Exonuclease III-Assisted Target Recycling Amplification Coupled with

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Exonuclease III-Assisted Target Recycling Amplification Coupled with Liposome-Assisted Amplification: One-Step and Dual-Amplification Strategy for Highly Sensitive Fluorescence Detection of DNA Fulin Zhou and Baoxin Li* Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China S Supporting Information *

ABSTRACT: Detection of ultralow concentration of specific DNA sequence is a central challenge in the early diagnosis of gene-related disease and biodefense application. Herein, we report a dualamplification strategy for highly sensitive fluorescence detection of DNA. In this proposed strategy, a dumbbell-shaped DNA probe is designed to integrate target binding, magnetic separation, and signal response. In the presence of specific DNA target, the multifunctional dumbbell probe can initiate exonuclease III (Exo III)-aided target recycling amplification, and, in the meantime, generate a large number of fluorescein (FAM)-encapsulated liposomes. The developed method offers very high sensitivity due to primary amplification via numerous FAM from a liposome and secondary amplification via target recycling amplification. The detection limit of the proposed method can reach 4 aM, which is much lower than that of the Exo III-aided target recycling technique applied for DNA quantification without FAM-encapsulated liposomes amplification. Moreover, the dual-signal amplification process can be completed one-step in this system. Therefore, this method provides a simple, isothermal, and low-cost approach for sensitive detection of DNA and holds a great potential for early diagnosis in gene-related diseases.

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termini. It is not active on 3′-overhang ends of double-stranded DNA or single-stranded DNA. Thus, Exo III provides a more versatile platform for amplification detection of DNA. In the reported Exo III-assisted signal amplification strategies, the signaling probes are selectively digested by Exo III with target cycling, generating multiple signaling events and achieving signal amplification.7−13 However, most of the Exo III-based signal amplification strategies can only achieve the detection limits in pM range.7−9,12 To further upgrade the detection sensitivity to make it rival the sensitivity of PCR, Exo III-based dual signal amplification14,15 and Exo III-based cascade signal amplification11,13,16−18 have recently been proposed. Indeed, the advanced techniques can significantly improve the detection sensitivity. However, the relatively high number of working steps in the advanced Exo III-based strategies makes them complex and time-consuming, as well as cost- and laborintensive. Thus, the development of a convenient and simple Exo III-based strategy for sensitive detection of DNA is still highly desirable. Liposomes, which are spherical phospholipid bilayer vesicles, have been widely applied as carriers of molecules in the fields of nanodevices, drug delivery, and gene delivery, as well as a mimic for cell membranes.19,20 Liposomes have attracted great attention for application in the bioassay field over several decades as a means to amplify the signal.21 Liposomes can

n order to profile the low abundance of target DNA to serve the early stage clinical diagnosis and medical treatment for some major diseases (for example, cancers), the development of amplification strategies to fabricate ultrasensitive DNA detection has recently received much effects.1−3 Target amplification and signal amplification are two commonly used approaches to increase the detection sensitivity. As the classical target amplification method, polymerase chain reaction (PCR) is the most widely used method for the detection of special DNA sequences, because of its excellent amplification efficiency, improving the sensitivity to the extent that the single-molecule detection limit is often realized.4 However, PCR-based methods require special thermal cycling equipment and DNA polymerase, making the assay procedure complicated and time-consuming.5 Besides, PCR is prone to yielding falsepositive results from the artifact amplification, which may have effect on the detection specificity.6 In order to overcome this problem, the nuclease-assisted signal amplification strategies, in which one target molecule leads to many cycles of targetdependent nuclease digestion of reporter molecules, have been developed for sensitive DNA detection.1−3,7−9 The target recycling strategies can directly amplify the amounts of signal reagents, which holds great promise as a substitute for PCR, because of its easy operation and isothermal reaction.9 Among the nuclease-based signal amplification methods, exonuclease III (Exo III) is employed more widely, because it does not require a specific recognition site.7−13 Exo III can only catalyze the stepwise removal of mononucleotides from 3′-hydroxyl termini of double-stranded DNA with blunt or recessed 3′© XXXX American Chemical Society

