Boronate Affinity Fluorescent Nanoparticles for FÃ¶rster Resonance

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Boronate Affinity Fluorescent Nanoparticles for Förster Resonance Energy Transfer Inhibition Assay of cis-Diol Biomolecules Shuangshou Wang, Jin Ye, Xinglin Li, and Zhen Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04507 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 18, 2016

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Analytical Chemistry

Boronate Affinity Fluorescent Nanoparticles

for Förster

Resonance Energy Transfer Inhibition Assay of cis-Diol Biomolecules Shuangshou Wang, Jin Ye, Xinglin Li, and Zhen Liu*

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China

AUTHOR INFORMATION

* Corresponding Author Tel.: +86 25 8968 5639; fax: +86 25 8968 5639. E-mail address: [email protected] (Z. Liu).

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Abstract: Förster resonance energy transfer (FRET) has been essential for many applications, in which an appropriate donor-acceptor pair is the key. Traditional dye-to-dye combinations remain the working horses, but are rather non-specifically susceptive to environmental factors (such as ionic strength, pH, oxygen, etc.). Besides, to obtain desired selectivity, functionalization of donor or acceptor is essential but usually tedious. Herein, we present fluorescent poly(m-aminophenylboronic acid) nanoparticles (poly(mAPBA) NPs) synthesized via a simple procedure and demonstrate a FRET scheme with suppressed environmental effects for the selective sensing of cis-diol biomolecules. The NPs exhibited stable fluorescence properties, resistance to environmental factors and Förster distance comparable size, making them ideal donor for FRET applications. By using poly(mAPBA) NPs and adenosine 5’-monophosphate modified graphene oxide (AMP-GO) as a donor and an acceptor, respectively, an environmental effects-suppressed boronate affinity-mediated FRET system was established. The fluorescence of poly(mAPBA) NPs was quenched by AMP-GO while it was restored when a competing cis-diol compounds was present. The FRET system exhibited excellent selectivity and improved sensitivity toward cis-diol compounds. Quantitative inhibition assay of glucose in human serum was demonstrated. As many cis-diol compounds such as sugars and glycoproteins are biologically and clinically significant, the FRET scheme presented herein could find more promising applications. Keywords:

Förster resonance energy transfer, boronate affinity, sensing, cis-diol

biomolecule, nanoparticles

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The theory of Förster resonance energy transfer (FRET) has developed as an important tool for many applications such as interaction analysis,1-3 in-vitro assay,4,5 in-vivo imaging,6-8 and single molecule detection.9-11 An appropriate donor-acceptor pair is the key in FRET. Traditional dye-to-dye combinations remain working horses for many FRET-based applications. However, small molecular dyes are rather non-specifically susceptive to environmental factors, such as ionic strength, oxygen species, pH, and so on, which occur through short-range interactions, and thus dye-to-dye FRET is often interfered by environmental factors.12-15 Nanoparticles (NPs), as a typical nanomaterial, has been gaining increasing attentions in FRET. A large variety of NPs, such as metal NPs,16,17 quantum dots (QDs),18-20 persistent-luminescence NPs,21,22 silica NPs23-25 and organic polymer NPs,26-28 have been prepared as promising FRET donors or acceptors. Fluorescent nanoparticles with appropriate size could be a good solution to avoid environmental effects, because of two aspects of reason. For one thing, a large portion of fluorophores are encapsulated or embedded within the NPs and their contacts with substances outside of the NPs are blocked, thereby the NPs could exhibit good stability of fluorescence properties. In fact, this aspect has been well supported by many works.29-33 For another, the center-to-center distances between nanoparticles with surrounding substances are significantly increased as compared with dye-to-dye combinations; therefore, fluorescent NPs should be unfavorable to short-range interactions, meanwhile it should be favorable to

