Magnetic Separation-Assistant Fluorescence Resonance Energy

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Magnetic Separation-assistant Fluorescence Resonance Energy Transfer Inhibition for High Sensitive Probing of Nucleolin Yan-Ran Li, Qian Liu, Zhangyong Hong, and He-Fang Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03064 • Publication Date (Web): 11 Nov 2015 Downloaded from http://pubs.acs.org on November 17, 2015

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Analytical Chemistry

Magnetic Separation-assistant Fluorescence Resonance Energy Transfer Inhibition for High Sensitive Probing of Nucleolin

Yan-Ran Li, Qian Liu, Zhangyong Hong, He-Fang Wang*

College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology (Nankai University), Tianjin Key Laboratory of Molecular Recognition and Biosensing, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, 94 Weijin Road, Tianjin 30071, China *E-mail: [email protected]

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Abstract For the widely-used “off-on” fluorescence (or phosphorescence) resonance energy transfer (FRET or PRET) system, the separation of donors and acceptors species was vital for enhancing the sensitivity. To date, separation of free donors from FRET/PRET inhibition systems was somewhat not convenient, whereas separation of the target-induced far-between acceptors has hardly been reported yet. We presented here a novel magnetic separation-assistant fluorescence resonance energy transfer (MS-FRET) inhibition strategy for high sensitive detection of nucleolin using Cy5.5-AS1411 as donor and Fe3O4-polypyrrole core-shell (Fe3O4@PPY) nanoparticles as NIR quenching acceptor. Due to hydrophobic interaction and π-π stacking of AS1411 and PPY, Cy5.5-AS1411 was bound onto the surface of Fe3O4@PPY, resulting in 90% of fluorescence quenching of Cy5.5-AS1411. Owing to the much stronger specific interaction of AS1411 and nucleolin, the presence of nucleolin could take Cy5.5-AS1411 apart from Fe3O4@PPY and restore the fluorescence of Cy5.5-AS1411. The superparamagnetism of Fe3O4@PPY enabled all separations and fluorescence measurements complete in the same quartz cell, and thus allowed the convenient but accurate comparison of the sensitivity and fluorescence recovery in the cases of separation or non-separation. Compared to non-separation FRET inhibition, the separation of free Cy5.5-AS1411 from Cy5.5-AS1411-Fe3O4@PPY solution (the first magnetic separation, MS-1) had as high as 25-fold enhancement of the sensitivity, whereas further separation of the nucleolin-inducing far-between Fe3O4@PPY from the FRET inhibition solution (the second magnetic separation, MS-2) could further enhance the sensitivity to 35-fold. Finally, the MS-FRET inhibition assay displayed the linear range of 0.625-27.5 µg L-1 (8.1-359 pM) and detection limit of 0.04 µg L-1 (0.05 pM) of nucleolin. The fluorescence intensity recovery (the 1


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percentage ratio of the final restoring fluorescence intensity to the quenched fluorescence intensity of Cy5.5-AS1411 solution by 0.09 g L-1 Fe3O4@PPY) was enhanced from 36% (for non-separation) to 56% (for two magnetic separations). This is the first accurate evaluation for the effect of separating donor/acceptor species on FRET inhibition assay.

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Introduction Fluorescence (or phosphorescence) resonance energy transfer (FRET or PRET) was the widely-used strategy for the design of optical sensors and probes.1-16 Therein, the “off-on” systems with low signal-background based on FRET/PRET and then FRET/PRET inhibition were especially popular.4,5,9,11,12 The FRET/PRET systems are usually composed of donors with fine photoluminescence signals, and acceptors with absorbance in the wavelength range of the donors’ emission. If donors and acceptors are close enough (≤ 10 nm), the emissions of donors will be quenched by acceptors (“off” state). The “on” state is achieved via the target-induced FRET/PRET inhibition, usually through changing absorption of acceptors to the wavelength range beyond the donors’ emission,8,10 or increasing the distance between acceptors and donors.4,5,11,12 The latter is easier achievable than the former, so most FRET/PRET inhibition is realized by pulling away the acceptors from the donors. For the “off-on” FRET/PRET inhibition systems, the separation of donors and acceptors species was vital for enhancing the sensitivity. First, the coexisted free donors would exhibit some photoluminescence signals (P0 value), and compete with the acceptor-binding donors to interact with the inhibitors, thus resulting in less sensitive photoluminescence change (∆P/P0). Second, the coexisted far-between acceptors in the presence of FRET/PRET inhibitors would still absorb the excitation and/or emission energy of donors, and even scatter excitation and emission light when acceptors are suspension nanoparticles, resulting in limited photoluminescence recovery, scattering interference and “background FRET/PRET”.17,18 To date, separation of free donors from FRET/PRET inhibition systems have been implemented in some literatures, mainly through gel filtration chromatography and reverse phase HPLC (for molecular beacon),19 centrifugation and 3


