Multi-pedal DNA walker biosensors based on catalyzed hairpin

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Multi-pedal DNA walker biosensors based on catalyzed hairpin assembly and isothermal strand-displacement polymerase reaction for the chemiluminescent detection of proteins Ningxing Li, Mingyuan Du, Yucheng Liu, Xinghu Ji, and Zhike He ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00129 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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Multi-pedal DNA walker biosensors based on catalyzed hairpin assembly and isothermal strand-displacement polymerase reaction for the chemiluminescent detection of proteins

Ningxing Li, Mingyuan Du, Yucheng Liu, Xinghu Ji, Zhike He*

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China

*Corresponding author. Tel: +86 27-68756557; E-mail: [email protected] (Z. He)

ABSTRACT In this study, two kinds of sensitive biosensors based on multi-pedal DNA walker along a 3-D DNA functional magnet particles track for the chemiluminescent detection of streptavidin are constructed and compared. In the presence of SA, multi-pedal DNA walker has been constructed by biotin-modified catalyst as a result of the terminal protection for avoiding the digestion by exonuclease I. Then through toehold-mediated strand exchange, a ‘leg’ of multi-pedal DNA walker would interact with a toehold of CHA-H1 coupled with magnetic microparticles (MMPs) and opens its hairpin structure. The newly open stem in CHA-H1 would be hybridized with a toehold of biotin-labeled H2. Via the strand displacement process, H2 displaces one ‘leg’ of multi-pedal DNA walker, and the other ‘leg’ would continue interacting with neighboring H1 to initiate next cycle. In order to solve the high background caused by the hybridization between CHA-H1 and H2 without CHA-catalyst, the other model

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has been designed. The principle of the other model (ISDPR DNA walker) is similar to the above one. After the terminal protection of SA, a ‘leg’ of multi-pedal DNA walker triggers to open the hairpin of ISDPR-H1 conjugated with MMPs. Then the biotin-modified primer could hybridize with the newly exposed DNA segment, triggering the polymerization reaction with the assistance of dNTPs/polymerase. As the extension of the primer, the ‘leg’ of multi-pedal DNA walker is displaced so that the other ‘leg’ could trigger proximal H1 to go on the next cycle. Due to its lower background and stronger signal, multi-pedal DNA walker based on ISDPR has a lower limit of detection for SA. The limit of detection (LOD) for SA is 6.5 pM. And for expanding the application of the method, the detections of folate receptor and thrombin have been explored. What’s more, these DNA walker methods have been applied in complex samples successfully.

KEYWORDS: DNA walker; magnet particles; chemiluminescence; streptavidin detection; catalyzed hairpin assembly; isothermal strand-displacement polymerase reaction

Proteins play a key role in most life activities, such as metabolism, heredity and immunization.1,2 Motivated by these important effects, protein detection has attracted lots of attention from clinical diagnostics, forensic analysis, food safety and drug screening.3-6 For instance, Li et al. have applied binding-induced DNA strand displacement for homogeneous protein assay and successfully detected streptavidin (SA) and growth factor BB.4 Moreover, a controllable amplified surface plasmon resonance imaging strategy is proposed by Chen’s group for detecting human IgG, ovalbumin and α-fetoprotein, reaching a sensitive detection level.7 Therefore, the detection for protein is particularly important. Terminal protection is often used in protein

detection

since

Yu’s

group

discovered

the

phenomenon

that

small-molecule-linked ssDNA was protected from digestion by nucleases when the ACS Paragon Plus Environment

