Near-Infrared-Fluorescent Probes for Bioapplications Based on Silica

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Near-Infrared-Fluorescent Probes for Bioapplications based on Silica-Coated Gold Nanobipyramids with Distance-Dependent Plasmon-Enhanced Fluorescence Caixia Niu, Quanwei Song, Gen He, Na Na, and Jin Ouyang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03034 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 14, 2016

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Analytical Chemistry

Near-Infrared-Fluorescent Probes for Bioapplications based on Silica-Coated Gold Nanobipyramids with Distance-Dependent PlasmonEnhanced Fluorescence Caixia Niu,† Quanwei Song,‡,§ Gen He,† Na Na,† and Jin Ouyang*,†

†

Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China ‡ State Key Laboratory of Petroleum Pollution Control, Beijing, 102206 § CNPC Research Institute of Safety and Environmental Technology, Beijing, 102206 * Jin Ouyang Fax: 86 010-62799838; Tel: 86 010-58805373; E-mail: [email protected]

ABSTRACT: Optical antennas with anisotropic metal nanostructures are widely used in the field of fluorescence enhancement based on localized surface plasmons (LSPs). They overcome the intrinsic defects of low brightness of near-infrared (NIR) dyes and can be used to develop sensitive NIR sensors for bioapplications. Here, we demonstrate a novel NIR plasmon-enhanced fluorescence (PEF) system consisting of elongated gold nanobipyramids (Au NBPs) antennas, silica, and NIR dyes. Silica was chosen as the rigid spacer to regulate the distance between the metal nanostructures and dyes. Maximum enhancement was observed at a distance of approximately 17 nm. The enhanced fluorescence could be quenched by Cu2+ and recovered by pyrophosphate (PPi) owing to the strong affinity between PPi and Cu2+. Thus, the Au NBP@SiO2@Cy7 nanoparticles (NPs) detect PPi via “switch-on” fluorescence signals, with a detection limit of 80 nM in the aqueous phase. The probe not only detects PPi in living cells but also can be used for a microRNA assay with a detection limit of 8.4 pM by detecting PPi in rolling circle amplification (RCA). Additionally, gold nanorods (Au NRs) with the same longitudinal plasmon resonance wavelength (LPRW) as the Au NBPs were prepared to synthesize Au NR@SiO2@Cy7 NPs for comparison. The experimental and finite-different time-domain (FDTD) simulation results indicate that the stronger electric fields of Au NBPs contribute to a fluorescence enhancement that is several times higher than that of Au NRs, confirming the superior properties of Au NBPs as novel ideal substrates to develop PEF biosensors.

prepare PEF sensors due to their simple preparation methods and tunable longitudinal plasmon resonance wavelengths (LPRWs) from visible to infrared regions.6, 16, 17 The distancedependent PEF by Au NRs has been well studied and reported.18,19 The concentrated electromagnetic fields generated from the ends of Au NRs facilitate the fluorescence enhancement. However, the end shape of Au NRs is rounded, which induces a relatively small electric field for PEF effects.20 In addition, most NR-based sensors are in solids, and only a few sensors in solution have been reported,17, 21 which limits the bioapplications of PEF sensors. Thus it is meaningful to explore PEF sensors in the aqueous phase using Au nanocrystals with sharper tips, which can produce stronger electric fields. In recent years, gold nanobipyramids (Au NBPs) have been developed with analogous but better optical properties than Au NRs.22, 23 Theoretical and experimental results have revealed that the PEF effects are influenced by various factors, including the appropriate distance between the metal and fluorophore,17, 24 the matching between the plasma band and a fluorophore absorbance/emission spectra,6 and the strong electric fields induced by anisotropic metal nanostructures with sharp tips.13 According to the lightning-rod effect, Au NBPs have

INTRODUCTION Near-infrared fluorescence (NIR) is an excellent method for bioapplications due to its high penetration depth and low photothermal damage. However, NIR fluorescence suffers from the defect of low brightness, which limits the use of NIR dyes in bioapplications.1-4 Optical antennas composed of anisotropic metal nanostructures are widely used for fluorescence enhancement based on localized surface plasmons (LSPs).5 They can serve as the substrates to produce plasmon-enhanced fluorescence (PEF), which has been developed into an effective platform to enhance the quantum yield and emission intensity of most dyes.6 PEF results from the interactions between dyes and the localized surface plasmon resonance (LSPR)7, 8 which refers to the collective oscillation of noble metal surface conduction electrons when the metal nanostructure dimensions are smaller than the wavelength of incident light.9 PEF sensors have gained increased attention for use in many bioapplications. Among the reported PEF fluorophoremetal nanostructures,1,10-13 gold nanomaterials are excellent optical antennas for their superior features of good biocompatibility, biosafety, inoxidizability, and facile preparation.14, 15 In particular, gold nanorods (Au NRs) are an ideal material to

