2.1 Materials and chemicals

The DNA sequences (shown in Table S1) used in the experiment were synthesized and purified by Sangon Biotechnology Co. ltd. (Shanghai, China). N-Hydroxylsuccinimide sodium salt (NHS) and 1-ethyl-3-(3-(dimethylamino) propyl) carbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich (St. Louis, USA). Three PEG-capped and carboxyl-modified CdSe/ZnS QDs (the maximum emission wavelength at 525 nm, 585 nm and 632 nm, the excitation wavelength at 340 nm) were provided by Suzhou Xingshuo Nanotech Co., ltd. (Suzhou, China). GO was purchased from Nanjing Xianfeng Nanomaterials Technology Co., ltd. (Nanjing, China). The other reagents used were of analytical grade, and all the solutions were prepared with twice-distilled water.

2.2 Instruments

The morphologies of all samples were characterized by high-resolution transmission electron microscope (HR-TEM) (JEM-2100, Hitach, Japan). Fourier transform infrared spectroscopy was performed on a Magna-560 spectrometer (Nicolet, Madison, WI). The UV–vis absorption spectra were monitored using a Shimadzu UV-29100 spectrophotometer (Tokyo, Japan). All fluorescence spectra were measured on a Cary Eclipse fluorescence spectrophotometer (Varian American Pty ltd., USA) with a 1 cm path-length quartz cell. The excitation wavelength was 340 nm, and the silt widths of excitation and emission were set at 10 nm.

2.3 Preparation of ssDNA modified QDs

Three ssDNA modified QDs were prepared as follows: 23.0 μL EDC (10 mg mL⁻¹) and 40 μL QDs (10 μmol/L) were added to 200 μL PBS buffer (100 mmol/L, pH = 6.0). The solution was continuously and gently agitated for 30 min to activate the carboxyl group. Subsequently, the pH of the above solution was adjusted immediately to 8.0 with PBS (pH = 8.5) prior to the addition of 13.8 µL NHS (10 mg mL⁻¹) and a certain concentration of complementary DNA. The mole ratio of complementary DNA to QDs was 40:1. After gently shaking for 3 h at room temperature, the resulting solutions were placed in a refrigerator at 4 ℃ overnight. Then, an ultrafilter tube (30 k_D) was used to centrifuge the solution to remove excess ssDNA, EDC, and NHS. The final products were diluted to 5 mL for the subsequent experiments.

2.4 Synchronous screening of hepatitis virus DNA

The as-prepared QDs@C_HAV-DNA, QDs@C_HBV-DNA, and QDs@C_HCV-DNA solution with the same concentration was mixed together in equal proportions. The mixed solution was abbreviated as QDs@C_DNA. Then, 60 µL QDs@C_DNA mixed solution and 500 µL Tris-HCL buffer (20 mmol/L Tris-HCL, 0.1 mol/L NaCl, pH = 7.4) were added to a series of 5.0 mL colorimeter tubes. And then HAV-DNA, HBV-DNA and HCV-DNA solution with different concentrations were added and a constant volume of 1 mL was achieved using distilled water. After mixed thoroughly and hybridized for 60 min in a thermostat water bath at 37 ℃, 150 µL GO (0.5 mg mL⁻¹) was added into the above mixed solution, and the solution volume was diluted to 2.5 mL with distilled water and incubated for 30 min at room temperature. The fluorescence spectra were recorded from 400 nm to 700 nm at an excitation wavelength of 340 nm, and the slit widths for excitation and emission were set at 10 nm.

2.5 Screening of hepatitis virus DNA in real sample

The serum samples were purchased from Sigma-Aldrich Company, which were diluted 10 times with distilled water before use. Then,16 nM and 64 nM of the target hepatitis virus DNA were added to the samples, respectively. The labeled samples with different standard additions were examined at an excitation wavelength of 340 nm under the optimized conditions, and the fluorescence spectra of the QDs@C_DNA and GO/DNA system were observed in the range of 400–700 nm.