Received: March 15, 2015 Accepted: June 25, 2015

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DOI: 10.1021/acs.analchem.5b00993 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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EXPERIMENTAL SECTION Chemicals and Materials. All oligonucleotides designed in this study were commercially synthesized by Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China), and purified using high-performance liquid chromatography (HPLC). The sequences of oligonucleotides are listed in Table 1. Amine group-modified magnetic beads (MBs) were

encapsulate various signal markers, including colorimetric dye,22 fluorophores,23,24 electrogenerated chemiluminescent markers,25,26 electrochemical markers27 and chemiluminescent markers,28 enzymes,29 and DNA.30 In these assay systems, the signal amplification is achieved by detecting the large amount of marker molecules released from liposomes. In addition, the ability to modify the liposome surface structure to perform recognition functions with a wide range of types of analytes is an important aspect of their use in bioanalysis. In this study, a simple ultrasensitive fluorescence biosensing platform for DNA detection has been developed based on fluorescein-encapsulated liposomes as primary signal amplification strategy and Exo III-assisted target recycling as secondary signal amplification strategy. The designed strategy is depicted in Scheme 1. A magnetic bead and a liposome are covalently

Table 1. Sequences of Oligonucleotides oligonucleotide

sequence

probe DNA

5′-CHO-(CH2)6-GGG TTT CTG TTT CTT CTG CTG AGG AGG TTT GTT CTT CGG TGC TAG TGT-(CH2)6-NH2-3′ 5′-ACA CTA GCA CCG TTA A-3′ 5′-ACA CTG GCA CCG TTA A-3′ 5′-ACT CTA GCA GCG TTA A-3′ 5′-CGT TCG AAC GTA TTA A-3′

target DNA T1 T2 Tn

Scheme 1. Schematic Illustration of the Dual-Amplification Strategy for DNA Detection through Exonuclease IIIAssisted Target Recycling Amplification Coupled with Liposome-Assisted Amplification

obtained from Shaanxi Lifegen Co., Ltd. (Xi’an, China). Dipalmitoyl phosphoethanolamine (DPPE), dipalmitoyl phosphocholine (DPPC), cholesterol, glycollic acid, glutaraldehyde, fluorescein (FAM), 1-ethyl-3 (3-(dimethylamino)-propyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were obtained from Sigma−Aldrich (St. Louis, MO, USA). Phosphate buffered saline (PBS) consisted of 0.1 M NaH2PO4, 0.1 M Na2HPO4, 0.1 M KCl, and 10 mM MgCl2 (pH 7.4). PBS-T consisted of 0.1 M NaH2PO4, 0.1 M Na2HPO4, 0.1 M KCl, 10 mM MgCl2, and 0.02% Tween-20 (pH 7.4). All other chemicals were of analytical-reagent grade and obtained from standard reagent suppliers. Millipore Milli-Q water (18 MΩ cm−1) was used in all experiments. Preparation of FAM-Encapsulated Liposomes. Signal reagent-encapsulated liposomes were prepared according to the procedure from the literature,30,31 with slight modifications. DPPC, cholesterol, and DPPE (6:6:1 molar ratio, 65 μmol and 0.0375 g in total) were dissolved in 4 mL of chloroform, followed by the addition of 1 mL of 0.1 M PBS buffer (pH 7.4) containing 10 mM FAM. After sonicating for 5 min, the organic solvent was removed by rotary evaporation under reduced pressure at 40 °C to form a thin lipid film, leaving a yellow viscous film of liposomes. Afterward, the film was hydrated in 1 mL of 0.1 M PBS for 1 h with vigorous shaking at 40 °C, and then sonicated for 10 min using a bath-type sonicator. Finally, the liposome fraction was dialyzed to remove the unencapsulated reagents using a membrane with a molecular weight cutoff of 3500 (MD34−3500) against 0.1 M PBS (pH 7.4) for 24 h at 4 °C, and stored in 0.1 M PBS (pH 7.4) at 4 °C for further use. Preparation of Magnetic Beads−DNA−Liposomes Dumbbell Probes. The procedure for preparation of magnetic beads−DNA−liposomes dumbbell probes is shown in Figure S1 in the Supporting Information. First, 40 μL of amine group-modified magnetic beads (1 mg/mL) was mixed with 20 μL of glycollic acid (0.5 mM), 20 μL of EDC (5 M), and 10 μL of NHS (2.5 M); the mixture then was shaken fully for 1 h. After being rinsed three times with PBS, the MBs were resuspended in 200 μL of PBS. Second, 46 μL of probe DNA (10 μM) was added into the aforementioned MBs solution, followed by shaking for 2 h at room temperature. The resulting MBs−DNA then were rinsed two times with PBS-T, two times with PBS, and resuspended in a final volume of 200 μL of PBS and stored at 4 °C. Third, FAM-encapsulated liposome was covalently coupled to the resulting MB−DNA probe, according