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FRET providing that its size is comparable to the Förster distance (typically in the range of 2-10 nm). However, to the best of our knowledge, such an aspect has not been well confirmed and NPs-based FRET systems with suppressed environmental effects have not been reported yet. On the other hand, in many FRET-based applications, both donors and acceptors are usually functionalized to acquire desired recognition selectivity. To this end, biomolecules, such as antibodies,34,35 aptamers,36,37 and lectins,38,39 have been widely used as the recognition ligands. However, these biomolecules are often associated with poor availability and poor storage stability40-42 meanwhile the functionalization step is usually tedious.43,44 Therefore, easy-to-obtain and stable affinity ligands and straightforward functionalization approaches are highly important. Boronate affinity, which relies on reversible covalent reaction between boronic acids and cis-diol containing compounds, has developed into an appealing chemistry for a range of important applications such as sensing40-44 and separation45-47 of cis-diol biomolecules over non-cis-diol compounds as well as the construction of biomimetic materials.48-52 As many cis-diol compounds such as sugars and glycoproteins are of highly biological and clinical values,53-55 boronate affinity-mediated FRET systems are of great importance. Compared with other fluorescent probes, boronic acid functionalized fluorescent probes displayed several improved properties, such as better water solubility and selectivity,56-59 as well as higher sensitivity.60,61 Recently, boronic acid-modified fluorescent dye-based FRET

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revealed that boronate ester formation significantly enhanced the FRET efficiency.62 Despite these, boronate affinity-mediated FRET systems have not been reported yet. Herein we present boronate affinity fluorescent nanoparticles with high resistance to environmental factors and its usefulness as a donor for FRET inhibition assay of cis-diol biomolecules. The nanoparticles were prepared facilely using m-aminophenylboronic acid (mAPBA) as the monomer through in-water self-polymerization followed by two-step ultrafiltration. Because a portion of boronic acid moieties were located on the surface of the obtained fluorescent NPs, no additional step for functionalization was necessary. As compared with fluorescent boronic acids, the prepared poly(mAPBA) NPs exhibited stable fluorescence properties and apparent resistance to environmental factors, such as amine-rich compounds, salt, pH and oxygen species. The particle size was 3-9 nm, just within the typical range of Förster distance (2-10 nm). Therefore, the prepared poly(mAPBA) NPs can be an ideal fluorescent nanoparticle model for the establishment of a FRET system with suppressed environmental effects. As a proof of the concept, a boronate affinity-mediated FRET system was established in this study, using the prepared poly(mAPBA) NPs as an donor while using adenosine 5’-monophosphate modified graphene oxide (AMP-GO) as an acceptor. Figure 1 schematically illustrates the fluorescence spectral property of poly(mAPBA) NPs and the principle of boronate affinity-mediated FRET for the selective sensing of cis-diol biomolecules over non-cis-diol compounds. AMP-GO was selected because of two reasons. On one hand, GO exhibits

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desirable features including high energy transfer efficiency, water-solubility and facile post-modification.63-65 On the other hand, the binding of AMP (which contains a cis-diol group) with the boronate affinity NPs is moderate due to the electrostatic repulsion between the phosphate group in AMP and the boronic acid moiety of the NPs (both are negatively charged under conditions to be used). As a result, its binding with the NPs can be replaced by a cis-diol compound with higher affinity toward the boronate affinity NPs. The fluorescence of poly(mAPBA) NPs is quenched by AMP-GO through long-range energy transfer, but when a competing cis-diol compound is present, AMP-GO bound with the NPs is competitively displaced and the fluorescence of the NPs is consequently restored. As the FRET and the inhibition are all governed solely by boronate affinity interaction, the FRET platform can allow for the selective sensing of cis-diol compounds. We experimentally confirmed the expected fluorescence properties of the fluorescent NPs and its usefulness in boronate affinity mediated FRET system with suppressed environmental effects. We further demonstrated quantitative assay of glucose in human serum by the FRET inhibition assay.

