Biomolecular Recognition and Enzyme Kinetics - ACS Publications

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Plasmon Resonance Energy Transfer Based Spectroscopy on Single Nanoparticles: Biomolecular Recognition and Enzyme Kinetics Shan-Shan Li, Qing-Ying Kong, Miao Zhang, Fan Yang, Bin Kang, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04467 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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Analytical Chemistry

Plasmon Resonance Energy Transfer Based Spectroscopy on Single Nanoparticles: Biomolecular Recognition and Enzyme Kinetics

Shan-Shan Li, Qing-Ying Kong, Miao Zhang, Fan Yang, Bin Kang,* Jing-Juan Xu,* and Hong-Yuan Chen*

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, 210023, China *Corresponding author. Tel/Fax: +86-25-89687294; E-mail address: [email protected] (B. Kang), [email protected] (J.J. Xu), [email protected] (H.Y. Chen)

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Analytical Chemistry 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

ABSTRACT The small absorption cross-sections of most molecules led to the low sensitivity of traditional optical absorption spectroscopy. This obstacle might be overcome by applying the near-field plasmon resonance energy transfer (PRET) between plasmonic nanoparticle and surrounding molecules. In this work, we utilized PRET based spectroscopy on single gold nanostars to study the specific biomolecule recognition and enzyme kinetics choosing biotin-SA pair and DNase I as models. By analyzing the changes of absorption spectra for black hole quencher 3 (BHQ3), derived from spectra difference, we explored the kinetics of specific biomolecule recognition and enzyme digestion in different physiological environment, and found that the viscosities of media and the sizes of molecules play vital role in biomolecular recognition and enzyme digestion. Compared with the traditional optical absorption spectroscopy techniques, PRET based spectroscopy offers a nanoscopic resolution owing to the small size of the probe, is more sensitive achieving detection on the order of hundreds or even dozens of molecules, and can achieve high selectivity due to the specific biomolecular recognition. This method might be used in the fields of molecular diagnostics, drug discovery, cell systems and clinical diagnostics.

KEYWORDS: plasmon resonance energy transfer based spectroscopy, single gold nanostars, biomolecule recognition, enzyme kinetics

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INTRODUCTION In past decades, fluorescence and absorption spectroscopy have become popular analytic techniques in a large range of applications in chemistry and biology.1-4 Especially in the past ten years, fluorescence techniques have been highly developed, and fluorescence emission from even a single dye molecule could be detected.5,6 Generated from the radiative relaxation of excited state electrons, fluorescence emission cross-section usually highly depends on external environments, like pH, temperature, light intensity, solution viscosity and dye concentration.7,8 These properties offer fluorescence probes many possibilities for sensing the change of local environments and have promoted many valuable applications.7,9 However, from another point of view, this nature also lead to large signal fluctuation (e.g. blinking and bleaching), and even resulted in critical nondeterminacy in microscale determination.8,10-12 Compared with fluorescence emission, molecular absorption, which originate from transition of ground state electrons, gives much more stable and definite signals since the absorption cross-section almost does not depend on the external environments or molecule concentration. In principle, absorption might provide many attractive strengths better than fluorescence for microscale and single molecule analysis. But in fact, the absorption cross-sections of most molecules are extremely small (~10-29 m2), thus it usually requires a large number of molecules (~109) to generate a detectable signal.13-15 Such intrinsic limitation makes it very difficult to improve sensitivity for traditional optical absorption spectroscopy. Fortunately, this obstacle could be overcome by applying the near-field energy 3



transfer between plasmonic nanoparticle and surrounding molecules. In 2007, plasmon resonance energy transfer (PRET) from single gold nanosphere to conjugated cytochrome c was discovered, which has been verified by follow-up reports.8,10,16-20 Only when the electronic resonance peak of chemical or biological molecules overlap with the plasmon resonance peak of the metallic nanoparticle, the resonance energy transfers from the nanoparticle to the adsorbed molecules generating “quantized quenching dips” in the scattering spectrum of the nanoparticle, which enhances the sensitivity of absorption spectroscopy by several orders of magnitude from hundreds of molecules on the surface of a single nanoparticle. The plasmonic effect could be utilized to realize the biosensing including DNA and other biomolecules by employing the single gold nanoparticles.21-23 The most using gold nanoparticles, like nanospheres or nanorods, usually exhibit a narrow plasmon resonance band at either visible or near-infrared region on a specific platform, which can only detect the molecules with absorption peaks at a narrow region.8,10,16,18,24 By designing the intrinsic parameters such as size and geometrical shape, the plasmon resonance band of nanoparticles, like gold nanostars, could be tuned to cover a wide range from visible to the near-infrared region (650 ~ 1000 nm) called “biological window” allowing for near-infrared absorption labeling,25-27 which makes it possible to study molecular behavior in near-infrared region. Herein, we utilized PRET based spectroscopy on single gold nanostars with a wide plasmon resonance band to analyze the specific biomolecule recognition and enzyme kinetics choosing biotin-streptavidin (SA) pair and DNase I as models. Black 4


