Plasmon-Amplified Endogenous Fluorescence Nanospectroscopic

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Plasmon-Amplified Endogenous Fluorescence Nanospectroscopic Sensor Based on Inherent Elastic Scattering for Ultra-Trace Ratiometric Detection of Capsaicinoids Suresh Kumar Chakkarapani, Seungah Lee, Boyeon Park, Hye-Young Seo, and Seong Ho Kang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00058 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 10, 2019

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Plasmon-Amplified Endogenous Fluorescence Nanospectroscopic Sensor Based on Inherent Elastic Scattering for Ultra-Trace Ratiometric Detection of Capsaicinoids Suresh Kumar Chakkarapani,† Seungah Lee,† Boyeon Park,‡ Hye-Young Seo,‡,* and Seong Ho Kang†,* †

Department of Applied Chemistry and Institute of Natural Sciences, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea ‡ World Institute of Kimchi, Kimchiro 86, Namgu, Gwangju 61755, Republic of Korea

Keywords: endogenous fluorescence; plasmon-fluorescence enhancement; capsaicinoids; ratiometric quantification; singlemolecule detection ABSTRACT: Endogenous fluorescence imaging techniques are key for modern single-molecule quantification without the use of additional labeling probes. However, the drawback of weak fluorescence signal is the primary challenge in meeting the ever-increasing demands of single-molecule detection. Here, we report a simple and reliable method that provides up to 100-fold uniform fluorescence enhancement of endogenous fluorescence of the capsaicinoid molecule. The method is based on a single nanoparticle plasmon-amplified endogenous fluorescence nanospectroscopic sensor (PAEFS). This work demonstrated the applicability of PAEFS in refining sensitivity at the single-molecule level by showing ultra-low limits of detection (106 times lower than previous reports) of fluorescence-based capsaicinoids with a wide range of linear response (18 zM to 85 pM). Spectrally overlapped capsaicinoid analogues were quantified ratiometrically to detect the analogue percentages in real samples. The novel endogenous fluorescence enhancement approach presented here represents a universal sensor for enhanced detection of single molecules using existing techniques without altering the original molecular features or using add-on labeling probes.

Fluorescence probe-coupled, single-molecule imaging approaches have been universally employed for high precision detection and quantification.1,2 Endogenous (native) fluorescencebased techniques have reversed the complexity of single-molecule imaging by reducing the use of additional labeled probes.3 However, the weak signal of endogenous fluorescence has been a formidable problem in quantitative single-molecule fluorescence imaging techniques. Considering the label-free advantage of endogenous fluorescence imaging, there remains an urgent need for ultra-sensitive signal enhancement without much complexity.4 Overcoming this fundamental challenge without significant modification from the basic detection technique is important for singlemolecule detection to be broadly adopted via detection of endogenously fluorescent molecules.5,6 Capsaicin (CAP) and dihydrocapsaicin (DICAP) are endogenous fluorescence materials that are also the major capsaicinoid analogs, accounting for 90% of the pungency in pepper.7 Pungency level differs with capsaicinoid analog level and genotype.8,9 Lower concentrations of capsaicinoids have been broadly used as analgesic creams and higher concentrations for neuropathic pain, postoperative pain, and cluster headache treatment.10–12 Capsaicinoids are also known for anti-platelet and anti-coagulant activity depending on concentration at the target organ site.13 However, the pharmacological effect of capsaicinoids is largely

limited by the irritation caused by their concentration-dependent pungency. This has driven the search for an analytical tool for quantification of capsaicinoid analogs at low concentrations without inhibiting their molecular properties.14,15 The weak endogenous fluorescence intensity of capsaicinoids makes them difficult to detect at low concentrations. In the recent past, the intensity of the labelling fluorophore has been enhanced by photonic crystal, multi-fluorophore labeling, and rolling cycle amplification.16–18 However, plasmonics have proven to be effective and reliable fluorescence enhancers.19,20 Near-field plasmon-fluorophore enhancement is attributed to (i) plasmon fluorophore energy transfer, (ii) radioactive deactivation rate of the fluorophore, and (iii) scattering of the plasmon to affect the coupling efficiency of the fluorescence emission.21–25 To date, various fluorophore immunoassay plasmonic substrates have been used for fluorescence enhancement.24,26,27 These techniques have been limited to practical applications such as (i) increasing the signal to noise ratio of multiple labeled fluorophores that were subsequently applied in extracted samples. This leads to multiple labeling of the detection molecules, causing ambiguity in the specificity and selectivity of detection. (ii) In addition, complex conjugation with the detecting molecule and metal surfaces causes complications by altering the native molecular structure, and assay procedures are technically challenging in widespread applications.28 Plasmon scatteringA

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amplified endogenous fluorescence techniques can address these challenges and expand endogenous fluorescence techniques to a broader application base. Herein, we demonstrate a simple, reliable, and highly sensitive endogenous fluorescence enhancement technique based on single nanoparticles that can be used for detecting a wide range of endogenous fluorescence materials. This is the first demonstration of single-molecule capsaicinoid detection by their own endogenous fluorescence enhancement with plasmon single nanoparticles. In contrast with the reported fluorescence enhancement technique that requires significant modification of the molecular structure and multiple labeling, the current approach enhances endogenous fluorescence of the detecting molecule.