EXPERIMENTAL SECTION Materials and Chemicals. Adenosine, 2-deoxyadenosine, mAPBA monohydrate (98%), and 6-aminopyridine-3-boronic acid (APDBA, 97%) were purchased from Alfa Aesar (Tianjin, China). N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) was from Aladdin Industrial Corporation (Shanghai, China). Adenosine 5’-monophosphate

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monohydrate (AMP), 4-vinyl-phenylboronic acid (VPBA), apo-transferrin human and lysozyme from chicken egg white) were from Sigma Aldrich (St. Louis, MO, USA). Glucose and N-hydroxysccinimide (NHS) and pyrimidine-5-boronic acid (PMBA, 97%) were from J&K Chemical (Shanghai, China). Fresh healthy human serum was obtained from Shuangliu Zhenglong Chemical and Biological Research Laboratory (Chengdu, China). Graphite powder was purchased from Sinopharm Chemical Reagent (Shanghai, China). Sulfuric acid (98%), hydrochloric acid (37%), potassium permanganate, potassium persulfate, phosphorus pentoxide, hydrazine monohydrate, hydrogen peroxide (30%), sodium phosphate and sodium dihydrogen phosphate were purchased from Nanjing Chemical Reagent (Jiangsu, China). All other reagents were of analytical grade unless otherwise noted. All ultrafiltration tubes were purchased from Millipore (Milford, MA, USA). Water used in all experiments was purified by a Milli-Q Advantage A10 water purification system (Millipore, Milford, MA, USA).

Apparatus. Transmission electron microscopy (TEM) was performed on a Tecnai G2 F30 S-TWIN TEM instrument (Hillsboro, Oregon, USA). Dynamic light scattering (DLS) was performed on a BI-200SM (Brookhaven Instrument Corporation, Holtsville, USA). All fluorescence experiments were performed on a RF-5301 (PC) S instrument (Shimadzu China, Shanghai, China). Unless otherwise specified, excitation wavelength was set at 350 nm while emission wavelength was 450 nm. The slit widths for excitation and emission

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were set at 3 or 5 nm. Energy-dispersive X-ray spectroscopy was performed on a Hitachi FE-SEM S-4800 equipped with EDX energy spectrum analysis instrument. UV-vis absorption characterization was performed on a Nanodrop-2000C instrument (Thermo Fisher Scientific, Shanghai, China). FT-IR characterization was performed on a Nicolet iS10 instrument (Thermo Fisher Scientific, Shanghai, China).

Preparation of Poly(mAPBA) NPs. At first, 400 mg mAPAB was dissolved in 80 mL phosphate solution (0.1 M, pH 10.5), followed by adding with 120 mL H2O2. After reaction for 5 h at 30 °C, the solution was ultra-filtered twice by tubular ultrafiltration membrane with a 50, 000 and 3, 000 cut-off MW, respectively. Then the tubular ultrafiltration membrane with a 3, 000 cut-off MW was washed with ultrapure water and ultra-filtered three times each. Finally, the particles remained on the membrane was collected and freeze-dried. The yield was measured to be 7%.

Preparation of Graphene Oxide. Graphene oxide was synthesized from natural graphite powder according to a modified Hummers’ method.66,67 3.0 g graphite powder was put into a mixture solution containing 12 mL concentrated H2SO4, 2.5 g K2S2O8, and 2.5 g P2O5. The mixture was kept in oil bath at 80 °C for 4.5 h. Then the mixture was diluted with 0.5 L ultrapure water and placed at room temperature overnight, and then the mixture was filtered and washed with ultrapure water. The product was dried under ambient condition overnight.

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This pre-oxidized graphite was then subjected to oxidation as described as follows. Pretreated graphite powder was put into cold (0 °C) concentrated H2SO4 with a volume of 120 mL. Followed by adding with 15 g KMnO4 gradually under stirring and the temperature of the mixture was kept to be below 20 °C. After that, the mixture was stirred at 35 °C for 2 h, and then diluted with 250 mL ultrapure water in an ice bath to keep the temperature below 50 °C. Then additional 0.7 L ultrapure water was added. Shortly after that, 20 mL H2O2 was added, and the color of mixture changed into brilliant yellow along with bubbling. Then the mixture was centrifuged and washed with 10% (v / v) hydrochloric acid solution and ultrapure water, respectively. Then the solution was sonicated for half an hour, followed by centrifuging at 4, 000 rpm for 30 min. The supernatant was purified by dialysis for one week. Finally, the solution was stored at 4 °C for use.