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dialysis.20 For the highly dispersable donor-acceptor couples with weak interactions (not the chemical bonding), however, the separation of free donors from FRET/PRET inhibition systems was so difficult that some literatures ended up without separation.4,5,21,22 The separation of the target-induced far-between acceptors, to the best of our knowledge, has hardly been reported yet. Therefore, it is vital to develop new strategies for convenient and efficient separation of free donors and target-induced far-between acceptors in FRET/PRET inhibition systems, especially in the cases of donor-acceptor couples with weak interactions. Herein, we represented a novel magnetic-separation assistant fluorescence resonance energy transfer (MS-FRET) inhibition assay with the greatly-simplified operation, greatly-improved sensitivity and fluorescence recovery (Figure 1). To demonstrate the proof-of-concept, human nucleolin was chosen as target for its shuttle nature in tumor diagnosis and signal transduction.23-32 The Cy5.5 labeled AS1411 (Cy5.5-AS1411) aptamer was used as the donor and Fe3O4-polypyrrole core-shell (Fe3O4@PPY) nanoparticles with magnetic separation33 capability and near infrared (NIR) absorbance34-38 were involved as the acceptor (Figure 1). Due to hydrophobic interaction and π-π stacking of AS1411 and PPY, Cy5.5-AS1411 was bound onto the surface of Fe3O4@PPY, resulting in fluorescence quenching of Cy5.5-AS1411 (FRET). Upon the presence of nucleolin, however, Cy5.5 was taken apart from Fe3O4@PPY owing to the much stronger specific interaction of AS1411 and nucleolin23-31. As a result of FRET inhibition, the fluorescence of Cy5.5-AS1411 was recovered.

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Figure 1. The principle of magnetic-separation assistant fluorescence resonance energy transfer (MS-FRET) inhibition assay of human nucleolin. MS-1 was the magnetic separation of free donors (Cy5.5-AS1411) from FRET system, and MS-2 was the magnetic separation of human nucleolin-induced far-between Fe3O4@PPY from the FRET inhibition system. Two magnetic separations were involved in MS-FRET inhibition assay. The first (MS-1) was the magnetic separation of free donors (Cy5.5-AS1411) from FRET system, and the second (MS-2) was the magnetic separation of human nucleolin-induced far-between Fe3O4@PPY from the FRET inhibition system. The MS-1 operation made the fluorescence background further decrease and eliminated the competition of free donors with acceptor-binding ones, whereas MS-2 made the nucleolin-inducing released Fe3O4@PPY not absorb the excitation and emission energy of the released donors again. Both magnetic separations displayed great enhancement for the sensitivity and fluorescence recovery. Although some FRET sensors have used the purified probes (i. e., separation of free donors), yet how much the purification on the sensitivity enhancement has not been reported. In this MS-FRET inhibition system, the high saturation magnetization and superparamagnetism of Fe3O4@PPY enabled all separations and fluorescence measurements complete in the same quartz cell, and thus allowed the convenient but accurate comparison of the sensitivity and fluorescence recovery in the cases of separation or non-separation (for example, 5


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MS-1 and MS-2 was measured with the same solution, just using magnet to absorb Fe3O4@PPY down the bottom of quartz cell before MS-2 measurement and no need for any solution transform). To the best of our knowledge, this is the first accurate evaluation for the effect of separating donor/acceptor species on FRET inhibition assay.

