Ultrasensitive Lipopolysaccharides Detection Based on Doxorubicin

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Ultrasensitive lipopolysaccharides detection based on doxorubicin conjugated N-(aminobutyl)-N-(ethylisoluminol) as electrochemiluminescence indicator and self-assembled tetrahedron DNA dendrimers as nanocarriers Shunbi Xie, Yongwang Dong, Yali Yuan, Yaqin Chai, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00276 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 20, 2016

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Analytical Chemistry

Ultrasensitive lipopolysaccharides detection based on doxorubicin conjugated N-(aminobutyl)-N-(ethylisoluminol) as electrochemiluminescence indicator and self-assembled tetrahedron DNA dendrimers as nanocarriers Shunbi Xie,†, ‡ Yongwang Dong,† Yali Yuan,† Yaqin Chai,∗, † Ruo Yuan∗, † †

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest

University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China ‡

Chongqing

Key

Laboratory

of

Environmental

Materials

&

Remediation

Technologies (Chongqing University of Arts and Sciences), Chongqing 400715, PR China; E-mail addresses: [email protected], [email protected]. fax: +86 23 68253172 ABSTRACT The preparation of self-assembled DNA nanostructure with different sizes and structures has been one of the most promising research areas in recent years, while the application of these DNA nanostructures in biosensors is far from fully developed. Here, we presented a novel carrier system to construct an electrochemiluminescence (ECL) aptasensor for ultrasensitive determination of lipopolysaccharides (LPS) on the basis of self-assembled tetrahedron DNA dendrimers. Doxorubicin (Dox), a well-known intercalator of double stranded DNA (dsDNA), was conjugated with the ECL luminophore of N-(aminobutyl)-N-(ethylisoluminol) (ABEI) to form a new type of ECL indicators (Dox-ABEI), which could noncovalently attach to dsDNA through intercalation. Based on this property, self-assembled tetrahedron DNA dendrimers

1

ACS Paragon Plus Environment


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were employed as an efficient nanocarrier to achieve a high loading efficiency for Dox-ABEI with significantly amplified ECL signal output. Streptavidin (SA) and biotin, a typical ligand-receptor pair, has been chosen to anchor the tetrahedron DNA dendrimers on the electrode surface. Moreover, by converting LPS content into DNA output, catalyzed hairpin assembly (CHA) target recycling signal amplification strategy was also adopted to enhance the sensitivity of the ECL aptasensor. With combining the loading power of the tetrahedron DNA dendrimers for ECL indicators, the inherent high sensitivity of ECL technique and target recycling for signal amplification, the proposed strategy showed a detection limit of 0.18 fg/mL for LPS. KEYWORDS:

Electrochemiluminescence

aptasensor,

lipopolysaccharides,

doxorubicin conjugated N-(aminobutyl)-N-(ethylisoluminol), tetrahedron DNA dendrimers INTRODUCTION Lipopolysaccharides (LPS, also known as endotoxins), the major component of the outer membrane of Gram-negative bacteria1-3, is the primary cause of septic shock4, pyrogenic reaction5, blood clotting6 and so on. Because LPS shows highly biologically active and toxic even at a low concentration of picomole per milliliter range7, 8, the specific and sensitive determination of LPS has great significance for medical and food security9, 10. Thus, various analysis methods on the basis of LPS binding proteins, peptides, and artificial affinity recognizing molecules have been reported for LPS detection11-13. However, these LPS assays are largely suffered from cross-binding to other species coexistence with LPS, because the specificity of the designer biomolecules or synthetic probes is not good enough for LPS capturing 14-16. Thereafter, the LPS binding aptamer (LBA), ligands with high affinity and specific for LPS, is selected by Kim group in 201217, and some LPS aptasensor have been 2