The 100 nm iron oxide nanoparticle was prepared by dissolving ferric nitrate and urea at a 1:3 ratio in isopropanol. Twenty-five milliliters of 25% ammonia was added to the solution under stirring at 700 rpm for 2 min. Immediately after, 15 mL of mixture was put into a Teflon-lined stainless autoclave, and the autoclave was heated to 180 °C and maintained for 12 h. Nanoparticle size and shape were determined from high-resolution scanning electron microscopy (SEM, Quanta FEG 650; FEI Co., Hillsboro, OR, USA). Capsaicinoid Conjugation on GNP. The surface of GNP was functionalized with 10 mM MUA and 30 mM MCH in ethanol. The solution was then sonicated for 20 min and incubated for 2.5 h to form a self-assembled monolayer of MUA-MCH. The 100 nm GNP-MUA-MCH monolayer was suspended in 50 mM MES and 0.1 M NaCl (pH 6.0) to generate a carboxylic acid-terminated alkanethiol monolayer on the surface. Next, 40 µg of EDC (2 mg/mL in 50 mM MES, pH 6.0) and 196 μg of NHSS (2 mg/mL in 1× PBS) were added to form GNP-MUA-MCH-NHSS. The EDC was added to optimize the distance between fluorophore and GNP. After stirring at room temperature for 40 min, the GNPMUA-MCH-NHSS was suspended in 1× PBS, and anti-HVA in PBS (pH 7.4) was added to a final concentration of 10 μg/mL. The GNP-anti-HVA was then centrifuged and washed with deionized water. The resulting GNP-anti-HVA (100 L of 2  105 particles/mL) complex nanotag was reacted with capsaicinoid standards, and 4 L of CAP-GNP was loaded placed on a cover slip for analysis at each concentration. The procedure was the same as for real sample analysis except that, instead of standards, the extracted capsaicinoid samples were reacted at the final step. Capsaicinoids and GNP Characterization. The samples were characterized by UV-Vis spectrometer (V-670 spectrophotometer, JASCO Co., Easton, MD, USA), transmission electron microscope (TEM) (2100F, JEOL Ltd, Tokyo, Japan) at a voltage of 200 keV using a Cu-grid (carbon coated, 200-mesh, Ted Pella, Inc., Redding, CA, USA), and SEM with an accelerating voltage of 30 kV. The surface topology of the gold-nanopatterned chip was obtained using atomic force microscopy (AFM) (NanoScope microscope, Digital Instruments, Santa Barbara, CA, USA) equipped with a type J scanner (scan size, 100-μm) and operating in tapping mode for air imaging. The resonance frequency of the silicon tips (Olympus Co. Ltd., Tokyo, Japan) was 300 kHz. These 150–160 µm short cantilevers had a nominal force constant (k) of 42 N/m and a tip with a radius of curvature < 10 nm. The AFM images were obtained by means of NanoScope software (Version 8.10). Lab-Made TIR-Based Detection System. The scattering and fluorescence signals from the plasmon-amplified endogenous fluorescence nanospectroscopic sensor (PAEFS) were collected using a lab-made, wavelength-dependent TIR system (Figure S1). The system setup was a modification of a previously reported setup.29 Briefly, the setup of the detection system was based on an upright Olympus BX53 microscope (Olympus). Two 671-nm, solid-state, continuous wave lasers (50 mW, SDL-671-040T, Shanghai Dream Lasers Technology Co., Ltd., Shanghai, China) for detection of gold nanoparticles (GNPs) and a 532-nm, solidstate, continuous wave laser (70 mW, SDL-532-LM-100T Shanghai Dream Lasers Technology Co., Ltd.) were the light sources for the fluorescence detection of capsaicinoids. A dichroic mirror (DC mirror, SWP-45UNP-R488/532-T633-PW-1025-C, Edmund Optics Inc., Barrington, NJ, USA) was placed after the laser sources. Two reflection mirrors (MM2-311-12.5, Semrock, Rochester, NY, USA) were placed for controlling the laser direction. Scattering and fluorescence signals were collected with three