attached at the 5′ and 3′ ends of a short linear DNA, respectively, to construct a dumbbell probe. This dumbbell probe contains a magnetic separation portion, a recognition portion, and a signal reporter portion. So, the dumbbell probe integrates target binding, magnetic separation, and signal response. In this strategy, Exo III-assisted target recycling signal amplification and liposomes-based signal amplification can be completed in one tube. As a result, the dualamplification method can achieve a detection limit as low as 4 aM for DNA, with excellent selectivity. Thus, this approach provides a potential platform for highly sensitive fluorescence detection of various DNA for early disease diagnostic applications. B


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Figure 1. (A) TEM image of fluorescein-encapsulated liposome. (B) Fluorescence image of fluorescein-encapsulated liposome.

to the glutaraldehyde coupling method.29,30 Twenty microliters (20 μL) of glutaraldehyde (0.25 mM) was added into 200 μL of FAM-encapsulated liposome prepared above, and the mixture was stirred gently for 2 h at 25 °C; the excess glutaraldehyde solution then was removed by dialysis overnight in PBS buffer at 4 °C, using a MD34-1 Da molecular weight cutoff membrane. And then, 130 μL of dialyzed liposome suspension was added into 150 μL of the MBs−DNA probe prepared above and incubated for 2 h. Finally, the resulting MBs−DNA− liposome dumbbell probes were rinsed three times with PBS to remove those components that are nonhybridized, and resuspended in a final volume of 100 μL of PBS to a concentration of 3.45 μM, and the prepared MBs−DNA− liposome dumbbell probes were stored at 4 °C for further use. Procedure for DNA Assay. Six microliters (6 μL) of the MBs−DNA−liposome dumbbell probes were mixed with 30 μL of target DNA (varying concentrations) and 15 μL of Exo III (1 U/μL), and the mixture then was diluted to a final volume of 190 μL with 100 mM PBS (pH 7.4). After incubation for 1 h at 37 °C, the unreacted MBs−DNA− liposome dumbbell probes were twice separated from the final reaction solution by a controllable magnetic field. Then, 10 μL of 1% Triton X-100 was added into the supernatant solution and maintained for 50 min to lyse the FAM-encapsulated liposomes. The fluorescence of the as-obtained solution was measured by a Hitachi Model F-4600 fluorometer (Tokyo, Japan) at the excitation wavelength of 490 nm, and the spectra were recorded in the range from 500 nm to 570 nm. Slit widths for the excitation and emission were set at 10 nm. The fluorescence emission intensity was measured at 518 nm. The target DNA concentration was quantified by the fluorescence intensity. The background signal (F0) was the fluorescence intensity of the system, in the absence of target DNA.

