A one-step fluorescent biosensing strategy for highly sensitive detection of HIV-related DNA based on strand displacement amplification and DNAzymes

Sensitive and specific detection of HIV-related DNA is of great importance for early accurate diagnosis and therapy of HIV-infected patients. Here, we developed a one-step and rapid fluorescence strategy for HIV-related DNA detection based on strand displacement amplification and a Mg2+-dependent DNAzyme reaction. In the presence of target HIV DNA, it can hybridize with template DNA and activate strand displacement amplification to generate numerous DNAzyme sequences. With the introduction of Mg2+, DNAzyme can be activated to circularly cleave the substrate DNA, which leads to the separation of fluorophore reporters from the quenchers, resulting in the recovery of the fluorescence. Under the optimal experimental conditions, the established biosensing method can detect target DNA down to 61 fM with a linear range from 100 fM to 1 nM, and discriminate target DNA from mismatched DNA perfectly. In addition, the developed biosensing strategy was successfully applied to assay target DNA spiked into human serum samples. With the advantages of fast, easy operation and high-performance, this biosensing strategy might be an alternative tool for clinical diagnosis of HIV infection.


Introduction
Human immunodeciency virus (HIV), a retrovirus, can lead to acquired immunodeciency syndrome (AIDS) which destroys the infected patient's immune system. 1 There are currently 36.7 million people living with HIV worldwide, but almost 60% of infected adults don't have access to antiretroviral treatment mostly due to the failure of early diagnosis. 2 It is reported that the mortality rate of AIDS has decreased signicantly with the wide application of combined antiretroviral therapy. 3 Therefore, early diagnosis and therapy of HIV infection can effectively prevent HIV transmission and prolong survival period. 4 Immunological methods such as enzyme-linked immunosorbent assay (ELISA) and western blot (WB) are common methods for clinical diagnosis of HIV infection. 5,6 However, it takes a period of time for the body to produce the corresponding antibodies aer infection with the HIV virus. Therefore, there is a "window period" for HIV detection, which makes the early diagnosis of HIV infection full of challenges. 7,8 In recent years, nucleic acid assays have attracted increasing attention because they can prompt for viral infection even in the absence of HIV antibodies. 9 With high sensitivity and speci-city, polymerase chain reaction (PCR) has evolved as the most commonly used technique for nucleic acid detection. Nevertheless, it is not suitable for short-length DNA detection due to the complexity in primer design. 10 Moreover, PCR-based methods need thermal cycler and specialized person which limit their use in resource-poor and impoverished areas. Thus, it is highly desirable to develop universal and cost-effective approaches for sensitive and specic detection of HIV-related DNA.
So far, various biosensing methods have thrown light on HIV-related DNA detection, such as colorimetry, 11 electrochemistry, 12 surface plasmon resonance (SPR), 13 and uorescence resonance energy transfer (FRET) etc. 14 Among these methods, uorescent method usually takes precedence as an alternative platform owing to its advantages of high sensitivity and simple procedure. To further improve the performance of the biosensors, different isothermal amplication strategies have been developed for genetic test and medical diagnosis, such as rolling circle amplication (RCA), 15-18 hybridization chain reaction (HCR), 19,20 catalytic hairpin assembly (CHA), 21,22 exonuclease III aided signal amplication, 23,24 and strand displacement amplication (SDA). 25 Combining polymerasemediated strand extension with nicking enzyme-assisted single strand cleaving process, SDA is an isothermal molecular chain reaction with high amplication efficiency. Due to the DNA biological circuit with feedback design, the target DNA switches to exponentially generate numerous secondary DNA molecules. Herein, SDA-based signal amplication techniques show great potential for highly sensitive and effective determination of target DNA. 26 When coupled with other signal amplication strategies, it can be applied to construct versatile biosensing system. DNAzyme, as an alternative tool enzyme to protein enzyme or ribozyme, is a kind of functional catalytic nucleic acid sequence selected by systematic evolution of ligands by exponential enrichment (SELEX) in vitro. 27 Horseradish peroxidase (HRP) mimicking DNAzyme possesses a special G-quadruplex structure which exhibited peroxidase-like activity aer binding to hemin and catalyzed to generate a colorimetric or chemiluminescent signal. [28][29][30] Although these colorimetric or chemiluminescent approaches based on HRP-mimicking DNAzyme are wellestablished, they usually involve complicated operation process, long assay time and insufficient sensitivity. Differing from the HRP-mimicking DNAzyme, metal ion-dependent DNAzyme is a class of RNA-cleaving DNAzyme which can catalyze the cleavage of RNA substrates or the ribonucleotides embedded in a chimeric DNA substrate with metal ion as cofactors. 31 Due to the inherent advantages of signicant catalytic efficiency, excellent biocompatibility and good stability, metal ion-dependent DNAzyme is much more amenable to combing with other signal amplication strategies and has attracted substantial research in various detection platforms. Particularly, because of its cyclic cleavage property, the metal ion-dependent DNAzyme can also be applied as signal amplication unit. [32][33][34] Thus, metal ion-dependent DNAzyme is frequently combined with other signal amplication strategies 35,36 to further improve the detection performance of constructed biosensors.
Herein, a facile, rapid and one-step homogeneous uorescence strategy was developed for HIV-related DNA detection by integrating SDA with Mg 2+ -dependent DNAzyme catalytic reaction. The proposed method exhibited good performance for HIV-related DNA detection with high sensitivity and excellent mismatch distinguishing ability. More importantly, the combination of SDA signal amplication and Mg 2+ -dependent DNAzyme catalytic recycling in our strategy ensures the entire reaction to be performed by one-step operation and reduce the detection time to 60 min. In addition, it could also be applied to assay the concentration of target HIV DNA spiked into human serum samples. Therefore, the proposed uorescence biosensing strategy presents an excellent platform towards HIV-related DNA analysis, which has great application potential for biomedical research and clinical diagnosis of HIV infection.