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hole quencher 3 (BHQ3) was utilized as near-infrared absorption labeling, and its spectral features were obtained from the quench dip on the scattering spectra of gold nanostars caused by near-field energy transfer from plasmonic polariton to surrounding molecular dipole. By analyzing the absorption-like spectra of BHQ3, derived from spectra difference, we studied the kinetics of specific biomolecule recognition for biotin-SA and enzyme digestion for DNase I by simulating different physiological environment. It suggested that the viscosities of media and the sizes of molecules play very important role in biomolecular recognition and enzyme digestion. Due to the near-field resonance energy transfer between plasmonic nanoparticle and surrounding molecules, the absorption cross-sections of molecules could be enlarged. Compared with the traditional optical absorption spectroscopy techniques, PRET based spectroscopy offers a high sensitivity achieving detection on the order of hundreds or even dozens of molecules. This PRET based spectroscopy on single nanoparticles might be used in the fields of molecular diagnostics, drug discovery, clinical diagnostics, pharmacology and medicine. EXPERIMENTAL SECTION Materials and Reagents. Tetrachloroauric acid trihydrate (HAuCl4·3H2O) and polyvinylpyrrolidone (PVP, MW 55000) were purchased from Sigma-Aldrich Inc. (Shanghai, China). Diethylene glycol (DEG) and dimethylamine (DMA, 40 wt. %) were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). N, N-dimethylformamide (DMF), hydrochloric acid (HCl, 37%) and ethanol were purchased from Nanjing Reagent Co. Ltd. (Nanjing, China). All chemicals were used 5



without further purification. Bovine serum albumin (BSA), phosphate buffered solution (PBS, 10 mM pH 7.4), Dulbecco’s Modified Eagles Medium (DMEM, 10 mM pH 7.4), fetal bovine serum (FBS, Gibco), and Gel Green were purchased from KeyGen Biotechnology Co. Ltd. (Nanjing, China). DNase I endonuclease (RNase-free, EN0521) and 100 mM tris-HCl (pH 7.5) were purchased from Thermo Fisher Scientific Co. Ltd. (Shanghai, China). 6× Loading Buffer, and 20 bp DNA Ladder (Dye Plus) were purchased from Takara Biomedical Technology Co. Ltd. (Beijing, China). Precast-GLgel 15% Native-PAGE, streptavidin (SA) and the oligonucleotides were purchased from Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China). Detailed DNA sequences are: DNA-BHQ3: 5’-biotin-TCA ACA TCA GTC TGA TAA GCT A-BHQ3-3’; DNA-SH: 5’-biotin-TCA ACA TCA GTC TGA TAA GCT A-SH-3’. Ultrapure water was used throughout the experiments. All other reagents were of analytical grade and used without further purification. Characterization. The morphology of the gold nanostars was characterized using transmission electron microscopy (TEM, JEOL JEM-2100). The absorption spectra were collected with a UV-3600 UV/Vis/NIR spectrometer and a nanodrop spectrometer. Raman spectra were obtained using a Renishaw inVia-Reflex Raman spectrometer, equipped with a 633 nm excitation laser, and a microscope with a 50× objective lens. DNA gel electrophoresis were obtained by a Bio-Rad Gel Imager. Synthesis of Symmetric Gold Nanostars. Symmetric gold nanostars were 6


