Quantifying the Degree of Aggregation from Fluorescent Dye

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Quantifying the Degree of Aggregation from Fluorescent DyeConjugated DNA Probe by Single Molecule Photobleaching Technology for the Ultra-sensitive Detection of Adenosine Xingbo Shi, Yu He, Wenli Gao, Xiaoying Liu, Zhongju Ye, Hua Liu, and Lehui Xiao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05317 • Publication Date (Web): 22 Feb 2018 Downloaded from http://pubs.acs.org on February 23, 2018

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Analytical Chemistry

Quantifying the Degree of Aggregation from Fluorescent DyeConjugated DNA Probe by Single Molecule Photobleaching Technology for the Ultra-sensitive Detection of Adenosine Xingbo Shi,1,2,* Yu He,1 Wenli Gao,1 Xiaoying Liu,4 Zhongju Ye,3 Hua Liu,3 and Lehui Xiao3,* 1

Hunan Provincial Key Laboratory of Food Science and Biotechnology, College of Food Science and Technology, Hunan Agricultural University, Changsha, 410128, China; 2 State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China; 3 State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China; 4 College of Science, Hunan Agricultural University, Changsha, 410128, China. Email: [email protected], [email protected] Fax: +86-022-23500201. ABSTRACT: In this work, we demonstrated a single molecule photobleaching-based strategy for the ultrasensitive detection of adenosine. A modified split aptamer was designed to specifically recognize individual adenosine molecules in solution. The specific binding of dye-labelled short strand DNA probes onto the elongated aptamer strand in the presence of adenosine resulted in a concentration-dependent self-aggregation process. The degree-of-aggregation (DOA) of the short DNA probes on the elongated aptamer strand could then be accurately determined based on the single molecule photobleaching measurement. Through statistically analyzing the DOA under different target concentrations, a well-defined curvilinear relationship between the DOA and target molecule concentration (e.g., adenosine) was established. The limit-of-detection (LOD) could down to 44.5 pM, which is lower than those recently reported results with fluorescence-based analysis. Owing to the high sensitivity and excellent selectivity, the sensing strategy described herein would find broad applications in biomolecule analysis under complicated surroundings.

Utilizing aggregation-caused signal change to develop novel sensors has attracted considerable attention owing to its easy implementation.1-4 Generally, there are two approaches to achieve this goal: one is based on noble metal nanoparticles as colorimetric probes, which exhibit colour or absorptivity changes after the aggregation process.4-6 The other is based on fluorescent probes, such as quantum dots (QDs), nanoclusters, carbon dots and fluorescent dyes, which display either increased or quenched fluorescence intensity in response to the target objects.3,7-14 However, these strategies are essentially performed on the ensemble averaged level. Normally, it is difficult to notice subtle changes in such signals, greatly limiting the sensitivities of these methods. Assessing the degree-of-aggregation (DOA) of nanoparticles or dyes at single-object levels is a promising strategy to improve the detection sensitivity of the method. However, for plasmonic probes, it is normally difficult to perform DOAbased sensing because the DOA is hard to quantitatively assess without electron microscopy (e.g., scanning electron microscopy and transmission electron microscopy). Basically, the DOA of fluorescent dyes can be accurately determined by real-time observation of the number of photobleaching steps from single clusters.9,15-18 This technique is termed as single-molecule photobleaching (SMPB) and has been used extensively in a variety of areas, including the study of molecular localization,19 assessing the extent of pro-