3.1 Construction of multicolor fluorescence system

Three kinds of QDs have good dispersion and spherical morphology (Fig. S1A-C). The average hydrodynamic size of QDs(5 2 5), QDs(5 8 5), and QDs(6 3 2) is 11.62 nm, 12.54 nm, and 13.44 nm, respectively (Fig. S1D-F). In order to verify the feasibility of constructing multicolor fluorescence system using QDs with different maximum emission wavelength, the fluorescence emission spectrums of QDs(5 2 5), QDs(5 8 5), and QDs(6 3 2) were firstly measured with the same concentration at the exciting wavelength of 340 nm. As presented in Fig. 1A, the maximum emission intensities of QDs(5 2 5), QDs(5 8 5), and QDs(6 3 2) corresponded to 525 nm, 585 nm, and 632 nm, respectively. It is consistent with the instruction manual provided by Suzhou Xingshuo Nanotech Co., ltd, and can be found on the company website. Besides, it also could be found that there is no obvious overlap between the emission spectrum of three QDs, which indicated that the emission signal of each QDs can be detected individually in a mixed system of QDs(5 2 5), QDs(5 8 5), and QDs(6 3 2). Moreover, it was exciting that no significant change was observed in the fluorescence intensity of QDs(5 2 5) after QDs(5 8 5) and QDs(6 3 2) were added in succession, and the emission spectrum of QDs(5 2 5) did not show obvious blue-shift and red-shift (Fig. 1B). The similar results were also observed even if the sampling sequence of the QDs was swapped (Fig. 1C, D).

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Fig. 1

(A)The emission fluorescence spectra of pure QDs(5 2 5), QDs(5 8 5) and QDs(6 3 2). (B)-(D) Comparison of the fluorescence intensity of a single QDs and that of mixture QDs.

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In order to realize the selective recognition of three hepatitis viral DNA, QDs(5 2 5), QDs(5 8 5), and QDs(6 3 2) was modified by complementary ssDNA strands of HAV-DNA, HBV-DNA and HCV-DNA, respectively. The modified QDs were referred to as QDs@C_HAV-DNA, QDs@C_HBV-DNA, and QDs@C_HCV-DNA. Compared with the unmodified QDs, typical DNA absorption peaks between 260 and 280 nm can be observed in UV–vis absorption spectrums of the QDs@C_HAV-DNA, QDs@C_HBV-DNA, and QDs@C_HCV-DNA (Fig. S2), indicating the ssDNA strands were successfully modified on the surface of QDs. As shown in Fig. 2A, the fluorescence intensity of modified QDs did not change obviously relative to those of unmodified QDs. The solution of QDs@C_HAV-DNA, QDs@C_HBV-DNA, and QDs@C_HCV-DNA presented green, yellow and pink fluorescence under UV illumination, respectively (Fig. 2B). The kinetic analysis of the change of fluorescence intensity of three modified QDs showed that the fluorescence intensity of QDs@C_HAV-DNA, QDs@C_HBV-DNA, and QDs@C_HCV-DNA remained relatively stable within 60 min (Fig. S3A-C). The fluorescence spectrums of QDs@C_DNA remained almost unchanged (inset in Fig. S3 A-C). The quantum yield of QDs(5 2 5), QDs(5 8 5), and QDs(6 3 2) was 83.4 %, 87.3 %, 89.5 %, respectively. After modification with HAV-DNA, HBV-DNA, and HCV-DNA, the quantum yield of QDs@C_HAV-DNA, QDs@C_HBV-DNA, and QDs@C_HCV-DNA was 80.1 %, 83.1 %, 84.6 %, respectively. It indicated that ssDNA modification does not change the fluorescence properties of QDs(5 2 5), QDs(5 8 5), and QDs(6 3 2). These results indicated that the QDs@C_HAV-DNA, QDs@C_HBV-DNA, and QDs@C_HCV-DNA have strong fluorescence properties and good optical stability.

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Fig. 2

(A)The emission fluorescence spectra of the modified QDs and the unmodified QDs. (B) Photograph of the QDs@C_HAV-DNA, QDs@C_HBV-DNA, and QDs@C_HCV-DNA under UV illumination. (C) The change of fluorescence emission spectra of QDs@C_DNA in presence of GO (30 μg mL⁻¹). (D) Fluorescence emission spectra of QDs@C_DNA/GO with different concentrations of HAV-DNA (1.0 nM and 128 nM).