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reported later18. Although the specificity for LPS has been improved, these electrochemical aptasensors showed unsatisfactory sensitivity to meet the requirement of detecting LPS ultra-sensitively19. In this regard, it is of great significance to choose predominant analytical technique and effective signal amplification strategies for the determination of LPS at ultralow levels. Electrochemiluminescence (ECL) technology is competitive with conventional assays due to its high sensitivity, rapid response and low background 20-23. As the most classic ECL luminescence reagents, luminol and its derivatives have been got a wide range of applications for their high emission yields, low oxidation potential as well as strong luminescence24, 25. A series of luminol-based ECL sensors, in which luminol is put in the detection solution, have been fabricated and applied in the early stage26-29. While in recent years, considerable efforts are being made for developing solid-state ECL sensors by adopting appropriate immobilization approaches such as doping or crosslinking30-32. Compared with putting luminophores in detection buffer, solid-state ECL has attractive advantages with saving reagents and improving the ECL luminous efficiency by decreasing the interaction distance between luminophores and the electrode33,

34

. However, current immobilization strategies often suffer from

complicated preparation procedure and limited loading amount, which can greatly affect the sensitivity and practicality of the constructed biosensor. Therefore, a means to reliably and robustly load luminol or its derivatives as well as avoid cumbersome operation steps is necessary. To achieve these requirements, we synthesized a novel ECL indicator (Dox-ABEI), which was obtained by covalently linking doxorubicin (Dox) with N-(aminobutyl)-N-(ethylisoluminol) (ABEI). Dox is a well-known intercalator of double stranded DNA (dsDNA)

35

. ABEI is widely used luminol’s derivative due to 3



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there is no significant reduction in luminescence activity when this reagent labeled with other appropriate reagent36. Therefore, the synthesized Dox-ABEI can noncovalently attach to dsDNA through intercalation and is no significant reduction in luminescence activity of ABEI. Thereafter, another major effort is focus on finding an efficient nanocarrier for the loading of Dox-ABEI complex effectively. Recently, DNA nanostructures with programmable functional moieties and divinable morphology

have

emerged

as

an

important

engineering

paradigm

for

nanotechnology37-41. Among them, dendritic DNA nanostructures have aroused great concern in the past decade42-47. The recent study by Yang al. demonstrated the construction of tetrahedron DNA dendrimers by incorporating tetrahedron DNA monomers with a step-by-step process48. Their monodispersity, excellent stability, highly branched, porous structures as well as the outstanding specificity of G-C and A-T hydrogen-bonding interactions49, make tetrahedron DNA dendrimers to serve as particularly promising nanocarriers for the binding of Dox-ABEI complex. Herein, a novel “signal on” aptasensor for ultrasensitive detection of LPS was designed by utilizing Dox-ABEI complex as ECL indicators and tetrahedron DNA dendrimers as effective nanocarriers. The Dox-ABEI complex, which was synthesized by combining a DNA intercalator of Dox and the luminophor of ABEI, could intercalate into tetrahedron DNA dendrimers and realize solid-state ECL detection. Besides, in our detection approach, catalyzed hairpin assembly (CHA) target recycling signal amplification strategy was adopted to enhance the sensitivity of ECL aptasensor. A typical LPS detection process was performed as follows: the hybrid of LBA and its partial complementary strand (cDNA) were first assembled onto the surface of Au@Fe3O4 magnetic nanocomposites. With the addition of LPS, cDNA was dissociated from the hybrid and released into the solution. The cDNA obtained 4


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though magnetic separation could hybridize with the hairpin probe H1 on the surface of electrode to open its hairpin structure. And then the cDNA could be further displaced from the H1-cDNA duplex with the introduction of another biotin-labeled hairpin DNA Biotin-H2. In this way, the recycling of cDNA was realized to amplify the response signal, and much H1-Biotin-H2 duplex with biotin could be obtained after the cyclic process. Thereafter, the H1-Biotin-H2 duplex with biotin was further exploited to bind with streptavidin (SA), followed by the binding of biotinylated tetrahedron DNA dendrimers (G2) which was intercalated with numerous Dox-ABEI (G2-Dox-ABEI) to achieve quantitative detection of LPS. Scheme 1 illustrated the construction process of the ECL aptasensor and the detection principle for LPS analysis.