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with a limit of detection of 80 nM. The detection mechanism is supported by the quenching effects of Cu2+ on Cy7 dyes and the strong affinity between PPi and Cu2+ (Scheme 1B).38, 39 Considering the biocompatibility of the sensor, we also applied the sensor to the Cu2+ and PPi imaging in living Hela cells. With the aid of RCA technology, the Au NBP@SiO2@Cy7 NPs were further developed for use in the microRNA assay (Scheme 1C). The limit of detection was calculated to be 8.4 pM. To our knowledge, this work represents the first study of a distance-dependent PEF-based NIR sensor based on silica-coated Au NBPs in solution for the PPi assay.

two sharper apexes as opposed to the rounded ends of Au NRs, which contributes to the several times larger local electricfield enhancement.25 In addition, similar to Au NRs, Au NBPs possess tunable LPRWs, which can be tuned by changing the aspect ratio.20 As such, Au NBPs have promise as a type of nanomaterial for use in PEF sensors. In addition, exploring solution-based PEF sensors is important because the most biomolecules retain their physiological activities only in the aqueous phase. Pyrophosphate (PPi) plays essential roles in numerous biological processes, such as energy transformation and metabolism by enzymatic reactions, among others.26, 27 PPi is the byproduct of the process of nucleic acid amplification; hence, the detection of PPi concentrations can represent important signals for DNA replication.28, 29 More importantly, PPi detection has been developed into an effective method for real-time DNA sequencing, namely pyrosequencing.30 However, most reported PPi sensors require a complicated synthesis process, and the limit of detection is not quite satisfactory. In addition, the applications of the PPi assay are still limited. Hence, there is great need to develop sensitive methods for the PPi assay and broaden the range of its application. Theoretically, the detection of PPi will be applied to the microRNA assay with the aid of rolling circle amplification (RCA), an isothermal nucleic acids replication technique. The advantages of RCA include high sensitivity, high specificity, high throughput and easy operation.31, 32 In the process of nucleic acid amplification, the dNTPs are converted into nucleic acid and PPi molecules are produced as by-products.33 Thus, PPi can serve as an important marker and represents a potential alternative to evaluate the processes and results of RCA.34 MicroRNAs are wellknown to play a vital role in gene transcription and expression, even in cancer diagnosis.35, 36 Herein, we propose using the different concentrations of microRNA as the primers for RCA to develop a sequence-dependent microRNA assay through the quantitative detection of PPi. In this work, we describe a versatile “switch-on” NIR PEF biosensor for the PPi assay based on silica-coated Au NBP antennas in the aqueous phase. First, we prepared the Au NBPs with plasma maxima at 750 nm, at which point the spectral overlap is the strongest with Cy7 dyes absorbing at 746 nm and emitting at 776 nm. The overlap between plasma band and absorption/emission spectra of the dye will increase the excitation rate and radiative decay rate.9, 37 Silica was chosen as the dielectric spacer to control the distance, and the NIR dye Cy7 molecules were bound to Au NBP@SiO2 nanoparticles (NPs) to prepare the Au NBP@SiO2@Cy7 NPs (Scheme 1A). To demonstrate the advantages of Au NBPs, Au NRs with the same LPRW at 750 nm were synthesized to prepare Au NR@SiO2@Cy7 NPs for comparison. The fluorescence enhanced factor maximum of Au NR@SiO2@Cy7 NPs is 4.5fold at a silica thickness of 17 nm, which is smaller than the value of 10.7-fold for Au NBP@SiO2@Cy7 NPs. Further, the finite-different time-domain (FDTD) simulation results indicate that the electric field of Au NBP is nearly 5 times stronger than that of the the Au NRs used in this work at an excitation wavelength of 750 nm, which contributes to the stronger PEF effects. Both the experimental and theoretical results confirm that Au NBPs are a better PEF substrate than Au NRs. The Au NBP@SiO2@Cy7 NPs were further applied to detect PPi through “turn-on” fluorescence signals in aqueous solutions