Preparation of AMP Modified Graphene Oxide. Freeze-dried powder of above prepared GO (11 mg) was dissolved into 20 mL phosphate buffer solution (0.1 M, pH 7.4), followed by adding with EDC and NHS (11 mg each). After reaction for 2 h at 30 °C, 11 mg AMP was added and the reaction was kept for 10 h. Then the solution was centrifuged at 15, 000 rpm for 1 h. The resulting precipitation was washed with ultrapure water and centrifuged three times each. Finally, the precipitate was dispersed into phosphate solution (0.1 M, pH 10.5) with a concentration of 2 mg/mL and stored at 4 °C for use.

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Environmental Effects on the Spectral Properties of Poly(mAPBA) NPs and Boronic Acids. A serious of phosphate solutions (0.1 M, pH 10.5) containing 0.25 mg/mL poly(mAPBA) NPs, mAPBA, VPBA, APDBA or PMBA were respectively added with different final concentrations of fructose (0, 0.2, 0.4, 0.8, 1.5, 3.0, 5.0, 10, 15 and 20 mg/mL), glucose (0, 0.2, 0.4, 0.8, 1.5, 3.0, 5.0, 10, 15 and 20 mg/mL), lysozyme (0, 1, 2, 3, 4 and 5 mg/mL), aniline (0, 0.2, 0.4, 0.6, 0.8, 1.0 and 2.0 mg/mL), adenosine (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL), NaCl (0, 0.2, 0.5, 1.0, 2.0 and 3.0 M), and H2O2 (0, 0.3%, 0.5%, 0.7%, 1% and 2% (v / v)). After incubation for 30 min at room temperature, fluorescence intensity was measured separately and compared. For influence of pH on the stability of spectral properties, phosphate solution with different pH (2.0, 4.5, 7.5, 10.5 or 12.5) was used to dissolve poly(mAPBA) NPs and boronic acids, and the fluorescence intensity was measured, respectively.

Environmental Effects on the Spectral Properties of Poly(mAPBA) NPs/AMP-GO FRET System. A series of phosphate solutions (0.1 M, pH 10.5) containing 0.25 mg/mL poly(mAPBA) NPs and 0.025 mg/mL AMP-GO were respectively added with different final concentrations of lysozyme (0, 1, 2, 3, 4 and 5 mg/mL), aniline (0, 0.1, 0.2, 0.5, 1.0 and 2.0 mg/mL), deoxyadenosine (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL), NaCl (0, 0.2, 0.5, 1.0, 2.0 and 3.0 M), and H2O2 (0, 0.3%, 0.5%, 0.7%, 1% and 2% (v / v)). After incubation for 30 min at room temperature, fluorescence intensity was measured separately and

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compared. To investigate the influence of pH on the stability of spectral properties, the fluorescence intensity of a series of 0.1 M phosphate solutions containing 0.25 mg/mL poly(mAPBA) NPs and 0.025 mg/mL AMP-GO at pH 2.0, 4.5, 7.4, 10.5 and 12.5 was measured.

Storage Stability of Poly(mAPBA) NPs. Phosphate solution (0.1 M, pH 10.5) containing poly(mAPBA) NPs with a concentration of 1.0 mg/mL was stored at room temperature after sealing. Fluorescence intensity of the solution was tested once per week and tested for 5 weeks.

Fluorescence of Poly(mAPBA) NPs and mAPBA Monomer Quenched by AMP-GO. Seven aliquots of 3 mL phosphate solutions (0.1 M, pH 10.5) containing 0.25 mg/mL poly(mAPBA) NPs were added with AMP-GO with different final concentrations (0, 0.01, 0.025, 0.05, 0.1, 0.2 and 0.5 mg/mL). After incubation for 30 min at room temperature, the fluorescence intensity (slit width for excitation, 3 nm; slit width for emission, 5 nm) of each solution was measured separately. For mAPBA monomer, five aliquots of 3 mL phosphate solutions (0.1 M, pH 10.5) containing 1.0 mg/mL mAPBA were added with AMP-GO with different final concentrations (0, 0.005, 0.01, 0.05 and 0.2 mg/mL). After incubation for 30 min at room temperature, the fluorescence intensity (excitation wavelength, 300 nm; emission

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wavelength, 375 nm; slit width for excitation, 3 nm; slit width for emission, 3 nm) of each solution was measured separately.