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synthesized by a seed-mediated growth method.28,29 For the preparation of icosahedral gold seeds, 0.1 g PVP was dissolved in 25 mL of DEG and heated until refluxing for 5 min. Then, 2 mL of DEG containing 20 mg HAuCl4·3H2O was quickly injected. Refluxing for 10 min, the reaction was stopped and cooled to room temperature. The gold seeds was centrifuged (6000 rpm, 30 min) and washed with DMF twice, and finally dispersed in 27 mL of DMF. In the syntheses of symmetric gold nanostars, 1.2 g PVP was dissolved in 15 mL of DMF. Then, 50 µL of 40% DMA and 80 µL of 2.5 M HCl solution were added with mild stirring. Later on, 1 mL of icosahedral gold seeds was added, followed by the addition of 20 µL of 0.5 M HAuCl4 solution. Ultimately, the reaction solution was gently stirred at 80 °C for 4 h. The product was purified by centrifugation (6000 rpm, 30 min) with ethanol twice and finally dispersed in 2 mL of PBS. Preparation of Plasmonic Probes. Streptavidin-coated symmetric gold nanostars (SA-stars) were prepared referring literature procedure with some modification.30,31 500 µL of 0.0167 mM SA was added to 500 µL of gold nanostars suspension (1 nM, pH 6.4, adjusted by 0.1 M HCl), followed by incubation at room temperature for 30 min. The conjugated SA-stars were centrifuged at 8000 rpm for 30 min and dispersed in 1 mL of PBS containing 2% BSA, and stored at 4 °C for further use. According to the standard curve of absorption spectra for SA and the absorption spectra of SA before and after modification, we can estimate the number of SA attached to a single star was about 1.15×103 (Figure S13). The prepared SA-stars solution was diluted using PBS to reach a certain concentration, and dropped onto the positively charged 7



glass microscope slide (22 × 40 × 0.1 mm, ShiTai Co., Jiangsu, China). After seeing the deposition of SA-stars under dark-field microscope, the glass slide was washed with water and then different medium (10 mM PBS, DMEM or Tris-HCl) with different concentrations of DNA-BHQ3 or DNase I was dropped in situ for followed experiments. Dark-Field Microscope Setup. A single-particle dark-field spectral microscope, borrowing from the literature,32 with a wavelength resolution of 2.43 nm per pixel (after calibration) was employed in the experiments. The setup was composed of an inverted microscope (Olympus IX73, Japan) equipped with a dark-field condenser (0.8 < NA < 0.92) and a 40× objective lens (NA 0.6). The sample slide was immobilized on a platform, and a light emitting plasma (LEP) provided white light source with broad spectrum to excite the gold nanostars to generate plasmon resonance scattering light. The scattering light was collected by a true-color charge-coupled devices (CCD, Olympus DP80, Japan) to generate the dark-field color images, and split by a transmission grating and recorded by an electron multiplying charge-coupled devices (EMCCD, photometrics, USA). In this work, the scattering spectra of the nanoparticles were extracted by Image J. The pseudo-first order rate constants were obtained by fitting the dynamic changes of absorption peaks with exponential equation. Finite-Difference Time-Domain (FDTD) Simulation. The optical property of gold nanostar was simulated by FDTD via the package of Lumerical FDTD Solutions 2015a. The dielectric constant of gold was from Johnson and Christy33 and the 8


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computational domain was bounded by perfectly matched layer (PML). The gold nanostar was excited with a quasi nonpolarized light, consisted of x-polarized and y-polarized incident plane wave propagating along the z-axis. The meshing size used in the simulating region was set as 1.5 nm.

RESULTS AND DISCUSSION Design of Plasmonic Probes. The underlying concept of our detection system for analyzing the specific biomolecule recognition and enzyme kinetics is illustrated schematically in Scheme 1. Gold nanostars with near-infrared broad scattering bands are conjugated with SA, which can recognize biotin, and the electronic resonance absorption peak of BHQ3 is matched well with the plasmon resonance scattering peak of the gold nanostar. During the specific biomolecule recognition for biotin-SA, the absorption spectra of BHQ3 derived from spectra difference exhibit an uptrend, and then show a decline in the enzyme digestion for DNase I period. The symmetric gold nanostars were prepared following a seed-mediated growth method.28,29 As shown in Figure S1A and S1B, the average diameter of icosahedral gold seeds is about 200 nm and the plasmon resonance peak is around 575 nm. TEM image of symmetric gold nanostars shows an average size about 408 nm and the localized electronic field is stronger at the tip (Figure 1A, 1B, S1C and S1D). Plasmon resonance scattering peak of a single gold nanostar is around 650 nm, which is matched well with the absorption peak of BHQ3 (Figure 1C and 1D), so the process of PRET can take place resulting in resonant quenching dip in plasmonic scattering spectrum of the nanoparticle. To 9



