenase have been the enzymes commonly used in the design of l-lactate biosensors due to the favorable kinetics of the enzymatic reactions, the stability of the immobilized enzymes, and the simple design of the biosensors. In fact, LOx is usually preferred over LDH because its reaction involves O2 and H2O2 that can be amperometrically detected. However, the oxidation of H2O2 requires a high overpotential. A strategy employed to overcome this problem is the addition of redox mediators to the solution, and in order to simplify the systems, an improved strategy is to include the mediator in the device development.
-lactate oxidase is a globular flavoprotein with a mean diameter of 5 nm that can be obtained with an acceptable degree of purity from various microorganisms, such as Pediococcus
and Aerococcus viridians
]. In the presence of molecular oxygen, the enzyme catalyzes the oxidation of l
-lactate to pyruvate, yielding hydrogen peroxide, which is electrochemically active and can be oxidized or reduced on the electrode surface giving rise to a current which is proportional to the concentration of lactate present in the sample [31
]. As can be seen in Scheme 1
, the electrons involved in the reaction are transferred from the substrate (l
-lactate) to the oxidized form of the enzyme cofactor (Flavin Adenine Dinucleotide, FAD) present in the enzyme structure. To regenerate the enzyme catalytic site, molecular oxygen takes the electrons from the reduced cofactor, turning into hydrogen peroxide. The flow of electrons from the substrate to the electrode surface can be enhanced by the presence of highly conductive nanomaterials in the sensing interface, which can act as tiny conductive centers, allowing kinetic barriers to be eliminated, and yielding more sensitive electrochemical responses [33
Recently, we reported the use of a multi-tasking N
′-Bis(3,4-dihydroxybenzylidene) -1,2-diaminobenzene Schiff base tetradentate ligand (3,4DHS) as reductant, stabilizer, and catalyst in a new concept of gold nanoparticles (AuNPs) synthesis [34
]. This ligand contains the quinone/hydroquinone functional group and is capable of reducing HAuCl4
in water, also acting as a capping agent for the generation of stable colloidal suspensions of Schiff base ligand–AuNPs assemblies of controlled size by providing a robust coating to AuNPs within a unique reaction step. These 3,4DHS–AuNPs assemblies—deposited on carbon electrodes—show a potent electrocatalytic effect towards hydrazine oxidation and hydrogen peroxide oxidation/reduction [34
]. Following these previous studies, the present work takes the preparation of 3,4DHS–AuNPs assemblies one-step further by using them for the construction of a new l
-lactate biosensor. This is based on the co-immobilization of l
-lactate oxidase along with 3,4DHS–AuNPs onto a screen-printed carbon electrode giving rise to a reagent-less biosensor, in an effort to simplify the development of point-of-care lactate analysis systems.
The biosensor response was optimized in terms of enzyme loading, solution pH, and the effect of potentially interfering substances. Finally, the developed biosensor has been applied to the determination of lactate in food and beverage samples. The results were validated by comparing with those obtained with a commercial enzymatic kit.