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DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing
Journal Pre-proof DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing Yu Xin Liu, Xue Xiao, Chun Hong Li, Chen Men, Qi Chao Ye, Wen Yi Lv, Yuan Fang Li, Cheng Zhi Huang, Shu Jun Zhen PII:
S0039-9140(20)30021-7
DOI:
https://doi.org/10.1016/j.talanta.2020.120730
Reference:
TAL 120730
To appear in:
Talanta
Received Date: 17 October 2019 Revised Date:
6 January 2020
Accepted Date: 8 January 2020
Please cite this article as: Y.X. Liu, X. Xiao, C.H. Li, C. Men, Q.C. Ye, W.Y. Lv, Y.F. Li, C.Z. Huang, S.J. Zhen, DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing, Talanta (2020), doi: https://doi.org/10.1016/j.talanta.2020.120730. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Graphical abstract for
DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing Yu Xin Liu,a Xue Xiao,c Chun Hong Li,b Chen Men,a Qi Chao Ye,a Wen Yi Lv,a Yuan Fang Li,a Cheng Zhi Huang and Shu Jun Zhen a, *
a
a
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, 400715, Chongqing, P. R. China. b
College of Pharmaceutical Sciences, Southwest University, 400715, Chongqing, P. R. China.
c
College of Chemistry and Environment Protection Engineering, Southwest Minzu University, Chengdu 610041, China *
Corresponding author. Tel: (+86)-23-68254059.
E-mail addresses: [email protected] (S. J. Zhen).
DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing Yu Xin Liu,a Xue Xiao,c Chun Hong Li,b Chen Men,a Qi Chao Ye,a Wen Yi Lv,a Yuan Fang Li,a Cheng Zhi Huang a, b and Shu Jun Zhen a, * a
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of
Chemistry and Chemical Engineering, Southwest University, 400715, Chongqing, P. R. China. b
College of Pharmaceutical Sciences, Southwest University, 400715, Chongqing, P. R. China.
c
College of Chemistry and Environment Protection Engineering, Southwest Minzu University, Chengdu 610041, China
ABSTRACT: Recently, various inorganic nanomaterials have been used as fluorescence anisotropy (FA) enhancers for biosensing successfully. However, most of them are size-uncontrollable and possess an intensive fluorescence quenching ability, which will seriously reduce the accuracy and sensitivity of FA method. Herein, we report a two-dimensional DNA nanosheet (DNS) without fluorescence quenching effect as a novel FA amplification platform. In our strategy, fluorophore-labelled probe DNA (pDNA) is linked onto the DNS surface through the hybridization with the handle DNA (hDNA) that extended from the DNS, resulting in the significantly enhanced FA value. After the addition of target, the pDNA was released from the DNS surface due to the high affinity between the hDNA and target, and the FA was decreased. Thus, target could be detected by the significantly decreased FA value. The linear range was 10-50 nM and the limit of detection was 8 nM for the single-stranded DNA detection. This new method is general and has been also successfully applied for the detection of ATP and thrombin sensitively. Our method improved the accuracy of FA assay and has great potential to detect series of biological analytes in complex biosensing systems.
Keywords: DNA nanosheet; fluorescence anisotropy; single-strand DNA; ATP; thrombin.
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INTRODUCTION Fluorescence Anisotropy (FA) is a homogeneous, attractive and robust method in biochemical analysis system [1-3]. It is directly related to the rotation motion and mass of the fluorescently labeled molecules when the temperature and the viscosity of the solution are fixed [4-6]. FA has been widely used to analyze a large number of targets such as nucleic acids [7-10], metal ions [5, 11, 12], small molecules[13-16] and so forth. However, since most targets have small molecular weights, it is a great challenge to produce significant FA changes when adding targets. In order to overcome this limitation, a wide variety of nanomaterials, including gold nanoparticles [17], silver nanomaterials [18], metal organic frameworks (MOFs) [19], metal organic gel (MOG) [20] and carbon nanomaterials [21-23] have been successfully used as FA enhancers. However, most of these nanomaterials possess an intensive fluorescence quenching ability through various mechanisms. For example, some nanomaterials efficiently quench the fluorescence of fluorescent dye by means of electron transfer (ET) [24], and the others possess broad and strong absorption within the visible light range which overlaps with the emission of commonly used fluorescent probe [25] and have been regarded as a kind of excellent acceptors of fluorescence resonance energy transfer (FRET) [26-30] to quench the fluorescence of fluorophore. Under the condition of severe fluorescence quenching, the FA data measurement can be influenced by the scattered light easily, making the obtained FA value be greater than 0.4 [31]. Generally, if the measured FA for a randomly oriented sample is bigger than 0.4, one can confidently infer the presence of scattered light in addition to fluorescence. To reduce the fluorescence quenching effect, several FA detection systems [32-34] have been developed. These systems reduce the fluorescence quenching efficiency by controlling the distance between fluorescent groups and amplification nanomaterials, However, the farther the fluorescent group is from the nanomaterial, the less restricted the fluorescent group is suffered, which will decrease FA signal and thus reduce the sensitivity. Moreover, it is difficult to control the number of probes connected to the surface of nanomaterials due to uneven size and shape of these nanomaterials. Furthermore, there is a strong adsorption between single-stranded DNA (ssDNA) and these nanomaterials through non-covalent or covalent interactions, which will result in low detection sensitivity. Therefore, finding an ideal FA amplifier which possess the properties including without fluorescence quenching effect, controlled size and shape, easy to link and release probes with controlled numbers is highly desirable. In recent years, a variety of excellent signal amplification methods have been developed to improve the sensitivity of biosensing, such as ion concentration polarization [35], isotachophoresis [36], DNA nanostructure-based approaches [37, 38] and so on. Especially, DNA nanostructures have aroused great attentions due to their facile synthesis, good biocompatibility and easy multiple modification. Compared with the traditional inorganic nanomaterials, DNA nanostructure can be used as a new FA amplifier because of its following amazing properties. First, owing to the chemical structural characteristics of nucleotides, DNA cannot form large conjugated structure, which leads us to speculate that DNA nanostructure has a weak absorption in the long wavelength range. Thus, the fluorescence will not be quenched by DNA nanostructure through FRET because there may be weak spectral overlap between the absorption of DNA nanostructure and the emission of common fluorescent probes. Thus, the accuracy of FA detection can be improved by the use of DNA nanostructure. Second, DNA nanostructures have well-defined geometry and superior addressability, so that probes can be modified on the surface of DNA nanostructures in a precisely controlled manner. Therefore, the reproducibility of FA detection can be guaranteed by the number-controlled probes on DNA nanostructure. Third, it is well known that after the formation of the double helix structure, the electronic system of nucleobases of DNA is shielded inside the hydrophilic double helix backbone [39]. So, there is a weak π-stacking interaction between DNA nanostructure and ssDNA, which will significantly reduce the adsorption of ssDNA on DNA nanostructure and improve the sensitivity of detection. In additional, the rotation rate of two-dimensional (2D) nanomaterials is slower than that of zero-dimensional (0D) spherical nanomaterials with the same surface area [40, 41]. Therefore, in this contribution, we established an universal and simple method by utilizing a DNA nanosheet (DNS), a 2D DNA nanostructure, as a novel FA amplification platform for the detection of ssDNA about chronic myelogenous leukemia (CML, type b3a2) [42-44], ATP and thrombin successfully.