Fluorescence Recovery of Poly(mAPBA) NPs when Transferrin was Present. Ten aliquots of 3 mL phosphate solutions (0.1 M, pH 10.5) containing 0.25 mg/mL poly(mAPBA) NPs and 0.1 mg/mL AMP-GO were added with different final concentrations of transferrin (0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL). After incubation for 30 min at room temperature, fluorescence intensity (slit width for excitation, 5 nm; slit width for emission, 5 nm) of each solution was measured, respectively.

Sensing Selectivity of the Poly(mAPBA) NPs/AMP-GO FRET System. Six aliquots of 3 mL phosphate solutions (0.1 M, pH 10.5) containing 0.25 mg/mL poly(mAPBA) NPs and 0.1 mg/mL AMP-GO or 1.0 mg/mL mAPBA and 0.05 mg/mL AMP-GO were added with different substrates (including glucose, aniline, adenosine, deoxyadenosine, transferrin and lysozyme) each with a concentration of 5 mg/mL. After incubation for 30 min at room temperature, fluorescence of each solution was measured separately and compared.

Dependence of Fluorescence Intensity of the Poly(mAPBA) NPs/AMP-GO FRET on the Concentration of Glucose. Eight aliquots of 3 mL poly(mAPBA) NPs/AMP-GO conjugate solutions (0.25 mg/mL poly(mAPBA) NPs and 0.025 mg/mL AMP-GO dissolved

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in 0.1 M phosphate solution, pH 10.5) were added with different concentrations of glucose (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL). After incubation for 30 min at room temperature, fluorescence intensity of each solution was measured and compared. The obtained data were used to plot a calibration curve at pH 10.5 for quantitation. A calibration curve at pH 7.4 was also established with the same procedure except that the pH of the solutions was fixed at 7.4.

Dependence of Fluorescence Intensity of the Poly(mAPBA) NPs/GO FRET on the Concentration of Glucose. Six aliquots of 3 mL poly(mAPBA) NPs/GO conjugate solutions (0.25 mg/mL poly(mAPBA) NPs and 0.025 mg/mL GO dissolved in 0.1 M phosphate solution, pH 10.5) were added with different concentrations of glucose (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL). After incubation for 30 min at room temperature, fluorescence intensity of each solution was measured and compared.

Pretreatment of Healthy Human Serum. Fresh healthy human serum was centrifuged at 10,000 rpm for 1 h in a tubular ultrafiltration membrane with a 3, 000 cut-off MW, and then the solution in tubular ultrafiltration membrane was collected and stored at 4 °C for use.

Sensing of Glucose in Human Serum. Five aliquots of 1 mL of above treated human serum were firstly freeze-dried separated. Then the obtained freeze-dried powder was

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added with 3 mL phosphate solution (0.1 M, pH 10.5 or 7.4) containing 0.25 mg/mL poly(mAPBA) NPs and 0.025 mg/mL AMP-GO. After incubation for 30 min at room temperature, fluorescence intensity of each solution was measured and the concentration of glucose was finally deduced from above mentioned calibration curves.

RESULTS AND DISCUSSION Preparation and Characterization of Poly(mAPBA) NPs. The preparation of the poly(mAPBA) NPs was straightforward through self-polymerization of mAPBA in the presence of H2O2 at alkaline condition followed by two-step ultrafiltration (Scheme S1A). The possible chemical structure of the resulting polymer is shown in Scheme S1B according to the mechanism of polymerization.68-70 The prepared poly(mAPBA) NPs exhibited good water dispersibility (Figure S1), which is favorable to real applications. Conditions for the preparation of poly(mAPBA) NPs, including polymerization time and the volume fraction of H2O2, were optimized. The optimal polymerization time was found to be 5 h while the optimal volume fraction of H2O2 was 60% (Figure S2). TEM images and DLS characterization shown in Figure 2A and 2B reveal that poly(mAPBA) NPs exhibited uniform particle size distribution. The size distribution range was found to be 3-9 nm and 5.5-9 nm according to the TEM images and the DLS histogram, respectively. Such particle size is just within the typical range of Förster distance (2-10 nm). UV-vis absorption, fluorescence excitation and emission spectra of mAPBA monomer and