MATERIALS AND METHODS Reagents. 6-Mercapto-1-hexanol (MCH, 97%), 2(morphozlino)ethanesulfonic acid (MES), dimethyl sulfoxide (DMSO, 99.5%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 11-mercaptoundecanoic acid (MUA, 95%), glycine, phosphate-buffered saline (PBS), capsaicin (CAP), and dihydrocapsaicin (DICAP) were purchased from SigmaAldrich (St. Louis, MO, USA). Dithiobis(succinimidyl propionate) (DSP) was obtained from Pierce (Rockford, IL, USA). Tris-HCl was purchased from J.T. Baker Company (Phillipsburg, NJ, USA), and 10 - 250 nm gold nanoparticles (GNPs) were obtained from BBITM Solutions (Crumlin, UK). Anti-homovanillic acid polyclonal antibodies (Anti-HVA) was purchased from MyBioSource (San Diego, CA, USA). N-Hydroxysulfosuccinimide (NHSS) was acquired from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Sample Preparation. Capsaicinoids were prepared from kimchi, baechu (Chinese cabbage), and red pepper powder. Kimchi was prepared according to the method used by World Institute of Kimchi (Table S1). The baechu (Chinese cabbage) was purchased from the Gwangju agricultural products market, and the red pepper powder was purchased from Dasan-food Co., Ltd (Yeongyang-gun, Gyeongbuk, Korea). For capsaicinoid extraction, glass beads (size 4) were added to each of the 2.5 g samples of separately homogenized kimchi, red pepper powder, and baechu, each in 15 mL of methanol. Samples with their glass beads were added to 22 mL capped clear vials equipped with polytetrafluoroethylene (PTFE) liners (Supelco, St. Louis, MO, USA). The vials were heated at 90 °C for 1 h. The extracted samples were cooled at room temperature, transferred to 25 mL volumetric flasks, and then filled to the mark with methanol. The supernatant of each sample was filtered through a disposable syringe with a 0.2 μm membrane filter (Milipore, Billerica, MA, USA). The filtered supernatants were used as analysis samples to quantify capsaicinoids. A gold-nanopatterned chip was fabricated as per the previously reported procedure.2 A glass wafer (WinWin Tech., Hwaseong, Korea) was the base of the spot and was washed with piranha solution (7:3 = H2SO4:H2O2, v/v), after which polymethylmethacrylate (PMMA) was spin-coated on the surface of the glass wafer to a 150-nm-thick layer. After exposure to electron beam lithography (Elionix E-beam system, 100 keV/100 pA), Au/Cr (20/5 nm thickness) was deposited and lifted-off (Korea Advanced Nano Fab Center, Suwon, Korea) by dichloromethane. Finally, the GNC was constructed into a 4  4 Au nanoarray (100 nm, 250 nm, and 500 nm in diameter of each spot) on the 10 mm2 glass wafer. B

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band-pass filters, 561/14 nm, 615/20 nm, and 680/10 nm (Semrock), and a numerical aperture 0.6-1.3 objective lens (UPlanFLN, 100, Olympus). HPLC-FLD Analysis. For HPLC analysis, a Lachrom Ultra C18 (50 mm  2.0 mm, 2 μm; Hitachi, Tokyo, Japan) column and an Agilent 1260 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a fluorescence detector (FLD) were used. The chromatographic conditions were as follows: mobile phase A, 0.1% acetic acid; mobile phase B, acetonitrile; isocratic condition A:B = 6:4; flow rate, 0.6 mL/min; excitation wavelength, 280 nm, emission wavelength, 325 nm; injection volume, 2 μL.

enhance fluorescence intensity through plasmon scattering, it is important to choose the optimal size of nanoparticles, as extinction depends on size and shape. GNPs with sizes ranging from 10 nm to 250 nm were used for optimization. The angular scattering intensities of the nanoparticle at the peak emission wavelength would vary from 500 nm to 800 nm by Mie scattering theory (Figure S3).33 However, considering the absorbance of capsaicinoids, Mie scattering in the 532 nm region is most important for selection of appropriately sized nanoparticles. The Mie scattering simulation of each size of nanoparticle showed maximum absorption for 100 nm GNP, and conversely, that the elastic scattering frequency was higher for 100 nm GNP than for the other sizes (Figure S3). The GNPs were functionalized with carboxyl groups and conjugated with capsaicin to detect the fluorescent enhancement at each nanoparticle size. Conjugation of capsaicin on each nanoparticle was confirmed with a red shift in UVVis absorbance after conjugation (Figure S4B).

RESULTS AND DISCUSSION Plasmon-Amplified Endogenous Fluorescence. The elastic scattering frequency of the plasmon nanoparticle plays the key role in enhancement of fluorescence at close proximity as it is the electromagnetic enhancement source for the fluorophores. The quantum yield (Qy) of a fluorophore was determined by the relative rates of the radiative (kr) and non-radiative (knr) pathways that deactivate the fluorescence.30

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When the fluorophore was conjugated at the vicinity of the plasmon nanoparticle, Qy was amplified by the increased excitation caused by radiative scattering of the plasmon nanoparticle. 31 Therefore, the absorption wavelength of the endogenous fluorescence should match the elastic scattering frequency of the plasmon nanoparticle (Figure 1). The UV-Vis spectrum of CAP shows absorbance at 280 nm and 532 nm (Figure S2). (A)

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Here, 100 nm GNP showed greater enhancement than the rest of the GNPs (Figure 2). The intensity of 250 nm GNP predominated and cannot be used to detect the small differences in fluorescence intensity at lower concentrations. Each CAP-conjugated gold nanoparticle (CAP-GNP) was detected on cover glass by a 532 nm wavelength laser. There was no scattering observed with 10 nm, 20 nm, 40 nm, 60 nm, or 80 nm GNPs. However, after conjugation with CAP, fluorescent intensity was observed with each nanoparticle size, and there was a considerable enhancement of scattering signal for the 100 nm and 250 nm GNPs (Figure 2). The fluorescence intensity over time was observed, and there was consistent photobleaching of each nanoparticle, as is characteristic of CAP (Figure 2B). From the above observations, it was concluded that 100 nm GNP would be optimal for endogenous fluorescence enhancement of capsaicin. It has been reported that fluorescence should occur at the optimum distance from the GNP for better enhancement.19 Therefore, the GNP was modified with MUA with an HVA linker to obtain an optimum distance between the fluorophore and the GNP surface (Figure S5A). The conjugation was confirmed using SEM and AFM data (Figure S5). The fabricated 100 nm gold-nanopatterned chip was conjugated with CAP using the same procedure used to conjugate CAP on GNP. The SEM data show capsaicin layer formation on the surface of gold nanospots, and AFM data show increased height of gold nanospots after conjugating with CAP. The distance between