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Scheme 1. Schematic illustration of fluorescence anisotropy amplification strategy based on 2D DNSs. (Double arrows represent the possible rotational contributions to the FA change).
EXPERIMENTAL SECTION Materials. All oligonucleotides (table S1 and table S2) used in this work were synthesized and HPLC purified by Sangon Biotechnology Co., Ltd. (Shanghai, China). Adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP) and uridine triphosphate (UTP) were purchased from Sangon Biotech (Shanghai, China). Thrombin, hemoglobin, albumin, amylase and pepsase were obtained from Shanghai Yuanye Biological Technology Co., Ltd.
All the chemicals were of analytical
grade and used without further purification. All stock solutions and buffers were prepared with Milli-Q ultrapure water (18.2 MΩ· cm-1). All procedures that required temperature control were measured by a thermomixer C (Eppendorf, Germany). Solutions were evenly blended by a vortex mixer MX-E (Haimen, China). The UV-vis absorption spectra were analyzed with a UV-3010 spectrophotometer (Daojin, Japan). General procedure. First, DNA nanosheet (DNS) was prepared by mixing 46 strands in a buffer at equal molar ratio at 100 nM. The mixture was then allowed to the following thermal annealing program: 85 ºC for 10 min, 80 ºC to 24 ºC, approximately 16 h. Then, dye-labeled probe was added to well-prepared DNSs under constant shaking at room temperature for 1 h. Last, target was added to the mixture and the final volume was made to 200 µL by adding buffer. After the addition of target, the mixture incubated for 2 h under constant shaking at 37 ºC. Then, FA was measured. The assembled DNSs were allowed to store at 4 °C for at least 1 week for further use. FA measurement. Fluorescence intensities were measured on F-2500 fluorescence spectrophotometer with a polarization filter (Hitachi, Tokyo, Japan). Excitation wavelength was set at 560 nm, and emission wavelength was recorded at 581 nm. Slits for the excitation and the emission were both set at 5 nm. The integration time was set at 0.4 s. The quartz cuvette was employed as the sample cell and the FA measurement experiments were all carried out at room temperature. The fluorescence anisotropy (r) was calculated by equations as follows: r=
ூ ିீൈூౄ
ூ ାଶீൈூౄ
(1)
and ூ
G= ౄ
ூౄౄ
(2)
3
where IVV and IVH are the fluorescence intensities of the vertically and horizontally polarized emission
after excitation by
vertically polarized light, respectively. The instrumental correction factor G corrects for the different detection efficiencies of perpendicular and parallel emission paths. Native Polyacrylamide Gel Electrophoresis. DNSs were subjected to 8% native polyacrylamide gel (gel was prepared in 2 mL of 40% acrylamide/bis-acrylamide gel solution (29:1) supplemented with 10 mM MgCl2, 1.6 mL 5× TBE, 56 µL 10% ammonium persulfate (APS) and 8 µL N, N, N', N'-tetramethylethylenediamine (TEMED), and the final volume was fill with water to 9 mL). Electrophoresis was conducted in 1× TBE at 120 V for 90 min or 160 V for 60 min. Once finished running, the gel was soaked in 0.005% (v/v) ethidium bromide (EtBr) solution for 10 min and then scanned using a WD-9403E portable UV apparatus (Beijing Liuyi Biotechnology Co., Ltd., China). Atomic force microscopy imaging. Atomic force microscopy (AFM) images were obtained in air under the tapping mode using Dimension Icon (Bruker). 10 µL of sample was deposited onto the surface of freshly cleaved mica chip and left in dry ambience air to allow for absorption. Then, the products were washed with 10 µL of Milli-Q ultrapure water three times to remove excess salt. The results were analyzed with NanoScope Analysis software.
RESULTS AND DISCUSSIONS Principle of the Design. In this study, taking full advantages of DNA nanotechnology, a two-dimensional rectangular DNA nanosheet (DNS) (the dimensions were 32 nm long and 24 nm wide calculated by theory) prepared according to the methods of Dong M. Shin et al [45] was used as a novel platform for enhancing FA. As depicted in Scheme 1, the fluorescent dye carboxytetramethylrhodamine (TAMRA) modified probe DNA (pDNA) was free in the absence of DNSs, which can bring about a low FA signal since the ssDNA binding to the TAMRA has a low molecular weight. In the presence of DNSs, pDNA was indirectly immobilized on DNSs surface through hybridization with a handle DNA (hDNA) extended from DNSs, which can cause enhanced FA signals by restricting the rotational diffusion of the TAMRA as a result of significant increased molecular weight. Because DNS cannot quench the fluorescence of TAMRA, TAMRA was designed as close as possible to the DNS surface to limit the rotation of TAMRA with the entire framework to achieve high FA. Feasibility of the DNS enhanced FA assay. First, AFM images (Fig. S1) confirmed the successful preparation of DNSs. The average length of DNSs was 30 ± 2 nm and the average width of DNS was 21 ± 2 nm (n=28), which were basically consistent with the theoretic results, indicating that the DNSs were prepared with anticipatory dimensions and patterns. Then, the assembly of DNS was confirmed with 8% native polyacrylamide gel electrophoresis (Native-PAGE). The products all displayed a low mobility (lanes 1-4, Fig. S2), indicating the successful synthesis of DNSs with protruding strands of different sequences. In order to investigate the fluorescence quenching ability of DNSs, we evaluated the spectral properties of the DNSs. The absorption of the DNSs and the fluorescence spectra of the (TAMRA)-conjugated DNAs were shown in Fig. 1A. As we anticipated, DNSs have no obvious absorption after 300 nm over a wide range of wavelengths, and there is weak spectral overlap between the emission of TAMRA and the absorption of DNSs, which effectively avoids the fluorescence quenching through the means of FRET and ensures the accuracy of the FA values. We next tested FA amplifying ability of DNSs. As show in Fig. 1B, there is a low FA signal in the absence of DNSs and a high FA signal in the presence of DNSs, which clearly confirms that the DNSs equip an outstanding capability for amplifying FA. Then, we studied the total fluorescence intensity in the absence and presence of DNSs. The weak change in fluorescence intensity (Fig. 1C) confirmed that the DNSs didn’t quench the fluorescence of TAMRA. The experimental results show no significant spectral shift in fluorescence spectra and small change in the shape of the fluorescence spectra of the unidirectional fluorescence spectra (HH, HV, VH, and VV) in the presence (Fig. 1D) and absence of DNSs (inset of Fig. 1D), further