The encapsulation efficiency of FAM in the FAMencapsulated liposomes and the loading capacity of FAM molecules per liposome are important parameters for the liposome function. The encapsulation efficiency was estimated using the ratio of the amount of encapsulated FAM in the liposomes to the amount of FAM added in the preparation of the liposomes. The added FAM had a volume of 1 mL and a concentration of 10 mM. The amount of encapsulated FAM in the prepared liposomes was calculated using fluorescence to measure the concentration of FAM, which was released from the prepared liposomes with 1% Triton X-100, according to the calibration curve of a standard FAM solution (Figure S2 in the Supporting Information). After calculation, the amount of encapsulated FAM was 6.45 × 10−6 mol. Therefore, the encapsulation efficiency was calculated to be 64.5%. The loading capacity of FAM molecules per liposome was calculated according to previous literature reports.25 We assumed that the liposomes are unilamellar and take the area per molecule for DPPC and DPPE to be 0.71 nm2 and that for cholesterol to be 0.19 nm2, and the surface area of the liposome was estimated to be 1.2 × 105; accordingly, there were ∼100 000 DPPC molecules per liposome. In addition, the total amount of DPPC in the synthesis of liposome was 30 μmol. Therefore, the amount of synthesized liposomes was 1.806 × 1014. According to the previous section, the amount of encapsulated FAM was 3.883 × 1018. Therefore, the loading capacity was calculated to be 2.15 × 104 FAM molecules/liposome. These results indicated that FAM-encapsulated liposomes prepared in this work have a high effective loading. The stability of the FAM-encapsulating liposomes during storage was studied by monitoring the increase in the fluorescence signal of FAM during the release from the vesicles. FAM molecules are presented inside the liposomes at relatively high concentration, so the fluorescence of FAM is self-quenched.32 When FAM is released to the surrounding medium, self-quenching decreases and fluorescence signals increase. The percent release of FAM was determined using the following equation:33

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RESULTS AND DISCUSSION Characterization of Signal Reagent-Encapsulated Liposome and Optimization of the Releasing Agent. In this work, FAM was employed as the fluorescence signal reagent to be encapsulated into liposomes. The FAM-encapsulated liposomes synthesized in this work were characterized by TEM and fluorescence microscopy. The TEM images of signal reagent-encapsulated liposome demonstrated that the size of the liposome is ∼200 ± 30 nm (as shown in Figure 1A). Fluorescence images showed FAM-encapsulated liposomes were spherical in shape, remarkably bright, and uniform in size (Figure 1B). The fluorescence micrograph visually indicated that the fluorescence dye was successfully encapsulated inside liposomes.

⎛ I − I0 ⎞ percent release = ⎜ ⎟ × 100% ⎝ Imax − I0 ⎠

where I0 is the initial fluorescence signal, I the fluorescence signal at a definite time point, and Imax the fluorescence signal after disrupting the vesicles with 20 μL of 1% Triton X-100 solution (in PBS pH 7.4). As shown in Figure S3 in the Supporting Information, the leakage percentages were determined to be 5.0% and 6.4%, respectively, after storage for 30 and 45 days at 4 °C. Furthermore, we detected the same C


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Analytical Chemistry concentration of target DNA (1 × 10−11 M) using FAMliposomes stored for different time periods. As shown in Figure S4 in the Supporting Information, compared with freshly made FAM−liposomes, the relative error of the relative fluorescence intensity (F − F0) was 4.8% after 30 days. This indicated that the liposomes were stable for 1.0 month, and the leakage of the encapsulated fluorescent molecule was more severe when the liposomes were stored for more than one month. Thus, the fluorescein-encapsulated liposomes could be used in one month. Several commonly used release agents (such as Triton X100,32 ethanol,26 melittin,34 and a 60 °C water bath35) can disrupt the liposomes and release the fluorescent dye from the liposome’s core, which results in fluorophore dequenching and an increase in fluorescence signal. In order to increase the release efficiency of FAM from the liposome, we optimized the releasing agent among several release agents, including 3% (v/ v) methanol, 3% (v/v) ethanol, 1% (v/v) Triton X-100, and a 60 °C water bath. As shown in Figure 2, when ethanol or