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Figure 1. Schematic representation of PAEFS. (A) Schematic representation of total internal reflection microscopy. (B) Capsaicinoids (CAP) conjugated with GNP via a thiol-HVA linker within 10 nm of the nanoparticle. (C) Endogenous fluorescence spectra of capsaicinoid. (D) Jablonski diagram of CAP fluorescence emission and fluorescence enhancement emission through elastic scattering of plasmon nanoparticles. (E) Schematic representation of the fluorescence enhancement after conjugating with gold nanoparticles.

The molar absorptivity coefficient of CAP was 3720 M−1cm−1 at 280 nm and 0.3756 M−1cm−1 at 532 nm.32 Though the molar absorptivity is higher at 280 nm, the detection of single molecules at this wavelength would not be cost-effective and deviates vastly from existing simple fluorescence detection methods. Therefore, 532 nm was chosen as the optimum wavelength; however, the weak fluorescence intensity hinders high sensitivity detection. To C

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GNP and CAP was calculated from AFM data that correlate with previously reported AFM results.34 The resultant fluorescence enhancement was observed to be around 100 times greater than the original fluorescence signal of the CAP (Figure S6). The enhanced florescence was compared with that of highly stable, silica-coated magnetic nanoparticles conjugated with rhodamine B isothiocyanate dye (MNP@SiO2RITC),35 as the fluorescence spectra matches that of capsaicin. However, capsaicin showed a shift in spectrum on conjugation with GNP, and the enhanced intensity was near that of MNP@SiO2RITC. The result shows successful enhancement of single-molecule quantification of capsaicinoids. Single-Nanoparticle Fluorescence Enhancement. For highly precise quantification of endogenous fluorescence on GNPs, the fluorescence signal was detected from individual nanoparticles rather than from an aggregated sample. A single GNP was observed by matching the Born-Wolf optical model of full width half maxima (FWHM) for the individual point spread function (PSF) with the acquired images.36 The PSF of wide-field microscopy has been demonstrated by the Born-Wolf model.37 This model describes the scalar-based diffraction that occurs in the microscope but neglects the aberrations of the refractive index (n) from the immersion medium and the substrate.

the FWHM of the PSF will be 261.2 nm and 248.4 nm (xy), respectively (Figure 3B). The PSF was approximated around 3 to 4 pixels (Figure 3B). With help of the trackmate algorithm,38 the exact spots fitting the desired PSF were analyzed (Figures 3C and 3D). The algorithm determines the appropriate threshold, enabling accuracy in single-molecule measurement. The intensity of each spot in the 3D image was significant in showing the reliability of the proposed method. Ratiometric Quantification of Capsaicinoid Analogues. For quantification of capsaicinoids, CAP and DICAP standards were conjugated on 100 nm GNPs through anti-HVA. Scattering intensity from bare 100 nm GNP was recorded and detected as background from the enhanced fluorescence signal of CAP and DICAP. To probe the enhancement in detection sensitivity, serial dilutions of known concentrations (18 zM to 85 pM) of CAP and DICAP in acetonitrile were employed as standards. Fluorescence images obtained on individual nanoparticles showed strong fluorophore enhancement even at low concentrations (Figure 4). (A)

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Figure 3. Single nanoparticle detection. (A) Fluorescence spectra of CAP and DICAP conjugated with 100 nm GNP. (B) Born-Wolf optical simulation of PSF at the imaging condition for CAP and DICAP along with the PSF value in pixels. (C) Fluorescence image of CAP-conjugated 100 nm GNP and (D) spotting the PSF with average pixels and signal threshold. (E) Relative fluorescence intensity (RFI) of each spot numbered in (D).

Figure 4. Ratiometric quantification. (A) Standard calibration curve of CAP and (B) respective fluorescence images at each concentration. (C) Ratiometric fluorescence intensity difference of CAP and DICAP with a 561/14 band pass filter. (D) Standard calibration curve of CAP and (E) ratiometric fluorescence intensity difference of CAP and DICAP with a 615/20 band pass filter. Mean ± standard deviation of three measurements (n = 3).

