A Label-Free Fluorescence Aptasensor Based on G-Quadruplex/Thioflavin T Complex for the Detection of Trypsin

Abstract

A novel, label-free fluorescent assay has been developed for the detection of trypsin by using thioflavin T as a fluorescent probe. A specific DNA aptamer can be combined by adding cytochrome c. Trypsin hydrolyzes the cytochrome c into small peptide fragments, exposing the G-quadruplex part of DNA aptamer, which has a high affinity for thioflavin T, which then enhances the fluorescence intensity. In the absence of trypsin, the fluorescence intensity was inhibited as the combination of cytochrome c and the DNA aptamer impeded thioflavin T's binding. Thus, the fluorescent biosensor showed a linear relationship from 0.2 to 60 μg/mL with a detection limit of 0.2 μg/mL. Furthermore, the proposed method was also successfully employed for determining trypsin in biological samples. This method is simple, rapid, cheap, and selective and possesses great potential for the detection of trypsin in bioanalytical and biological samples and medical diagnoses.

Keywords: DNA aptamer; cytochrome c; label-free; thioflavin T; trypsin.

Figure 1

Schematic illustration of the fluorometric assay for detecting trypsin.

Figure 2

Feasibility of the proposed method: Fluorescence intensity of DNA aptamer-ThT; DNA aptamer-ThT with and without adding trypsin. All reactions were performed in 50 mmol/L Tris and 1 mmol/L KCl buffer at pH 7.5. In total, 50 μmol/L DNA aptamer, 1 mmol/L ThT, 10 mmol/L cyt c, 10 mg/mL trypsin, and 30 min of incubation time were employed. Fluorescence intensity was measured using an F-2700 with excitation at 490 nm, and the emission spectra were collected in the range of 450 to 550 nm.

Figure 3

Optimizing the experimental conditions: (a) concentration of different experimental components, including DNA chain (1 mM ThT, 10 mM cyt c, and 10 mg/mL trypsin; 30 min of incubation time and a different concentration of DNA aptamer were employed), cytochrome c (250 nM DNA aptamer, 1 mM ThT, and 10 mg/mL trypsin; 30 min of incubation time and a different concentration of cyt c were employed), and ThT (250 nM DNA aptamer, 80 μM cyt c, and 10 mg/mL trypsin; 30 min of incubation time and a different concentration of ThT were employed); (b) reaction time for trypsin and cytochrome c (250 nM DNA aptamer and 80 μM cyt c with or without 10 mg/mL trypsin incubated within a different time range and then 10 μM ThT added for testing) and reaction time for the DNA chain (250 nM DNA aptamer with or without 80 μM cyt c incubated within a different time range and then 10 μM ThT added for testing).

Figure 4

(a) Fluorescence emission spectra upon addition of trypsin at different concentrations, from 0 to 140 μg/mL. (b) Fluorescence signal in response to different concentrations of trypsin, showing the linear curve of the enhanced fluorescence intensity to the concentration of trypsin. The inset shows the linearity of the fluorescence intensity with respect to trypsin concentrations.

Figure 5

Selectivity of the assay. Selectivity of the proposed trypsin assay toward trypsin, HSA, GSH, Actin, PKA, ALP, and thrombin (60 μg/mL each).