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poly(mAPBA) NPs are shown in Figure 2C and 2D. The maximum excitation and maximum emission of poly(mAPBA) NPs was at 350 and 455 nm, respectively. As compared with mAPBA monomer, a red-shift of 50 nm in the excitation spectrum and a red-shift of 80 nm in the emission spectrum were observed for poly(mAPBA) NPs. Such spectral shift was assigned to π-π stacking between aromatic rings in the nanoparticles. As poly(mAPBA) is a linear polymer, poly(mAPBA) NPs were probably formed through folding of the polymer chain(s) and therefore π-π stacking is rich in the NPs. Besides, the absorption spectra differed from the excitation spectra, especially for the monomer. This is an indication that more than one light absorbing species were present in the samples: different species or a single species in different states. Considering that the monomer was of relatively high purity (98%) and un-polymerized species were removed during the synthesis of the NPs, a single or limited species with different states was more possible, such as in a monomer-dimer equilibrium, protonation equilibrium, solvent association equilibrium, and so on.

Preparation and Characterization of AMP-GO. AMP-GO was synthesized as an effective acceptor for pairing with poly(mAPBA) NPs. GO containing abundant active functional groups was first synthesized and then modified with AMP through covalent reaction between primary amines of AMP and carboxyl or epoxy groups of GO. GO and the prepared AMP-GO were characterized by TEM, Fourier transform infrared spectroscopy

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(FT-IR), UV-vis absorption and fluorescence emission spectrum (Figure S3). TEM characterization showed almost no difference in morphology between GO and AMP-GO, which indicates that the integrity of morphology was kept during the process of AMP functionalization. GO and AMP-GO were irregular nanosheets with some wrinkles in morphology and the size was estimated to be within hundreds of nm to 2 µm, according to the TEM images. Newly emerged peaks for carbon-nitrogen double-bond (1715 cm-1) and phosphorus-oxygen double-bond (1217 cm-1, 1175 cm-1) for AMP-GO indicate that GO was successfully modified with AMP, which is also confirmed by the appearance of N element in energy dispersive X-ray spectroscopy (EDX) characterization (Table S1). In the UV-vis spectrum of AMP-GO, the major absorption peaks of GO remained, but the absorption of AMP-GO was higher and broader, which is favorable for energy transfer. In addition, good overlap between the emission spectra of poly(mAPBA) NPs (Figure 2D) and the absorption spectra of AMP-GO (Figure S3D) indicated that the quenching of the fluorescence of poly(mAPBA) NPs by AMP-GO would follow an energy transfer mechanism. However, as GO and AMP-GO emitted nearly no fluorescence (Figure S3E and S3F), the energy transfer would be non-radiative.

Tolerance of Poly(mAPBA) NPs to Environmental Effects. The effects of a variety of environmental factors, including fructose, glucose, adenosine, lysozyme, aniline, sodium chloride, pH and H2O2, on the fluorescence of poly(mAPBA) NPs were investigated and

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compared with those on the fluorescence of free boronic acids, including mAPBA, VPBA, APDBA and PMBA. Fructose, glucose and adenosine were selected because they are cis-diol compounds and can bind with boronic acids through boronate affinity interaction (covalent). Lysozyme and aniline were selected because they are both amine-rich compounds, which can involve in electron transfer or charge transfer. Sodium chloride was selected to adjust the ionic strength of surrounding solution while H2O2 was selected as a representative of oxygen species. As shown in Figure 3, except that there are few exceptions (for instance, PMBA was insensitive to the presence of lysozyme and aniline), generally to say, the fluorescence of the free boronic acids were apparently influenced by all environmental factors. In comparison, the fluorescence of poly(mAPBA) NPs was much more stable; fluctuation of the fluorescence intensity was the slightest under the same environment effects. Besides, fluorescence fluctuation of poly(mAPBA) NPs was less than 8.8% within storage in aqueous solution for 5 weeks at room temperature (Figure S4), which indicates excellent storage stability.