The fluorescence emission spectra of the GNP-conjugated CAP and DICAP show emission around 600 nm and 580 nm, respectively (Figure 3A). According to the Born-Wolf optical model, if the point source was detected at 600 nm and 580 nm wavelengths with 100x magnification and 1.4 NA and 160 nm pixel length, then

The 100-fold fluorescence signal enhancement gave rise to 30 zM and 90 zM as respective LODs for CAP and DICAP. Capsaicin has not been reported below nM concentrations by fluorescence detection. The LOD acquired is 106 times lower than that in previous reports.39 The wide linear dynamic range shows excelD

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lent reproducibility. One of the major problems in detecting the capsaicinoid analogues was fluorescence spectral overlap. Even though the spectra differ after conjugation with GNP (Figure 3A), the difference was inadequate to separate the signals with band pass filters. Therefore, we employed a fluorescence intensitybased ratiometric quantification method for detecting capsaicinoid analogues. Both bandpass filters were used at their center wavelengths to image both CAP and DICAP at known concentrations (Figures 4C and 4F). The fluorescence contribution of each was compared to estimate the percentages of CAP and DICAP by comparing to the standard calibration curves (Figures 4A and 4D). The ratiometric quantification was based on the difference in fluorescence intensity ratio of CAP and DICAP. The fluorescence emission shift was different for CAP and DICAP with overlapped emission spectra (Figure S7). Different bandpass filters were used to avoid maximum overlap with fluorescence emission. For ratiometric quantification, both CAP and DICAP were imaged with the two bandpass filters at the same concentrations. The concentrations of CAP and DICAP were determined by the following equation:

Therefore, we applied the developed PAEFS method to detect the endogenous fluorescence of CAP and DICAP from extracts of red pepper powder and kimchi. The samples were spiked (standard addition method) with standards and imaged with both CAP and DICAP wavelengths (Figure 5). The difference in analytical response between the spiked and unspiked samples was due to the amount of capsaicin and dihydrocapsaicin in the spike. This provides a precise determination of the capsaicin and dihydrocapsaicin concentration in the original samples. The results show that the concentration of capsaicinoids in red pepper powder from the calibration curve is 316.43 ± 2.07 mg/kg, with 61.3% CAP and 36.4% DICAP. The respective value for kimchi is 12.14 ± 0.64 mg/kg with 61.1% CAP and 38.6% DICAP, and that for baechu is 4.232  10-9 ± 0.001  10-9 mg/kg with 97.8% CAP and 0.8% DICAP. The type of pepper used in the experiment was Capsicum annuum L. According to the content of capsaicinoids of red pepper powder (T034) and kimchi (T020) presented in the standard of traditional food in Korea, the spice grade is distinguished (Table S2). The red pepper powder used in this experiment and in the manufacture of kimchi are the same material. According to the addition ratio of raw materials in Table 1, red pepper powder was added at a rate of 3.96% when manufacturing kimchi. Therefore, capsaicinoid content was about 11.66 mg/kg in kimchi when using red pepper powder with an average capsaicinoid content of 305.19 mg/kg. The method was validated with the results from HPLC-FLD detection. The limits of detection for CAP and DICAP in various samples ranged from 0.02 to 0.06 mg/kg and 0.02 to 1.38 mg/kg, respectively, whereas the limits of quantification for these capsaicinoids were respectively between 0.05 to 1.83 mg/kg and 0.06 to 4.19 mg/kg (Figure S8). In red pepper powder, capsaicin and dihydrocapsaicin were in the ranges of 185.49-188.96 mg/kg and 115.58-118.39 mg/kg, respectively. The average total content of capsaicinoids was 305.19 ± 3.57 mg/kg. In kimchi, the capsaicin and dihydrocapsaicin were in the ranges of 6.71-7.51 mg/kg and 4.31-4.78 mg/kg, respectively. The average total content of capsaicinoids was 11.66 ± 0.63 mg/kg. The comparison of results from the two methods shows (Table 1) similarities in quantification. However, although capsaicin and dihydrocapsaicin were not detected in baechu by HPLC-FLD (Figure S9), they were detected with PAEFS. Paired t test was used to determine the significance of capsaicinoid detection by PAEFS with an HPLC-FLD method. The average of the differences (| |) between the two results was 11.24. The standard deviation (sd) was calculated to be 4.31. By

(3) where Kd is the dissociation constant, F is the actual fluorescence at the chosen wavelength, R is the fluorescence ratio at the two wavelengths Fλ2/Fλ1, Rmin is the minimum ratio value, Rmax is the maximum ratio value, and Fλ1max/Fλ1min is a scaling factor. Practical Application and Comparison of PAEFS with HPLC-FLD. Kimchi, a traditional staple in South Korean cuisine, has gained attention throughout the world for its health benefits. Generally, kimchi is fermented with probiotic lactic acid bacteria to a state of mild pungency. Capsaicinoid analogues are the main reason for the pungency in kimchi and red peppers. (A)