Scheme 1. Schematic illustration of (A) the synthesis of Au NBP@SiO2@Cy7 NPs, the detection of (B) PPi and (C) microRNA. EXPERIMENTAL SECTION Materials. All reagents used were analytical grade, and they were used as received without further purification. Sodium borohydride (NaBH4), ascorbic acid, cetyltrimethyl ammonium bromide (CTAB), cetyltrimethyl ammonium chloride (CTAC) in water, tetraethoxysilane (TEOS), pyrophosphate (PPi), 3-(N-morpholino) propanesulfonic acid (MOPS) and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (Shanghai, China). Silver nitratewere (AgNO3) and (3aminopropyl)triethoxysilane (APTES) were obtained from Alfa Aesar (Shanghai, China). Hydrochloric acid (HCl), ammonia solution, hydrogen peroxide solution and sodium citrate were purchased from Beijing Chemical Works. Chloroauric acid (HAuCl4) was obtained from Sinopharm Chemical Reagent Co. (China). Cy7 NHS ester was purchased from Tianjin Biolite Biotech Co., Ltd. The HeLa human cervical cancer cell line, fetal bovine serum (FBS) and penicillin–streptomycin solution (PSS) were obtained from Peking Union Medical College Hospital. The DNA template and microRNA were synthesized by Takara Biotechnology Co., Ltd. (China). The sequences of the DNA template and microRNA are listed in Table S1. DNA polymerase (phi29) was purchased from New England Biolabs. Apparatus. Fluorescence (FL) spectra were measured on a Cary Eclipse (Varian, America) spectrophotometer. Ultravio-

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Analytical Chemistry were kept for 24 h at 30 °C to allow the CTAB bilayer to adsorb on the Au NBP surface. Briefly, NaOH (0.1 M, 0.2 mL) was added to each Au NBP solution to adjust the pH to 10~11, and the solutions were stirred for 0.5 h. Then, TEOS (20% in methanol, 0.08 mL) was added slowly and allowed to react with Au NBPs for 24 h at 30 °C. The resulting solutions were centrifuged at 11000 rpm for 15 min and washed with water and ethanol three times. The precipitates from the six tubes were each re-dispersed in 10 mL ethanol. Synthesis of Au NBP@SiO2@Cy7 NPs. To synthesize the Au NBP@SiO2@Cy7 NPs, the core-shell Au NBP@SiO2 NPs were modified with amino groups. Ammonium hydroxide (25%, 100 µL) was added to Au NBP@SiO2 solutions, and the mixtures were stirred for 0.5 h at room temperature. Then, APTES (50 µL) was injected, and the resulting solutions were stirred for 3 h at 35 °C and 1 h at 65 °C. The Au NBP@SiO2NH2 NPs were centrifuged and washed with ethanol three times. To bond the Cy7 dyes to Au NBP@SiO2-NH2, Cy7 NHS ester was dissolved in HEPES buffer (pH 7.4) at 3×10-3 M and added to 4 mL of the Au NBP@SiO2-NH2 NPs solutions mentioned above. The mixtures were stirred for 10 h at room temperature to react. The prepared Au NBP@SiO2@Cy7 NPs were collected by centrifugation and washed, re-dispersed in HEPES buffer (pH 7.4), and stored at 4 °C for further use. Synthesis of Au NRs. Au NRs were prepared using a modified seed-mediated growth method. The seed solution was made by adding a freshly prepared, ice-cold NaBH4 solution (0.01 M, 0.6 mL) to a mixing solution composed of HAuCl4·3H2O (0.01 M, 0.25 mL) and a CTAB solution (0.1 M, 9.75 mL). The seed solution was stirred for 15 min and aged at 30 °C for 1 h before use. The growth solution was prepared by the sequential addition of HAuCl4·3H2O (0.01 M, 12.5 mL), AgNO3 (0.01 M, 2.5 mL) and ascorbic acid (0.1 M, 1.375 mL) to a CTAB solution (0.1 M, 237.5 mL). Then, the seed solution (0.3 mL) was added under vigorous stirring. When the color of the solution changed to light pink, the stirring was stopped, and the solution was stored undisturbed at 30 °C overnight. The Au NRs were purified by centrifugation at 12480 rpm for 20 min and washed with water and ethanol three times. The Au NRs were suspended in water before use. Synthesis of core-shell Au NR@SiO2@Cy7 NPs. The synthesis of core-shell Au NR@SiO2@Cy7 NPs followed the same to the method for preparing core-shell Au NBP@SiO2@Cy7 NPs. Synthesis of SiO2@Cy7 NPs. The synthesis of SiO2@Cy7 NPs is similar way for preparing core-shell Au NBP@SiO2@Cy7 NPs. PEF studies of Au NBP@SiO2@Cy7 NPs. To study the PEF effects more accurately, the total concentration of Cy7 in Au NBP@SiO2@Cy7 NPs should be ensured to be the same as that in SiO2@Cy7 NPs. Firstly, the number of Cy7 molecules in Au NBP@SiO2@Cy7 NPs and SiO2@Cy7 NPs should be calculated using the absorption spectra.18 The ratio was estimated from NCy7/NNP=[ACy7·VCy7·εNP]/ [ANP·VNP·εCy7] (Supporting Information, section S2). Secondly, through dilution, the concentration of conjugated Cy7 in Au NBP@SiO2@Cy7 NPs is controlled to be the same as that in SiO2@Cy7 NPs.

