Amiloride-modulated phosphorescence turn-off/on method for the detection of abasic site-containing dsRNA based on uridine triphosphate-capped Mn-doped ZnS quantum dots

2.1 Materials and chemicals

All kinds of reagents related to RNA and DNA (see SI for sequence) were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Uridine 5′-triphosphate trisodium salt dihydrate, zinc sulfate heptahydrate, manganese chloride, and sodium sulfide nonahydrate were purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China). Phosphoric acid, boric acid, acetic acid, and sodium hydroxide were used to prepare Britton–Robinson (BR) buffer solution (J&K scientific Ltd., Beijing, China). All chemicals were analytical grade and were used as received without further purification. Ultrapure water (18.2 MΩ cm) was prepared via a Milli-Q water integration system (Merck KGaA, Darmstadt, Germany).

2.2 Apparatus

The microstructure was characterized using a laser particle size analyzer (Zetasizer Nano ZS, Malvern, UK), a transmission electron microscope (TEM, Tecnai G2 F20, FEI, US), an atomic force microscope (AFM, SPA400, Seiko, Japan), and an X-ray diffractometer (XRD, D8 ADVANCE, Bruker, Germany). Material composition data were obtained via X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo, US). Surface group properties were determined using an infrared spectrophotometer (IR, Varian 640, US) with KBr pellets as the sample matrix in the frequency range of 500–4000 cm⁻¹. Establishment of the detection system was mainly achieved by setting the fluorescence spectrophotometer (Eclipse, Varian, US) to phosphorescence mode at the excitation wavelength of 300 nm, and the slit widths of excitation and emission were 10 and 20 nm, respectively. An ultraviolet spectrophotometer (UV-3600, Shimadzu, Japan) was used for auxiliary characterization.

2.3 Synthesis of UTP-capped Mn-ZnS QDs

UTP-capped Mn-doped ZnS QDs were synthesized through a one-step wet-chemical method. Added 2.5 mL ZnSO₄ (0.1 M) and 1 mL MnCl₂ (0.01 M) to 50 mL UTP solution (0.01 M) in sequence. The reaction was stirred for 30 min under an argon atmosphere. 2.5 mL Na₂S (0.1 M, the solution was prepared upon use) was quickly added after the UTP stably combined with Zn²⁺ and Mn²⁺, and then stirring was continued for 20 min. The reaction solution was then transferred to a beaker and bathed at 50 °C for 2 h under air atmosphere. Centrifuged (3000 rpm, 5 min) and washed (ethanol, v/v = 1:1) three times, then vacuum dry to obtain the desired UTP-QD product.

2.4 Measurement procedure

BR buffer solution (0.5 mL, 0.1 M, Ph 5.8), UTP-QDs (0.5 mL, 0.12 mg mL⁻¹), NaCl (0.5 mL, 0.1 M), and equal volumes of AMI standard solutions of different concentrations were sequentially added to a 10 mL calibrated test tube (final concentration range of 10–625 nM) to examine the quenching behavior of AMI on the RTP of UTP-QDs.

BR buffer solution (0.5 mL, 0.1 M, pH 5.8), UTP-QDs (0.5 mL, 0.12 mg mL⁻¹), NaCl (0.5 mL, 0.1 M), and AMI (500 μL, 6 mM) were sequentially added to a 10 mL calibrated test tube to detect AP-dsRNA based on its RTP recovery, which was quenched by AMI-induced. After the mixture was left for 5 min, AP-dsRNA standard solution (or real sample) was added. Then, left for another 10 min to measure RTP.

2.5 Biological sample treatment

Serum samples were collected at a local hospital (The Second People’s Hospital, Linfen, China), and plant lysate samples, which came from tobacco (Nicotiana tabacum, K326) leaves infected with cucumber mosaic virus, were provided by Biofavor Biotech Services Ltd. (Wuhan, China). An appropriate (150-fold) dilution of serum and plant lysate was made before detection.

3.1 Characterization of UTP-capped Mn-doped ZnS QDs

Morphological characterization results showed that the QDs have an average particle size of 3.6 nm (Fig. 1A) and a thickness of 3.9 nm (Fig. 1B). The XRD pattern of QDs (Fig. 1C) showed obviously (1 1 1), (2 2 0), and (3 1 1) planes of typical zinc blend structure. The XPS pattern of UTP-QDs exhibited the peaks of Zn (2p) at 1022 and 1045 eV (Fig. 1D), which are attributed to the presence of Zn²⁺ that matches the Zn–S bonding energy (Shin et al., 2011). Weak Mn 2p spectra in the range of 630–660 eV shown in Fig. 1D (inset) prove the existence of Mn²⁺ in the form of zinc atom substitutes or interstitial impurities (Yang et al., 2001).