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analyze the specific biomolecule recognition and enzyme kinetics, the gold nanostars were first modified by SA denoted as SA-stars, which was confirmed by UV-vis and Raman spectra (Figure 1E and 1F). The SA-stars were dispersed in PBS containing 2% bovine serum albumin (BSA) to prevent nonspecific binding on stars and the aggregation of stars due to steric effect.30,34 BSA was widely used in the molecular biorecognition of SA and biotin, and it seems that there was little or nearly no effect of BSA on the recognition events.27,34,35 Dark-field images and scattering spectral images of individual gold plasmonic probes were acquired by using a dark-field spectral microscope equipped with a true-color imaging charge-coupled device (CCD) and an electron multiplying charge-coupled devices (EMCCD) (Figure S2). Kinetics of Specific Biomolecular Recognition. Biomolecular recognition schemes rely on specific biomolecular recognition reactions, e.g. biotin-SA, which has been widely used in a variety of biological and analytical systems, such as cell imaging, clinical

diagnostics

and

pharmacology.27,30,34,36-39

Streptavidin

(SA)

is

a

non-glycosylated neutral protein secreted by Streptomyces avidinii displaying strong binding property to biotin, similar to avidin.37,38,40 Biotin, coupled to various reporters or probes such as fluorescent dyes, enzymes or DNA, can recognize SA coupled to various low or high molecular weight substances.30,34,37-39 Thus, we chose biotin-SA as the receptor-ligand pair utilizing PRET based spectroscopy to analyze the kinetics of the specific biomolecule recognition. Figure 2A shows the schematic illustration of specific biomolecule recognition for biotin-SA. Firstly, SA-stars were immobilized on glass slide and covered by 100 µL 10 mM DMEM with 8 µM biotin. Then, we 10


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collected real-time scattering spectra of an individual SA-star at different time intervals during the molecular recognition of biotin-SA (Figure 2B). The scattering peak at ~820 nm derived from the light emitting plasma (LEP) self-stimulated line was used as interior label to normalize the data. The corresponding absorption spectra of BHQ3 on a single SA-star were obtained by spectra difference, i.e. scattering spectrum of an individual SA-star at 0 min minus those at different time intervals (Figure 2C). Scattering spectra of SA-stars and corresponding absorption spectra of BHQ3 at different time intervals in DMEM containing different concentrations (2, 4, 6 and 10 µM) of biotin were shown in Figure S3. There are no noticeable spectral shift in absorption spectra of BHQ3 and the absorption peaks at 670 nm over time for different concentrations of biotin in DMEM, fitted by exponential equation, exhibit various uptrend to final balances (Figure 2D). The pseudo-first order reaction rate constants (k’) were obtained, which are proportional to the second-order reaction rate constant (k), and then we could get the second-order reaction rate constant in DMEM (Figure 2E, (7.31 ± 0.99) × 10-4 min-1 µM-1). To simulate different physiological environment, we collected real-time scattering spectra of SA-stars at different time intervals in 100 µL 10 mM PBS containing different concentrations (2, 4, 6, 8 and 10 µM) of biotin during the molecular recognition of biotin-SA and the corresponding absorption spectra of BHQ3 were also presented (Figure S4). The absorption peaks of BHQ3 at 670 nm over time for different concentrations of biotin in PBS were fitted by exponential equation (Figure 2F). According to the obtained pseudo-first order reaction rate constants, we could get the second-order reaction rate constant in PBS 11



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(Figure 2G, (1.21 ± 0.17) × 10-3 min-1 µM-1). The second-order reaction rate constant in PBS is two times higher than that in DMEM, which might be attributed to the discrepancy of viscosity, because DMEM not only includes some inorganic salts, but also contains many amino acids. To ensure that the observed uptrend of absorption peaks is not caused by medium itself (PBS or DMEM), we performed supplementary control experiments and there are no noticeable variation trend in absorption peaks at 670 nm over time (Figure S5). We collected the scattering spectra of single stars without SA modified in PBS containing 8 µM biotin for 180 min, and found that there was no changing trend in absorption peaks at 670 nm over time (Figure S6), which indirectly confirms that SA still retain the same epitope presentation for recognition of biotin after adsorption on the surface of the nanoparticles. Furthermore, to demonstrate if the presence of BSA affects the recognition events, we collected scattering spectra of single SA-stars modified by 10% or 20% BSA (Figure S7). The pseudo-first order reaction rate constants (0.01183 ± 0.00191 min-1 and 0.01185 ± 0.00335 min-1) for 10% and 20% BSA modified SA-stars are nearly the same as that of 2% BSA modified SA-star (0.01196 ± 0.00186 min-1), indicating that there was little or nearly no effect of BSA on the recognition events. Enzyme Kinetics. Understanding of enzyme kinetics is important for pharmacology, medicine,