let (UV) absorption was measured with a Nicolet 380 (Nicolet, America) spectrophotometer. Scanning electron microscopy (SEM) images were obtained on an S-4800 (Hitachi, Japan) scanning electron microscope. Transmission electron microscope (TEM) micrographs were obtained on a JEOL 2010 transmission electron microscopy. The zeta potential measurements were performed on a Nano-ZS Zetasizer ZEN3600 (Malvern Instruments Ltd., U.K.). Fluorescence cell imaging experiments were performed with an A1r laser scanning confocal microscope A1r (Nikon, Japan). The RCA experiments were performed on a GE 9612 PCR instrument (China). Synthesis of gold Au NBPs. The Au NBPs were prepared using a modified seed-mediated growth method. Specifically, the seed solution was made by mixing HAuCl4·3H2O (0.01 M, 0.125 mL), sodium citrate (0.01 M, 0.25 mL), and deionized water (9.625 mL). Then, freshly prepared, ice-cold NaBH4 solution (0.01 M, 0.15 mL) was added 10 minutes later, and the mixed solution was stirred vigorously for 15 min. The seed solution was aged at 30 °C for 2 h before use. The growth solution was prepared by adding HAuCl4 (0.01 M, 10 mL), AgNO3 (0.01 M, 2 mL), HCl (1 M, 4 mL), and ascorbic acid (0.1 M, 1.6 mL) sequentially into an aqueous CTAB solution (0.1 M, 200mL). Then the seed solution (6 mL) was added into the growth solution and the solution was gently mixed for 15 s. The reaction solution was kept undisturbed overnight at 30 °C. Purification of Au NBPs. The as-grown Au NBPs (approximately 220 mL) were centrifuged at 12480 rpm for 20 min. The precipitate was collected and re-dispersed into a CTAC solution (0.08 M, 150 mL). Then, AgNO3 (0.01 M, 50 mL) and ascorbic acid (0.1 M, 15 mL) were subsequently added to the CTAC solution. The mixed solution was kept at 65 °C for 6 h to allow Ag to overgrow on the Au NPs and Au NBPs. The resulted Ag/Au products were centrifuged at 12480 rpm for 20 min. The precipitate was re-dispersed into a CTAB solution (0.1 M, 150 mL) and left undisturbed overnight at 30 °C. The supernatant was removed carefully, and the remaining black Au/Ag NRs were redispersed in water (100 mL). Then, ammonia (25%, 1.65 mL) and hydrogen peroxide solutions (30%, 0.75 mL) were added to the solution. The resultant mixture was kept undisturbed for 3 h; during this time, the color changed from green to brown, and white AgCl precipitated at the bottom of the container. The supernatant was carefully removed and centrifuged at 1000 rpm for 10 min and washed with water three times. The precipitate was redispersed in water (60 mL) for further use. The LPRW of the asgrown Au NBPs was around 750 nm. To obtain purified Au NPs, appropriate amounts of ammonia and hydrogen peroxide solutions were added to the supernatant, and the solution was kept for 3 h. The resulting solution was centrifuged at 1000 rpm for 10 min and washed with water three times. The precipitate was collected and re-dispersed in water to obtain the Au NP solution. Synthesis of core-shell Au NBP@SiO2 NPs. Core-shell Au NBP@SiO2 NPs were prepared according to the reported preparation method for Au NR@SiO2 as reported with slight modifications. The as-prepared Au NBP solution (60 mL) was divided into six 10 mL containers, and then, a CTAB solution (0.1 M, 0.02 mL, 0.06 mL, 0.12 mL, 0.2 mL, 0.4 mL, or 0.6 mL) was added into each of the six containers. The solutions

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776 nm) of Cy7 (Figure S2). The chemical structure of Cy7 NHS ester is shown in Figure S3.