The tolerance of the poly(mAPBA) NPs/AMP-GO FRET system to environmental effects was also investigated. As shown in Figure S5, the fluorescence of poly(mAPBA) NPs in the presence AMP-GO was insensitive to environmental factors (cis-diol compounds such as glucose and adenosine will recover the quenched fluorescence in this case and therefore they were not considered as environmental factors). Particularly, the presence of lysozyme

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or aniline did not significantly influence the fluorescence of the FRET system, which suggests that the quenching behavior of AMP-GO toward poly(mAPBA) NPs did not obey an electron transfer or charge transfer mechanism.

Quenching and Recovery of Fluorescence of Poly(mAPBA) NPs. The quenching properties of AMP-GO toward free mAPBA and poly(mAPBA) NPs were investigated separately. As shown in Figure S6 and Figure 4A and 4B, the quenching efficiency increased as increasing the concentration of AMP-GO and the quenching ratio almost reached 100% when the concentration of AMP-GO was 0.5 and 0.2 mg/mL for poly(mAPBA) NPs and mAPBA, respectively. This suggests that AMP-GO is an effective acceptor and the stability of spectral properties of poly(mAPBA) NPs is better than that of free mAPBA. Comparison between the quenching efficiency of AMP-GO and GO under the same concentration (Figure S7) reveals that the modification of GO with AMP is favorable for efficient quenching due to boronate affinity binding. The feasibility of the poly(mAPBA) NPs/AMP-GO combination for the competitive assay of cis-diol biomolecules was investigated. As shown in Figure 4C and 4D, the fluorescence of poly(mAPBA) NPs was restored when the glycoprotein transferrin was present and the restored fluorescence intensity increased linearly with increasing the concentration of transferrin within a certain concentration range. The mechanism of fluorescence recovery of poly(mAPBA) NPs herein was due to the competitive displacement of APM-GO by the

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glycoprotein. Binding force between AMP and boronic acid groups was relatively weak because of the coexistence of electrostatic repulsion effect (both AMP and poly(mAPBA) NPs were negatively charged at the pH used) and boronate affinity. Therefore, when cis-diol compounds with stronger boronate affinity was present, poly(mAPBA) NPs were released from AMP-GO and consequently the fluorescence of poly(mAPBA) NPs was restored. There are two linear regions in the response curve in Figure 4D, which was possibly due to that the transferrin molecule contains two glycan chains.

Selectivity toward cis-Diol Biomolecules over non-cis-Diol Compounds. The sensing selectivity of the poly(mAPBA) NPs/AMP-GO FRET system toward cis-diol biomolecules over non-cis-diol compounds was investigated. As shown in Figure 5, the poly(mAPBA) NPs/AMP-GO FRET system exhibited apparent response toward cis-diol biomolecules, including glucose, adenosine and transferrin, but limited response toward non-cis-diol compounds, such as aniline, deoxyadenosine and lysozyme. These results demonstrated excellent sensing selectivity of the designed FRET system toward cis-diol biomolecules over non-cis-diol compounds. In contrast, when mAPBA was used as the donor, almost no selectivity was observed because it responded to not only cis-diol biomolecules but also non-cis-diol compounds. Besides, the poly(mAPBA) NPs/AMP-GO FRET system provided apparently stronger responses toward cis-diol biomolecules as compared with free mAPBA, indicating better sensitivity of poly(mAPBA) NPs than free mAPBA.

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Analysis of Real Samples. Fluorescence restoration of poly(mAPBA) NPs versus the concentration of glucose after quenched by AMP-GO was investigated under two different pH values. As shown in Figure 6A and Figure S8, the response curves are linear within the concentration range of 0.05 to 1.2 mg/mL (R2 = 0.979 for pH 10.5, R2 = 0.977 for pH 7.4, n = 8). The linear concentration range covers normal fasting blood-glucose range for healthy human (0.7-1.1 mg/mL).71 The feasibility of the poly(mAPBA) NPs/AMP-GO FRET system for real sample application was investigated using human serum of a healthy individual as a representative sample. In order to eliminate possible interference from glycoproteins, the sample was centrifuged with an ultrafiltration tube with a cut-off molecular weight of 3,000 prior to the FRET inhibition assay. The interference from other small cis-diol compounds, such as nucleosides and catecholamines can be ignored since they are usually present at a very low concentration in blood.72 The glucose concentration of the sample was determined to be 0.93 ± 0.08 mg/mL (n = 5) at pH 10.5 and 0.98 ± 0.05 mg/mL (n = 4) at pH 7.4, which are in good agreement with each other and also match well with the concentration of glucose in healthy human serum.73 This indicates that this FRET system is feasible for the selective sensing of glucose under different pHs. Although non-functionalized GO could also quench the fluorescence of poly(mAPBA) NPs, the response of the poly(mAPBA) NPs/GO system was almost constant under the investigated concentration range of glucose (Figure 6B), which suggests that the functionalization of