Materials and reagents
All oligonucleotide sequences were synthesized and purchased from Sangon Inc. (Shanghai, China). Table S1 in ESI † shows the sequences of the oligonucleotides used in the study. All oligonucleotides were HPLC-puried and dissolved in tris-ethylenediaminetetraacetic acid (TE) buffer (pH 8.0, 10 mM Tris-HCl, 1 mM ethylene diamine tetraacetic acid) and stored at À20 C, which were diluted in appropriate buffer prior to use. DL20 DNA marker was purchased from Takara (Dalian, China). Gold view (GV) was purchased from SBS Genetech (Beijing, China). Klenow fragment and Nb.BbvCI nicking enzyme were purchased from New England Biolabs Inc. (Beverly, MA, USA). Deoxynucleotide triphosphates (dNTPs) were obtained from Sangon Inc. (Shanghai, China). All other chemicals not mentioned here were of analytical reagent grade. Millipore-Q water ($18 MU) was used in all experiments. Human serum samples were obtained from the First Affiliated Hospital of Chongqing Medical University.

Isothermal amplication system
The strand displacement amplication and DNAzyme cleavage reaction were simultaneously performed in onestep. The reaction mixture contained 0.5 mM template, 0.25 mM uorophore/quencher-functionalized substrates, various concentrations of target DNA, 0.5 mM dNTPs, 5 unit Nb.BbvCI nicking enzyme and 3 unit Klenow fragment in 1Â CutSmart™ buffer (20 mM pH 7.9 Tris-acetate, 500 mM potassium acetate, 10 mM magnesium acetate, 100 mg mL À1 BSA) as well as 1Â Klenow fragment buffer (100 mM pH 7.5 Tris-HCl, 70 mM MgCl 2 , 1 mM DTT) to yield a nal volume of 20 mL, then the mixture was incubated at 37 C for 60 min.

Fluorescence measurement
Aer the strand displacement amplication and DNAzyme cleavage reaction, 80 mL H 2 O was added to the mixture to yield a nal volume of 100 mL. Then, all uorescence measurements were performed on a Cary Eclipse Fluorescence spectrophotometer (Agilent, California), using a quartz uorescence cell with an optical path length of 1.0 cm. The uorescence emission spectra were recorded in the region from 500 to 600 nm in a quartz cuvette at an excitation wavelength of 492 nm. The maximum uorescence emission intensity was obtained at 518 nm. Both the excitation and emission slit widths were set at 5 nm at room temperature. Prior to each experiment, all cuvettes were washed with 70% ethanol and distilled water.

Gel electrophoresis
The feasibility of the SDA reaction was explored by 8% native polyacrylamide gel electrophoresis (PAGE), which was conducted on DYY-6C electrophoresis analyzer (Liuyi Instrument Company, China) in 1Â TBE buffer (90 mM Tris-HCL, 90 mM boric acid, 2 mM EDTA, pH 7.9) at a 120 V constant voltage for 30 min. The gel was stained with gold view (GV) for 30 min and photographed by gel imaging analysis system (Bio-Rad, USA).