Cytotoxicity and cell imaging of Au NBP@SiO2@Cy7 NPs. The cytotoxicity of Au NBP@SiO2@Cy7 NPs on HeLa cells was evaluated using the MTT assay. HeLa cells were plated on a 96-well plate at 37°C and 5% CO2 for 24 h. Then, different doses of Au NBP@SiO2@Cy7 NPs were added and incubated with the cells for 24 h. The cell viabilities were determined by using an enzyme-labeling instrument to measure the absorbance at 490 nm and calculating the value according to a formula. Three independent experiments were carried out under the same conditions. For fluorescence imaging, HeLa cells were first cultured in DMEM medium with 10% FBS and 0.01% PSS in an incubator at 37 °C and 5% CO2. Then, the cells were dispersed in a confocal dish with standard culture medium and allowed to adhere for 24 h. Au NBP@SiO2@Cy7 NPs were then added, followed by incubation in culture medium at 37 °C and 5% CO2 for 12 h. After being washed with PBS (0.01 M, pH 7.4) three times and rinsed with methanol, the cells were ready for confocal fluorescence imaging. Rolling circle amplification. Different concentrations of let-7d as primers and 1 nM circular template were mixed in MOPS buffer. The tubes containing the mixtures were heated at 95 °C for 5 min and cooled down to room temperature for 4 h. During this time, the primer hybridized with the circular template completely. Then, 500 µM dNTPs and 100 U/mL phi29 polymerase were added to each tube. All of the mixtures were reacted at 30 °C for 13 h and terminated at 65 °C for 10 min in a GE 9612 PCR instrument. The products were stored at 4 °C until use. RESULTS AND DISCUSSION Synthesis and purification of Au NBPs. First, the synthesis and purification of Au NBPs were performed (Figure 1A). The as-grown Au NBPs were prepared using a seed-mediated growth method using CTAB as stabilizer. The LPRWs of the Au NBPs samples can be controlled in the near-infrared region by tuning the volumes of the seed solution.20 The as-grown Au NBPs were not pure, as there were nearly 50% Au NPs in the samples (Figure 1B). The samples were re-dispersed into CTAC solutions and incubated with AgNO3 at 65 °C, during which time the Ag accumulated on the Au NBPs and Au NPs to generate Au/Ag NRs and Au/Ag NPs, respectively. They were separated according to their different depletion interactions between surfactant micelles.40-42 The Au NBPs remained in the center of the precipitated Au/Ag NRs (Figure 1C), whereas the Au NPs exist in the Au/Ag NPs (Figure 1D). It is worth noting that the concentration of CTAB is key in the separation process. A high CATB concentration (0.2 M) will induce Au/Ag NPs to precipitate together with Au/Ag NRs, whereas a low concentration (0.05 M) will result in a small amount of precipitated Au/Ag NRs. Only an appropriate concentration (0.1 M) will result in a satisfactory separation (Figure S1). After the Ag corrosion step with H2O2 and NH3•H2O, the purified Au NBPs and Au NPs were generated (Figure 1E, 1F). One important factor to produce maximum PEF is the matching between the absorption/emission of the dye and extinction maxima of the metal nanostructures.6, 8, 43 In this study, we chose Au NBPs with a maximum plasma around 750 nm in aqueous solution to achieve the highest overlap of the LPRW of Au NBPs with the absorption/emission spectra (746 nm,

Figure 1. (A) Schematic illustration of the purification of Au NBPs; TEM images of (B) Au NPs and Au NBPs; (C) Au/Ag NRs; (D) Au/Ag NPs; (E) purified Au NPs; (F) purified Au NBPs and (G) Extinction spectra of the Au NPs and Au NBPs (blank line), purified Au NPs (blue line) and purified Au NBPs (red line).