clinical

diagnostics,

drug

discovery

and

industrial

bioprocess

technology.41-43 DNase I is an endonuclease, which can digest single-strand DNA and double-strand DNA leading to the hydrolytic cleavage of phosphodiester bond. Herein, we selected DNase I to digest single-strand DNA as a model utilizing PRET based 12


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spectroscopy to analyze the enzyme kinetics. 1 µg DNA fully digested by 1 U DNase I only needs 10 min at 37 °C. Our experiments were performed at 25 °C, so we firstly investigated the time required for the enzyme digestion by gel electrophoresis. As exhibited in Figure S8, there is no stripe in lane 2 (DNA-BHQ3) which is different from lane 2’ (DNA-SH), because the emission spectrum of Gel Green exactly locates in the absorption spectrum of BHQ3 resulting in quenching of Gel Green. The lanes of DNA-BHQ3 digested by DNase I (lane 3, 4 and 5) and DNA-SH digested by DNase I (lane 3’ 4’ and 5’) for different times are different from those of DNA-BHQ3 and DNA-SH (lane 2 and 2’) demonstrating that DNase I took effect. Considering that the lanes of DNA-BHQ3 digested by DNase I (lane 3, 4 and 5) for different times are abreast meaning that the digestion tends to balance at 60 min, so we collected real-time scattering spectra for 60 min in future experiments. Figure 3A exhibits the schematic illustration of enzyme digestion for DNase I. To eliminate the interference from medium itself, we conducted control experiment in 10 mM tris-HCl without DNase I. First of all, SA-stars were immobilized on glass slide to collect the scattering spectrum of an individual SA-star in PBS (Figure S9A), and then excess DNA-BHQ3 was added to PBS in situ. After 180 min, the SA-stars were fully transformed into SB-stars via biomolecule recognition of biotin-SA. Subsequently, we changed the PBS to 100 µL 10 mM tris-HCl without DNase I to collect real-time scattering spectra of an individual SB-star at different time intervals until 60 min (Figure S9B). The absorption spectra of BHQ3 on a single SB-star were obtained by spectra difference, i.e. scattering spectrum of the individual SA-star 13



minus those of the corresponding individual SB-star at different time intervals, and there is no changing trend in absorption peaks of BHQ3 at 670 nm over time (Figure S9C and S9D). To simulate different physiological environment, we changed the viscosity of medium by the addition of 25% fetal bovine serum (FBS) to tris-HCl and performed similar experiment to exclude the interference from medium itself (Figure S9E-S9H). Then, we added different concentrations (0.8, 0.2, 0.4, 0.6 and 0.9 U µL-1) of DNase I to tris-HCl and collected real-time scattering spectra of SB-stars at different time intervals during enzyme digestion for DNase I, and gave the corresponding absorption spectra of BHQ3 at different time intervals (Figure 3B, 3C and S10). There are no noticeable spectral shift in absorption spectra of BHQ3 and the absorption peaks at 670 nm over time for different concentrations of DNase I in tris-HCl, fitted by exponential equation, exhibit various decline to final balances (Figure 3D). The pseudo-first order reaction rate constants (k’) were obtained and then we could get the second-order reaction rate constant (k) in tris-HCl (Figure 3E, (5.53 ± 0.15) × 10-2 min-1 U-1 µL). Similarly, we added different concentrations (0.2, 0.4, 0.6, 0.8 and 0.9 U µL-1) of DNase I to tris-HCl + 25% FBS and collected real-time scattering spectra of SB-stars at different time intervals during enzyme digestion for DNase I, and gave the corresponding absorption spectra of BHQ3 at different time intervals (Figure S11). The absorption peaks of BHQ3 at 670 nm over time for different concentrations of DNase I in tris-HCl + 25% FBS were fitted by exponential equation (Figure 3F). Based on the obtained pseudo-first order reaction rate constants, we could get the second-order reaction rate constant in tris-HCl + 25% FBS (Figure 14