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small molecule moiety is bound to its protein target.8 And also, the detections of SA and thrombin based on it are successful.9, 10 Fluorescence, electroanalysis and mass spectrometry are widely used for protein detection.11-13 Compared with the others, chemiluminescence (CL) has its unique characteristics of lower background for not needing external light source.14 The detection systems have introduced luminal which is one of the most widely used CL reagents15, acridinium ester or peroxalate ester as luminous substrate16-18 according to their different features. Among above CL systems, the reaction of luminol-H2O2 with p-iodophenol, which was catalyzed by horseradish peroxidase (HRP), metal ions or DNAzyme,19,20 such as G-quadruplex DNAzymes,21 is pretty common in DNA and protein detections for the steady-state light signal and higher sensitivity.14 Moreover, during the past years streptavidin-horseradish peroxidase (SA-HRP) based on conjugation between streptavidin and biotin was generally applied in biotin-labeled biosensors.22 Therefore, luminol-H2O2 reaction catalyzed by HRP was employed in this work. By further introduction, functionalized magnetic microparticles (MMPs) have been used to remove superfluous SA-HRP effectively. Aimed to duplicate macroscopic machinery functions at the molecular level, molecular machines relying on DNA building blocks have provided an avenue for the wide development of DNA nanotechnology for the remarkable locomotion and controllability.23-25 Due to the characteristics of DNA machines, a plenty of DNA walking appliances, such as DNA robots, have been designed.26-28 Nowaday, three-dimensional (3-D) DNA nanomachines with the advantage of high DNA loading capacity on the surface have attracted considerable attention.29 For example, Li et al. have reported a DNA nanomachine, built from a DNA-functionalized gold nanoparticle, which moves a DNA walker along a 3-D DNA-AuNP track and executes the task of releasing payloads.30 Our prior work has also designed a sensitive DNA walker biosensor for DNA detection.31 Generally, versatile signal amplification strategies such as traditional linear rolling circle amplification (LRCA), hybridization chain reaction (HCR), primer-generation rolling circle amplification (PG-RCA), exponential amplification reaction (EXPAR), polymerase chain reaction (PCR), ACS Paragon Plus Environment

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loop-mediated

isothermal

amplification

(LAMP),

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and

helicase-dependent

amplification (HAD) strategies have been driven by DNA sequences and enzymes,32-37 which could been effectively employed in DNA walkers. Catalyzed hairpin assembly (CHA), as a kind of the most promising nonenzymatic amplification methods, could form stable duplex DNA nanoscale assemblies as a result of an initiation of a single-stranded catalyst.38 And Ellington’s group has constructed a 3-D DNA walker which moves on the surface of microparticle based on it.39 Similar to CHA, isothermal strand-displacement polymerase reaction (ISDPR), triggered by DNA polymerase to repeat a DNA template, is one of the mature enzymatic amplification strategies.14 Considered the advantage of CHA and ISDPR that strand displacement could propel the movement of DNA walker, the construction of 3D-DNA walker biosensors has attracted our interest. Herein, we have designed two kinds of biosensors based on multi-pedal DNA walker along a 3-D DNA functional magnet particles track for the detection of SA. Also, the DNA walker based on ISDPR is better than that based on CHA. As shown in scheme 1A, in the presence of SA, biotin-linked catalyst can be protected from digestion by exonuclease I (exo I) as a result of terminal protection, and multi-pedal DNA walker is constructed. Due to the hybridization between multi-pedal DNA walker and a toehold on CHA-H1 coupled with MMPs, the hairpin structure of CHA-H1 has been opened through toehold-mediated strand exchange. The newly exposed DNA segment of CHA-H1 would interact with a toehold of biotin-labeled H2, and a tripartite complex of these DNA strands is formed via branch migration. As the process of strand displacement, this complex could turn into the most thermodynamically favorable configuration. When one ‘leg’ of DNA walker is displaced by H2, the other ‘leg’ would still interact with the adjacent H1 to trigger more displacement reaction. Finally, the SA-HRP, as the catalyst, is introduced in the DNA walker products. However, the high background caused by the hybridization between CHA-H1 and H2 without CHA-catalyst has influenced the LOD of SA, and the other model has been constructed to solve this. The principle of scheme 1B is similar to 1A. After terminal protection of SA, the multi-pedal DNA walker acts as a ACS Paragon Plus Environment

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trigger, leading to stem separation of ISDPR-H1 conjugated with MMPs. Following this, the biotin-modified primer could hybridize with the open segment, triggering the polymerization reaction with the assistance of dNTPs and polymerase. As primer extension process, one ‘leg’ of the multi-pedal DNA walker is displaced and the other ‘leg’ could induce proximal H1 to the next cycle. These two models have been compared, and we also simply analyzed the merit and demerit of them. And the results of detections for folate receptor and thrombin are satisfactory. Therefore, the DNA walker biosensors can lay the foundation for the future development of protein detections.

