Poly(m-phenylenediamine) nanospheres and nanorods: selective synthesis and their application for multiplex nucleic acid detection

Abstract

In this paper, we demonstrate for the first time that poly(m-phenylenediamine) (PMPD) nanospheres and nanorods can be selectively synthesized via chemical oxidation polymerization of m-phenylenediamine (MPD) monomers using ammonium persulfate (APS) as an oxidant at room temperature. It suggests that the pH value plays a critical role in controlling the the morphology of the nanostructures and fast polymerization rate favors the anisotropic growth of PMPD under homogeneous nucleation condition. We further demonstrate that such PMPD nanostructures can be used as an effective fluorescent sensing platform for multiplex nucleic acid detection. A detection limit as low as 50 pM and a high selectivity down to single-base mismatch could be achieved. The fluorescence quenching is attributed to photoinduced electron transfer from nitrogen atom in PMPD to excited fluorophore. Most importantly, the successful use of this sensing platform in human blood serum system is also demonstrated.

Figure 1. Instrumental analysis of the precipitate thus formed.

Low magnification SEM images of the PMPD nanostructures formed using (A) water and (C) NMPD as reaction solvent, (B) and (D) corresponding to the high magnification SEM images.

Figure 2. Performance of target DNA detection and determination of detection limit.

(A) Fluorescence emission spectra of P_HIV (50 nM) at different conditions: (a) P_HIV; (b) P_HIV+300 nM T₁; (c) P_HIV+PMPD nanorods; (d) P_HIV+PMPD nanorods+300 nM T₁. Curve e is the emission spectra of PMPD nanorods. Inset: fluorescence intensity change (F/F ₀–1) of P_HIV–PMPD nanorods complex (where F ₀ and F are the fluorescence intensity without and with the presence of T₁, respectively) plotted against the logarithm of the concentration of T₁. (B) (a) Fluorescence emission spectra of P_HIV (500 pM), (b) fluorescence quenching of P_HIV (500 pM) by 1-µL PMPD nanorods, and (c) fluorescence recovery of (b) by T₁ (50 pM). Inset in Figure 2B: the corresponding fluorescence intensity histograms with error bar. Excitation was at 480 nm, and the emission was monitored at 522 nm. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg²⁺ (pH: 7.4). 10-µL PMPD nanorods were used in each measurement.

Figure 3. Illustration of the sensing process and fluorescence quenching mechanism.

(A) A schematic (not to scale) illustrating the fluorescence-enhanced nucleic acid detection using PMPD nanorod as a sensing platform and (B) a schematic illustrating the photo-induced electron transfer (PET) process to explain the mechanism of fluorescence quenching.

Figure 4. Influence of pH value on the fluorescence quenching.

Fluorescence quenching histograms of of P_HIV (50 nM) by PMPD nanorods at different pH values (where F and F ₀ are the fluorescence intensity with and without the presence of PMPD). 10-µL PMPD nanorods were used in each measurement.

Figure 5. Kinetic behaviour study of fluorescence quenching and recovery at different temperatures.

(a) Fluorescence quenching of P_HIV (50 nM) by PMPD nanorods and (b) fluorescence recovery of P_HIV–PMPD by T₁ (300 nM) as a function of incubation time (A: 25°C, B: 50°C). Excitation was at 480 nm, and the emission was monitored at 522 nm. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg²⁺ (pH: 7.4). 10-µL PMPD nanorods were used in each measurement.

Figure 6. Evaluation of discrimination ability at different temperatures.

(A) Fluorescence emission spectra of P_HIV (50 nM) at different conditions: (a) P_HIV–PMPD complex; (b) P_HIV–PMPD complex+300 nM T₁; (c) P_HIV–PMPD complex+300 nM T₂; (d) P_HIV–PMPD complex+300 nM T₃; (e) P_HIV–PMPD complex+300 nM T₄; (f) P_HIV–PMPD complex+300 nM T₅. Inset: fluorescence intensity histogram with error bar. (B) Fluorescence signal enhancement of P_HIV–PMPD complex upon incubation with 300 nM T₁ and 300 nM T₂ at 25 and 50°C, respectively. 10-µL PMPD nanorods were used in each measurement.

Figure 7. Evaluation of discrimination ability using (A) shorter probe or (B) longer target.

(A) Fluorescence emission spectra of P_s (50 nM) at different conditions: (a) P_s–PMPD complex; (b) P_s–PMPD complex+300 nM T_s1; (c) P_s–PMPD complex+300 nM T_s2; (d) P_s–PMPD complex+300 nM T_s3. (B) Fluorescence emission spectra of P_HIV (50 nM) in the presence of PMPD nanorods at different conditions: (a) P_HIV–PMPD complex; (b) P_HIV–PMPD complex+300 nM T_L1; (c) P_HIV–PMPD complex+300 nM T_L2. Inset: fluorescence intensity histograms with error bar. Excitation was at 480 nm, and the emission was monitored at 522 nm. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg²⁺ (pH: 7.4). 10-µL PMPD nanorods were used in each measurement.

Figure 8. Performance of single-base mismatch discrimination in the presence of blood serum.

The histograms of fluorescence intensity ratio of F(T₂)/F(T₁), where F(T₁) and F(T₂) are the fluorescence intensities in the presence of different amount of blood serum spiked with T₁ and T₂, respectively. ([T₁] = 300 nM; [T₂] = 300 nM). Excitation was at 480 nm, and the emission was monitored at 522 nm. The total volume of each sample was 300 µL in Tris-HCl buffer (pH 7.4) containing 5 mM Mg²⁺. 10-µL PMPD nanorods were used in each measurement.

Figure 9. Multiplex DNA detection.

Fluorescence intensity histograms of the probe mixture ([P_HIV] = [P_HBV] = [P_K167] = 50 nM) toward different target combinations in the presence of PMPD nanorods under excitation/emission wavelengths of 480/522, 587/601, and 643/660 nm/nm. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg²⁺ (pH: 7.4). 10-µL PMPD nanorods were used in each measurement.