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Fig. 1. (A) UV-vis absorption spectrum (red) of DNSs and fluorescence spectra of the TAMRA-labeled pDNA. Concentration: DNSs, 50 nM; p-d0, 50 nM. (B) FA responses with or without DNSs. (C) Fluorescence spectra in the absence and presence of DNSs. (D) The unidirectional fluorescence spectra (HH, HV, VH, VV) in the presence of DNSs. The inset is the unidirectional fluorescence spectra in the absence of DNSs. Concentration: DNSs, 97 nM; p-d0, 30 nM. certifying that the accuracy of the FA values is ensured. In contrast, the fluorescence was almost quenched and the FA value was exceed 0.4 (Fig. S4) when GO or MOF (MIL-101) was employed for FA amplification. All of these experimental results confirmed that DNSs could be used as efficient FA amplifiers. Next, to identify the interaction mechanism between DNSs and TAMRA-labeled probe, the handle-free DNSs are mixed with ssDNA and double-strand DNA (dsDNA) respectively. Then we removed DNSs through centrifugal ultrafiltration (molecular weight cut-off 100KDa) and measured the fluorescence intensity of the solution in the collection tube. The centrifugal ultrafiltration experimental results confirmed that DNSs can scarcely absorb ssDNA or dsDNA (Fig. 2A & 2B). Thus, when the pDNA is released from DNSs in the presence of target, it will not bind to DNSs again and the loss of sensitivity caused by non-specific binding could be significantly reduced. Subsequently, we investigated the influence of the distance between TAMRA and DNSs and the position of TAMRA on DNSs surface on the enhancement of FA. In order to explore the factor of distance, we keep the position of the extended chain on the DNSs unchanged, then modify the sequence of extended chain correspond to h-d0, h-d4.42, h-d7.14, h-d9.86 , and h-d13.26, respectively (the pDNA are p-d0, p-d4.42, p-d7.14, p-d9.86, and p-d13.26 in table S1). The experimental results (Fig. 2C) showed ∆r response (∆r = r0-r, where r0 is the FA values in presence of DNSs and r is the FA values in the absence of DNSs) decreased dramatically once TAMRA leaves away from DNSs surface, suggesting that when the distance between TAMRA and DNSs is short, the integration degree of the rotational movement of the formed complex with that of TAMRA is high. To explore the factor of position, we keep the length of the extended chain on the DNSs unchanged (the distance between TAMRA and DNSs is 0), and make the transformation sequence correspond to DNSs with h-S1, h-S2, h-S3, and h-S4, respectively (the pDNA is p-d0 in table S1). According to the ∆r response, the location of fluorophore on DNSs (Fig. S3) showed small influence on FA value (Fig. 2D). Detection of CML-DNA. The short ssDNA sequence of Type b3a2, a frequent BCR/ABL fusion gene existing in chronic myelogenous leukemia (CML), was selected as a proof-of-concept target DNA (tDNA). As shown in Fig. 3A, a single-stranded
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Fig. 2. (A) The interaction mode between handle-free DNSs and ssDNA. 1, ssDNA; 2, handle-free DNSs mixed with ssDNA; 3, subnatant of handle-free DNSs mixed with ssDNA after ultrafiltration. (B) The interaction mode between handle-free DNSs and dsDNA. 1, dsDNA; 2, handle-free DNSs mixed with dsDNA; 3, subnatant of handle-free DNSs mixed with dsDNA after ultrafiltration. Concentration: DNSs, 50 nM; p-d0, 50 nM. (C) ∆r response of different distance between DNSs and TAMRA. The inset is FA signal in the presence (orange column) or absence (green column) of DNSs at the corresponding distance between the DNSs and TAMRA. (D) ∆r response of different position between DNSs and TAMRA. The inset shows FA signal to TAMRA on the different position of the DNSs in the presence (orange column) or absence (green column) of DNSs. Experiments were performed in the presence of 97 nM DNSs, 30 nM of p-d0, p-d4.42, p-d7.14, p-d9.86, and p-d13.26, respectively. handle extended from the DNSs surface (hDNA1) whose target recognition region was designed to complementary with tDNA and the probe load region was used to combine with pDNA1. Before the tDNA was added, the pDNA1 was combined with DNSs which has restricted the rotation of TAMRA and achieved a high FA response. After the addition of tDNA, the pDNA1 was released from the DNSs surface through toehold-mediated DNA strand exchange reaction, resulting in the obviously decreased FA value. Thus, the tDNA could be detected by the decreased FA value. We investigated FA responses and fluorescence intensity responses in the absence and presence of tDNA with/without subsequent incubation with DNSs. A low FA signal in the absence of DNSs and a high FA signal in the presence of DNSs were observed (Fig. 3B). The total fluorescence intensity before or after the addition of tDNA in the presence of DNSs (Fig. S5) has barely changed and no observable peak shift in the unidirectional fluorescence spectra (Fig. S6A & S6B) without or with tDNA in the presence of DNSs. These results further confirmed that the DNS is an excellent FA amplifier. In order to achieve the best experimental performance, we investigated the effects of the concentration of DNSs firstly. If the concentration of DNSs was low, small number of the pDNA1 could be immobilized on the surface of DNSs, resulting in a low FA value. However, when the concentration of DNSs was too high, there will be extra DNSs without pDNA1 connections since the number of pDNA1 is constant, and the tDNA would hybridize with hDNA1 directly instead of releasing the pDNA1 through toehold-mediated DNA strand exchange reaction, so that the pDNA1 on DNSs were unable to release from DNSs surface easily, leading to the small change of FA. The experimental results (Fig. S7) showed that when the concentration of DNSs was 40 nM, ∆r (∆r = r0-r, where r0 is the FA values in absence of tDNA and r is the FA values in the presence of tDNA) reached its maximum
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Fig. 3. (A) Schematic illustration of fluorescence anisotropy amplification strategy based on 2D DNSs for the detection ssDNA. (B) FA signal in the absence and presence of tDNA with or without DNSs. (C) ∆r response with the increasing concentration of tDNA. Inset: linear correlation of ∆r with different concentrations of tDNA. (D) Selectivity test for tDNA detection. Experiments were performed in the presence of 40 nM DNSs, 25 nM pDNA1, 40 nM tDNA and 40 nM of the all mismatched DNA, respectively. value. Then, we tested the influence of incubation temperature and time on the detection. The experimental results (Fig. S8) showed that the most suitable incubation conditions of pDNA1 connected on DNSs were 25 ºC for 1h. The optimized incubation temperature and time for the toehold-mediated strand displacement reaction between the complex of DNSs/pDNA1 and tDNA were 37 ºC for 2 h. We further examined the number of toehold base domain (N) on hDNA1. Four types of pDNA