DNA probe, which includes the complementary base sequence for target DNA. The DNA probe is modified at its 5′-end with an aldehyde group and at its 3′-end with an amine group. The 5′ terminus of the probe DNA was conjugated onto the surface of the amine group-modified magnetic beads, and the 3′ terminus was covalently coupled to the FAM-encapsulated liposomes via the glutaraldehyde coupling method. So, the MBs−DNA−liposomes dumbbell probe integrates target binding, magnetic separation, and signal response. In the presence of target DNA, the dumbbell probe hybridizes with target DNA to form a duplex DNA. Exo III can catalyze the stepwise removal of mononucleotides of double-stranded DNA from 3′ to 5′ with no requirement of specific recognition sequence, and it is less active on single-stranded DNA or 3′ protruding termini of double-stranded DNA. Therefore, in this system, Exo III can catalyze the stepwise removal of mononucleotides from the blunt 3′ termini of liposome-labeled probe DNA, which results in the release of the target DNA and the liposome. The released target DNA can hybridize with another dumbbell probe and then initiate the next round of cleavage. Eventually, each target DNA can undergo many cycles to trigger the digestion of the MB−DNA−liposome dumbbell probes, releasing a large number of FAM-encapsulated liposomes. On the other hand, each liposome holds a large amount of FAM. Furthermore, the superfluous MB−DNA− liposomes dumbbell probes in the system are removed by magnetic separation in order to decrease the background. When these liposomes are disrupted, tens of thousands of FAM are released. Particularly, the 3′ terminus of our probe was covalently coupled to large FAM-encapsulated liposomes, which may block the 3′-end of the probe. In order to reduce or eliminate the block of liposomes, we adopted two ways to solve this problem. First, we inserted “−(CH2)6 −” between the last base of 3′-end of the probe DNA and the modified amine group. Second, the 3′ terminus amine group was covalently coupled to the liposomes via the glutaraldehyde coupling method. In this case, the last base of 3′-end of the probe DNA and liposome were connected by “−(CH2)6−NC−(CH2)3− CN−” group, which was long enough and could prevent the block caused by the large liposome. Thus, dual signal amplification strategy (fluorescein-encapsulated liposomes and Exo III-assisted target recycling) can result in a great amplified increase of fluorescence signal. To illustrate the feasibility of this proposed sensing strategy, the fluorescence emission spectra of the sensing system were recorded under the different conditions. As shown in Figure 3, the MB−DNA−liposome dumbbell probes in the system were removed by magnetic separation in the absence of target DNA and Exo III, so a weak fluorescence signal was observed (curve a in Figure 3). When both target DNA and Exo III were added into the sensing system, high fluorescence emission was observed (curve e in Figure 3). However, in the absence of Exo III (curve b in Figure 3) or with heat-inactivated Exo III as the control experiment (curve d in Figure 3), no fluorescent signal could be detected, suggesting that the Exo III can catalyze the cleavage of duplex DNA (target DNA/probe DNA), releasing the target DNA and the fluoresceinencapsulated liposomes. Note that, as a control experiment, in the absence of target DNA, no fluorescence change was observed (curve c in Figure 3), which was almost the same as that of the synthesized probe. This strongly indicated that only target DNA could trigger the cleavage process by the Exo III to release the intact target DNA and the fluorescence reagent-

Figure 2. Fluorescence spectra of FAM-encapsulated liposome in 0.10 M PBS (0.1 M KCl, 10 mM MgCl2, and pH 7.4), before release (spectrum a), after release with 3% (v/v) ethanol (spectrum b), 3% (v/v) methanol (spectrum c), 60 °C water bath (spectrum d), and 1% (v/v) Triton X-100 (spectrum e).

methanol was used as a releasing agent, the fluorescence peak intensity increased from 1850 to 4161 (3% ethanol) or 4339 (3% methanol), and the signal increased ∼2-fold, while the heating and use of 1% Triton X-100 resulted in a 3-fold increase in fluorescence (fluorescence intensity increased from 1850 to 4874 and 5108, respectively). The result indicated that 1% Triton X-100 was the best releasing agent. In addition, we captured the fluorescence images and corresponding TEM image of what liposome was incubated with Triton X-100 for different times (see Figure S5 in the Supporting Information). When liposome was mixed with Triton X-100, in the original 10 min, the release agent opened an exit on the liposome; therefore, the fluorescent molecules inside the liposome could flow outward (Figure S5a in the Supporting Information). Then, more fluorescent dye flows outward as time goes on (Figure S5b in the Supporting Information), and, finally, all of the fluorescent dye was released into the solution (Figure S5c). Principle of Dual-Amplification Fluorescence Detection of DNA. The strategy for dual signal amplification fluorescence detection of DNA is depicted in Scheme 1. A dumbbell probe is designed, and the probe contains three domains: a magnetic separation portion, a recognition portion, and a signal reporter portion. The recognition portion is the D