Preparation and characterization of Au NBP@SiO2@Cy7 NPs. The preparation process for Au NBP@SiO2@Cy7 NPs is schematically illustrated in Scheme 1A. The distance between the metal particles and dye is another important criterion for producing PEF. The distance between Cy7 and Au NBPs is controlled by coating with silica and changing the silica thicknesses. Silica has good biocompatibility and low cytotoxicity, and its surface can be modified easily to bond to dyes.44 Silica coating also prevents the NPs from aggregating and contributes to their good dispersibility in solvents. 45 These excellent properties of silica have made it a popular option for coating NPs and producing PEF nanocomposites. Then, the silica-coated Au NBP (Au NBP@SiO2) NPs were amino functioned to allow the Cy7 NHS ester molecules to covalently bond to the surface of SiO2, thus completing the preparation of the Au NBP@SiO2@Cy7 NPs. The amount of Cy7 molecules covalently bond to per Au NBP@SiO2 is calculated to be about 3900 through the reported method (Supporting Information, section S2). 46, 47 Silica coating was performed with a modified Stöber method using TEOS.24 By varying the concentrations of CTAB, we successfully controlled the silica thickness to range from 9 nm to 25 nm (Figure 2). The concentration of CTAB is pivotal for controlling the silica thicknesses. As the concentration of CTAB increases, the silica thicknesses decreases (Figure 3A). As the silica thickness increases, their extinction peaks red shift a little, indicating that the Au NBP@SiO2 NPs are not aggregated (Figure 3B).

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Analytical Chemistry (Figure S5). The fluorescence intensities of Au NBP@SiO2@Cy7 NPs were stronger than those of SiO2@Cy7 NPs. They were influenced by the silica thickness, indicating that the fluorescence of the dye-metal structures is distance dependent. As the silica thickness increased from 9 nm to 17 nm, the FL enhanced factor changed from 1.8 fold to 10.7 fold, whereas when the silica thickness became increasingly thicker, the FL enhanced factor decreased to 1.4 fold. Thus, 17 nm was found to be the best distance between Au NBP and Cy7 to produce the strongest PEF (Figure 3D). Preparation and PEF studies of Au NR@SiO2@Cy7 NPs. It is reported that the sharper apexes of metal nanomaterials produce stronger electric fields.20 In order to confirm the advantages of Au NBPs, we also synthesized Au NR@SiO2@Cy7 NPs based on the Au NRs with an extinction maximum around 750 nm for comparison (Figure 4A). The matching between the absorbance/emission maximum of Cy7 and maximum plasma of Au NRs is responsible for the PEF effect (Figure 4C). The SiO2 thickness was controlled from 9 nm to 21 nm using the same method to coat Au NBPs (Figure S6). The fluorescence of Au NR@SiO2@Cy7 NPs was excited and recorded under the same conditions. Interestingly, the strongest PEF of Au NR@SiO2@Cy7 NPs was also recorded at around 17 nm (Figure 4B, S7). The enhanced fluorescence factor of Au NR@SiO2(17nm)@Cy7 NPs was 4.5 fold, which was smaller than the value of 10.7 fold for Au NBP@SiO2(17nm)@Cy7 NPs (Figure 4D). Additionally, the finite-difference time-domain (FDTD) simulations showed that the electric field of Au NBP was nearly 5 times stronger than that of Au NR at the same excitation wavelength of 746 nm, which theoretically contributes to a stronger fluorescence signal (Figure S8). The simulation results are were consistent the experimental results, indicating that Au NBPs are better PEF materials than Au NRs.

Figure 2. TEM images of silica-coated Au NBPs with silica thicknesses of (A) 9 nm, (B) 11 nm, (C) 14 nm, (D) 17 nm, (E) 21 nm, and (F) 25 nm.

Figure 3. (A) The silica shell distances versus CTAB concentrations; (B) The extinction spectra of Au NBP@SiO2 NPs with different SiO2 thicknesses; (C) The zeta potential of Au NBPs coated in CTAB, Au NBP@SiO2 NPs and amino-functioned Au NBP@SiO2 NPs; (D) Fluorescence enhanced factor of Au NBP@SiO2@Cy7 NPs with different SiO2 thicknesses.

The amino-modification of Au NBP@SiO2 NPs was performed with APTES. The zeta potential results show that the Au NBP@SiO2 NPs are electronegative and that the Au NBPs in CTAB and amino-functioned Au NBP@SiO2 NPs are positively charged, confirming the successful synthesis of aminofunctioned Au NBP@SiO2 NPs (Figure 3C). The Cy7 molecules are bond to Au NBP@SiO2-NH2 NPs through the formation of amide bonds produced by amino groups and NHS ester. The SiO2@Cy7 NPs were prepared for control using the same method. The prepared SiO2@Cy7 NPs were uniformed with an averaged diameter of 50 ± 5 nm (Figure S4). It is worth mentioning that linking Cy7 molecules to the surface of SiO2 NPs cannot significantly affect the fluorescence of Cy7.24 In addition, it also allow us to detect the fluorescence enhancement factors for Cy7 molecules in equivalent chemical environments. Therefore, choosing SiO2@Cy7 NPs as control samples is a suitable and convincing method to observe the PEF effects.18, 47, 48 PEF studies of Au NBP@SiO2@Cy7 NPs. The fluorescence intensities of Au NBP@SiO2@Cy7 NPs and SiO2@Cy7 NPs were recorded at an excitation wavelength of 746 nm