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, the t value was 4.48, less than the t

table40 value (6.97) for 98% confidence and 2 degree of freedom (n). Therefore, the results obtained with the two methods were not significantly different; hence, capsaicinoid detection by PAEFS was validated. The LOD was compared with that of a nonenhancing iron oxide nanoparticle. The molar absorptivity of capsaicin on iron oxide nanoparticle was 0.3876 M−1cm−1 at 532 nm that was similar to the molar absorptivity of standard capsaicin. Whereas, the molar absorptivity of capsaicin on gold nanoparticle was 4582 M−1cm−1. The LOD obtained with PAEFS was 106 lower than that obtained with an iron oxide nanoparticle. The results show the sensitivity and advantage of PAEFS for single-molecule endogenous fluorescence detection. The endogenous capsaicinoids were detected as model material, but the technique is not limited to one or two endogenous fluorescence materials. The method can be applied to weak endogenous fluorescence molecules such as (1) analytes: DNA, RNA, a-mouse IgG, streptavidin, human IgG, (2) biomarkers: f-PSA, TNF-α, troponin I, Creactive protein, UL16-binding protein, and (3) pathogens and

0.5

Figure 5. Quantification of capsaicinoids in real samples. Fluorescence spectra of red pepper powder, kimchi, and baechu (A) before and (B) after conjugation with GNP by anti-HVA linker. Single-particle ratiometric fluorescence detection of capsaicinoids in (C) red pepper powder, (D) kimchi, and (E) baechu at three different dilutions of stock solution. The concentration of capsaicinoids in red pepper powder from the calibration curve is 316.43 ± 2.07 mg/kg with 61.3% CAP and 34.2% DICAP, that in kimchi is 12.14 ± 0.64 mg/kg with 61.1% CAP and 38.6% DICAP, and that in baechu is 4.23210-9 ± 0.00110-9 mg/kg with 97.8% CAP and 0.8% DICAP. Mean ± standard deviation of three measurements (n = 3).

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(1) Gustavsson, A. K.; Petrov, P. N.; Lee, M. Y.; Shechtman, Y.; Moerner, W. E. 3D single-molecule super-resolution microscopy with a tilted light sheet. Nat. Commun. 2018, 9 (1), 123-130. (2) Ju, S.; Lee, S.; Chakkarapani, S. K.; Kim, K.; Yu, H.; Kang, S. H. One-shot dual-code immunotargeting for ultra-sensitive tumor necrosis factor-α nanosensors by 3D enhanced dark-field super-resolution microscopy. Anal. Chem. 2018, 90 (8), 5100-5107. (3) Stringari, C.; Abdeladim, L.; Malkinson, G.; Mahou, P.; Solinas, X.; Lamarre, I.; Brizion, S.; Galey, J.-B.; Supatto, W.; Legouis, R.; Pena, A.-M.; Beaurepaire, E. Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing. Sci. Rep. 2017, 7 (1), 3792-3802. (4) Stadler, C.; Rexhepaj, E.; Singan, V. R.; Murphy, R. F; Pepperkok, R.; Uhlén, M.; Simpson, J. C; Lundberg, E. Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells. Nat. Methods 2013, 10 (4), 315-323. (5) Brennan, D. J.; O'connor, D. P.; Rexhepaj, E.; Ponten, F.; Gallagher, W. M. Antibody-based proteomics: fast-tracking molecular diagnostics in oncology. Nat. Rev. Cancer 2010, 10 (9), 605-617. (6) Mancias, J. D.; Wang, X.; Gygi, S. P.; Harper, J. W.; Kimmelman, A. C. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature, 2014, 509 (7498), 105-109. (7) Reyes-Escogido, M. D. L.; Gonzalez-Mondragon, E. G.; VazquezTzompantzi, E. Chemical and pharmacological aspects of capsaicin. Molecules 2011, 16 (2), 1253-1270. (8) Contreras-Padilla, M.; Yahia, E. M. Changes in capsaicinoids during development, maturation, and senescence of chile peppers and relation with peroxidase activity. J. Agric. Food Chem. 1998, 46 (6), 2075-2079. (9) Estrada, B.; Bernal, M. A.; Díaz, J.; Pomar, F.; Merino, F. Fruit development in capsicum annuum: changes in capsaicin, lignin, free phenolics, and peroxidase patterns. J. Agric. Food Chem. 2000, 48 (12), 6234-6239. (10) O'Neill, J.; Brock, C.; Olesen, A. E.; Andresen, T.; Nilsson, M.; Dickenson, A. H. Unravelling the mystery of capsaicin: a tool to understand and treat pain. Pharmacol. Rev. 2012, 64 (4), 939-971. (11) Luo, X. J.; Peng, J.; Li, Y. J. Recent advances in the study on capsaicinoids and capsinoids. Eur. J. Pharmacol. 2011, 650 (1), 1-7. (12) Sharma, S. K.; Vij, A. S.; Sharma, M. Mechanisms and clinical uses of capsaicin. Eur. J. Pharmacol. 2013, 720 (1-3), 55-62. (13) Adams, M. J.; Ahuja, K. D.; Geraghty, D. P. Effect of capsaicin and dihydrocapsaicin on in vitro blood coagulation and platelet aggregation. Thromb. Res. 2009, 124 (6) 721-723. (14) Iida, T.; Moriyama, T.; Kobata, K.; Morita, A.; Murayama, N.; Hashizume, S.; Fushiki, T.; Yazawa, S.; Watanabe, T.; Tominaga, M. TRPV1 activation and induction of nociceptive response by a nonpungent capsaicin-like compound, capsiate. Neuropharmacology, 2003, 44 (7), 958-967. (15) Macho, A.; Lucena, C.; Sancho, R.; Daddario, N.; Minassi, A.; Muñoz, E.; Appendino, G. Non-pungent capsaicinoids from sweet pepper. Eur. J. Nutr. 2003, 42 (1), 2-9. (16) Woodbury, R. L.; Varnum, S. M.; Zangar, R. C. Elevated HGF levels in sera from breast cancer patients detected using a protein microarray ELISA. J. Proteome res. 2002, 1 (3), 233-237. (17) Schweitzer, B.; Roberts, S.; Grimwade, B.; Shao, W.; Wang, M.; Fu, Q.; Shu, Q.; Laroche, I.; Zhou, Z.; Tchernev, V. T.; Christiansen, J.; Velleca, M.; Kingsmore, S. F. Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 2002, 20 (4), 359-365. (18) Ali, M. M.; Li, F.; Zhang, Z.; Zhang, K.; Kang, D.-K.; Ankrum, J. A.; Le, X. C.; Zhao, W. Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine. Chem. Soc. Rev. 2014, 43 (10), 3324-3341. (19) Luan, J.; Morrissey, J. J.; Wang, Z.; Derami, H. G.; Liu, K.-K.; Cao, S.; Jiang, Q.; Wang, C.; Kharasch, E. D.; Naik, R. R.; Singamaneni, S. Add-on plasmonic patch as a universal fluorescence enhancer. Light Sci. Appl. 2018, 7 (1), 29-41. (20) Chen, T.; Dong, B.; Chen, K.; Zhao, F.; Cheng, X.; Ma, C.; Lee, S.; Zhang, P.; Kang, S. H.; Ha, J. W.; Xu, W.; Fang, N. Optical superresolution imaging of surface reactions. Chem. Rev. 2017, 117 (11), 7510-7537.