Experimental Details Chemical and Materials. All reagents, at least of analytical grade, were used directly. Iron(III) chloride hexahydrate (FeCl3⋅6H2O, 97%), sodium acetate anhydrous (99%), diethylene glycol (DEG) and ethylene glycol (EG) were purchased from Aladdin (Shanghai, China). Sodium acrylate (99%) was brought from Heowns (Tianjin, China) and pyrrole was from Alfa Aesar (Tianjin, China). Cy5.5-labelled AS1411 aptamer (sequence: 5’-Cy5.5-TTG GTG GTG GTG GTT GTG GTG GTG GTG G-3’) with HPLC purification was synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China). Bovine serum albumin (BSA), human serum albumin (HSA) and bovine thrombin were from Bioing Biotechnology Co. Ltd. (Shenyang, China), and horse radish peroxidase (HRP), trypsin and cytochrome C were from Sigma (St. Louis, America). Human nucleolin peptide was purchased from Abcam trading Co. Ltd. (Shanghai, China). 10 mM PBS buffer (pH 7.0, containing 100 mM NaCl, 5 mM KCl and 4 mM MgCl2) was used in all experiments. The human serum was collected in a local hospital.

Apparatus. The X-ray powder diffraction (XRPD) patterns were recorded on a Rigaku D/max-2500 X-ray diffractometer (Rigaku, Japan) equipped with Cu Kα radiation (λ = 1.5418 Å). The Fourier transform infrared (FT-IR) spectra (4000-400 cm–1) were recorded with a Nicolet MAGNA-560 FTIR spectrometer (Nicolet, Madison, WI). The thermogravimetric analysis (TGA) was conducted with PTC-10A TG-DTA thermo-analyzer (Rigaku, Japan). The magnetic properties 6



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(M-H curve) were measured on a LDJ 9600-1 vibrating sample magnetometer (LDJ Electronics Inc., Troy, MI, USA) by cycling the ﬁeld from −6 to 6 kOe at room temperature. The transmission electron microscope (TEM) of products were taken on a JEOL 100 CXII (JEOL, Tokyo, Japan) operating at a 200 kV accelerating voltage. The fluorescence spectra were measured on an F-4600 fluorescence spectrophotometer (Hitachi Ltd. Japan) with a xenon lamp and a quartz cell (1 cm × 1 cm) using 689 nm as the excitation wavelength. The UV-Vis spectrum was obtained from a UV-3650 UV-Vis-NIR spectrometry (Shimadzu, Japan). The confocal fluorescence images were taken with a TCS SP8 laser confocal scanning microscope (Leica Co., Germany) at the excitation of 630 nm.

Synthesis of Fe3O4 and Fe3O4@PPY Nanoparticles. The Fe3O4 nanoparticles were synthesized according to our previous procedure.39,40 For the synthesis of Fe3O4@PPY, the mixture of 25 mg Fe3O4 nanoparticles and 10 mL of ultrapure water was ultrasonically dispersed for 30 min to get a homogeneous brown solution, then 100 µL of pyrrole was added under stirring, and the resultant mixture was stirring for 1 h. The aqueous solution of FeCl3·6H2O (0.2 g in 5 mL ultrapure water) was added dropwise into the above mixture and then the reaction was set in ice bath for 12 h. The final black product was washed with ultrapure water and ethanol for three times respectively, and dried in vacuum for further use.

Fluorescence Measurements. All fluorescence measurements were achieved on F-4600 fluorescence spectrophotometer with the slits of 10 nm (excitation slit), 5 nm (emission slit) and PMT voltage of −700 V. Typically, to 2 mL of PBS buffered 25 nM Cy5.5-AS1411 solution, Fe3O4@PPY was added at the final concentration of 0.09 g L-1, and the mixture was vortexed for 8 min, then the free Cy5.5-AS1411 was removed and the Cy5.5-AS1411-Fe3O4@PPY was 7


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magnetically harvested after three washes of PBS buffer. The Cy5.5-AS1411-Fe3O4@PPY was re-dispersed in 2 mL of fresh PBS (or 2 mL of serum for evaluation the response of the probe in complex sample matrix), and the fluorescence spectra was recorded in the absence or presence of various amounts of human nucleolin (the incubation time of human nucleolin and the re-dispersed Cy5.5-AS1411-Fe3O4@PPY was 5 min, and the temperature was set at 37 oC). The direct measurements of fluorescence spectra of the mixed solution of Cy5.5-AS1411-Fe3O4@PPY and human nucleolin were marked as MS-1 series, while the fluorescence spectra recorded after the magnetic separation of Fe3O4@PPY species were signed as MS-2 series. The direct measurements without any magnetic separation were the MS-0 series. For optimization of FRET system, Fe3O4@PPY with final concentration of 0.01-0.1 g L-1 was added into 2 mL of PBS buffered 25 nM Cy5.5-AS1411 solution, incubating for 1-10 min. For evaluating the interaction time of target protein nucleolin and the Cy5.5-AS1411-Fe3O4@PPY system, 5 µg L-1 nucleolin was added to the FRET system at various incubation time of 1-7 min.