Scheme 1 (A) Preparation of the tetrahedron DNA dendrimer (G2)-Dox-ABEI complex; (B) Fabrication process of the electrochemiluminescence aptasensor for LPS determination based on DNA tetrahedron dendrimers for the ECL indicators 5



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binding and CHA target recycling strategy for signal amplification. EXPERIMENTAL SECTION Reagents and apparatus. Doxorubicin (Dox), human serum albumin (HSA), lipopolysaccharides (LPS), gold chloride (HAuCl4), Streptavidin (SA), hemoglobin (Hb), and hexanethiol (HT) were purchased from Sigma (St. Louis, MO, USA). N-(aminobutyl)-N-(ethylisoluminol) (ABEI) was obtained from TCI Development Co., Ltd. (Shanghai, China). Amido-modified Fe3O4 (Fe3O4-NH2) magnetic microspheres were bought from Tianjin BaseLine ChromTech Research Centre (Tianjin, China). All synthetic DNA sequences in Table S1 (See the Supporting Information) were provided by Sangon Inc. (Shanghai, China). The binding buffer was 20 mM Tris-HCl (containing 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2, pH 7.4). The working buffer was 0.1 M phosphate-buffered solution (PBS, containing 10 mM KCl and 2 mM MgCl2, pH 8.0). Electrochemical impedance spectroscopy (EIS), cyclic voltammetric (CV), and electrochemical deposition were performed on a CHI 660E electrochemistry workstation (CHI Instruments of Shanghai, China). The ECL emissions were measured with a MPI-A ECL analyzer (Xi’an Remex Electronics, China). The gels electrophoresis was carried out on a Molecular Imager Gel DocTM XR+ with Image LabTM software (Bio-Rad, Canada). The atomic force microscopy (AFM) was performed on a Multimode 8 (Bruker, USA). The transmission electron micrograph (TEM) was performed on a Tecnai G2 F20 microscope (FEI Co, U.S.A). The UV-vis absorption spectra were recorded with a UV-2550 spectrophotometer (Shimadzu, Japan). Synthesis of Au@Fe3O4 magnetic nanocomposites. Firstly, 16 nm gold nanoparticles were prepared according to the reference50. Then, 1 mL Fe3O4-NH2 6


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magnetic microspheres were collected on a magnet and rinsed with ultrapure water at least three times. After that, 5 mL synthesized gold nanoparticles (AuNPs) was mixed with the purified Fe3O4-NH2 and stored for 1 h. During this process, AuNPs could assemble on Fe3O4 surface via the Au-N bonding. The resulting Au@Fe3O4NPs nanocomposites were isolated with magnet and dispersed in 2 mL ultrapure water. Preparation of the Dox-ABEI compounds. The process of Dox-ABEI compounds preparation was shown schematically in Figure 1. Briefly, 0.5 mL of Dox solution (0.015 M) was mixed with 0.5 mL of ABEI solution (0.01 mM) through ultrasonic, and then 0.5 mL of GA (1 wt %) which work as cross-linking agent was dropped into the above mixture with constant stirring for 12 h to obtain the Dox-ABEI compounds. The UV-vis characterizations of the Dox-ABEI compounds were shown in Figure S1.

Figure 1 The synthetic route of the Dox-ABEI compounds Preparation

of

tetrahedron

DNA

dendrimer-Dox-ABEI

complex.

Tetrahedron DNA dendrimers were synthesized through a step-by-step assembly and enzyme-free strategy according to the reference48. Three types of tetrahedron DNA monomers T0, T1 and Biotin-T2 were first prepared as building blocks for the construction of tetrahedron DNA dendrimers. T0 with four same sticky ends was used as the core of the tetrahedron DNA dendrimers. T1 as the first layer was assembled onto T0 through hybridization. One sticky end of T1 could hybridize with T0, and the 7