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Fig. 1

Characterization of UTP-capped Mn-doped ZnS QDs: (A) TEM image and particle size distribution (inset); (B) AFM image and height distribution (inset); (C) XRD patterns; (D) XPS images and Mn 2p spectra (inset).

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As an inorganic material, Mn-doped ZnS QDs has relatively few infrared characteristic peaks; hence, the infrared characteristic peak at 1000–4000 cm⁻¹ is mainly contributed by UTP (Fig. 2). Two distinct characteristic peak shifts, including the carbonyl group on amide shifting from 1702 cm⁻¹ to 1637 cm⁻¹ and the PO₂⁻ peak shifting from 1255 cm⁻¹ to 1236 cm⁻¹, were observed by comparing UTP with UTP-QDs. This shifting indicates that amide and phosphate bonds are involved in the modification of UTP-capped Mn-doped ZnS QDs (Ren and Yan, 2012). The attribution of detailed infrared characteristic peaks is shown in Table S1.

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Fig. 2

Infrared spectra of UTP-QDs and UTP.

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RTP characterization results showed that the largest excitation peak and the largest emission peak of the UTP-QDs were 300 and 590 nm, respectively, with an RTP lifetime of 2.54 ms (Fig. S1) and an absolute quantum yield of 14.78% (Fig. S2). The mechanism of RTP generation is attributed to the energy transfer from the band gap of ZnS to the Mn²⁺ dopant and the subsequent transition from the triplet state (⁴T₁) to the ground state (⁶A₁) of the Mn²⁺ incorporated into the ZnS host lattice (Chung et al., 2001) (Fig. S3). The maximum phosphorescent excitation peak of UTP-QDs slightly blue-shifted from 290 nm to 300 nm compared with other materials that modified Mn-doped ZnS QDs, suggesting that UTP binds to the lower energy sites of the QDs (Sakthivel et al., 2015).

In summary, these results confirm that this method successfully prepares UTP-capped Mn-doped ZnS QDs with RTP and uniform morphology.

3.2 AMI turns-off the RTP of UTP-QDs

The RTP quenching of UTP-QDs occurred rapidly within 5 min after adding AMI and reached a stable value, which was dependent on pH (Fig. S4). Meanwhile, the RTP lifetime of QDs slowly decreased with the increase in AMI concentration (Fig. S1). Two classical quenching models, namely, dynamic and static models (Valeur, 2003), were analyzed to explain the quenching process of photoluminescence. The inverse of the RTP variable (P₀ − P)⁻¹ (P₀ and P represent the RTP intensity of UTP-QDs in the absence and presence of AMI, respectively) to the inverse of the concentration of AMI was plotted (Fig. 3A) and found to be consistent with Lineweaver–Burk equation (Eq. (1)) (Lakowicz and Weber, 1973).

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Fig. 3

(A) Lineweaver–Burk plot of interaction between UTP-QDs (60 mg L⁻¹) and AMI (0–625 nM) in BR buffer (50 mM, pH 5.8) containing 100 mM NaCl; (B) UV absorption of the correlation between UTP-QDs, AMI and AP-dsRNA; (C) RLS spectra of the correlation between UTP-QDs, AMI and AP-dsRNA; (D) TEM image of the precipitate formed by UTP-QDs and AMI.

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This result implies that the RTP quenching process followed the static quenching (Lakowicz, 2013).

Two main causes contributed to this static quenching model. (1) The UV–vis spectrum characterization results showed that the absorption peak of AMI extensively overlapped with the RTP excitation peak of UTP-QDs, indicating that the internal filtering effect easily occurred (Fig. 3B). (2) Resonance light scattering (RLS) spectra (Fig. 3C) and TEM images (Fig. 3D) showed that obvious RLS signals appeared in the system with the addition of AMI, indicating the generation of aggregates. This finding is in line with theoretical expectations that UTP on the surface of QDs can coordinate with AMI from a non-luminous complex precipitate in the presence of NaCl (Pettis et al., 2004).

3.3 AP-dsRNA-induced RTP restoration of AMI-modulated UTP-QDs

The RTP intensity of UTP-QDs was almost unchanged in the presence of AP-dsRNA (Fig. S5), meaning that the interaction between AP-dsRNA and UTP-QDs is weak due to steric hindrance in AP-dsRNA (Varshavsky, 2006), which makes it difficult for UTP-QDs to bind to it. However, AP-dsRNA was able to recover the AMI-quenched RTP (Fig. 4). Both the considerable variation in UV (Fig. 3B) and reduction in RLS (Fig. 3C) after adding AP-dsRNA to the solution of UTP-QDs indicated the de-aggregation of QDs in the presence of AMI, which was also verified by TEM image (Fig. S6).