sequences (pDNA1-1, pDNA1, pDNA1-2, and pDNA1-3 in table S1) with different lengths were designed. When the pDNA was too short, the pDNA was unable to hybridize with hDNA tightly, while it could not be released from DNSs upon the addition of tDNA if the pDNA was too long. Thus, the optimal base number of toehold domain was confirmed as 8 (Fig. S9). Next, the FA changes after the addition of tDNA with different concentrations under optimal experimental conditions were measured. As evident from Fig. 3C, with the increasing concentration of tDNA from 1 nM to 60 nM, the ∆r dramatically increased, implying the increasing amount of the pDNA1 was released from the DNSs surface and dissociated in a solution through DNA toehold-mediated chain exchange reaction. ∆r exhibited a linear response (R2= 0.9933) with the concentration of tDNA in the range of 10-50 nM (inset of Fig. 3C) and the limit of tDNA detection was 8 nM (Fig. S10). To investigate the selectivity of this method, we compared the FA responses with tDNA and various mismatched targets including single-base mismatched DNA (t1), two-base mismatched DNA (t2), three-base mismatched DNA (t3), four-base mismatched DNA (t4) and random non-complementary DNA (nc). As shown in Fig. 3D, increasing in the number of mismatched bases followed by significantly decreasing in the ∆r, and no distinct FA changes induced by more than three base-mismatched DNA can be observed, certifying the excellent selectivity of our approach. General Applicability of the Strategy. To evaluate the generality of our method, we first explored it for the detection of ATP. As described in Fig. 4A, hDNA2 that contains the sequence of the anti-ATP aptamer was protruded from the surface of DNSs and
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Fig. 4. Detection of ATP and thrombin. (A) Schematic illustration of fluorescence anisotropy amplification strategy based on 2D DNSs for the detection of ATP and thrombin. (B) ∆r response with the increasing concentration of ATP. Inset: linear correlation of ∆r with different concentrations of ATP. (C) The relative ∆r response of ATP, GTP, CTP, and UTP (350 µM respectively). (D) ∆r response with the different concentration of thrombin. Inset: linear correlation of ∆r with different concentrations of thrombin. (E) Selectivity of the FA assay of thrombin over pepsase, trypsin, hemoglobin, albumin (0.68 µM respectively). Experiments were performed in the presence of 40 nM DNSs, 25 nM pDNA2 and 25 nM pDNA3. hybridizes with pDNA2 to make TAMRA close onto the DNS surface to obtain an increasing FA value. In the presence of ATP, the pDNA2 could be released from the DNSs surface through high specific interactions between aptamer and ATP, resulting in decreased FA. The experimental results (Fig. S11) showed that there are small changes of the total fluorescence intensity and no obviously spectral shift in the unidirectional fluorescence spectra without or with ATP in the presence of DNSs (Fig. S12A & S12B), further suggesting that DNSs didn’t quench fluorescence. As shown in Fig. 4B, the FA value increased as the concentration of the ATP increased from 25 µM to 600 µM. A good linear correlation (R2= 0.9922) was obtained between ∆r (∆r = r0-r, where r0 and r are the FA values in the absence and presence of ATP, respectively) and the amount of ATP in the range of 150-450 µM (inset of Fig. 4B). The detection limit was found to be 43 µM (3σ). In order to evaluate selectivity of the approach for ATP detection, we investigated the FA responses toward three nucleotides, including CTP, GTP, and UTP, under the same experimental conditions. As shown in Fig. 4C, an obvious FA changes were induced by ATP while other ATP analogues can hardly cause the changes of FA, confirming the excellent selectivity of our proposal. The binding affinity for the ATP and hDNA2/pDNA2 was 33.6 µM [46], implying the reaction was in progress toward the direction in our design. Next, we apply this strategy for the detection of thrombin by using an extended single-stranded handle named hDNA3 that contains anti-thrombin aptamer (Fig. 4A). As shown in Fig. 4D, the ∆r response increased with the increased concentration of thrombin and there was a good linear relationship (inset of Fig. 4D) between the changes of FA signal (R2= 0.9945) and the amount of thrombin in the range of 0.41-0.95 μM. As shown in Fig. 4E, an obvious increase of FA changes was clearly observed in the presence of thrombin whereas there are no obvious FA changes with other types of analytes. These results demonstrated that the developed method has sufficient selectivity to thrombin detection. To verify the degree of the reaction, we explored the binding affinity between hDNA3/pDNA3 and thrombin [47], and 0.32 µM was obtained (Fig. S13), which illustrated the strong binding between hDNA3/pDNA3 and thrombin and the reaction was in progress toward the direction in our design. Subsequent results (Fig. S14-S15) demonstrated that our strategy was successfully used to detect thrombin with high accuracy.
CONCLUSIONS
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In summary, we have designed a novel FA method by using the 2D DNS as a new amplification platform. Compared with the previous FA amplification strategy, using DNS as an FA enhancer contains the following advantages: first, it can tremendously improve the detection accuracy because the fluorescence of dye on probe DNA cannot be quenched by DNSs; second, the sensitivity of detection was guaranteed due to the weak interaction between DNSs and ssDNA; third, as we have successfully applied this new method in the detection of CML-DNA, ATP and thrombin, it is an universal sensing platform and can be applied to detect other targets by adapting the corresponding recognition sequence. Lastly, as DNSs possess excellent biocompatibility, we believe that this strategy has a promising future for diagnoses in biosensing systems.
AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], Tel: 86-23-68254059; Fax: 86-23-68367257.
Notes The authors declare no competing financial interest.
ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21974109), the fund of Chongqing Fundamental and Advanced Research Project (cstc2017jcyjBX0068) and the Fundamental Research Funds for the Central Universities (XDJK2019TY003).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at:
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Highlights (1) The detection accuracy was tremendously improved because the fluorescence of dye-labeled probe DNA cannot be quenched by DNA nanosheets (DNSs). (2) The sensitivity of detection was guaranteed due to the weak interaction between DNSs and single-strand DNA. (3) It could be generalized to any kind of target detection based on the use of an appropriate aptamer.