toxins: aflatoxin M1, E. coli157, SARS-CoV, S-OIV, anthrax protective antigen.31 CONCLUSIONS Simple and innovative inelastic scattering of plasmon was used as an electromagnetic field enhancer for ultra-trace and high sensitivity detection of endogenous fluorophores. The absorbance of fluorescence molecules matched with the elastic scattering frequency of the plasmon to produce a 100-fold fluorescence enhancement. The proposed method pioneers a new approach for endogenous fluorescence detection, as it does not require additional toxic labelling probes. Capsaicinoid analogues were used as model endogenous fluorophores; the method can be applied to any given endogenous fluorescence molecule of weak intensity. Additionally, the method represents a simple, cost-effective approach for single-molecule detection. The primary focus of the work was to show the effects of enhancement parameters and influence endogenous fluorescence for label-free, ultra-trace detection. The work also demonstrated highly sensitive detection of capsaicinoid analogues and its application in practical samples. It is important to note that this method has wide applications in bioimaging, biosensing, and methods demanding a single-molecule fluorescence approach. Due to the minimal complexity and excellent reproducibility, this novel technique can be easily adopted for single-molecule endogenous fluorescence detection.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional statistical data analyses, HPLC-FLD data, and the single-molecule imaging setup.

AUTHOR INFORMATION Corresponding Author 

E-mail address: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ORCID Seungah Lee: 0000-0002-4254-7018 Seong Ho Kang: 0000-0003-2101-4113

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by a grant from the World Institute of Kimchi (KE1803-3) funded by the Ministry of Science, ICT, and Future Planning, Republic of Korea, and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2018R1D1A1B07042362). We thank Dr. H. Yu (Korea Research Institute of Standards and Science, Republic of Korea) for providing us with SEM and AFM images of a gold-nanopatterned chip.

REFERENCES F

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(21) Flauraud, V.; Regmi, R.; Winkler, P. M.; Alexander, D. T. L.; Rigneault, H.; Van Hulst, N. F.; García-Parajo, M. F.; Wenger, J.; Brugger, J. In-plane plasmonic antenna arrays with surface nanogaps for giant fluorescence enhancement. Nano Lette. 2017, 17 (3), 17031710. (22) Tam, F.; Goodrich, G. P.; Johnson, B. R.; Halas, N. J. Plasmonic enhancement of molecular fluorescence, Nano Lett. 2007, 7 (2), 496501. (23) Kinkhabwala, A.; Yu, Z.; Fan, S.; Avlasevich, Y.; Müllen, K.; Moerner, W. E. Large single-molecule fluorescence enhancements produced by a bowtie nanoantenna. Nat. Photonics 2009, 3 (11), 654657. (24) Tabakman, S. M.; Lau, L.; Robinson, J. T.; Price, J.; Sherlock, S. P.; Wang, H.; Zhang, B.; Chen, Z.; Tangsombatvisit, S.; Jarrell, J. A.; Utz, P. J.; Dai, H. Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range. Nat. Commun. 2011, 2, 466-474. (25) Chakkarapani, S. K.; Sun, Y.; Lee, S.; Fang, N.; Kang, S. H. Three-dimensional orientation of anisotropic plasmonic aggregates at intracellular nuclear indentation sites by integrated light sheet superresolution microscopy. ACS Nano 2018, 12 (5), 4156-4163. (26) Zhang, B.; Kumar, R. B.; Dai, H.; Feldman, B. J. A plasmonic chip for biomarker discovery and diagnosis of type 1 diabetes. Nat. Med. 2014, 20 (8), 948-953. (27) Zhang, B.; Pinsky, B. A.; Ananta, J. S.; Zhao, S.; Arulkumar, S.; Wan, H.; Sahoo, M. K.; Abeynayake, J.; Waggoner, J. J.; Hopes, C. Tang, M.; Dai, H. Diagnosis of Zika virus infection on a nanotechnology platform. Nat. Med. 2017, 23 (5), 548-550. (28) Wang, C.; Tadepalli, S.; Luan, J.; Liu, K.-K.; Morrissey, J. J.; Kharasch, E. D.; Naik, R. R.; Singamaneni, S. Metal‐organic framework as a protective coating for biodiagnostic chips. Adv. Mater. 2017, 29 (7), 1604433-1604439. (29) Chakkarapani, S. K.; Zhang, P.; Ahn, S.; Kang, S. H. Total internal reflection plasmonic scattering-based fluorescence-free nanoimmunosensor probe for ultra-sensitive detection of cancer antigen 125. Biosens. Bioelectron. 2016, 81, 23-31.