Human serum samples preparation
The human serum samples were diluted 25 times prior to detection. Then, the target DNA was detected in these human blood plasma samples following the same procedure.

Principle of the uorescence assay
In this work, a signal-on uorescence method was developed for HIV-related DNA detection based on strand displacement amplication and Mg 2+ -dependent DNAzyme catalytic reaction. Fig. 1 shows the working principle. The system includes templates, ribonucleobase-containing substrate DNA functionalized with a uorophore/quencher pair, Nb.BbvCI nicking enzyme, Klenow fragment, deoxynucleotide solution mixture (dNTPs) and Mg 2+ ions. The template consists of two nucleic acid scaffolds that include recognition site for the target DNA and replication track that yields the nicking domain for Nb.BbvCI and Mg 2+ -dependent DNAzyme sequence. In the presence of target DNA, the specic hybridization between target DNA and the corresponding domain of the template forms a partial duplex. Then, along the template the target DNA is extended to form a complete duplex with the help of Klenow fragment and dNTPs. Subsequently, the nicking enzyme can specically recognize the newly formed duplex nicking site, cleaving the upper extended DNA strand and exposing a new replication site for polymerase. This results in the secondary replication of the strand, while displacing the DNAzyme sequence. Thus, a large number of DNAzyme sequences are produced through the continuously extension, cleavage and strand displacement amplication. Aer introduction of cofactor Mg 2+ ions, DNAzyme sequence can be activated to circularly cleave the uorophore/quencherfunctionalized nucleic-acid substrates. Aer cleavage, uorophore/quencher pairs of substrates are separated from each other, resulting in great amplication uorescence signal.

Feasibility assay of the sensing strategy
To verify whether the target DNA could initiate the reaction, leading to a detectable uorescence signal, we explored the feasibility of this proposed method through comparative experiments, in which uorescence emission spectra of different mixtures were recorded. As shown in Fig. 2A, the mixture without target DNA showed very weak uorescence intensity at 518 nm due to the efficient quenching of FAM by the closely positioned BHQ-1 (curve d). The control experiment without Klenow fragment (curve b) or Nb.BbvCI (curve c) also exhibited weak uorescence intensity, indicating that exclusion of either the nicking endonuclease or polymerase could prohibit the strand displacement amplication. On the contrary, the uorescence intensity had signicant enhancement upon the addition of target DNA to the mixture containing both the Klenow fragment and Nb.BbvCI (curve a). Thus, the cooperative polymerization by the polymerase and scission by the nicking enzyme were indispensable to self-assemble the DNAzyme sequences.
Furthermore, a native polyacrylamide gel electrophoresis (PAGE) was also carried out to verify the strand displacement reaction products. As shown in Fig. 2B, the newly produced band, representing the products of SDA, was observed with the addition of target DNA (lane 1). However, the corresponding products could not be observed in the control group without the addition of Nb.BbvCI (lane 2) or Klenow fragment (lane 3). These results were in line with our previous uorescence results. Consequently, our results demonstrated that SDA could be initiated in the presence of target HIV DNA and effectively generated a large number of DNAzyme sequences.

Optimization of experiment conditions
In order to obtain the best analytical performance of the proposed strategy, several parameters were optimized, such as Klenow fragment concentration, Nb.BbvCI nicking enzyme amount, substrate concentration and total reaction time (Fig. 3). The uorescence intensity was used to assess the performance. Firstly, it was clear that strand displacement amplication played crucial role for high sensitivity detection of target in this strategy. Therefore, the two key factors of Klenow fragment and Nb.BbvCI nicking endonuclease concentration in this process were optimized. Fig. 3A presented the effect of Klenow fragment concentration on uorescence response. With increasing polymerase concentration from 1.0 to 3.0 units, the uorescence intensity increased gradually, and the uorescence signal tended to be a steady value at 3.0 units. Therefore, 3.0 units were selected as the optimal polymerase concentration in the subsequent experiments. The effect of Nb.BbvCI nicking endonuclease concentration on uorescence response was also investigated, and the results were illustrated in Fig. 3B. When the concentration of Nb.BbvCI varied from 2 to 5 units, the uorescence intensity improved correspondingly, further enhancing Nb.BbvCI concentration, the uorescence intensity tended to level off. Therefore, 5 unit was selected as the optimal concentration. Secondly, the inuence of functionalized substrate concentrations was investigated. Fig. 3C showed the effect of the concentration of uorophore/ quencher-functionalized substrates. The uorescence signal reached maximum when the concentration of substrates probe was 250 nM and decreased at higher concentrations. Therefore, we set the concentration of substrates as 250 nM throughout this work. Finally, the impact of the total reaction time was illustrated in Fig. 3D. As the reaction time was extended from 20 to 60 min, the uorescence intensity increased sharply and reached maximum at 60 min, then it tended to decrease aer 60 min. Consequently, 60 min was chosen as the optimal reaction time.