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GO with AMP was critical for the FRET inhibition assay.

CONCLUSIONS We have presented fluorescent poly(mAPBA) NPs with high tolerance to environmental factors and demonstrated a boronate affinity-mediated FRET scheme for the selective sensing of cis-diol biomolecules via using the fluorescent NPs as a donor. The synthesis procedure of the NPs was simple and straightforward. The NPs were water-solubility and post-modification free. More importantly, they exhibited expected properties, i.e., stable fluorescence properties, resistance to environmental factors and Förster distance comparable particle size, making them an ideal donor for FRET-based applications. The poly(mAPBA) NPs/AMP-GO FRET was solely mediated by boronate affinity interaction, and thereby exhibited excellent selectivity and improved sensitivity toward cis-diol compounds. To the best of knowledge, such a NPs-based and environmental effects-suppressed FRET scheme has not been reported in literature yet so far. It can be a general FRET platform for the selective sensing of cis-diol compounds. As many cis-diol compounds are biologically or clinically significant, this new FRET platform can find more promising applications in many fields such as clinical diagnostics.

ACKNOWLEDGEMENT

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We acknowledge the financial support of the National Science Fund for Distinguished Young Scholars (No. 21425520) from the National Natural Science Foundation of China. This project is also supported by the program B for outstanding Ph. D. candidates of Nanjing University.

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Captions: Figure 1. Schematic of a FRET scheme based on poly(mAPBA) NPs and AMP-GO for the selective sensing of cis-diol biomolecules. Different color pictograms represent different environmental factors, such as amine-rich compounds, ionic strength, oxygen species, pH and so on. Figure 2. A, B) TEM images of poly(mAPBA) NPs at different magnifications (inset in A is the particle size distribution of poly(mAPBA) NPs characterized by dynamic light scattering); C, D) UV-vis absorption (black), fluorescence excitation (red) and emission

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spectra (green) of mAPBA monomer and poly(mAPBA) NPs, respectively. Figure 3. Fluorescence intensity of poly(mAPBA) NPs and different monomers as a function of different environmental factors. Right Y-axis is for curves in blue, left Y-axis is for curves in other colors. F0 represents the initial fluorescence intensity while F represents the fluorescence intensity in presence of different environmental factors. Figure 4. A, B) Dependence of the fluorescence intensity of poly(mAPBA) NPs on the concentration of AMP-GO; C, D) Dependence of the restored fluorescence intensity of poly(mAPBA) NPs/AMP-GO on the concentration of competing transferrin. Excitation wavelength was set at 350 nm. The slit widths used for A, B and C, D were different (See the Experimental Section for the details). Figure 5. Response of A) the poly(mAPBA) NPs/AMP-GO and B) mAPBA /AMP-GO conjugates toward different compounds. F0 represents the fluorescence intensity quenched by AMP-GO while F represents the fluorescence intensity when a certain competing compound was present. The excitation and emission wavelengths for poly(mAPBA) NPs/AMP-GO were set at 350 and 450 nm, respectively; while the excitation and emission wavelengths for mAPBA/AMP-GO were 305 and 375 nm, respectively. Figure 6. Dependence of restored fluorescence of poly(mAPBA) NPs quenched by A) AMP-GO and B) GO on the concentration of glucose at pH 10.5.

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Figure 1

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lysozyme

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Boronate Affinity Fluorescent Nanoparticles for FÃ¶rster Resonance

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