1
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
S0039-9140(20)30021-7
DOI:
https://doi.org/10.1016/j.talanta.2020.120730
Reference:
TAL 120730
To appear in:
Talanta
Received Date: 17 October 2019 Revised Date:
6 January 2020
Accepted Date: 8 January 2020
Please cite this article as: Y.X. Liu, X. Xiao, C.H. Li, C. Men, Q.C. Ye, W.Y. Lv, Y.F. Li, C.Z. Huang, S.J. Zhen, DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing, Talanta (2020), doi: https://doi.org/10.1016/j.talanta.2020.120730. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Graphical abstract for
DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing Yu Xin Liu,a Xue Xiao,c Chun Hong Li,b Chen Men,a Qi Chao Ye,a Wen Yi Lv,a Yuan Fang Li,a Cheng Zhi Huang and Shu Jun Zhen a, *
a
a
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, 400715, Chongqing, P. R. China. b
College of Pharmaceutical Sciences, Southwest University, 400715, Chongqing, P. R. China.
c
College of Chemistry and Environment Protection Engineering, Southwest Minzu University, Chengdu 610041, China *
Corresponding author. Tel: (+86)-23-68254059.
E-mail addresses: [email protected] (S. J. Zhen).
DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing Yu Xin Liu,a Xue Xiao,c Chun Hong Li,b Chen Men,a Qi Chao Ye,a Wen Yi Lv,a Yuan Fang Li,a Cheng Zhi Huang a, b and Shu Jun Zhen a, * a
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of
Chemistry and Chemical Engineering, Southwest University, 400715, Chongqing, P. R. China. b
College of Pharmaceutical Sciences, Southwest University, 400715, Chongqing, P. R. China.
c
College of Chemistry and Environment Protection Engineering, Southwest Minzu University, Chengdu 610041, China
ABSTRACT: Recently, various inorganic nanomaterials have been used as fluorescence anisotropy (FA) enhancers for biosensing successfully. However, most of them are size-uncontrollable and possess an intensive fluorescence quenching ability, which will seriously reduce the accuracy and sensitivity of FA method. Herein, we report a two-dimensional DNA nanosheet (DNS) without fluorescence quenching effect as a novel FA amplification platform. In our strategy, fluorophore-labelled probe DNA (pDNA) is linked onto the DNS surface through the hybridization with the handle DNA (hDNA) that extended from the DNS, resulting in the significantly enhanced FA value. After the addition of target, the pDNA was released from the DNS surface due to the high affinity between the hDNA and target, and the FA was decreased. Thus, target could be detected by the significantly decreased FA value. The linear range was 10-50 nM and the limit of detection was 8 nM for the single-stranded DNA detection. This new method is general and has been also successfully applied for the detection of ATP and thrombin sensitively. Our method improved the accuracy of FA assay and has great potential to detect series of biological analytes in complex biosensing systems.
Keywords: DNA nanosheet; fluorescence anisotropy; single-strand DNA; ATP; thrombin.
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INTRODUCTION Fluorescence Anisotropy (FA) is a homogeneous, attractive and robust method in biochemical analysis system [1-3]. It is directly related to the rotation motion and mass of the fluorescently labeled molecules when the temperature and the viscosity of the solution are fixed [4-6]. FA has been widely used to analyze a large number of targets such as nucleic acids [7-10], metal ions [5, 11, 12], small molecules[13-16] and so forth. However, since most targets have small molecular weights, it is a great challenge to produce significant FA changes when adding targets. In order to overcome this limitation, a wide variety of nanomaterials, including gold nanoparticles [17], silver nanomaterials [18], metal organic frameworks (MOFs) [19], metal organic gel (MOG) [20] and carbon nanomaterials [21-23] have been successfully used as FA enhancers. However, most of these nanomaterials possess an intensive fluorescence quenching ability through various mechanisms. For example, some nanomaterials efficiently quench the fluorescence of fluorescent dye by means of electron transfer (ET) [24], and the others possess broad and strong absorption within the visible light range which overlaps with the emission of commonly used fluorescent probe [25] and have been regarded as a kind of excellent acceptors of fluorescence resonance energy transfer (FRET) [26-30] to quench the fluorescence of fluorophore. Under the condition of severe fluorescence quenching, the FA data measurement can be influenced by the scattered light easily, making the obtained FA value be greater than 0.4 [31]. Generally, if the measured FA for a randomly oriented sample is bigger than 0.4, one can confidently infer the presence of scattered light in addition to fluorescence. To reduce the fluorescence quenching effect, several FA detection systems [32-34] have been developed. These systems reduce the fluorescence quenching efficiency by controlling the distance between fluorescent groups and amplification nanomaterials, However, the farther the fluorescent group is from the nanomaterial, the less restricted the fluorescent group is suffered, which will decrease FA signal and thus reduce the sensitivity. Moreover, it is difficult to control the number of probes connected to the surface of nanomaterials due to uneven size and shape of these nanomaterials. Furthermore, there is a strong adsorption between single-stranded DNA (ssDNA) and these nanomaterials through non-covalent or covalent interactions, which will result in low detection sensitivity. Therefore, finding an ideal FA amplifier which possess the properties including without fluorescence quenching effect, controlled size and shape, easy to link and release probes with controlled numbers is highly desirable. In recent years, a variety of excellent signal amplification methods have been developed to improve the sensitivity of biosensing, such as ion concentration polarization [35], isotachophoresis [36], DNA nanostructure-based approaches [37, 38] and so on. Especially, DNA nanostructures have aroused great attentions due to their facile synthesis, good biocompatibility and easy multiple modification. Compared with the traditional inorganic nanomaterials, DNA nanostructure can be used as a new FA amplifier because of its following amazing properties. First, owing to the chemical structural characteristics of nucleotides, DNA cannot form large conjugated structure, which leads us to speculate that DNA nanostructure has a weak absorption in the long wavelength range. Thus, the fluorescence will not be quenched by DNA nanostructure through FRET because there may be weak spectral overlap between the absorption of DNA nanostructure and the emission of common fluorescent probes. Thus, the accuracy of FA detection can be improved by the use of DNA nanostructure. Second, DNA nanostructures have well-defined geometry and superior addressability, so that probes can be modified on the surface of DNA nanostructures in a precisely controlled manner. Therefore, the reproducibility of FA detection can be guaranteed by the number-controlled probes on DNA nanostructure. Third, it is well known that after the formation of the double helix structure, the electronic system of nucleobases of DNA is shielded inside the hydrophilic double helix backbone [39]. So, there is a weak π-stacking interaction between DNA nanostructure and ssDNA, which will significantly reduce the adsorption of ssDNA on DNA nanostructure and improve the sensitivity of detection. In additional, the rotation rate of two-dimensional (2D) nanomaterials is slower than that of zero-dimensional (0D) spherical nanomaterials with the same surface area [40, 41]. Therefore, in this contribution, we established an universal and simple method by utilizing a DNA nanosheet (DNS), a 2D DNA nanostructure, as a novel FA amplification platform for the detection of ssDNA about chronic myelogenous leukemia (CML, type b3a2) [42-44], ATP and thrombin successfully.