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As aforementioned, GO can effectively adsorb ssDNA via hydrophobic and π-π stacking interactions between DNA nucleobases and sp²-hybridized atoms of GO nanosheets, but it has low affinity toward double-strand DNA (dsDNA) (Liu et al., 2016a; Wang et al., 2018). Besides, GO can effectively quench the fluorophore of QDs through energy resonance transfer. Here, one of our concerns is that whether the fluorescence of QDs with three emission wavelengths can be synchronously quenched. Consequently, the fluorescence quenching properties of GO for QDs@C_HAV-DNA, QDs@C_HBV-DNA, and QDs@C_HCV-DNA were investigated. From Fig. S4 it could be seen that the GO is transparent due its single layered structure and has irregular edges. As illustrated in Fig. 2C, the fluorescence intensity of the mixed system significantly reduced after the addition of GO, and the fluorescence quenching efficiency for QDs@C_HAV, QDs@C_HBV and QDs@C_HCV was approximately 79.71 %, 78.04 %, and 84.56 %, respectively. Thus, the fluorescence of QDs@C_HAV, QDs@C_HBV and QDs@C_HCV could be quenched simultaneously by GO.

As shown in Fig. 2D, when HAV-DNA was incubated with QDs@C_HAV-_DNA for 60 min prior to the addition of GO, the fluorescence intensity of QDs@C_HAV-_DNA was obviously recovered at 525 nm, but there was no obvious change of the fluorescence intensity at 585 nm and 632 nm. If the HBV-DNA or HCV-DNA was incubated with QDs@C_HBV-_DNA or QDs@C_HCV-_DNA before adding GO, the fluorescence intensity of the QDs@C_HBV-_DNA and QDs@C_HCV-_DNA at 585 nm, and 632 nm also recovered obviously, illustrating the proposed system can effectively recognize and distinguish three kinds of target DNA(Fig. S5A-B). If the DNA_HAV, DNA_HBV, and DNA_HCV were mixed in equal proportion and incubated with QDs@C_DNA together, the fluorescence intensity was recovered simultaneously at 525 nm, 585 nm and 632 nm (Fig. S5C). This further confirmed that current conception is feasible and can be used for constructing a synchronous screening system for three kinds of hepatitis virus DNA.

3.2 Optimization of conditions for synchronous screening of hepatitis viral DNA

The effect of GO concentration on the fluorescence intensity of the multicolor fluorescence system was investigated. As shown in Fig. S6A, the fluorescence intensity corresponding to 525 nm, 585 nm and 632 nm decreased gradually with the increase of GO concentration. From the response curve of the fluorescence intensity ratio (F/F₀) to GO concentration (where F and F₀ refer to the fluorescence intensity of the multicolor system in the presence and absence of GO, respectively), it could be seen that the fluorescence intensity did not change when the GO concentration was higher than 30 μg mL⁻¹ (Fig. S6 B). Thus, 30 μg mL⁻¹ GO was selected for the subsequent experiments.

As we know, weak alkaline medium is favorable for DNA hybridization. The effect of pH on fluorescence intensity of the multicolor fluorescence system was investigated over the pH range of 6.0–8.5 (Fig. S6C). The fluorescence quenching efficiency of GO for three kinds of QDs@C_DNA was relatively high at pH 7.4, but decreased at pH higher than 7.4. In the presence of target DNA, the maximum fluorescence recovery ratio was achieved at pH = 7.4. In order to ensure the high sensitivity of the system, Tris-HCl buffer with pH 7.4 was selected for the subsequent experiments.

Fig. S6D displayed the effects of incubation time on analytical performance of the multicolor fluorescence system. The fluorescence intensity of the system in the presence of GO gradually decreased with the prolonged incubation time and plateaued within 30 min at room temperature. Thus, 30 min was selected as the reaction time between QDs@C_DNA and GO. The hybridization reaction time of QDs@C_DNA/GO and target DNAs was further optimized. It could be seen that the fluorescence intensity ratio of the system gradually increased with the extension of hybridization time, and did not obviously change after 60 min (Fig. S6E). Thus, 60 min was selected as the hybridization time.

The ionic strength played an important role in DNA hybridization reaction. the fluorescence recovery ratio F/F₀ was optimal when the NaCl concentration in hybridized buffer was 50 mM (Fig. S6F). Therefore, 50 mM NaCl solution was used in this study.