Cell Fluorescence Imaging. The nucleolin-positive MCF-7 cells were used for fluorescence imaging of nucleolin excreted during the growth of MCF-7 cells and the AD293 cells were as negative control. Typically, MCF-7 and AD293 cells were cultured respectively in DMEM medium and RPMI 1640 medium (Gibco) supplemented with 10% uninactivated fetal bovine serum (FBS, Sigma) and 1% antibiotics (10,000 units/mL penicillin and 10,000 µg/mL streptomycin, Hyclone) at 37oC and 5% CO2. The cells were routinely treated with 0.25% 4

Trypsin-EDTA (Gibco), and 5×10 of cells were seeded per well in 24-well culture plates (Costar) containing coverslips (14 mm in diameter) and cultured in complete medium for 24 h. Then the coverslips were washed with medium and incubated in 500 µL medium containing the purified 8



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Cy5.5-AS1411-Fe3O4 @PPY (magnetic harvesting part of 25 nM of Cy5.5-AS1411 and 0.09 g L-1 of Fe3O4@PPY) for 0.5, 2 and 6 h. After incubation, the cells were washed with sterile PBS and Fe3O4@PPY was removed to the well corner of by magnetic separation, then 300 µL of fixing solution (4% paraformaldehyde) and 300 µL of 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) were added successively to each well incubating for 15 min and 3 min, and washed with PBS respectively. The coverslips were then mounted onto microscope slides and photographs were taken using confocal fluorescence microscope.

Results and Discussion Characterization of Fe3O4@PPY Nanoparticles. The Fe3O4@PPY nanoparticles had similar XRPD patterns of Fe3O4 (Figure S1a in Supporting Information), which can be indexed to face center cubic phase of Fe3O4 (JCPDS No. 19-0629). The broad peaks around 19-28 degree were ascribed to the presence of amorphous PPY. The FTIR absorption peaks (Figure S1b in Supporting Information) around 3000-3500 cm-1 (N-H and C-H stretching vibration), 1544, 1458 cm-1 (the fundamental stretching vibration of pyrrole rings), 1297 cm-1 (C-N stretching vibrations) and 1040 cm-1 (C-H deformation vibrations), also indicated the presence of PPY in [email protected],42 The peaks at 580 cm-1 in both FTIR spectra of Fe3O4 and Fe3O4@PPY (Figure S1b), corresponding to the hallmark of Fe-O stretching vibration, demonstrated the existence of Fe3O4 in Fe3O4@PPY. The amount of PPY coating on the surface of Fe3O4 was further evaluated by the TGA curve (Figure S1c in Supporting Information). The 10% weight loss at about 250-300 o

C of Fe3O4 was ascribed to the capping of sodium acrylate, while around 60% weight loss at

250-480 oC was observed in Fe3O4@PPY (Figure S1c), where the extra 50% of weight loss was attributed to PPY coating. The prepared Fe3O4@PPY displayed supermagnetism, with the 9


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saturation magnetization of 23.72 emu g-1 and negligible coercivity and remanence at 300 K in the magnetic hysteresis loops (Figure S1d in Supporting Information). The decrease saturation magnetization compared to Fe3O4 (51.2 emu g-1) was caused by the nonmagnetic PPY coating. Such supermagnetism of Fe3O4@PPY enabled the convenient separation and redispersion of FRET acceptors in the subsequent FRET and FRET inhibition system. The core-shell structure of Fe3O4@PPY was ascertained by the TEM images (Figure S1e in Supporting Information), where the dark Fe3O4 nanoparticles were wrapped by the grey PPY coating. The thickness of PPY coating was about 20 nm calculating from the averaged diameter of Fe3O4@PPY around 120 nm and Fe3O4 around 80 nm.