2
Scheme 1. Schematic illustration of fluorescence anisotropy amplification strategy based on 2D DNSs. (Double arrows represent the possible rotational contributions to the FA change).
EXPERIMENTAL SECTION Materials. All oligonucleotides (table S1 and table S2) used in this work were synthesized and HPLC purified by Sangon Biotechnology Co., Ltd. (Shanghai, China). Adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP) and uridine triphosphate (UTP) were purchased from Sangon Biotech (Shanghai, China). Thrombin, hemoglobin, albumin, amylase and pepsase were obtained from Shanghai Yuanye Biological Technology Co., Ltd.
All the chemicals were of analytical
grade and used without further purification. All stock solutions and buffers were prepared with Milli-Q ultrapure water (18.2 MΩ· cm-1). All procedures that required temperature control were measured by a thermomixer C (Eppendorf, Germany). Solutions were evenly blended by a vortex mixer MX-E (Haimen, China). The UV-vis absorption spectra were analyzed with a UV-3010 spectrophotometer (Daojin, Japan). General procedure. First, DNA nanosheet (DNS) was prepared by mixing 46 strands in a buffer at equal molar ratio at 100 nM. The mixture was then allowed to the following thermal annealing program: 85 ºC for 10 min, 80 ºC to 24 ºC, approximately 16 h. Then, dye-labeled probe was added to well-prepared DNSs under constant shaking at room temperature for 1 h. Last, target was added to the mixture and the final volume was made to 200 µL by adding buffer. After the addition of target, the mixture incubated for 2 h under constant shaking at 37 ºC. Then, FA was measured. The assembled DNSs were allowed to store at 4 °C for at least 1 week for further use. FA measurement. Fluorescence intensities were measured on F-2500 fluorescence spectrophotometer with a polarization filter (Hitachi, Tokyo, Japan). Excitation wavelength was set at 560 nm, and emission wavelength was recorded at 581 nm. Slits for the excitation and the emission were both set at 5 nm. The integration time was set at 0.4 s. The quartz cuvette was employed as the sample cell and the FA measurement experiments were all carried out at room temperature. The fluorescence anisotropy (r) was calculated by equations as follows: r=
ூ ିீൈூౄ
ூ ାଶீൈூౄ
(1)
and ூ
G= ౄ
ூౄౄ
(2)
3
where IVV and IVH are the fluorescence intensities of the vertically and horizontally polarized emission
after excitation by
vertically polarized light, respectively. The instrumental correction factor G corrects for the different detection efficiencies of perpendicular and parallel emission paths. Native Polyacrylamide Gel Electrophoresis. DNSs were subjected to 8% native polyacrylamide gel (gel was prepared in 2 mL of 40% acrylamide/bis-acrylamide gel solution (29:1) supplemented with 10 mM MgCl2, 1.6 mL 5× TBE, 56 µL 10% ammonium persulfate (APS) and 8 µL N, N, N', N'-tetramethylethylenediamine (TEMED), and the final volume was fill with water to 9 mL). Electrophoresis was conducted in 1× TBE at 120 V for 90 min or 160 V for 60 min. Once finished running, the gel was soaked in 0.005% (v/v) ethidium bromide (EtBr) solution for 10 min and then scanned using a WD-9403E portable UV apparatus (Beijing Liuyi Biotechnology Co., Ltd., China). Atomic force microscopy imaging. Atomic force microscopy (AFM) images were obtained in air under the tapping mode using Dimension Icon (Bruker). 10 µL of sample was deposited onto the surface of freshly cleaved mica chip and left in dry ambience air to allow for absorption. Then, the products were washed with 10 µL of Milli-Q ultrapure water three times to remove excess salt. The results were analyzed with NanoScope Analysis software.
RESULTS AND DISCUSSIONS Principle of the Design. In this study, taking full advantages of DNA nanotechnology, a two-dimensional rectangular DNA nanosheet (DNS) (the dimensions were 32 nm long and 24 nm wide calculated by theory) prepared according to the methods of Dong M. Shin et al [45] was used as a novel platform for enhancing FA. As depicted in Scheme 1, the fluorescent dye carboxytetramethylrhodamine (TAMRA) modified probe DNA (pDNA) was free in the absence of DNSs, which can bring about a low FA signal since the ssDNA binding to the TAMRA has a low molecular weight. In the presence of DNSs, pDNA was indirectly immobilized on DNSs surface through hybridization with a handle DNA (hDNA) extended from DNSs, which can cause enhanced FA signals by restricting the rotational diffusion of the TAMRA as a result of significant increased molecular weight. Because DNS cannot quench the fluorescence of TAMRA, TAMRA was designed as close as possible to the DNS surface to limit the rotation of TAMRA with the entire framework to achieve high FA. Feasibility of the DNS enhanced FA assay. First, AFM images (Fig. S1) confirmed the successful preparation of DNSs. The average length of DNSs was 30 ± 2 nm and the average width of DNS was 21 ± 2 nm (n=28), which were basically consistent with the theoretic results, indicating that the DNSs were prepared with anticipatory dimensions and patterns. Then, the assembly of DNS was confirmed with 8% native polyacrylamide gel electrophoresis (Native-PAGE). The products all displayed a low mobility (lanes 1-4, Fig. S2), indicating the successful synthesis of DNSs with protruding strands of different sequences. In order to investigate the fluorescence quenching ability of DNSs, we evaluated the spectral properties of the DNSs. The absorption of the DNSs and the fluorescence spectra of the (TAMRA)-conjugated DNAs were shown in Fig. 1A. As we anticipated, DNSs have no obvious absorption after 300 nm over a wide range of wavelengths, and there is weak spectral overlap between the emission of TAMRA and the absorption of DNSs, which effectively avoids the fluorescence quenching through the means of FRET and ensures the accuracy of the FA values. We next tested FA amplifying ability of DNSs. As show in Fig. 1B, there is a low FA signal in the absence of DNSs and a high FA signal in the presence of DNSs, which clearly confirms that the DNSs equip an outstanding capability for amplifying FA. Then, we studied the total fluorescence intensity in the absence and presence of DNSs. The weak change in fluorescence intensity (Fig. 1C) confirmed that the DNSs didn’t quench the fluorescence of TAMRA. The experimental results show no significant spectral shift in fluorescence spectra and small change in the shape of the fluorescence spectra of the unidirectional fluorescence spectra (HH, HV, VH, and VV) in the presence (Fig. 1D) and absence of DNSs (inset of Fig. 1D), further