3.3 Synchronous screening of hepatitis viral DNA

The analytical performance of the constructed system was evaluated under the optimized conditions by adding three kinds of hepatitis viral DNA with the same proportion. As shown in Fig. 3A, the fluorescence intensities of the system at 525 nm, 585 nm, and 632 nm simultaneously increased with the increase of HAV-DNA, HBV-DNA, and HCV-DNA concentration. Fig. 3B showed a good linear relationship between the fluorescence intensity ratio F/F₀ (F and F₀ refer to the fluorescence intensity of the system in the presence and absence of target DNA, respectively) and the concentration of three hepatitis virus DNA. The F/F₀ presented a sensitive response to the concentration of HAV-DNA, HBV-DNA, and HCV-DNA in the range of 1.0–192 nM, 8.0–192 nM, and 1.0–128 nM, respectively. The standard curves for HAV-DNA, HBV-DNA and HCV-DNA were F/F₀ = 0.012C_(HAV-DNA) + 1.19 (R² = 0.992), F/F₀ = 0.0102C_(HBV-DNA) + 1.27(R² = 0.984) and F/F₀ = 0.0132C_(HCV-DNA) + 1.25 (R² = 0.985), respectively. Based on 3σ/S, the detection limit for HAV-DNA, HBV-DNA, and HCV-DNA were calculated as 0.46 nM, 1.53 nM, and 0.58 nM. The relative standard deviation (RSD) for five times repeated detection of 32 nM HAV-NDA, HBV-DNA, and HCV-DNA was about 3.07 %, 3.73 %, 3.45 %, respectively. It indicated acceptable reproducibility of the proposed multicolor fluorescence system.

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Fig. 3

(A) Fluorescence spectra of the multicolor fluorescence system with different concentrations of HAV-DNA, HBV-DNA, and HCV-DNA (0, 1.0, 8.0, 16.0, 32.0, 64.0, 128.0, 192.0 nM). (B)The relationships between fluorescence intensity ratio (F/F₀) and the concentration of HAV-DNA, HBV-DNA, and HCV-DNA.

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Table 1 shows the comparison between the proposed method and other reported methods for hepatitis virus DNA in terms of detection range and limit. The analytical parameters of the constructed system are comparable to or better than those of fluorescence methods (Chen et al., 2015; Zhu et al., 2015; Zeng et al., 2018; Fakih et al., 2017). Compared with single or double target detection, the constructed probe could rapidly detect three kinds of hepatitis viral DNA simultaneously. Although the sensitivity of our constructed system is inferior to those of some electrochemical methods and molecularly imprinted fluorescence sensor (Honorato Castro et al., 2014; Liu et al., 2016b; Luo et al., 2019), modifier preparation and electrode modification in these strategies are sophisticated and time consuming. In term of the operation process, the proposed system is convenient and time-efficient.

3.4 Specificity

The specific performance of the constructed multicolor fluorescence system was investigated by recognition of three hepatitis virus DNA in a coexistence system containing their single-base, double-base, and triple-base mismatched ssDNA strands. As displayed in Fig. 4, the fluorescence intensity of three hepatitis virus DNA was apparently recovered at 525 nm, 585 nm and 632 nm, which corresponded to high fluorescence intensity ratio (F/F₀). Compared with hepatitis virus DNA, their single-base, double-base, and triple-base mismatched ssDNA strands could not induce significantly recovery of the fluorescence. This is mainly due to the low hybridization capability of ssDNA towards its base mismatched complementary sequences. The constructed system can effectively distinguish and screen three kinds of hepatitis virus DNA from numerous ssDNA strands. To evaluate the anti-interference of the proposed multicolor fluorescence system, the effect of some coexistent substances (eg. biomolecules and ions) in the biological fluids on the detection of hepatitis virus DNA was investigated. As displayed in Table S2, 100 μM Na⁺, K⁺, and Ca²⁺ can hardly affect the detection of three hepatitis virus DNA. Moreover, 200 μM l-Cys, l-Gly, ATP, and BSA also had no obvious effect on the detection results. All of the observations above clearly indicated the high selectivity of the proposed method.

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Fig. 4

Selectivity investigation of the proposed method in the presence of target DNA and different base mismatch strands. The concentration of HAV-DNA, HBV-DNA, and HCV-DNA was 192 nM, 192 nM and 128 nM, respectively. The concentration of GO was 30 μg mL⁻¹. The concentration of base mismatched ssDNA was the same as the target DNA.

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3.5 Screening of hepatitis viral DNA in serum sample

In order to investigate the applicability of the proposed system, spiked recovery experiments were performed by adding three kinds of hepatitis virus DNA with two concentrations into the diluted serum sample. The results were presented in Table 2. The recoveries for HAV-DNA, HBV-DNA and HCV-DNA were 92.19 %-108.20 %, 94.13 %-105.13 %, and 91.18 %-94.56 %. These results demonstrated the potential applicability of the proposed system for diagnosis of viral hepatitis by screening viral DNA.