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other three same sticky ends could complementary to Biotin-T2. Biotin-T2 as the second layer was further assembled onto T1. One sticky end of Biotin-T2 could complementary to T1, living three same sticky ends that had been labeled with biotin for further use. Four different single strands assembled into one tetrahedron DNA monomer through hybridization. Taking the assembly of T0 as an example: mixing four single strands (T0a, T0b, T0c, T0d) with equal ratio in the Tris-HCl buffer to achieve a final concentration of 2 µM for each strand. After that, the mixture was heated to 95 oC for 2 min and then cooled to 4 oC slowly to obtain monomers T0. Monomers T1 and Biotin-T2 were prepared according to the same procedure. Different generations of DNA dendrimer (Gn) were prepared via stepwise assembly of tetrahedron DNA monomers, and the procedure was shown in Scheme 1A. In order to meet the need for signal amplification and simplify the experimental operation, here we chose generations of G2 DNA dendrimer as the nanocarrier in our system. In a typical procedure, T0 mixed with T1 at a 1:4 molar ratio and further incubated for 1 h at room temperature to form the first generation of DNA dendrimer (G1), the obtained G1 had twelve free sticky ends available. Then G1 further assembled with Biotin-T2 at a 1:12 molar ratio to construct the second generation of DNA dendrimer G2 in a similar way. After G2 tetrahedron DNA dendrimers was successfully assembled, 1 mL of the prepared Dox-ABEI compounds was added into the above tetrahedron DNA dendrimers solution and stored for 4 h. Then the above mixture was transferred into a dialysis tube and was dialyzed for 1 day against 400 mL of PBS with mild shaking to obtain G2-Dox-ABEI complex. Fabrication of the ECL aptasensor. Firstly, 20 µL Au@Fe3O4 magnetic nanocomposites and 20 µL of 2 µM amido-terminated LBA were mixed in an eppendorf tube and incubated overnight to obtain the Au@Fe3O4/LBA biocomplex. 8


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Then the Au@Fe3O4/LBA biocomplex was incubated with 20 µL of 2 µM cDNA solution and incubated at 37 ºC for 2 h. After removing non-hybridized cDNA via magnetic separation, 20 µL of LPS solution with different concentrations was mixed with the above Au@Fe3O4/LBA/cDNA biocomplex and incubated for 30 min. Finally, the solution containing different concentration of the released cDNA was collected with a magnetic field for further use. The GCE electrode was successively polished with 0.3 and 0.05 µm Al2O3 powder, follow by ultrasonically cleaning with water and ethanol, respectively. Thereafter, the clean GCE was immersed into 1 % HAuCl4 to deposit a thin layer of AuNPs at -0.2 V for 30 s. Then 20 µL H1 (2 µM) was dropped onto the AuNPs modified GCE for 12 h to obtain GCE/AuNPs/H1 electrode. Subsequently, the modified electrode was blocked the unspecific sites with 20 µL of HT (1.0 mM) for 30 min. After that, 20 µL solution containing 10 µL the preceding released cDNA and 10

µL

Biotin-H2

(4

µM)

were

dropped

onto

the

GCE/AuNPs/H1/HT/cDNA+Biotin-H2 electrode and incubated for 2 h at 37 °C. After washing, the above electrode was further reacted with 20 µL (2 µM) SA for 1 h. Ultimately, 20 µL of the prepared G2-Dox-ABEI complex was dropped onto the GCE/AuNPs/H1/HT/Biotin-H2/SA electrode for 2 h. After thoroughly rinsed with ultrapure water, the electrode was prepared for ECL detection. Agarose Gel Electrophoresis. All tetrahedron DNA monomers and tetrahedron DNA dendrimers were prepared at a concentration of 5 µM. Then 10 µL of each DNA nanostructure was subjected to gel electrophoresis in 2% agarose gels at 100 V for 1.5 h and then stained with ethidium bromide. Images were documented by utilizing a Molecular Imager Gel DocTM XR+ with Image LabTM software.

RESULTS AND DISCUSSION 9



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Characterization

of

tetrahedron

DNA

Page 10 of 23

dendrimers.

Agarose

gel

electrophoresis was employed to characterize the prepared tetrahedron DNA dendrimers. As shown in Figure 2A, the single strand DNA of T0a with the lowest molecular weight was run fastest on the agarose gel (lane 1). After the single strand DNA self-assembled into the monomers of the target dendrimer T0, T1, and Biotin-T2, three bright bands at a similar position were observed (lane 2-4). Specially, Biotin-T2 has a little higher molecular weight compared with T1 due to the labeling of the biotin on the single strand DNA. Then with tetrahedron DNA monomers gradually incorporated into the assembly process, the G1 tetrahedron DNA dendrimers and G2 tetrahedron DNA dendrimers run more and more slowly (lane 5, 6). The results demonstrated the excellent packing efficiency and the successful formation of the tetrahedron DNA dendrimers. AFM and TEM were further employed to observe the morphology of the assembled tetrahedron DNA dendrimers (G2). From Figure 2B and Figure 2C, morphology with circular DNA nanostructures was observed. These results suggested the successful formation of the tetrahedron DNA dendrimers.