FRET Between Cy5.5-AS1411 and Fe3O4@PPY. The Cy5.5-AS1411 has the strong emission in the wavelength range of 700-850 nm, while the Fe3O4@PPY displays the broad absorption at that wavelength range (Figure 2a). The massive spectra overlaps between the emission of Cy5.5-AS1411 and the absorption of Fe3O4@PPY (Figure 2a) made the FRET between the donor and the acceptor possible upon their distance was closed enough through hydrophobic interaction and π-π stacking of AS1411 and PPY. As shown in Figure 2b and 2c, the FRET between Cy5.5-AS1411 and Fe3O4@PPY did happen as the fluorescence intensity of Cy5.5-AS1411 was effectively quenched in the presence of Fe3O4@PPY. For the certain amount of Fe3O4@PPY (e. g., 0.01 g L-1), the quenched fluorescence intensity of Cy5.5-AS1411 reached stable within the incubation time of 8 min (Figure 2b), and in the given incubation time of 8 min, the quenched fluorescence intensity of Cy5.5-AS1411 kept invariable when the concentration of Fe3O4@PPY was no less than 0.09 g L-1. Consequently, 0.09 g L-1 of Fe3O4@PPY and 8 min incubation time was used for all the FRET experiments. It was worth to note that Fe3O4@PPY 10



could quench 90% of the fluorescence intensity at 711 nm of Cy5.5-AS1411, indicating that Fe3O4@PPY was a high-efficient NIR fluorescence quencher. Cy5.5-AS1411 Fe3O4@PPY

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Figure 2. (a) Normalized fluorescence emission spectrum of Cy5.5-AS1411 and absorption spectrum of Fe3O4@PPY. (b) Time-dependent fluorescence spectra of 25 nM Cy5.5-AS1411 in the presence of 0.01 g L-1 Fe3O4@PPY. (c) Fluorescence spectra of 25 nM Cy5.5-AS1411 in the presence of various amounts of Fe3O4@PPY (the incubation time of 25 nM Cy5.5-AS1411 and Fe3O4@PPY was 8 min). 11


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Merits of MS-FRET Inhibition for Nucleolin Detection. Owing to the high specific interaction of AS1411 aptamer and nucleolin, the FRET between Cy5.5-AS1411 and Fe3O4@PPY was inhibited in the presence of nucleolin, resulting in restoring the fluorescence intensity of Cy5.5-AS1411. To make sure the restored fluorescence intensity was only relevant to nucleolin via FRET inhibition, the fluorescence spectra of the relevant controls were measured. As shown in Figure 3a, both Fe3O4@PPY and nucleolin exhibited negligible fluorescence intensity in the wavelength range of 700-860 nm, and the nucleolin did not cause any increase of the fluorescence intensity of Cy5.5-AS1411 in the absence of Fe3O4@PPY. Therefore, the restored fluorescence intensity of Cy5.5-AS1411 in the subsequent experiments was only related to the nucleolin induced Cy5.5-AS1411-Fe3O4@PPY FRET inhibition. To evaluate the kinetics of nucleolin induced fluorescence recovery of Cy5.5-AS1411-Fe3O4@PPY system, the fluorescence spectra of the mixture of nucleolin (5 µg L-1) and Cy5.5-AS1411-Fe3O4@PPY at different incubation times were measured (Figure 3b). The fluorescence intensity was recovered gradually upon the addition of nucleolin, and stabilized after saturation at the incubation time of 5 min, so 5 min incubation time of nucleolin and Cy5.5-AS1411-Fe3O4@PPY was chosen in the subsequent experiments.

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nucleolin, Cy5.5-AS1411 in the presence of nucleolin, Fe3O4@PPY in the presence of nucleolin). (b) Time-dependent fluorescence spectra of Cy5.5-AS1411-Fe3O4@PPY upon the addition of nucleolin. The concentration of Cy5.5-AS1411, Fe3O4@PPY and nucleolin was 25 nM, 0.09 g L-1 and 5 µg L-1 respectively.

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Figure 4. Comparison of MS-FRET and non-separation FRET inhibition for nucleolin detection. (a-c) Nucleolin concentration-dependent fluorescence spectra of Cy5.5-AS1411-Fe3O4@PPY in the case of MS-2 (a, two times of magnetic separation), MS-1(b, one magnetic separation) and MS-0 (c, without magnetic separation). (d) The relationship of (F-F0)/F0 against the concentration of nucleolin in the case of MS-2, MS-1 and MS-0 (F and F0 represents the fluorescence intensity at 711 nm in presence and absence of nucleolin respectively).