Analytical performance of the biosensing method
The dynamic range and sensitivity of the proposed uorescence strategy was conrmed under the optimal experimental conditions. As shown in Fig. 4A, the uorescence intensity moved up with the increase of target HIV DNA concentration. In the range from 100 fM to 1 nM of target DNA concentration, the calibration plots demonstrated a good linear relationship between the uorescence intensity (F) (at 518 nm) and the logarithm of target DNA concentrations (C) (Fig. 4B). The resulting linear equation was F ¼ 17.4847 lg C (pM) + 72.7695 with a correlation coefficient of 0.9974 and detection limit of 61 fM from three times the standard deviation corresponding to the blank sample detection, which was much lower than previous reported methods for detecting HIV-related DNA (Table S2 †). The high sensitivity of the developed strategy could be attributed to the high amplication efficiency of strand displacement amplication as well as the Mg 2+dependent DNAzyme catalytic reaction, providing an alternative detection strategy for HIV-related DNA.

Specicity and reproducibility of the biosensing method
To investigate the selectivity of the developed method, the uorescence intensity for different targets (1 nM) were tested under the same experimental conditions, including target HIVrelated DNA, single base mismatched DNA-1, two-base mismatched DNA-2 and non-complementary DNA-3 (Fig. 5). The results showed that DNA-3 (curve d) caused negligible changes in uorescence signals against the blank test (curve e), the DNA-1 (curve b) and DNA-2 (curve c) caused only a slight increase. However, in the presence of HIV DNA, a dramatic increase in the uorescence signal was obtained (curve a). It could be ascribed to the fact that only target HIV DNA could hybridize with template DNA with high efficiency and activate strand displacement amplication to generate abundant Mg 2+ -dependent DNAzyme for signal transduction. This result also demonstrated that the designed method had good selectivity and single nucleotide difference discrimination ability. Five replicate measurements of target HIV DNA at 1 pM and 100 pM showed the variation coefficients of 4.6% and 1.5%, respectively.

Detection of HIV-related DNA in human serum samples
To investigate the interference of biological samples on the uorescence strategy, the proposed spiked assay was carried out by adding different concentrations (10 pM, 100 pM and 1 nM) HIV-related DNA to 25-fold-diluted serum sample. As shown in Table S3, † the recoveries for three detections were 92.5%, 115.7% and 97.7%, respectively, suggesting that complex  mixtures have little effect on the detection performance. Thus, the established strategy could become a potential tool for HIV DNA assay in real biological samples.

Conclusion
In summary, we have successfully demonstrated a one-step and rapid uorescence biosensing strategy for HIV-related DNA detection based on SDA and Mg 2+ -dependent DNAzyme catalytic reaction. Under the cyclic process of extension, cleavage and strand displacement reaction, low amount of target HIVrelated DNA could generate lots of Mg 2+ -dependent DNAzyme sequences with excellent cyclic cleavage property, which greatly improved the sensitivity for detection of target DNA down to 61 fM. Even more noteworthy, the combination of SDA signal amplication and Mg 2+ -dependent DNAzyme catalytic recycling in our strategy ensures the entire detecting process to be performed by one-step operation and only takes 60 min to get an excellent detection uorescence signal. Thus, the proposed uorescence biosensing strategy has great application potential for clinical diagnosis of HIV infection. More importantly, this uorescence method could be conveniently applied to other nucleic acids detection by adjusting the corresponding DNA sequences. Therefore, with one-step operating process and good analytical performance, the developed strategy offers great potential for point-of-care diagnostics of disease related DNA, miRNA as well as other biomarkers.

Conflicts of interest
The authors declare no conict of interest.