Figure 4. (A) TEM image of related Au NRs used in this study with LPRW at 750 nm; (B) TEM images of Au NR@SiO2 NPs with silica thickness of 17 nm; (C) Absorption spectra of Cy 7 (red line), emission spectra of Cy7 (blue line) and the extinction spectra of Au NRs (black line) with LPRW at 750 nm; (D) Fluorescence spectra of SiO2@Cy7 NPs, Au NR@SiO2(17 nm)@Cy7 NPs and Au NBP@SiO2(17 nm)@Cy7 NPs.

Fluorescence detection of Cu2+ and PPi using the Au NBP@SiO2@Cy7 NPs. Because the Au NBP@SiO2@Cy7

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97.9%, 95.1%, and 93.0% for the doses of 50, 150, 250, and 350 µL Au NBP@SiO2@Cy7 NPs, respectively, confirming the high biocompatibility of Au NBP@SiO2@Cy7 NPs with living cells (Figure S10). We demonstrated the feasibility of Au NBP@SiO2@Cy7 NPs for Cu2+ and PPi imaging using confocal fluorescence microscopy. The fluorescence intensity of HeLa cells incubated with Au NBP@SiO2@Cy7 NPs was quenched by Cu2+ and recovered with PPi (Figure 6). However, the restored fluorescence by PPi was weaker than the fluorescence of Au NBP@SiO2@Cy7 NPs, indicating that the affinity between PPi and Cu2+ is not strong enough to eliminate Cu2+ from Au NBP@SiO2@Cy7-Cu2+. The results show that Au NBP@SiO2@Cy7 NPs can be used to detect the Cu2+ and PPi in living cells.

NPs possess enhanced fluorescence in the aqueous phase, they potentially can be used as sensitive fluorescent biological sensors. Herein, we developed the Au NBP@SiO2@Cy7 NPs for the detection of PPi. The Au NBP@SiO2(17 nm)@Cy7 NPs exhibited the highest enhanced fluorescence, and the fluorescence can be quenched by copper ions. However, there is a stronger coordination affinity between PPi and copper ions, and the formation of Cu2+-PPi complexes will restore the fluorescence; thus, a quantitative assay for PPi can be developed based on these “switch-on” fluorescence signals (Scheme 1B).38 The detection process was carried out in HEPES buffer (5 mM, pH 7.4). As the Cu2+ concentration increased, the fluorescence of Au NBP@SiO2@Cy7 NPs was gradually quenched (Figure 5A). The linearity range of Cu2+ was 0.25 µM-3 µM, and the correlation coefficient (R) was 0.998 (Figure 5B). The limit of detection was found to be 76 nM. Based on the stronger affinity between Cu2+ and PPi, the quenched fluorescence can be gradually recovered with the addition of PPi (Figure 5C). The linearity range for PPi was from 2 µM to 14 µM with a correlation coefficient (R) of 0.978, and the limit of detection is calculated to be 80 nM using the formula of 3σb/s (Figure 5D). Then, we further determined the selectivity for PPi. We added some anions, including F-, Cl-, Br-, I-, SO42-, CO32-, NO3-, NO2-, H2PO4-, HPO42-, PO43-, and P2O74- to the Au NBP@SiO2@Cy7-Cu2+ solutions, and only PPi could markedly restore the fluorescence (Figure S9). Thus, these results indicate that Au NBP@SiO2@Cy7 NPs are an excellent NIR PEF biosensor for PPi.

Figure 6. (A) Confocal fluorescence microscopy images of Hela cells after incubation with Au NBP@SiO2@Cy7 NPs for 10 h. (B) Confocal fluorescence microscopy images of Hela cells after incubation with Au NBP@SiO2@Cy7 NPs for 10 h and Cu2+ for 2 h. (C) Confocal fluorescence microscopy images of Hela cells after incubation with Au NBP@SiO2@Cy7 NPs for 10 h, Cu2+ for 2 h and PPi for 2 h. The columns 1, 2, and 3 are fluorescence images, bright-field images, and merged images of Hela cells, respectively.