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Cy5.5-AS1411-Fe3O4@PPY upon the addition of various amount of nucleolin in the case of MS-FRET (Figure 4a and 4b) or non-separation FRET inhibition were recorded (Figure 4c). MS-2 and MS-1 referred the MS-FRET inhibition, with two times and one time of magnetic separation respectively (Figure 1), and MS-0 was non-separation FRET inhibition. As shown in Figure 4a-4c, in all cases, the fluorescence intensity was gradually recovered with the increase of nucleolin

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concentration, but with quite different background signals and sensitivity. Compared to the non-separation FRET inhibition (Figure 4c), the first time of magnetic separation (Figure 4b), i. e., the removal of free Cy5.5-AS1411 from the binding Cy5.5-AS1411-Fe3O4@PPY, had led to the ultra-low background signals in the absence of nucleolin (F0) and much more sensitive fluorescence recovery. The second magnetic separation, i. e., the separation of human nucleolin-induced far-between Fe3O4@PPY, had resulted in further enhancement of the restoring fluorescence intensity (Figure 4a vs. 4b). Figure 4d plotted the (F-F0)/F0 against the concentration of nucleolin in the case of MS-FRET and non-separation FRET inhibition assay, and Table 1 summarized the sensitivity, detection limit and percentage of fluorescence recovery (the percentage ratio of the final restoring fluorescence intensity to the quenched fluorescence intensity of Cy5.5-AS1411 solution by 0.09 g L-1 Fe3O4@PPY) in all cases. As shown in Table 1, the sensitivity of MS-1 was 25-fold of MS-0, and that of MS-2 was 35-fold of MS-0. Compared to MS-1, MS-2 further improved 1.4-fold in sensitivity. The most sensitive MS-2 FRET inhibition assay displayed the linear range of 0.625-27.5 µg L-1 (8.1-359 pM) and detection limit of 0.04 µg L-1 (0.05 pM) of nucleolin, which were much more sensitive than the those reported in literature.31 All these data indicated that the magnetic separation of the donor and acceptor species in FRET inhibition was promising for the improvement of sensitivity as well as the decrease of detection limit. The separation of the free donor from the FRET inhibition could enhance the sensitivity to 25-fold, suggesting the importance for purifying the FRET probes before the addition of the inhibitors. Besides, the magnetic separation of the donor and acceptor species in FRET inhibition could enhance the percentage of fluorescence recovery from 36% (for non-separation) to 56% (for two magnetic separations). 15


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Table 1

The Quantitative Comparison of the Analytical Figures of Merit of MS-FRET and Non-separation FRET

Inhibition for Nucleolin Detection Measurement

Calibration function a

Linear range (µg L-1)

Detection limit b -1

modes MS-2

∆F/F0=1.93CN+8.07

MS-1

∆F/F0=1.35CN +6.80

Fluorescence intensity

(µg L )

recovery (%) c

0.04

56

0.06

40

0.75

36

0.625-27.5 (R = 0.9978) 0.625-27.5 (R = 0.9949)

MS-0

∆F/F0=0.055 CN +0.042

2.5-27.5 (R = 0.9905)

a

ΔF= F−F0, where F and F0 is the fluorescence intensity at 711 nm of Cy5.5-AS1411-Fe3O4@PPY in the presence

and absence of nucleolin. CN refers to the concentration of nucleolin in µg L-1. b

Calculated as 3σ/k, where σ is the standard deviation of nine measurements of ∆F/F0 for the lowest concentration

of nucleolin in the linear range, and k is the slope of the calibration function (sensitivity). c

Calculated as the ratio of the final restored fluorescence intensity to the quenched fluorescence intensity of

Cy5.5-AS1411 solution (25 nM) by 0.09 g L-1 of Fe3O4@PPY.

20

(F-F0)/F0

15

10

5

A BS

Tr yp si n

P

Cy tC

R H

N uc le PB oli S n N u in cle se ol ru in m H SA Th ro m bi n

0

in

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Selectivity of MS-2 Cy5.5-AS1411-Fe3O4@PPY FRET inhibition for nucleolin assay. All proteins were of the concentration of 6.25 µg L-1. Nucleolin in PBS, HSA, Thrombin, HRP, Cyt C, Trypsin and BSA were measured in 2 mL of PBS buffer, whereas nucleolin in serum was measured in 2 mL serum.