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3G, (4.17 ± 0.16) × 10-2 min-1 U-1 µL), which is smaller than that in tris-HCl. The repeatability of the measurements were verified by Figure S12, and the three SB-stars show an averaged pseudo-first order reaction rate constants (k’ = 0.0394 min-1) with a standard deviation of 0.0006 min-1 in tris-HCl + 25% FBS containing 0.6 U µL-1 DNase I. The molecular weight of DNase I is much bigger than that of DNA-BHQ3, thus it may mean that the size of DNase I is larger than that of DNA-BHQ3 and the steric hindrance effect is more distinct for DNase I. According to the above study, we can say that the viscosities of media and the sizes of molecules play vital role in biomolecular recognition and enzyme digestion. In our measurement system, our research object is a single gold nanoplasmonic probe, and volume and molecular number where the signal originate from are much lesser than those of traditional optical absorption spectroscopy techniques. The detailed comparison of different absorption spectroscopy techniques were presented in Table 1. On account of these, PRET based spectroscopy offers a nanoscopic resolution owing to the small size of the probe, is much more sensitive than traditional absorption spectroscopy techniques achieving detection on the order of hundreds or even dozens of molecules, and can achieve high selectivity due to the specific biomolecular recognition and enzyme digestion leading to the quenching dip of the gold nanoplasmonic probe deepened and shoaled. CONCLUSIONS In summary, we utilized the near-field energy transfer between plasmonic nanoparticle and surrounding molecules to improve the sensitivity of absorption 15



spectroscopy denoted as near-infrared absorption labeling. The specific biomolecule recognition and enzyme kinetics were analyzed by utilizing plasmon resonance energy transfer (PRET) based spectroscopy on single gold nanostars. By analyzing the absorption spectra of BHQ3, derived from spectra difference, we studied the influence of physiological environments on the specific biomolecule recognition for biotin-streptavidin (SA) and enzyme digestion for DNase I and found that the viscosities of media and the sizes of molecules play very important role in biomolecular recognition and enzyme digestion. Compared with the traditional optical absorption spectroscopy techniques, PRET based spectroscopy offers a high sensitivity achieving detection on the order of hundreds or even dozens of molecules. By using nanoprobes with plasmon resonance wavelength range from the visible to the near infrared region, PRET based spectroscopy could be applied to study molecular behavior in confined spaces of living systems. We believe that PRET based spectroscopy will have huge impacts on the fields of molecular diagnostics, drug discovery, clinical diagnostics, pharmacology, medicine and industrial bioprocess technology. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: TEM image and extinction spectrum of icosahedral gold seeds; setup diagram of the high-throughput single-particle dark-field spectral microscope; Scattering spectra of 16


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single SA-stars and corresponding absorption spectra of BHQ3 on single SA-stars in DMEM or PBS containing different concentrations of biotin for 180 min; Scattering spectra of a single star without SA modified; Scattering spectra of single SA-stars modified by 10% or 20% BSA; DNA gel electrophoresis; Scattering spectra of single SB-stars and corresponding absorption spectra of BHQ3 on single SB-stars in tris-HCl or tris-HCl + 25% FBS containing different concentrations of DNase I for 60min; The standard curve of absorption spectra for SA. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected]; [email protected] Tel/Fax: +86-25-89687294 ORCID Jing-Juan Xu: 0000-0001-9579-9318 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21327902, 21535003, 21675081), State Key Laboratory of Analytical Chemistry for Life Science (5431ZZXM1715), and Priority Academic Program Development of Jiangsu Higher Education Institutions. REFERENCES (1) Zhou, Y.; Zhang, J. F.; Yoon, J. Fluorescence and Colorimetric Chemosensors for 17