Figure 2 (A) Agarose gel electrophoresis of different DNA nanostructure: lane 1, single stranded DNA; lanes 2, monomers T0; lanes 3, monomers T1; lanes 4, Biotin-T2; lane 5, tetrahedron DNA dendrimers of G1; lanes 6, tetrahedron DNA dendrimers of G2. (B) AFM image of the tetrahedron DNA dendrimers of G2. (C) TEM image of the

10


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tetrahedron DNA dendrimers of G2. The stepwise characterization of the sensing interface. Each step in the preparation of the sensing interface was confirmed with CV and EIS in 5.0 mM [Fe(CN)6] 3−/4−. As displayed in Figure 3 A, a reversible redox (curve a) was exhibited on the pretreated bare GCE. With AuNPs electrodeposited onto electrode, the perk current increased (curve b) owing to the excellent conductivity of AuNPs. After the H1 was immobilized onto the AuNPs modified electrode, the current response decreased (curve c) due to the electronic repulsion between the negatively charged DNA and [Fe(CN)6]3−/4−. After blocking with non-conductive HT, the peak current decreased again (curve d). As expected, a further decreased response was observed after incubating the mixture of cDNA and Biotin-H2 (curve e), because more negative charges has been introduced on the electrode surface. After binding with the biological macromolecule of SA, the current response decreased continuously (curve f). Subsequent assembling of G2-Dox-ABEI complex caused dramatic inhibition of peak current and significant separation of the peak gap (curve g), which attributed to the largely increased negative charges of the tetrahedron DNA dendrimers. Since EIS has been reported to be a very sensitive technique for characterizing electrode surface modification51,

52

, we used this technique to conform the

construction process of the biosensor. Figure 3B showed the impedance spectra at each modified steps. Comparing with the bare GCE (curve a), a decreased semicircle diameter was obtained for the AuNPs-deposited electrode (curve b) due to the conductivity of AuNPs. However, the semicircle diameter increased consecutively with the sequential incubation of negatively charged H1 (curve c) and non-conductive HT (curve d). The resistance increased further after the electrode incubated with the mixture of cDNA and Biotin-H2 (curve e). And after binding with biological 11



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macromolecule SA, the resistance increased sequentially (curve f). Finally, the semicircle diameter increased greatly after assembling the G2-Dox-ABEI complex (curve g). These results consistent with that observed in CV (Figure 3A), suggesting the successful fabrication of the ECL aptasensor according to Scheme 1.

Figure 3 The CV (A) and EIS (B) characterization of electrode at different modify stages: bare GCE (a); GCE/AuNPs (b); GCE/AuNPs/H1 (c); GCE/AuNPs/H1/HT (d); GCE/AuNPs/H1/HT/cDNA+Biotin-H2 (e); GCE/AuNPs/H1/HT/Biotin-H2/SA (f); GCE/AuNPs/H1/HT/Biotin-H2/SA/G2-Dox-ABEI complex (g). Detection buffer: 2 mL 5 mM Fe(CN)6]3−/4− (pH 7.0). Optimization of experimental conditions. H2O2 as the co-reaction reagent of the luminol and its derivatives ECL systems has great influence on the luminescence efficiency. To investigate the optimal concentration of H2O2, the ECL responses of the aptasensors incubated with 1.0 pg/mL LPS for different concentration of H2O2 were recorded. As can be seen from Figure 4A, with the increase of H2O2 concentration, a consecutive increased ECL intensity was observed from 0 to 5 mM, while further increasing the concentration of H2O2 did not induce an increased ECL response. Therefore, the optimized concentration of H2O2 was 5.0 mM in the detection buffer. The incubation time of the cDNA and Biotin-H2 for ligand displacement also influenced the performance of the ECL aptasensor. Figure 4B showed the effect of the 12


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incubation time of cDNA and Biotin-H2 on the electrode surface for 1.0 pg/mL LPS detection. The ECL intensity enhanced gradually with the augment of incubation time and then reached to a plateau after 2 h, suggesting that 2 h was enough for ligand displacement. Thus, 2 h was employed as the ligand displacement time between cDNA and Biotin-H2.