(30) Würth, C.; Grabolle, M.; Pauli, J.; Spieles, M.; Resch-Genger, U. Relative and absolute determination of fluorescence quantum yields of transparent samples. Nat. Protoc. 2013, 8 (8), 1535-1550. (31) Bauch, M.; Toma, K.; Toma, M.; Zhang, Q.; Dostalek, J. Plasmonenhanced fluorescence biosensors: a review. Plasmonics 2014, 9 (4), 781-799. (32) Bardhan, R.; Grady, N. K.; Halas, N. J. Nanoscale control of near‐infrared fluorescence enhancement using Au nanoshells. Small 2008, 4 (10), 1716-1722. (33) Bekshaev, A. Y.; Bliokh, K. Y.; Nori, F. Mie scattering and optical forces from evanescent fields: a complex-angle approach. Opt. express, 2013, 21 (6), 7082-7095. (34) Lee, S.; Yu, H.; Kang, S. H. Plasmonic metal scattering immunoassay by total internal reflection scattering microscopy with nanoscale lateral resolution. Chem. Comm. 2015, 51 (5), 945-947. (35) Phukan, G.; Shin, T. H.; Shim, J. S.; Paik, M. J.; Lee, J. K.; Choi, S.; Kim, Y. M.; Kang, S. H.; Kim, H. S.; Kang, Y.; Lee, S. H.; Mouradian, M. M.; Lee, G. Silica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro. Sci. Rep., 2016, 6, 29095-290106. (36) Wolf, E. (1959). Electromagnetic diffraction in optical systems-I. An integral representation of the image field. Proc. R. Soc. Lond. A, 253(1274), 349-357. (37) Kirshner, H.; Aguet, F.; Sage, D.; Unser, M. 3‐D PSF fitting for fluorescence microscopy: implementation and localization application. 2013, J. Microsc. 249 (1), 13-25. (38) Tinevez, J.-Y.; Perry, N.; Schindelin, J.; Hoopes, G. M.; Reynolds, G. D.; Laplantine, E.; Bednarek, S. Y.; Shorte, S. L.; Eliceiri, K. W. TrackMate: An open and extensible platform for single-particle tracking. Methods 2017, 115, 80-90. (39) Zak, A.; Siwinska, N.; Slowikowska, M.; Borowicz, H.; Szpot, P.; Zawadzki, M.; Niedzwiedz, A. The detection of capsaicin and dihydrocapsaicin in horse serum following long-term local administration. BMC Vet. Res. 2018, 14 (1), 193-198. (40) Harris, D. C. Quantitative Chemical Analysis, 8th ed.; W. H. Freeman and Company: New York, 2010.

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Table 1. Capsaicin and dihydrocapsaicin contents in red pepper powder, kimchi, and baechu by PAEFS and HPLC-FLD

Sample

Capsaicin (mg/kg)

Dihydrocapsaicin (mg/kg)

Capsaicinoids1 (mg/kg)

PAEFS

HPLC-FLD

PAEFS

HPLC-FLD

PAEFS

HPLC-FLD

Red pepper powder

194.78 ± 1.262

187.80 ± 2.00

115.46 ± 0.06

117.39 ± 1.57

316.43 ± 2.07

305.19 ± 3.57

Kimchi

7.42 ± 0.05

7.12 ± 0.40

4.69 ± 0.02

4.54 ± 0.23

12.14 ± 0.64

11.66 ± 0.63

Baechu

3.92410-9 ± 0.00110-9

ND3

0.21610-9 ± 0.00110-9

ND

4.23210-9 ± 0.00110-9

ND

1

Capsaicinoids = capsaicin + dihydrocapsaicin. Values are mean ± standard deviation of three measurements (n = 3). 3 ND: not detected.

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Table of Contents Cap-Au

Fluorescence Intensity

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EM CCD

Rotatory filter 100

Cap

Objective lens

Thiol-HVA linker

Cap

Au d 10 nm Prism

PAEF

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