Using N-doped Carbon Dots Prepared Rapidly by Microwave Digestion as Nanoprobes and Nanocatalysts for Fluorescence Determination of Ultratrace Isocarbophos with Label-Free Aptamers

Abstract

The strongly fluorescent and highly catalytic N-doped carbon dots (CD_N) were rapidly prepared by a microwave irradiation procedure and were characterized by electron microscopy (EM), laser scattering, infrared spectroscopy (IR), and by their fluorescence spectrum. It was found that the CD_N had a strong catalytic effect on the fluorescence reaction of 3,3',5,5'-tetramethylbenzidine hydroxide ((TMB)⁻H₂O₂) which produced the oxidation product of TMB (TMB_OX) with strong fluorescence at 406 nm. The aptamer (Apt) was adsorbed on the CD_N surfaces which weakened the fluorescence intensity due to the inhibition of catalytic activity. When the target molecule isocarbophos (IPS) was added, it reacted with the Apt to form a stable conjugate and free CD_N which restored the catalytic activity to enhance the fluorescence. Using TMB_OX as a fluorescent probe, a highly sensitive nanocatalytic method for determination of 0.025⁻1.5 μg/L IPS was established with a detection limit of 0.015 μg/L. Coupling the CD_N fluorescent probe with the Apt⁻IPS reaction, a new CD fluorescence method was established for the simple and rapid determination of 0.25⁻1.5 μg/L IPS with a detection limit of 0.11 μg/L.

Keywords: TMB; aptamer; carbon dot catalysis; fluorescence; isocarbophos.

Figure 1

Principle of carbon dot (CD) and 3,3′,5,5′-tetramethylbenzidine hydroxide oxidation product (TMB_OX) probes for isocarbophos (IPS) based on the aptamer (Apt) reaction.

Figure 2

Fluorescence (A) and excited (B) spectra of CD_N. (a) 0 mg/L CD_N; (b) 129.2 mg/L CD_N; (c) 265.2 mg/L CD_N; (d) 530.4 mg/L CD_N; (e) 1060.8 mg/L CD_N; (f) 2128.4 mg/L CD_N; (g) 4250 mg/L CD_N; (h) 8500 mg/L CD_N; (i) 17,000 mg/L CD_N.

Figure 3

Fluorescence (A) and excited (B) spectra of the Apt–IPS–CD_N system. (a) 0.21 μmol/L Apt + 11.28 mg/L CD_N + 0.027 mol/L NaH₂PO₄–Na₂HPO₄; (b) a + 0.25 μg/L IPS; (c) a + 0.5μg/L IPS; (d) a + 0.75μg/L IPS; (e) a + 1.0 μg/L IPS; (f) a + 1.25 μg/L IPS; (g) a + 1.5μg/L IPS.

Figure 4

Fluorescence of the CD_N catalytic system (A) Fluorescence spectra of the Apt–IPS–CD_N–H₂O₂–TMB catalytic analytical system, a: 31 nmol/L Apt + 0.45 mg/L CD_N + 0.053 mmol/L H₂O₂ + 0.017 mmol/L TMB + 0.13 mmol/L pH 3.6 HAc–NaAc; b: a + 0.025 μg/L IPS; c: a + 0.1μg/L IPS; d: a + 0.3μg/L IPS; e: a + 0.5μg/L IPS; f: a + 0.7μg/L IPS; g: a + 0.9μg/L IPS; h: a + 1.2μg/L IPS. (B) Excited spectra of A. (C) Fluorescence spectra of the CD_N–H₂O₂–TMB catalytic system, a: 0.13 mmol/L H₂O₂+33 μmol/L TMB + 0.13 mmol/L pH 3.6 HAc–NaAc; b: a + 0.028 mg/L CD_N; c: a + 0.057 mg/L CD_N; d: a + 0.113 mg/L CD_N; e: a + 0.17 mg/L CD_N; f: a + 0.34 mg/L CD_N. (D) Fluorescence spectra of the Apt–CD_N–H₂O₂–TMB system, a: 0.34 mg/L CD_N + 0.053 mmol/L H₂O₂ + 0.017 mmol/L TMB + 0.13 mmol/L pH 3.6 HAc–NaAc; b: a + 5.17 nmol/L Apt; c: a + 7.23 nmol/L Apt; d: a + 10.33 nmol/L Apt; e: a + 15.5 nmol/L Apt IPS; f: a + 20.67 nmol/L Apt; g: a + 25.83 nmol/L Apt; h: a + 31 nmol/L Apt.

Figure 5

Mechanism of catalytic reaction of nitrogen-doped carbon dots.

Figure 6

(A) SEM, (B) TEM, (C) laser scattering, and (D) IR of CD_N.