tein/DNA crosslinking20 and deriving the number of subunits in membrane-binding proteins.21 Therefore, using SMPB to develop a DOA-based sensing strategy is feasible, provided that a reasonable recognizing system can be identified. Aptamers, which are promising recognition elements, are specific single-stranded DNA fragments selected from random sequence libraries by SELEX (systematic evolution of ligands by exponential enrichment) technology.22-26 Owing to their distinctive properties, aptamers exhibit high recognition ability for numberous targets ranging from small molecules27-29 to cells.23,30 However, single-stranded aptamers are unitary probes and unsuitable for DOA-based sensing strategies owing to the lack of adequate sites for the formation of fluorescent dye aggregates. One solution to this problem is to design multinary probes. Some DNA aptamers can be split into two pieces, and the resultant split aptamer, comprising two single-stranded DNA fragments, constitutes a binary probe that can selectively bind to adjacent regions on a target.31,32 Split aptamers have been used to detect several targets, including adenosine,33 ATP,32 17b-estradiol34 and cocaine.35,36 Among these molecules, adenosine is a ubiquitous, endogenous purine involved in plenty of physiological and pathophysiological regulatory mechanisms, which has received great attention recently.29,37-39 Furthermore, adenosine is an essential drug for the treatment of a number of diseases, especially arrhythmia.40 Therefore,

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adenosine was chosen as a model target to verify the DOAbased sensing strategy.

Figure 1. Schematic diagram of (a) normal adenosine split aptamers (Cy5-Probe1 and Cy5-Probe2) recognizing adenosine and of (b) adenosine-induced aggregation of dye conjugated split aptamers (Cy5-Probe1 and Cy5-N-Probe2). Herein, we demonstrated a novel adenosine sensing strategy based on split aptamers and SMPB technology. Figure 1 illustrates the principles of the adenosine sensing strategy. The two fragments of the split aptamer (Probe1 and Probe2) were chosen for adenosine according to a previous report.41 The aptamer sequences are shown in Table 1. When adenosine was added into a solution containing the normal split aptamer labelled with Cy5 at the 5′ ends, the DOA is only 2 (as shown in Figure 1(a)). To extending the detection capacity during the target recognizing process, in this design (Figure 1(b)), one fragment of the probe was directly labelled with Cy5 at the 5′ end (Cy5-Probe1), and the other complimentary fragment was lengthened with multiple repeating fragments and was also labelled with Cy5 at the 5′ end (Cy5-N-Probe2, where N represents the number of repeating original fragments). In the presence of adenosine, a supramolecular aptamer complex would be generated, resulting in fluorescent dye aggregation. Then, the concentration of adenosine could be quantitatively determined based on the DOA of the fluorescent dyeconjugated DNA probe. Name

Sequence (from 5′ to 3′)

Cy5-Probe1

Cy5 – ACC TGG GGG AGT AT

Cy5-Probe2

Cy5 – TGC GGA GGA AGG TTT

Cy5-N-probe2

Cy5 – TGC GGA GGA AGG TTT – TGC GGA GGA AGG TTT – TGC G GA GGA AGG TTT – TGC GGA GGA AGG TTT – TGC GGA GGA AGG TTT – TGC GGA GGA AGG TTT – TGC GGA GGA AGG TTT – TGC GGA GGA AGG TTT

Table 1. Oligonucleotide sequences used in this work. Figure 2 shows the typical fluorescence image and photobleaching steps from eight typical fluorescence images of Cy5-labeled DNA aggregates. In these images, all of the fluorescent spots were irreversibly photobleached, which results in a step-by-step sudden intensity decrease during a period of continued illumination. The number of fluorescent dyes within the inset fluorescence image was determined by tracking the photobleaching steps (Figure 2(b–i)). Since individual dye was irreversibly photobleached step-by-step, each step in the track indicates one bleached dye molecule. In the sequence of Cy5-N-Probe2, there are eight repeating original units. The maximum number of photobleaching steps for the aggregate is 9 in this design. To avoid the underestimation of the total number of photobleaching steps, we did not include more re-

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peating units in the Cy5-N-Probe2, which is not limited principally. However, increasing the number of fluorescent dye units in an aggregate can increase the possibility of synchronous photobleaching of the fluorescent molecules. In the current study, more than 9 steps from single spot were rarely observed, indicating that very few fluorescent molecules aggregated through non-specific self-adsorption. The aggregation between two Cy5-N-Probe2s was also negligible.