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Fig. 4

Concentration-dependent RTP intensity of AMI (300 nM)-modulated UTP-QDs (60 mg L⁻¹) in the presence of AP-dsRNA at various concentrations and the corresponding calibration curve (inset). All solutions were prepared in 50 mM BR buffer at pH 5.8 containing 100 mM NaCl.

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The phosphorescence recovery intensity (ΔP) showed a good linearity against the AP-dsRNA concentration (C, in the range of 1.4–850 nM) with a linear regression equation of ΔP = 0.7325C + 25.8815 (R = 0.9988). The relative standard deviation for the 11 replicate detections of 300 nM AP-dsRNA was 2.1%, whereas the detection limit (3 s) for AP-dsRNA was 0.86 nM (Fig. 4). RTP suffers from less background interference by biological fluids; hence, this system can detect AP-dsRNA in real samples without complex pretreatments. A comparison of its analysis performance with other methods is shown in Table 1.

3.4 Factors affecting the detection system

The pH of the system affected the AMI-induced RTP quenching (Fig. S7A) and the subsequent RTP recovery by AP-dsRNA (Fig. S7B). The recovered RTP intensity remarkably increased with the increase in pH from 3.0 to 5.8, remained unchanged in the pH range of 5.8–6.8, and slowly decreased and fluctuated in the range of 6.8–9.0. The optimal pH range of 5.8–6.8 favored the AMI-induced RTP quenching of the UTP-QDs, and mono-cation formed by amidino (pK_a 8.7) protonation easily form ion pairs with abasic sites in dsRNA, thereby increasing RTP recovery. Moreover, the ionization of UTP (quaternary anion) is also minimal under this condition.

The concentration of NaCl can substantially enhance the binding affinity of AMI to UTP and AP-dsRNA; this binding affinity also plays an important role in quenching and recovering RTP in the system. When the concentration of NaCl is 100 mM, the quenching efficiency and recovery efficiency can be properly balanced in the detection system (Fig. S8). Accordingly, subsequent experiments were performed in 50 mM BR buffer containing 100 mM NaCl at pH 5.8. The optimal RTP-restoration by AP-dsRNA is reached in the concentration range of 250–350 nM of AMI (Fig. S9). The middle concentration (300 nM) was selected for subsequent experiments.

3.5 Selectivity of the RTP detection for AP-dsRNA

The potential interference of structural analogs and co-existing substances in the determination of AP-dsRNA was investigated to evaluate the practicability of the RTP method for RNA detection (Fig. S10). Results showed that AP-dsRNA has obvious recovery ability to the RTP of UTP-QD-AMI compared with other co-existing materials; therefore, this system has excellent selectivity. AP-dsDNA showed certain RTP recovery ability, but this recovery was limited by the different spatial conformation of RTP with AP-dsRNA, which determines the specificity of this detection system to AP-dsRNA. This can be attributed to differences in the double helix structure of DNA and RNA, that is, RNA is A-form and DNA is B-form, and the addition of UTP-QD-AMI can change the conformation of AP-dsRNA but not of AP-dsDNA. The larger inclination of the A-form than the B-form (1°) in nucleic acids (19°) allows more extensive base-to-base overlap and provides more efficient stacking interactions of nucleotides (Saenger, 2013), which translates into an efficient binding affinity for AP-dsDNA (Sato et al., 2012). This process explains why double-stranded nucleic acids (dsDNA and dsRNA) without AP-sites and single-stranded nucleic acids (ssDNA and ssRNA) with AP-sites do not effectively restore the quenched RTP.

The combination of AMI and UTP-QDs cannot be broken nor can the recovery of the quenched RTP of UTP-QDs by AP-dsRNA be affected; therefore, the detection performance of the system is less affected by other nucleotides and co-existing substances. These substances are found in low concentrations in biological fluids or can be appropriately diluted to eliminate interference.

3.6 Application of the RTP-recovery system in AP-dsRNA detection

The AMI-modulated UTP-capped Mn-doped ZnS QD-based turn-off–on RTP-system was used for AP-dsRNA detection in serum and plant lysate samples. The phosphorescence background of biological fluids samples was negligible after proper dilution (150 times), and obtained spiked recovery rates of 95%–103% (Table S2). The developed turn-off–on RTP systems can be employed in the detection of AP-dsRNA in biological fluids.