EXPERIMENTAL SECTION Materials and Reagents All reagents and apparatus are listed in the Supporting Information.

Modification of MMPs First, 500 µL of carboxyl-modified MMPs (10 mg mL-1) were taken into polystyrene tube and washed by using PBS buffer solution. And then, 425 µL of PBS buffer included a certain amount of DNA, 50 µL of NHS (0.05 M) and 25 µL of EDC (0.1 M) were mixed with the washed MMPs and stirring all the night. After three times washing by Tris-Tween buffer, the unreacted carboxylic acid in the conjugated MMPs were removed. Finally, the MMPs which were sealed with TE buffer containing 1% BSA were stored in refrigerator. Procedures for SA detection based on CHA DNA walker First of all, a certain concentration of SA and 10µL of CHA-catalyst are mixed in 80µL of exo I buffer solution. After 30 min, 5 units of exo I are added into the mixture reaction for a 60 min. When the enzyme reaction finished, the solution was heated in 80 °C of water bath pot for 15 min, and then kept 1 h at room temperature. Then, 5 µL


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of CHA-H1 modified MMPs, 10 µL of H2 and 85 µL of CHA buffer solution (NaCl 300 mM) were added into, followed by incubating for 1 hour at 37 °C under stirring. After washing three times, 50 µL of SA-HRP was mixed with the walker products for 30 min. In order to remove the redundant SA-HRP, the MMPs have been rinsed for three times. And the SA-HRP-MMPs were transferred into the polystyrene microplates. Then, 50 µL of solution of luminal and H2O2 was added and commixed with them. The polystyrene microplates were put into ChemiDoc XRD apparatus to record the intensity by the Quantity One software. Procedures for SA detection based on ISDPR DNA walker Similar with the procedures of SA detection based on CHA, a certain concentration of SA and 10µL of ISDPR-catalyst are mixed into 80µL of exo I buffer solution at first reaction for a 30 min. Then, 5 units of exo I are put into the tube. After 60 minutes incubation at 37 °C, the solution was heated in 80 °C of water bath pot for 15 min, and then kept 1 h at room temperature. 5 µL of ISDPR-H1 modified MMPs, 10 µL of primer, 1 mM dNTP, 10 units of polymerase and 87 µL of ISDPR buffer were added and stirred for 2 h at 37 °C. After washing three times, SA-HRP solution (50 µL) was added and incubated for 30 min. Through three times washing, the SA-HRP-MMPs were transferred into the wells and mixed with 50 µL of luminal and H2O2 solution. The CL imaging was recorded. SA detection in complex biological environment In order to explore the antijamming capability, the detection for SA in human serum was tested. First, various concentrations of SA and 10 µL of catalyst were added into 80 µL buffer reaction for 30 min, and then 5 units of exo I were mixed with them under shaking for 1 hour. After heated 15 min in 80 °C’s water bath and kept 1 hour at 25 °C, the other reactants for CHA and ISDPR were added into the PE pipe, respectively. After washing three times, 50 µL of SA-HRP was put into the tube. The SA-HRP-MMPs which rinsed three times were removed to the wells and


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commixed with 50 µL of luminol and H2O2 solution. The CL intensity of each polystyrene well was recorded. Gel electrophoresis Various buffer and DNA sequence were mixed together to react for different time. Then, SYBR Green I and bromophenol blue were added into the tube. After incubated for 15 min, the products agarose gel elelectrophoresis analysis (3%, 1 × Tris-borate-EDTA, pH 8.3, 110 V) were realized for 30 min, and imaged by using Bio Rad (Fig. S1).