Figure 2. Representative single molecule fluorescence image (a) and photobleaching steps from the adenosine aptamers with different DOAs (b-i). In order to establish a relationship for the quantification of the target molecules based on the photobleaching steps, an averaged DOA in the solution was defined as Y = CP/(CP – CT), where Y represents the DOA, CT represents the dosage of target molecules in the solution, and CP represents the total dosage of fluorescent probes in the solution. Without the introduction of target molecules (CT = 0), the statistically averaged photobleaching step should be one. When the target molecules were introduced, basically, the number of the whole counted photobleaching steps should be a constant value (i.e., CP), while the number of spots on the fluorescent image will decrease as a function of the target concentration (i.e., CP – CT). The averaged DOA is then a statistically averaged value, which could bypass the effect of non-uniform aggregation process. Depending on the concentration of the target molecules, principally, the detection limit of this design could reach to single-molecule level. As a proof-of-concept experiment, we firstly determined the concentration-dependent DOA as a function of target molecules (adenosine) in the concentration rang of nM. In detail, Cy5-N-Probe2 and Cy5-Probe1 were mixed together (1:1, v/v the final concentration in the reaction solution is 1 and 8 nM respectively). Then, the mixed solution was incubated with various concentrations of adenosine (in a final concentration of 1, 2, 3, 4, 5, 6, 7 and 8 nM). The photobleaching steps of the mixture dramatically increase with increasing adenosine concentration. At least 300 independent dye aggregates were analysed in each case, and the DOAs were calculated. The distribution histograms of DOA under different adenosine concentrations are shown in Figure S1. In Figure 3, the averaged DOA is plotted as a function of adenosine concentration. Non-specific interaction of adenosine with pure Cy5-Probe1 and Cy5-N-Probe2 was not found. The relationship between

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DOA and the concentration of adenosine could be well fitted by the equation of Y = CP/(CP – CT) with r2 of 0.99. Moreover, the fitted CP based on the equation is 9.28, which is in good agreement with the theoretical value of 9 (Cy5-N-Probe2 + Cy5-Probe1). These results confirmed that the formula proposed above is reliable for the quantitative estimation of the target concentration based on the statistically measured DOA. To further investigate the sensitivity of this sensor, for the convenient of curve fitting, we transformed the equation into 1/Y = 1 − CT/CP. The value of 1/DOA is linearly dependent on the concentration of adenosine from 0 to 8 nM (as shown in the inset plot of Figure 3). The linear regression equation can be expressed as y = 1 − 0.11x with a correlation coefficient of 0.99. Moreover, the limit-of-detection (LOD) was estimated to be 44.5 pM based on the 3σ/S calculation (σ is the standard deviation for a blank solution and S is the slope of the calibration curve), which is lower than those recently reported results with fluorescence based analysis, as shown in Table S1. It is worth to emphasize that the linear range of this design could be extended in a broad range which is dependent on the concentration of the target molecules as well as the probe concentration adopted. However, when the target concentration is higher than that of Cy5-Probe1, the DOA will be saturated (close to 9, Figure 3).

12

0.0

1/DOA

8

Cy5-probe1+ Cy5-N-probe2 Cy5-probe1 Cy5-N-probe2

0.2

10

DOA

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Analytical Chemistry

0.4

a

8

g

h

i

6 4 2

b

0.6 0.8

6

The selectivity of this sensing strategy for adenosine assay was also explored by using the adenosine analogues thymidine, cytidine, uridine, guanosine, ATP, ADP and AMP as the interference substrates. Figure 4 shows the DOA response after the addition of 8 nM adenosine, 1 µM thymidine, 1 µM cytidine, 1 µM uridine, 1 µM guanosine, 8 nM ATP, 8 nM ADP, 8 nM AMP and a blank sample under the same experimental conditions. Evidently, there is no significant DOA change on the addition of thymidine, cytidine, uridine and guanosine in comparison with that of the blank control, which implies that the engineered split aptamer retains high selectivity towards adenosine and is able to discriminate adenosine from its analogues. So far, many aptamer-based ATP biosensors have been developed. Although all of those aptamers show good selectivity toward adenosine, they typically show fewer and less recognition capability toward the sugar and especially the triphosphate moiety. Similar limitation was also found in the aptamer used here.42-43