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Table 2

Page 18 of 24

Effects of Coexisted Proteins on ∆F of 0.625 µg L-1 Nucleolin Concentration (µg L-1)

Change in ∆F (%)

HSA

31.3

−4.70

BSA

25.0

−2.16

Cyt C

37.5

−2.47

Trypsin

37.5

−3.07

Thrombin

62.5

−4.78

HRP

50.0

2.99

Substance

Selectivity of MS-2 Cy5.5-AS1411-Fe3O4@PPY FRET Inhibition for Nucleolin Assay. Besides the great improvement in sensitivity, the strategy of MS-2 Cy5.5-AS1411-Fe3O4@PPY FRET inhibition also displayed high selectivity for nucleolin detection (Figure 5). The MS-2 Cy5.5-AS1411-Fe3O4@PPY method only had sensitive fluorescence recovery against nucleolin, and other proteins, such as HSA, BSA, Cyt C, trypsin, thrombin and HRP, displayed very limited fluorescence recovery at the same concentration of 6.25 µg L-1. Even in the complex sample matrix, e. g. human serum, nucleolin still had sensitive response (about 77% of ∆F/F0 in pure PBS, comparable to the related literature21). The interference of the coexisted proteins for the nucleolin detection was also assessed (Table 2). The restored fluorescence intensity of 0.625 µg L-1 nucleolin was unaffected by 40-50 fold of BSA or HSA, 60-fold of Cyt C and trypsin, 100-fold of thrombin and 80-fold of HRP (an error of ±5% in the relative fluorescence intensity was considered as unaffected). All these data demonstrated that MS-2 Cy5.5-AS1411-Fe3O4@PPY FRET inhibition was a sensitive and selective method for nucleolin detection.

In Vitro Cell Imaging. For the real sample analysis, the Cy5.5-AS1411-Fe3O4@PPY purified via one magnetic separation (MS-1 in Figure 1) was explored for in vitro imaging of the nucleolin positive breast cancer cell MCF-7 (Figure 6). For comparison, the nucleolin negative cell line of AD293 was also involved (Figure S2 in Supporting Information). The purified Cy5.5-AS1411-Fe3O4@PPY probe was incubated with the adherent cells for different times (0.5, 2 17


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and 6 h), then the cells were washed with PBS buffer for three times and the Fe3O4@PPY was moved to the well corner via magnet (MS-2 in Figure 1) before fluorescence imaging. As shown in Figure 6, the fluorescence background of 0.5 h incubation was very low (Figure 6, 0.5 h), whereas the red fluorescence signals became more and more brightened as the incubation times went on (Figure 6, 2 h and 6 h vs 0.5 h). In contrast, the nucleolin negative cells AD293 did not displayed the red fluorescence during the incubation time as long as 6 h (Figure S2). All these data demonstrated that the purified Cy5.5-AS1411-Fe3O4@PPY was promising for the in-situ monitoring of nucleolin secreted by the nucleolin positive living cells.

Figure 6. Fluorescence images of MCF-7 cell lines incubated with Cy5.5-AS1411-Fe3O4@PPY for different times (the bars was 25 µm). Conclusion We have developed a novel MS-FRET inhibition strategy for high sensitive detection of 18



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nucleolin using Cy5.5-AS1411 as donor and Fe3O4@PPY as high efficient NIR quenching acceptor. The magnetic Fe3O4@PPY enabled the convenient separation of the donor and acceptor species in FRET inhibition system, and thus was beneficial for the accurate evaluation on the effect of the coexisted donors and acceptors on FRET inhibition assay. The results showed that the separation of free donors from FRET solution had as high as 25-fold enhancement of the sensitivity, whereas further separation of the inhibitor-induced far-between acceptors from the FRET inhibition solution could further enhance the sensitivity to 35-fold. The proposed MS-FRET inhibition strategy would be easily applicable to the high sensitive detection of other biomarkers and benefit for accurate evaluation of the solution chemistry of FRET and FRET inhibition systems. Acknowledgements

This work was supported by the National Basic Research Program of China (No. 2011CB707703), the National Natural Science Foundation of China (No. 21435001, 21575070, 21175073) and the Tianjin Natural Science Foundation (No.13JCYBJC17000).

Supporting Information Available Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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For TOC only

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Magnetic Separation-Assistant Fluorescence Resonance Energy

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