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Fig. 1. (A) UV-vis absorption spectrum (red) of DNSs and fluorescence spectra of the TAMRA-labeled pDNA. Concentration: DNSs, 50 nM; p-d0, 50 nM. (B) FA responses with or without DNSs. (C) Fluorescence spectra in the absence and presence of DNSs. (D) The unidirectional fluorescence spectra (HH, HV, VH, VV) in the presence of DNSs. The inset is the unidirectional fluorescence spectra in the absence of DNSs. Concentration: DNSs, 97 nM; p-d0, 30 nM. certifying that the accuracy of the FA values is ensured. In contrast, the fluorescence was almost quenched and the FA value was exceed 0.4 (Fig. S4) when GO or MOF (MIL-101) was employed for FA amplification. All of these experimental results confirmed that DNSs could be used as efficient FA amplifiers. Next, to identify the interaction mechanism between DNSs and TAMRA-labeled probe, the handle-free DNSs are mixed with ssDNA and double-strand DNA (dsDNA) respectively. Then we removed DNSs through centrifugal ultrafiltration (molecular weight cut-off 100KDa) and measured the fluorescence intensity of the solution in the collection tube. The centrifugal ultrafiltration experimental results confirmed that DNSs can scarcely absorb ssDNA or dsDNA (Fig. 2A & 2B). Thus, when the pDNA is released from DNSs in the presence of target, it will not bind to DNSs again and the loss of sensitivity caused by non-specific binding could be significantly reduced. Subsequently, we investigated the influence of the distance between TAMRA and DNSs and the position of TAMRA on DNSs surface on the enhancement of FA. In order to explore the factor of distance, we keep the position of the extended chain on the DNSs unchanged, then modify the sequence of extended chain correspond to h-d0, h-d4.42, h-d7.14, h-d9.86 , and h-d13.26, respectively (the pDNA are p-d0, p-d4.42, p-d7.14, p-d9.86, and p-d13.26 in table S1). The experimental results (Fig. 2C) showed ∆r response (∆r = r0-r, where r0 is the FA values in presence of DNSs and r is the FA values in the absence of DNSs) decreased dramatically once TAMRA leaves away from DNSs surface, suggesting that when the distance between TAMRA and DNSs is short, the integration degree of the rotational movement of the formed complex with that of TAMRA is high. To explore the factor of position, we keep the length of the extended chain on the DNSs unchanged (the distance between TAMRA and DNSs is 0), and make the transformation sequence correspond to DNSs with h-S1, h-S2, h-S3, and h-S4, respectively (the pDNA is p-d0 in table S1). According to the ∆r response, the location of fluorophore on DNSs (Fig. S3) showed small influence on FA value (Fig. 2D). Detection of CML-DNA. The short ssDNA sequence of Type b3a2, a frequent BCR/ABL fusion gene existing in chronic myelogenous leukemia (CML), was selected as a proof-of-concept target DNA (tDNA). As shown in Fig. 3A, a single-stranded
5
Fig. 2. (A) The interaction mode between handle-free DNSs and ssDNA. 1, ssDNA; 2, handle-free DNSs mixed with ssDNA; 3, subnatant of handle-free DNSs mixed with ssDNA after ultrafiltration. (B) The interaction mode between handle-free DNSs and dsDNA. 1, dsDNA; 2, handle-free DNSs mixed with dsDNA; 3, subnatant of handle-free DNSs mixed with dsDNA after ultrafiltration. Concentration: DNSs, 50 nM; p-d0, 50 nM. (C) ∆r response of different distance between DNSs and TAMRA. The inset is FA signal in the presence (orange column) or absence (green column) of DNSs at the corresponding distance between the DNSs and TAMRA. (D) ∆r response of different position between DNSs and TAMRA. The inset shows FA signal to TAMRA on the different position of the DNSs in the presence (orange column) or absence (green column) of DNSs. Experiments were performed in the presence of 97 nM DNSs, 30 nM of p-d0, p-d4.42, p-d7.14, p-d9.86, and p-d13.26, respectively. handle extended from the DNSs surface (hDNA1) whose target recognition region was designed to complementary with tDNA and the probe load region was used to combine with pDNA1. Before the tDNA was added, the pDNA1 was combined with DNSs which has restricted the rotation of TAMRA and achieved a high FA response. After the addition of tDNA, the pDNA1 was released from the DNSs surface through toehold-mediated DNA strand exchange reaction, resulting in the obviously decreased FA value. Thus, the tDNA could be detected by the decreased FA value. We investigated FA responses and fluorescence intensity responses in the absence and presence of tDNA with/without subsequent incubation with DNSs. A low FA signal in the absence of DNSs and a high FA signal in the presence of DNSs were observed (Fig. 3B). The total fluorescence intensity before or after the addition of tDNA in the presence of DNSs (Fig. S5) has barely changed and no observable peak shift in the unidirectional fluorescence spectra (Fig. S6A & S6B) without or with tDNA in the presence of DNSs. These results further confirmed that the DNS is an excellent FA amplifier. In order to achieve the best experimental performance, we investigated the effects of the concentration of DNSs firstly. If the concentration of DNSs was low, small number of the pDNA1 could be immobilized on the surface of DNSs, resulting in a low FA value. However, when the concentration of DNSs was too high, there will be extra DNSs without pDNA1 connections since the number of pDNA1 is constant, and the tDNA would hybridize with hDNA1 directly instead of releasing the pDNA1 through toehold-mediated DNA strand exchange reaction, so that the pDNA1 on DNSs were unable to release from DNSs surface easily, leading to the small change of FA. The experimental results (Fig. S7) showed that when the concentration of DNSs was 40 nM, ∆r (∆r = r0-r, where r0 is the FA values in absence of tDNA and r is the FA values in the presence of tDNA) reached its maximum
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Fig. 3. (A) Schematic illustration of fluorescence anisotropy amplification strategy based on 2D DNSs for the detection ssDNA. (B) FA signal in the absence and presence of tDNA with or without DNSs. (C) ∆r response with the increasing concentration of tDNA. Inset: linear correlation of ∆r with different concentrations of tDNA. (D) Selectivity test for tDNA detection. Experiments were performed in the presence of 40 nM DNSs, 25 nM pDNA1, 40 nM tDNA and 40 nM of the all mismatched DNA, respectively. value. Then, we tested the influence of incubation temperature and time on the detection. The experimental results (Fig. S8) showed that the most suitable incubation conditions of pDNA1 connected on DNSs were 25 ºC for 1h. The optimized incubation temperature and time for the toehold-mediated strand displacement reaction between the complex of DNSs/pDNA1 and tDNA were 37 ºC for 2 h. We further examined the number of toehold base domain (N) on hDNA1. Four types of pDNA sequences (pDNA1-1, pDNA1, pDNA1-2, and pDNA1-3 in table S1) with different lengths were designed. When the