The application of Au NBP@SiO2@Cy7 NPs to detect microRNA with RCA. We then applied the Au NBP@SiO2@Cy7 NPs for the detection of microRNA using RCA technology. In this study, let-7d was chosen as the targeted object to be detected. The circular template was designed to include sequences complementary to let-7d and let7d was used as the primer for RCA directly.32 In the presence of polymerase and dNTPs, only let-7d could trigger RCA, during which PPi is released from dNTPs (Scheme 1C). Different doses of let-7d produced different concentrations of PPi, and the restored fluorescence can be recorded. Increasing the concentration of let-7d resulted in increased fluorescence (Figure 7). A good linearity range for let-7d was recorded from 10 pM to 100 pM with a correlation coefficient (R) of 0.99, and the limit of detection was calculated to be 8.4 pM. The results indicate that the Au NBP-based PEF-PPi sensing

Figure 5. (A) Fluorescence spectra of the nanoprobe upon exposure to different concentrations of Cu2+; (B) Plot of I/I0 of the nanoprobe upon Cu2+ versus the concentrations of Cu2+; Insert represents the linear region of I/I0 to Cu2+; (C) Fluorescence spectra of the complex of nanoprobe-Cu2+ upon exposure to different concentrations of PPi; (D) Plot of I/I0 of the nanoprobe-Cu2+ upon PPi versus the concentrations of PPi; Insert represents the linear region of I/I0 to PPi.

Cytotoxicity and cell imaging of Au NBP@SiO2@Cy7 NPs. Considering the potential biocompatibility of Au NBP@SiO2@Cy7 NPs, we used Hela cells to confirm the feasibility of Au NBP@SiO2@Cy7 NPs for Cu2+ and PPi imaging using confocal fluorescence microscopy. First, MTT assays were performed in HeLa cells to evaluate the cytotoxicity of Au NBP@SiO2@Cy7 NPs. The viability was 99.2%,

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platform is an efficient and sensitive method for microRNA detection. Author Contributions The manuscript was written through contributions of all authors./ All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Figure 7. (A) Fluorescence spectra of the nanoprobe-Cu2+ upon PPi produced in the RCA process with different concentrations of let-7d. (B) Plot of I/I0 of the nanoprobe-Cu2+ upon PPi produced with concentrations of let-7d. Insert represents the linear region of I/I0 to let-7d.

The authors gratefully acknowledge the support from the National Natural Science Foundation of China (21475011, 21675014, 21422503), the National Grant of Basic Research Program of China (no. 2011CB915504), and the Fundamental Research Funds for the Central Universities.

CONCLUSIONS In summary, a NIR distance-dependent PEF biosensor based on silica-coated Au NBPs for PPi was developed for the first time by bonding Cy7 to Au NBP@SiO2 in the aqueous phase. The maximum fluorescence enhanced factor for Au NR@SiO2@Cy7 NPs was 4.5 fold at a silica thickness of 17 nm, which was less than the value of 10.7 fold for Au NBP@SiO2@Cy7 NPs. Further, the FDTD simulation results showed that the electric field of Au NBPs was nearly five times stronger than that of the Au NRs used in this work at an excitation wavelength of 750 nm, which contributed to the stronger PEF effects. Both the experimental and theoretical results confirm the superiority of Au NBPs as PEF substrates over Au NRs. The Au NBPs@SiO2@Cy7 NPs were further applied to detect PPi through “turn-on” fluorescence signals in aqueous solutions with a limit of detection of 80 nM. Because of their good biocompatibility, the Au NBP@SiO2@Cy7 NPs could also detect Cu2+ and PPi in living cells. In addition, the Au NBP-based PEF-PPi sensing platform was also developed into a sensitive microRNA assay. The limit of detection was calculated to be 8.4 pM. To our knowledge, this work represents the first study of a NIR distance-dependent PEF sensor based on Au NBP@SiO2 in solution for the PPi assay. This work proposes a new PPi sensor based on Au NBPs, expands the bioapplications of PEF sensors, and provides a new avenue for preparing more-sensitive PEF biosensors.

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ASSOCIATED CONTENT Supporting Information Fluorescence spectra, SEM images, TEM images, the chemical structure of Cy7 NHS ester, the selectivity to PPi, and the MTT assay results are included in the Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * Jin Ouyang Fax: 86 010-62799838; Tel: 86 010-58805373; E-mail: [email protected]

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Near-Infrared-Fluorescent Probes for Bioapplications Based on Silica

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