A Surface Acoustic Wave (SAW)-Based Lab-on-Chip for the Detection of Active α-Glycosidase

Abstract

Enzyme detection in liquid samples is a complex laboratory procedure, based on assays that are generally time- and cost-consuming, and require specialized personnel. Surface acoustic wave sensors can be used for this application, overcoming the cited limitations. To give our contribution, in this work we present the bottom-up development of a surface acoustic wave biosensor to detect active α-glycosidase in aqueous solutions. Our device, optimized to work at an ultra-high frequency (around 740 MHz), is functionalized with a newly synthesized probe 7-mercapto-1-eptyl-D-maltoside, bringing one maltoside terminal moiety. The probe is designed ad hoc for this application and tested in-cuvette to analyze the enzymatic conversion kinetics at different times, temperatures and enzyme concentrations. Preliminary data are used to optimize the detection protocol with the SAW device. In around 60 min, the SAW device is able to detect the enzymatic conversion of the maltoside unit into glucose in the presence of the active enzyme. We obtained successful α-glycosidase detection in the concentration range 0.15-150 U/mL, with an increasing signal in the range up to 15 U/mL. We also checked the sensor performance in the presence of an enzyme inhibitor as a control test, with a signal decrease of 80% in the presence of the inhibitor. The results demonstrate the synergic effect of our SAW Lab-on-a-Chip and probe design as a valid alternative to conventional laboratory tests.

Keywords: SAW; acoustic sensor; enzyme detection; α-glycosidase.

Figure 1

The proposed SAW-LoC biosensor: (a) assembled device, in which the sensors are covered with the microfluidic chamber, scale bar = 0.3 cm; (b) reaction scheme of the probe synthesis; (c) illustration of the sensor working principle: the maltoside moieties, indicated with a double blue circle, are enzymatically converted releasing glucose units.

Figure 2

In-cuvette characterization of probe and enzyme: enzymatic conversion of the probe at (a) 25 °C, (b) 40 °C and (c) 60 °C by varying the enzyme concentration; (d) enzyme activity of α-glycosidase in the presence of its inhibitor acarbose in different concentrations.

Figure 3

Results of QCM-D measures: (a) traces acquired during the experiments (black: ΔF, blue: ΔD) referred to the third overtone, events: (1) functionalization solution injection, (2) rinsing with water, (3) enzyme solution (150 U/mL) injection, (4) rinsing with water, reported traces are related to the experiment with enzyme concentration of 150 U/mL; (b) ΔF measured after rinsing with water for the functionalization for the selected overtones (n = 16 for each overtone); (c) ΔD measured after rinsing with water for the functionalization for the selected overtones (n = 16 for each overtone); (d) ΔF₃ measured after rinsing with water for the injection of different enzyme concentrations (n = 4 for each enzyme concentration), values are calculated as ΔF between events 4 and 3; (e) ΔD₃ measured after rinsing with water for the injection of different enzyme concentrations (n = 4 for each enzyme concentration), values are calculated as ΔD between events 4 and 3; (f) probe areal masses (ng cm⁻²) calculated from ΔF₃ using the Sauerbrey equation (Equation (1)). For this calculation, experiments giving outliers, indicated by the red crosses in plots b and c, after the functionalization, are eliminated (n = 4 in all cases except for the experiment with enzyme concentration 1.5 U/mL, for which n = 3), values are calculated as ΔF between events 4 and 1. Statistical tests are performed comparing the frequency shift due to functionalization and to samples, * indicates p < 0.1, obtained from a Mann–Whitney U-test.

Figure 4

Measures performed with the SAW-LoC device: (a) characteristic curve obtained analyzing samples containing α-glycosidase in concentrations ranging from 0.15 to 150 U/mL; (b) frequency shifts measured analyzing samples containing α-glycosidase (15 U/mL) and its inhibitor acarbose (2.5 mg/mL), compared to those obtained after the functionalization and the injection of samples containing only the enzyme (15 U/mL); n indicates the number of sensors used for each analysis; statistical tests are performed comparing the frequency shift from functionalization and from samples, * indicates p < 0.1, while ** indicates p < 0.01, obtained from a Mann–Whitney U-test.