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sensitivity for detecting target DNA. To save enzyme consumption, 15 U of Exo III was selected for the subsequent assay. Analytical Performance of Dual-Amplification Strategy for Fluorescence Detection of DNA. Under the optimized conditions, experiments were carried out by adding increasing concentrations of target DNA into the system to illustrate the sensitivity of the dual signal amplification strategy. Figure 4A depicts the fluorescence emission spectra obtained in the dual signal amplification assay, in response to different DNA concentrations. It was observed that the fluorescence intensity increased with increasing DNA concentration in the range from 5 aM to 700 aM. With the measurement of the fluorescence intensity, we quantitatively analyzed the fluorescence response with the concentration of target DNA. As shown in Figure 4B and Figure 4C, the change in fluorescence intensity (F − F0) of the system was a good linear fit to the concentration of target DNA, and the detection limit (taken to be 3 times the standard deviation in the blank solution) was calculated to be 4 aM, which was much lower than that of the recently reported Exo III-aided target recycling technique applied for DNA quantification without FAM-encapsulated liposomes amplification.9 This indicated that employing the liposomes as amplification carrier was feasible. The high sensitivity of this proposed method might be attributed to two factors: high amplification efficiency of FAM-encapsulated liposomes and Exo III−triggered regeneration of the target DNA. To test the specificity of the proposed strategy for DNA detection, other DNA molecules, including a single-base mismatched DNA (T1), two-base mismatched DNA (T2), and the noncomplementary DNA (Tn), were adopted in place of the target DNA. Figure 5 shows the changes in fluorescence intensity, relative to target DNA and other mismatched DNA strands. The completely complementary target DNA (T), T1, T2, and Tn were distinctly discriminated under the same detection conditions. Thus, this method exhibited a good performance to discriminate perfect complementary target and the base mismatched targets and had great potential in the area of DNA diagnostics and clinical analysis. Furthermore, to verify the general applicability of this assay for complicated biological samples, we imitated the same environment with real samples by spiking target DNA solution into 5% human serum. There is no target DNA in human serum, so the serum sample was spiked with 50 aM and 0.5 fM target DNA to test the performance of the assay, respectively. The spiked serum was detected by the present method, and the found concentration was obtained. The recoveries were calculated by the ratio of found concentration and added concentration. As shown in Table S1 in the Supporting Information, the recoveries were 94.2% ± 3.6% and 102.1% ± 2.7% (n = 3), indicating the potentiality of the proposed assay for DNA detection in real biological samples.

Figure 3. Fluorescence spectra of the FAM dye in different conditions: no target DNA, no Exo III (curve a); 0.1 pM target DNA, no Exo III (curve b); no target DNA, 20 U Exo III (curve c); 0.1 pM target DNA, 20 U inactive Exo III (curve d); and 0.1 pM target DNA, 20 U Exo III (curve e). Experimental conditions: 10 μL of synthesized biosensor probe (3.45 μM), 30 μL of target DNA.