RESULTS AND DISCUSSION Principle of SA detection based on CHA DNA walker In Scheme 1, the principle of SA detection based on CHA DNA walker has been shown. In the presence of SA, biotin modified CHA-catalyst could avoid the digestion by exo I because of terminal protection from the combination between SA and biotin. Briefly, multi-pedal DNA walker has been constructed. Through toehold-mediated strand exchange, the hairpin structure of CHA-H1 coupled with MMPs could be opened after hybridized with the ‘leg’ of multi-pedal DNA walker. And then a newly exposed DNA segment of CHA-H1 would interact with the loop in H2, inducing branch migration to come into being a tripartite complex between the ‘leg’ of multi-pedal DNA walker, H2 and CHA-H1. And this complex would resolve into the H1:H2 duplex which is the most thermodynamically steady state configuration via strand displacement, because the stronger intermolecular force between them. When H2 displaces one ‘leg’ of multi-pedal DNA walker, the neighboring H1 could still be hybridized by the other ‘leg’, and the displaced one can participate in subsequent reaction cycles achieving the target recycling and leading to H2 conjugated biotin enriching on the surface of MMPs. Ultimately, as the catalyst of CL system, SA-HRP has been introduced in the products of DNA walker through SA-biotin interaction.


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After the magnetic separation to get rid of needless SA-HRP, a strong CL signal is produced by adding CL substrate. While in the absence of SA, CHA-catalyst would be disintegrated by exo I so that the strand displacement proceeds would be much slower, presenting very weak CL signal. Sensitivity and selectivity of SA detection based on CHA DNA walker In order to get steady-state light signal and a higher sensitivity, the HRP-catalyzed H2O2-PIP-luminol system with PIP as a fortifier is used for CL imaging detection. Under the optimal condition (Fig. S2 and S3), we investigated the sensitivity of the method based on CHA with different SA concentrations. In these Figures, ∆I =I0-I, where ∆I means the relative CL intensity, I0 presents target’s signal and background signal is defined as I. As shown in Fig. 1A, the relative CL intensity increases gradually with the increase of the concentration of SA, and there is a nice linear relationship between them. The linear equation is ∆I=4.9773 C +18.0671 (R2=0.9928) when the concentrations are from 30.77 to 461.5, where C is SA’s concentration and ∆I means the relative CL intensity. On the basis of the equation CLOD=3S/k where k is the regression equation’s slope, S is standard deviation of the blank (N=5) and CLOD is the limit of detection (LOD), the limit of detection for SA is 11.1 pM. Also, we evaluate the specificity of this method. SA and other proteins, for example, BSA, trypsin and thrombin have been investigated. As presented in Fig. 1B, although other proteins’ concentrations are higher than SA’s, the SA’s relative CL intensity is stronger than other proteins’. The specificity between SA and biotin makes contribution for the high selectivity. Therefore, this proposed strategy expresses a wonderful capability for SA detection. Principle of SA detection based on ISDPR DNA walker For the sake of decreasing the high background of DNA walker based on CHA, we have designed a new model based on ISDPR. The principle of Scheme 2 is similar to Scheme 1 at the first step. With the presence of SA, ISDPR-catalyst could form the


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multi-pedal DNA walker and be protected from digestion by exo I. The ISDPR-H1 can recognize and hybridize with one ‘leg’ of the multi-pedal DNA walker and undergo a conformational change to separate stem. Then a biotin-modified primer could hybridize with the open stem, and a polymerization reaction would be triggered by polymerase and dNTPs. The ‘leg’ of multi-pedal DNA walker has been displaced as the polymerization reaction processes. After this other ‘legs’ could interact with another H1 to next cycle, leading to the biotin-modified primer’s multiplication for MMPs. After being loaded into the system, a signal trace would be afforded by SA-HRP for CL analysis of SA. In the absence of SA, the ISDPR-catalyst has been degraded through enzyme reaction and just very weak CL signal would be presented. Optimization of detection conditions of ISDPR DNA walker Many parameters of SA detection in Scheme 2 influencing CL intensity have been investigated. As presented in Fig. 2A, a series of concentrations of ISDPR-catalyst in this work are investigated to get the optimal one. When the concentration of ISDPR-catalyst increases, the relative CL intensity goes up fast at first, and then it is gradually stable after reaching the maximum. Therefore, 2 nM ISDPR-catalyst is chosen for the assay. Fig. 2B displays the relationship between the amount of exo I and the relative CL intensity. With the increase of amount of exo I, the relative CL intensity presents a peak. So 5 units of exo I is selected. The relationship between the relative CL intensity and the concentration of primer-9 in the range from 1 to 25 nM is shown in Fig. 2C. Thus, 10 nM primer-9 is used for the study. The concentration of SA-HRP is examined from 150 to 350 ng/mL, and Fig. 2D has described the results. With the variation of concentration, the relative CL intensity increases until reaching 250 ng/mL. Therefore, 250 ng/mL SA-HRP is selected as the best one. What’s more, the optimization for other parameters is in Fig. S4, S5 and S6.