Figure 4 (A) The optimum concentration of H2O2; (B) The optimum incubation time of the cDNA and Biotin-H2 for ligand displacement. All the ECL intensity was detected with 1.0 pg/mL LPS in 2 mL PBS (pH 8.0). Comparison of different labeled probes. In this work, the tetrahedron DNA dendrimers was employed as effective nanocarriers for the binding of Dox-ABEI complex. For comparing and demonstrating the advantages of the aptasensor with tetrahedron DNA dendrimers, the ECL intensity of the aptasensor incubated with different labeled probes was investigated in the same working buffer. The same batch of aptasensors were constructed for the detection of 1.0 pg/mL LPS, and then incubated with the different labeled probes. The changed ECL values (∆I) of the aptasensors with different labeled probes were displayed in the Figure 5. The ∆I of the aptasensor incubated with target G2-Dox-ABEI complex was 5629 a.u. (B), while that decreased to 1395 a.u. when the aptasensor was incubated with Biotin-T2-Dox-ABEI complex. Compared A with B in Figure 5, a remarkable increased ECL intensity was observed, indicating that the immobilized amount of Dox-ABEI was obviously 13



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improved by forming the tetrahedron DNA dendrimers.

Figure 5 The ECL intensity of the aptasensors with different labeled probes: (A) Biotin-T2-Dox-ABEI complex; (B) G2-Dox-ABEI complex in 2 mL PBS (pH 8.0) containing 5.0 mM H2O2. Analytical property of the ECL aptasensor for LPS detection. To investigate the potential quantitative application and the sensitivity of the proposal platform, various concentrations of LPS were detected by the prepared ECL aptasensor under the optimal conditions. Figure 6A depicted the strong correlation between the ECL intensity and the concentration of LPS. The calibration plot was illustrated in Figure 6B, the ECL response was proportional to the logarithm (lg) of LPS concentration from 1.0 fg/mL to 100 ng/mL with a regression equation expressed as I = 1680.1 lgc + 10735.9 and the correlation coefficient was 0.9971. A detection limit of 0.18 fg/mL for the target can be estimated (defined as LOD = 3SB/m). The analytical performance of the proposal method were compared with other reported LPS detection methods (Table S2, see the Supporting Information). As shown, the proposed aptasensor exhibited excellent analytical performance, which should be attributed to the large immobilized amount of Dox-ABEI complex and catalyzed hairpin assembly target recycling strategy for signal amplification.

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Figure 6 (A) ECL intensity of the aptasensors incubated with different concentrations of LPS (a-j): 1.0 fg/mL, 10 fg/mL, 0.1 pg/mL, 1.0 pg/mL, 10 pg/mL, 0.1 ng/mL, 1 ng/mL, 50 ng/mL, and 100 ng/mL. (B) The calibration plot of ECL intensity vs the logarithm of LPS concentration. The reproducibility, stability and selectivity of the aptasensor. To investigate the reproducibility, we studied the batch-to-batch precision and reproducibility of the biosensor toward three concentrations (low, middle, high), 0.1 pg/mL, 10 pg/mL and 50 ng/mL LPS, and the relative standard deviations (RSD) were 6.1%, 4.7%, and 5.3% (n = 3), respectively, suggesting acceptable accuracy and reproducibility of the ECL assay. The stability was monitored by consecutive cyclic potential scanning. As shown in Figure 7A, the aptasensor has an excellent stability with RSD of 0.9% by consecutive cyclic potential scans for 15 circles. To further explore the selectivity, Hb, glucose and HSA were chosen as interfering agents. According to Figure 7B, no significant ECL response of the Hb, glucose, HSA (0.1 ng/mL) were observed except that for target LPS (1.0 pg/mL). In addition, the ECL intensity of the mixture containing the above three interferents and LPS was almost the same as the value obtained from LPS only. This result indicated good selectivity of this strategy for LPS detection.