encapsulated liposomes. The results indicated that our proposed strategy is feasible for the detection of DNA. Optimization of Experimental Conditions. In order to achieve the best sensing performance, experimental conditions affecting synthesis of MB−DNA−liposome probe and quantification of target DNA were optimized, including the concentrations of glycollic acid and glutaraldehyde, the amounts of aldehydes liposome, MB−DNA−liposome probe and Exo III, and the release time. Taking into account the steric hindrance resulted by coupling the FAM-encapsulated liposome to the MB−DNA, a small number of DNA was coupled every MBs would be better. So, parts of amine groups of MB were first blocked by the acid−base reaction between amine group of amine-modified MBs and carboxy group of glycollic acid, and then the MBs conjugated DNA probe. We investigated the effect of glycollic acid concentration upon fluorescence signal. It was observed that the fluorescence peaks increased with increasing glycollic acid concentration within the range from 0.4 mM to 0.5 mM, followed by a decrease beyond the concentration of 0.5 mM. Thus, 0.5 mM glycollic acid was used in the subsequent research (see Figure S6 in the Supporting Information). The concentration of glutaraldehyde also has a great effect on the sensitivity. When the concentration is too low, the connecting efficiency between the MB−DNA and aldehydes liposome is low, resulting in low detection sensitivity. However, a concentration that is too high would lead to too much aldehyde on the surface of every liposome, and one liposome would couple with large quantities of DNA; therefore, with Exo III, it is difficult to hydrolyze all the DNA on the surface of one liposome to release the fluorescein-encapsulated liposomes, which would reduce the sensitivity. The optimal concentration of glutaraldehyde was 0.25 mM (Figure S7 in the Supporting Information). The experimental results (Figures S8, S9, and S10 in the Supporting Information) showed that the optimal amounts of aldehydes liposome, MB−DNA−liposome probe, and the release time were 130 μL, 6 μL, and 50 min, respectively. Another important factor in this assay system is the amount of Exo III, because the sensitivity of the assay could be dramatically increased by using Exo III-assisted amplification. The experimental results showed that the bigger the number of Exo III in the range of 5−20 U (see Figure S11 in the Supporting Information), the greater the

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CONCLUSION In summary, we designed the MB−DNA−liposome dumbbell probe, which integrates target binding, magnetic separation, and signal response. With this multifunctional dumbbell probes, a dual-signal amplification strategy in one tube was developed for detection of DNA with remarkably enhanced sensitivity. In this proposed strategy, Exo III-aided target recycling technique was combined with liposome-assisted amplification. With highly efficient amplification and excellent signal readout, the E


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Figure 5. Specificity of the assay for DNA detection by hybridizing probe DNA with different target DNA: completely complementary target DNA (T), single-base mismatched DNA (T1), two-base mismatched DNA (T2), and noncomplementary DNA (Tn ). Experimental conditions: 6 μL of MB−DNA−liposome probe (3.45 μM), 15 U Exo III, and 500 aM target DNA.

realized in one step, which greatly shortens the assay time. Therefore, we propose that this dual-signal amplification strategy holds a great potential for early diagnosis in generelated diseases.

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ASSOCIATED CONTENT

S Supporting Information *

Schemes for preparation of dumbbell probes, the stability and fluorescence image of liposomes, and optimization of conditions. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.analchem.5b00993.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +86-29-81530727. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported financially by the National Natural Science Foundation of China (Nos. 21275096 and 21475083), Shaanxi Provincial Natural Science Foundation (No. 2013SZS08-Z01), and Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28).

Figure 4. (A) Fluorescence spectra for the determination of DNA at different concentrations: 0 (curve a), 5.0 × 10−18 M (curve b), 1.0 × 10−17 M (curve c), 3.0 × 10−17 M (curve d), 5.0 × 10−17 M (curve e), 7.0 × 10−17 M (curve f), 1.0 × 10−16 M (curve g), 3.0 × 10−16 M (curve h), 5.0 × 10−16 M (curve i), and 7.0 × 10−16 M (curve j). (B) Linear relationship between the change in fluorescence intensity (F0 − F) and target DNA concentration from 5.0 aM to 70 aM. (C) Linear relationship between the change in fluorescence intensity (F0 − F) and target DNA concentration from 70 aM to 700 aM. Error bars represent the standard deviations of three independent measurements. Experimental conditions: 6 μL of MB−DNA−liposome probe (3.45 μM), 15 U Exo III, and 30 μL of target DNA; the total volume of the mixture was 200 μL.

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detection limit of the proposed method can reach attomolar, which has improved by as much as 3 orders of magnitude, compared with that of the Exo III-aided target recycling technique applied for DNA quantification without FAMencapsulated liposomes amplification.9 In addition, the current protocol is really simple, and dual-signal amplification can be F


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Exonuclease III-Assisted Target Recycling Amplification Coupled with

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