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Sensitivity and selectivity of SA detection based on ISDPR DNA walker Similarly, the sensitivity of the method based on ISDPR in Scheme 2 is also evaluated. When the concentration of SA increases, the relative CL intensity increases by degrees as Fig. 3A described, and there is a good liner relationship between them. The regression equation is ∆I=6.1456 C +175.0649 (R2=0.9921) in the linear ranging from 30.77 to 461.5 pM. The LOD for SA is 6.5 pM. In this study, the strategy’s specificity has been tested. The experiment is challenged with BSA, trypsin and thrombin. SA’s relative CL intensity is stronger than other proteins’ while concentrations of other proteins are higher than SA’s as Fig. 3B shown. Hence, this proposed method has a high selectivity. The comparisons between CHA DNA walker and ISDPR DNA walker The two kinds of multi-pedal DNA walker biosensors have their own advantages. The DNA walker based on CHA doesn’t need enzyme to propel it through the track, so that it is low-cost, time-saving and easy-operating. However, there is still a problem that the high background caused by the hybridization between H2 and CHA-H1 without CHA-catalyst. Hence, the other model has been designed. The DNA walker based on ISDPR has avoided the hybridization between ISDPR-H1 and primer-9 as a result of too few complementary bases between them. The LOD of CHA DNA walker is higher than ISDPR DNA walker’s. It’s possibly that the impetus provided by polymerase is more effective than H2, and the lower background of ISDPR DNA walker biosensor, for the interaction between CHA-H1 and H2 without multi-pedal DNA walker is stronger than it between ISDPR-H1 and primer without multi-pedal DNA walker. These differences could offer a broad platform for applying in various systems. SA detection in complex biological environment For the purpose of introducing the method in complex environment, the proposed


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methods have been developed for analyzing SA in 20 % human serum. As Fig. 4 described, there are slightly differences of the relative CL intensities between buffer solution and the diluted human serum, and CL intensities’ relative standard error between them is acceptable. Therefore, these methods are indicated that could be used in complex biological environment for SA detection. Folate receptor and thrombin detections For expanding the application of multi-pedal DNA walker based on ISDPR, the detections of folate receptor and thrombin have been explored. As Fig. 5A described, the relationship between folate receptor’s concentrations and the relative CL intensity presents a good liner. The LOD for folate receptor is calculated to be 17.03 pM. And Fig. 6A has presented the liner relationship of the relative CL intensity and thrombin’s concentrations. Based on the formula, the LOD for thrombin is 710 pM. And for the sake of comparing the LODs of DNA walker based on ISDPR and CHA, the results of detections for folate receptor and thrombin based on CHA DNA walker have been show in Fig. S7 and S8. Also, the LODs based on ISDPR DNA walker are lower than those based on CHA DNA walker. And the results are consistent with SA detection. Besides, the specificity of this method has been tested with different proteins. In Fig. 5B and Fig. 6B, the relative CL intensity of target protein is a great deal stronger than other proteins’ while the concentrations of others are higher. So this aforesaid method has a high selectivity.