pDNA was too short, the pDNA was unable to hybridize with hDNA tightly, while it could not be released from DNSs upon the addition of tDNA if the pDNA was too long. Thus, the optimal base number of toehold domain was confirmed as 8 (Fig. S9). Next, the FA changes after the addition of tDNA with different concentrations under optimal experimental conditions were measured. As evident from Fig. 3C, with the increasing concentration of tDNA from 1 nM to 60 nM, the ∆r dramatically increased, implying the increasing amount of the pDNA1 was released from the DNSs surface and dissociated in a solution through DNA toehold-mediated chain exchange reaction. ∆r exhibited a linear response (R2= 0.9933) with the concentration of tDNA in the range of 10-50 nM (inset of Fig. 3C) and the limit of tDNA detection was 8 nM (Fig. S10). To investigate the selectivity of this method, we compared the FA responses with tDNA and various mismatched targets including single-base mismatched DNA (t1), two-base mismatched DNA (t2), three-base mismatched DNA (t3), four-base mismatched DNA (t4) and random non-complementary DNA (nc). As shown in Fig. 3D, increasing in the number of mismatched bases followed by significantly decreasing in the ∆r, and no distinct FA changes induced by more than three base-mismatched DNA can be observed, certifying the excellent selectivity of our approach. General Applicability of the Strategy. To evaluate the generality of our method, we first explored it for the detection of ATP. As described in Fig. 4A, hDNA2 that contains the sequence of the anti-ATP aptamer was protruded from the surface of DNSs and
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Fig. 4. Detection of ATP and thrombin. (A) Schematic illustration of fluorescence anisotropy amplification strategy based on 2D DNSs for the detection of ATP and thrombin. (B) ∆r response with the increasing concentration of ATP. Inset: linear correlation of ∆r with different concentrations of ATP. (C) The relative ∆r response of ATP, GTP, CTP, and UTP (350 µM respectively). (D) ∆r response with the different concentration of thrombin. Inset: linear correlation of ∆r with different concentrations of thrombin. (E) Selectivity of the FA assay of thrombin over pepsase, trypsin, hemoglobin, albumin (0.68 µM respectively). Experiments were performed in the presence of 40 nM DNSs, 25 nM pDNA2 and 25 nM pDNA3. hybridizes with pDNA2 to make TAMRA close onto the DNS surface to obtain an increasing FA value. In the presence of ATP, the pDNA2 could be released from the DNSs surface through high specific interactions between aptamer and ATP, resulting in decreased FA. The experimental results (Fig. S11) showed that there are small changes of the total fluorescence intensity and no obviously spectral shift in the unidirectional fluorescence spectra without or with ATP in the presence of DNSs (Fig. S12A & S12B), further suggesting that DNSs didn’t quench fluorescence. As shown in Fig. 4B, the FA value increased as the concentration of the ATP increased from 25 µM to 600 µM. A good linear correlation (R2= 0.9922) was obtained between ∆r (∆r = r0-r, where r0 and r are the FA values in the absence and presence of ATP, respectively) and the amount of ATP in the range of 150-450 µM (inset of Fig. 4B). The detection limit was found to be 43 µM (3σ). In order to evaluate selectivity of the approach for ATP detection, we investigated the FA responses toward three nucleotides, including CTP, GTP, and UTP, under the same experimental conditions. As shown in Fig. 4C, an obvious FA changes were induced by ATP while other ATP analogues can hardly cause the changes of FA, confirming the excellent selectivity of our proposal. The binding affinity for the ATP and hDNA2/pDNA2 was 33.6 µM [46], implying the reaction was in progress toward the direction in our design. Next, we apply this strategy for the detection of thrombin by using an extended single-stranded handle named hDNA3 that contains anti-thrombin aptamer (Fig. 4A). As shown in Fig. 4D, the ∆r response increased with the increased concentration of thrombin and there was a good linear relationship (inset of Fig. 4D) between the changes of FA signal (R2= 0.9945) and the amount of thrombin in the range of 0.41-0.95 μM. As shown in Fig. 4E, an obvious increase of FA changes was clearly observed in the presence of thrombin whereas there are no obvious FA changes with other types of analytes. These results demonstrated that the developed method has sufficient selectivity to thrombin detection. To verify the degree of the reaction, we explored the binding affinity between hDNA3/pDNA3 and thrombin [47], and 0.32 µM was obtained (Fig. S13), which illustrated the strong binding between hDNA3/pDNA3 and thrombin and the reaction was in progress toward the direction in our design. Subsequent results (Fig. S14-S15) demonstrated that our strategy was successfully used to detect thrombin with high accuracy.
CONCLUSIONS
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In summary, we have designed a novel FA method by using the 2D DNS as a new amplification platform. Compared with the previous FA amplification strategy, using DNS as an FA enhancer contains the following advantages: first, it can tremendously improve the detection accuracy because the fluorescence of dye on probe DNA cannot be quenched by DNSs; second, the sensitivity of detection was guaranteed due to the weak interaction between DNSs and ssDNA; third, as we have successfully applied this new method in the detection of CML-DNA, ATP and thrombin, it is an universal sensing platform and can be applied to detect other targets by adapting the corresponding recognition sequence. Lastly, as DNSs possess excellent biocompatibility, we believe that this strategy has a promising future for diagnoses in biosensing systems.
AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], Tel: 86-23-68254059; Fax: 86-23-68367257.
Notes The authors declare no competing financial interest.
ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21974109), the fund of Chongqing Fundamental and Advanced Research Project (cstc2017jcyjBX0068) and the Fundamental Research Funds for the Central Universities (XDJK2019TY003).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at:
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Highlights (1) The detection accuracy was tremendously improved because the fluorescence of dye-labeled probe DNA cannot be quenched by DNA nanosheets (DNSs). (2) The sensitivity of detection was guaranteed due to the weak interaction between DNSs and single-strand DNA. (3) It could be generalized to any kind of target detection based on the use of an appropriate aptamer.
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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.