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Figure 7 (A) Continuous cyclic potential scans of the proposed aptasensor; (B) Specificity of the ECL aptasensor toward (a) blank control, (b) 0.1 ng/mL Hb, (c) 0.1 ng/mL Glucose, (d) 0.1 ng/mL HSA, (e) 0.1 ng/mL Hb+0.1 ng/mL glucose+0.1 ng/mL HSA+1.0 pg/mL LPS, (f) 1.0 pg/mL LPS. All the ECL intensity was detected 1.0 pg/mL of LPS in 2 mL PBS (pH 8.0) containing 5 mM H2O2. The analysis of LPS in samples. To test the viability of the proposed assay for application, the recovery experiments were performed. Human serum samples, which obtained from Xinqiao Hospital of Chongqing, were selected as real samples for LPS analysis by using the standard addition method. Prior to use, the serum was obtained by centrifugation clotted blood at 3000 rpm for 20 min, and then collected and stored at -20 °C for further use. After that, different concentrations of LPS were spiked into 10-fold-diluted serum samples, and the recoveries of LPS in the serum samples were monitored by the developed ECL aptasensor and a conventional enzyme-linked immunosorbent assay (ELISA) method. From Table 1, we could see that the data obtained by our method exhibited good accordance with that calculated by ELISA method, suggesting the reliability of our proposed strategy for LPS detection in complicated samples.

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Table 1 Detection LSP in serum samples. Samples

Amount of LPS added/(ng mL-1)

1

1

2

5

3

10

4

50

Amount of LPS found± ±SD, ng mL-1 and (recovery/%) Our method ELISA 0.102+4.37 -(102.0) 0.493+5.12 0.52+3.14 (98.6) (104.0) 0.949+2.13 1.03+2.51 (94.9) 108.5 5.32+3.35 5.24+2.73 (106.4) (104.8)

Relative error/% --5.19 -7.86 1.52

CONCLUSIONS In summary, we have synthesized a novel ECL indicator by combining DNA intercalator and luminophor in one molecule and prepared the tetrahedron DNA dendrimers to serve as an effective nanocarrier for the loading of massive ECL indicators. This solid-state ECL platform displays several fascinating features: (1)

facile self-assembly preparation of DNA nanostructures. The size-controllable DNA nanostructures used in this work were self-assembled by a step-by-step and enzyme-free strategy. (2) High load capacity. The hybridized double-stranded DNA configuration in tetrahedron DNA dendrimers also allows thousand-fold loading of ECL indicator without labeling or complex operation in the DNA nanostructure, which provided a new perspective for using DNA nanostructures as an effective carriers for signal amplification. (3) Ultrahigh sensitivity. The advantageous character of the DNA tetrahedron dendrimers for the ECL indicators binding combined with CHA target recycling signal amplification strategy provided an ultrasensitive method for LPS detection with a detection limit down to femtogram per milliliter level. (4)

Good biostability. DNA nanostructures exhibit good loading stability under physiological conditions. Taking these advantages into account, we expect that our constructed ECL aptasensor will find potential applications for detecting a wide range of molecular analytes. 17



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ACKNOWLEDGMENT This work was financially supported by the NNSF of China (21575116, 21275119, 51473136) and the Fundamental Research Funds for the Central Universities (XDJK2015A002), China.

AUTHOR INFORMATION Supporting Information The details of the DNA sequences, characterization of the Dox-ABEI compounds, electrochemical and ECL measurements and a comparison table of different analytical methods for LPS detection are provided. * Corresponding authors Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected], [email protected]. Notes The authors declare no competing financial interest. REFERENCES (1) Gutsmann, T.; Schromm, A.; Brandenburg, K.; Int. J. Med. Microbiol. 2007, 297, 341-352. (2) Therisod, H.; Labas, V.; Caroff, M.; Anal. Chem. 2001, 73, 3804-3807. (3) Li, J. J.; Purves, R. W.; Richards, J. C.; Anal. Chem. 2004, 76, 4676-4683. (4) Ding, S.; Chang, B.; Wu, C.; Chen, C.; Chang, H.; Electrochem. Commun. 2007. 9, 1206-1211. (5) Murai, T.; Ogawa, Y.; Kawasaki, H.; Kanoh, S.; Infect. Immun. 1987. 55, 18


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For TOC only

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