Fluoride-Ion Detection. Chem. Rev. 2014, 114, 5511-5571. (2) Jameson, D. M.; Ross, J. A. Fluorescence Polarization/Anisotropy in Diagnostics and Imaging. Chem. Rev. 2010, 110, 2685-2708. (3) Bordiga, S.; Groppo, E.; Agostini, G.; van Bokhoven, J. A.; Lamberti, C. Reactivity of Surface Species in Heterogeneous Catalysts Probed by in Situ X-ray Absorption Techniques. Chem. Rev. 2013, 113, 1736-1850. (4) Zaera, F. New Advances in the Use of Infrared Absorption Spectroscopy for the Characterization of Heterogeneous Catalytic Reactions. Chem. Soc. Rev. 2014, 43, 7624-7663. (5) Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Van Orden, A.; Werner, J. H.; Keller, R. A. Single Molecule Fluorescence Spectroscopy at Ambient Temperature. Chem. Rev. 1999, 99, 2929-2956. (6) Michalet, X.; Weiss, S.; Jäger, M. Single-Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics. Chem. Rev. 2006, 106, 1785-1813. (7) Han, J.; Burgess, K. Fluorescent Indicators for Intracellular pH. Chem. Rev. 2010, 110, 2709-2728. (8) Choi, Y.; Park, Y.; Kang, T.; Lee, L. P. Selective and Sensitive Detection of Metal Ions by Plasmonic Resonance Energy Transfer-Based Nanospectroscopy. Nat. Nanotechnol. 2009, 4, 742-746. (9) Kuimova, M. K.; Botchway, S. W.; Parker, A. W.; Balaz, M.; Collins, H. A.; Anderson, H. L.; Suhling, K.; Ogilby, P. R. Imaging Intracellular Viscosity of a Single Cell during Photoinduced Cell Death. Nat. Chem. 2009, 1, 69-73. 18


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Scheme 1. Schematic illustration of analyzing the specific biomolecule recognition and enzyme kinetics utilizing plasmon resonance energy transfer (PRET) based spectroscopy on single gold nonstars.

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Figure 1. Design of plasmonic probes. (A) Transmission electron microscopic (TEM) image of symmetric gold nanostars. (B) Finite-Difference Time-Domain (FDTD) simulated distribution of localized electronic field of gold nanostar. (C) Theoretical extinction spectrum, theoretical scattering spectrum, and experimental extinction spectrum of gold nanostars. (D) UV-vis spectrum of DNA-BHQ3. UV-vis spectra (E) and Raman spectra (F) of star, streptavidin (SA) and SA-star.

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Figure 2. Kinetics of specific biomolecule recognition. (A) Schematic illustration of specific biomolecule recognition for biotin-SA. (B) Scattering spectra of a single SA-star in DMEM containing 8 µM biotin for 180 min. Red star indicates the self-stimulated line from the light source used to normalize the data. (C) Corresponding absorption spectra of BHQ3 on a single SA-star in DMEM containing 26


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8 µM biotin. (D) The changes of absorption peaks for BHQ3 (at 670 nm) over time in DMEM containing different concentrations of biotin. (E) Pseudo first-order rate constant against concentration of biotin in DMEM. (F) The changes of absorption peaks for BHQ3 (at 670 nm) over time in PBS containing different concentrations of biotin. (G) Pseudo first-order rate constant against concentration of biotin in PBS.

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Figure 3. Enzyme kinetics. (A) Schematic illustration of enzyme digestion for DNase I. (B) Scattering spectra of a single SB-star in tris-HCl containing 0.8 U µL-1 DNase I for 60 min. Red star indicates the self-stimulated line from the light source used to normalize the data. (C) Corresponding absorption spectra of BHQ3 on a single SB-star in tris-HCl containing 0.8 U µL-1 DNase I. (D) The changes of absorption 28


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peaks for BHQ3 (at 670 nm) over time in tris-HCl containing different concentrations of DNase I. (E) Pseudo first-order rate constant against concentration of DNase I in tris-HCl. (F) The changes of absorption peaks for BHQ3 (at 670 nm) over time in tris-HCl + 25% FBS containing different concentrations of DNase I. (G) Pseudo first-order rate constant against concentration of DNase I in tris-HCl + 25% FBS.

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Table 1. Comparison of different absorption spectroscopy techniques.

Spectroscopy

UV-vis spectroscopy

Microscopic spectroscopy

Single particle spectroscopy

Signal volume (L)

10-6 ~ 10-3

10-9 ~ 10-6

~10-18

Signal molecular number (take 22 bases DNA as example)

1010 ~ 1012

108 ~ 1010

10 ~ 102

Minimum signal particle number (take 60 nm Au nanoparticle as example)

106 ~ 108

104 ~ 106

1

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TOC

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Biomolecular Recognition and Enzyme Kinetics - ACS Publications

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