DOA

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c

d

e

f

0 e e e e e k P P P sin Blan idin anin tidin ridin AT AD AM eno U ym Gu Cy Ad Th

1.0 0

2

4

6

8

Adenosine/nM

4 2 0 0

2

4

6

8

10 16

Adenosine/nM Figure 3. The relationship between DOA and adenosine concentration. The concentrations of adenosine were 1-15 nM. A curve (r2 = 0.99) was fitted using the formula of Y = CP/(CP – CT) from 1 to 8 nM. The inset shows the linear relationship between the reciprocal of the DOA and the adenosine concentration over the range of 0-8 nM. The control experiments to explore the non-specific interactions of adenosine with pure Cy5-N-Probe2 and Cy5-Probe1 were also performed. Error bars show the standard deviation of three independent experiments (n = 3). On this basis, we further increased the concentration of Cy5-Probe1 and Cy5-N-Probe2 solution to 800 and 100 nM respectively. The final adenosine concentrations in the reaction solution were added as 100, 200, 300, 400, 500, 600, 700 and 800 nM respectively. Figure S2 shows the relationship between DOA and adenosine concentration. For the convenient of single object fluorescence imaging, the final concentration of the reaction solution was diluted to nM level, which doesn’t affect the determination of DOA value. As illustrated in Figure S2, a well-defined curvilinear relationship was obtained under the concentration range of 0-800 nM with an r2 of 0.99.

Figure 4. Selectivity assay of the sensing method toward (a) adenosine, (b) blank sample, (c) thymidine, (d) guanosine, (e) cytidine, (f) uridine, (g) ATP, (h) ADP, and (i) AMP. Owing to the excellent selectivity as well as superior sensitivity of this sensing strategy toward adenosine, the practical applicability of this sensor was further explored. Firstly, water samples spiked with different concentrations of standard adenosine were analysed. As can be seen from Table S2, the recovery of added adenosine is in the range of 95.56-100.00% and the standard deviation is between 0.2% and 8.5%, which indicates that the proposed sensing strategy could be applied to accurately monitor adenosine in water samples with excellent reliability and reproducibility. In addition to the water sample assay, we further explored the capability of the sensor for serum sample analysis. Different concentrations of adenosine were added into the cattle serum sample and then the total concertation of adenosine was measured by the method as noted above. The actual adenosine content in cattle serum sample was determined by HPLC, which is 7.45 nM. As illustrated in Table S3, the recoveries for adenosine in cattle serum were in the range of 96.1-98.98%, indicating that our strategy could also be used to determine adenosine in biological sample. In conclusion, in this work, we proposed a novel sensing strategy for the ultrasensitive detection of adenosine on the basis of split aptamers and quantitative single molecule photobleaching technology. Analysis of the DOA of fluorescent probes at single-molecule level allows us to determine subtle differences in signal change, thus improving the detection sensitivity. The proposed sensing strategy achieved an LOD of

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44.5 pM which is far below the previously reported results. Moreover, the design of our sensor is convenient and practical, thus avoiding the drawbacks of complicated aptamer response procedures that other fluorescent methods are subject to. Owing to its high sensitivity and excellent selectivity, the sensing strategy described herein affords an attractive platform for the detection of other biomolecules in the future. However, it has to mention that the data analysis method presented above is time-consuming because those data points were extracted oneby-one in the image. Automated program for data extraction and analysis should greatly facilitate the potential application of this method.44,45

ASSOCIATED CONTENT Supporting Information The experimental section, supporting figures and tables. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]

Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (NSFC, Project no. 21522502 and 31301484), Natural Science Foundation of Hunan Province (2015JJ3082) and the Open Foundation of State Key Laboratory of Chemo/Biosensing and Chemometrics ( Z2015025 ).

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