CONCLUSION In conclusion, two kinds of sensitive biosensors based on multi-pedal DNA walker along a 3-D DNA functional magnet particles track for SA detection were constructed. And the ISDPR DNA walker has lower LOD than CHA DNA walker. It is proved that these biosensors for SA detection are of high sensitivity and selectivity. Moreover, the comparison between two models provided us a broad platform, so that we could do further exploration in the future. And these two biosensors have been


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used for folate receptor and thrombin detections successfully. Also, in 20% human serum, the results of detection of SA are satisfactory. Therefore, the multi-pedal DNA walker exhibits great potential in application of it in versatile biosensing.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21675119, 21475101).

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Scheme 1. Schematic illustration of the principle for the sensitive detections of SA based on CHA DNA walker. Schematic illustration of the principle for the sensitive detections of SA based on ISDPR DNA walker.

Figure 1. (A) Relationship between the relative CL intensity and the concentration of SA. Inset: CL images of SA at different concentrations for Scheme 1A. (B) Specificity of the assay for SA detection with different proteins for Scheme 1A. Experimental


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conditions: 5 µg of MMPs, 2 nM CHA-catalyst, 5 U of exo I, 5 nM H2, and 250 ng/mL SA-HRP.

Scheme 2. Schematic illustration of the principle for the sensitive detections of SA based on ISDPR DNA walker.

Figure 2. Effects of (A) the concentrations of ISDPR-catalyst, (B) the amount of exo I, (C) the concentrations of primer-9 (D) the concentration of SA-HRP on the relative CL intensity of the sensing system based on ISDPR. ACS Paragon Plus Environment

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Figure 3. (A) Relationship between the relative CL intensity and the concentration of SA. Inset: CL images of SA at different concentrations for Scheme 1B. (B) Specificity of the assay for SA detection with different proteins for Scheme 1B. Experimental conditions: 5 µg of MMPs, 2 nM ISDPR-catalyst, 5 U of exo I, 10 nM primer-9, 10 U of Klenow, and 250 ng/mL SA-HRP.


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Figure 4. The comparisons of the sensing system for SA detection (A) CHA multi-pedal DNA walker biosensor, (B) ISDPR multi-pedal DNA walker biosensor in buffer solution and 20% diluted human serum samples, respectively.


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Figure 5. (A) Relationship between the relative CL intensity and the concentration of folate receptor based on ISDPR DNA walker. Inset: CL images of folate receptor at different concentrations. (B) Specificity of the assay for folate receptor detection with different proteins.


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Figure 6. (A) Relationship between the relative CL intensity and the concentration of thrombin based on ISDPR DNA walker. Inset: CL images of thrombin at different concentrations. (B) Specificity of the assay for thrombin detection with different proteins.


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Supporting Information Available: [The following files are available free of charge.] Supplementary Information For Multi-pedal DNA walker biosensors based on catalyzed hairpin assembly and isothermal strand-displacement polymerase reaction for the chemiluminescent detection of proteins Brief description of contents. Materials and Reagents Figure S1. The result of gel electrophoresis for DNA walker products. Figure S2. Effects of (A) the concentrations of CHA-catalyst, (B) the amounts of exo I, (C) the concentrations of H2 (D) the concentration of SA-HRP on the relative CL intensity of the sensing system based on CHA. Figure S3. Effects of the amounts of MB-CHA-H1 on the relative CL intensity of the sensing system based on CHA DNA walker. Figure S4. Effects of different primers on the relative CL intensity of the sensing system based on ISDPR DNA walker. Figure S5. Effects of the amounts of MMPs on the relative CL intensity of the sensing system based on ISDPR DNA walker. Figure S6. Effects of the amounts of Klenow on the relative CL intensity of the sensing system based on ISDPR DNA walker. Figure S7. (A) Relationship between the relative CL intensity and the concentration of folate receptor based on CHA DNA walker. Inset: CL images of folate receptor at different concentrations. (B) Specificity of the assay for folate receptor detection with different proteins. Figure S8. (A) Relationship between the relative CL intensity and the concentration of thrombin based on CHA DNA walker. Inset: CL images of thrombin at different concentrations. (B) Specificity of the assay for thrombin detection with different proteins.


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Multi-pedal DNA walker biosensors based on catalyzed hairpin

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