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Ultrasensitive Detection of Alpha-Fetoprotein by Total Internal Reflection Scattering-based Super-Resolution Microscopy for Super-Localization of Nano-Immunoplasmonics Sujin Ahn, Peng Zhang, Hyunung Yu, Seungah Lee, and Seong Ho Kang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03069 • Publication Date (Web): 25 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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Analytical Chemistry

Ultrasensitive Detection of Alpha-Fetoprotein by Total Internal Reflection Scattering-based Super-Resolution Microscopy for SuperLocalization of Nano-Immunoplasmonics Sujin Ahn,† Peng Zhang,† Hyunung Yu,‡ Seungah Lee,§ and Seong Ho Kang*,†,§ †

Department of Chemistry, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea Nanobio Fusion Research Center, Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea § Department of Applied Chemistry and Institute of Natural Sciences, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea ‡

ABSTRACT: Super-localization of immunoplasmonic nanotags on antibody-bound gold-nanoislands (GNIs) along the x, y coordinates was determined using total internal reflection scattering-based super-resolution microscopy (TIRS-SRM) at subdiffraction limit resolution. Individual immunoplasmonic nanotags (20-nm silver nanoparticles) and 100-nm GNIs were selectively acquired in the evanescent field layer by wavelength-dependent plasmonic scattering using two illumination lasers (405 nm and 635 nm, respectively). Alpha-fetoprotein (AFP), a liver cancer-related model protein, was immobilized as a target molecule on the GNI arrays. The centroid position of a localized immunoplasmonic nanotag on the GNI was resolved at less than 10 nm of spatial resolution by applying 2D Gaussian fitting to its point spread function. This method showed enhanced sensitive quantification with a limit of detection (LOD) of 7.04 zM (1–2 molecules of AFP/GNI), which was 100–5,000,000,000 times lower than detection limits obtained with previous APF detection methods. Furthermore, the method was also successfully applied to quantify AFP molecules at the single-molecule level in human serum samples. The wavelength-dependent TIRS-SRM method was demonstrated to be an effective tool for super-localizing individual protein molecules and interactions in nanoscale regions and was a reliable method for the ultrasensitive quantitative detection of disease-related protein molecules as a nanosensor and for diagnosis at the single-molecule level.

Single-molecule localization microscopy (SMLM) is a powerful tool to elucidate many biological phenomena,1 enabling the study of fine structural details and dynamics.2-4 SMLM compensates for the resolution drawback of conventional microscopy and offers subdiffraction spatial resolution to the nanometer scale.5-7 The localized position can be used to track the probe, to characterize the dynamic and elastic properties of the investigated sample,8,9 or to image the sample. Plasmonic nanometals are photostable, providing unlimited photon collection to observe molecular binding over arbitrarily long time intervals.10-12 In addition, nanometals strongly scatter light at their plasmon peak wavelengths and can be easily functionalized with antibodies. These advantages enable noble plasmonic nanometals to serve as extremely intense probes for immunoassays13-15 and biochemical sensors.16-21 In addition, the common detection methods measured signal from the accumulated sum of all tags present in the target region, including contributions from both specific and nonspecific binding events. Therefore, the super-localization analysis in immunoassay is necessary to get the specific binding information with high accuracy. Plasmonic nanometals have been detected and imaged with a variety of techniques including bright-field microscopy,22,23 dark-field microscopy,24,25 photo-thermal interference contrast techniques,26-28 differential interference contrast microscopy,29,30 confocal Raman microscopy,31 and dynamic surfaceenhanced Raman spectroscopy.32 In particular, total internal reflection scattering (TIRS) spectroscopy is one of the most pow-

erful techniques for highly sensitive imaging with greatly reduced background noise, strong signals, and high angular resolution.33-35 Kang et al. introduced optically controlled TIRS36 and localization of molecules along the axial-direction in an immunoassay37 using TIRS. However, localization along the lateral-direction of a confined nanoscale region using TIRS-based super-resolution microscopy (TIRS-SRM) at subdiffraction limit resolution has not yet been reported for nanosensors or on biochips. Alpha-fetoprotein (AFP) is an acid glycoprotein that exists in the early fetal development of the liver and yolk sac. Generally, the AFP concentration in a healthy adult is less than 25 ng/mL, whereas the value may exceed 400 ng/mL in the pathological changes due to liver cancer.38 Thus, it could be of great significance to develop rapid and sensitive analytical methods to quantify AFP for early detection and diagnosis. Accordingly, numerous immunological methods for determining the concentration of AFP have been reported.39-41 The classic enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods for the determination of AFP in biological fluids based on spectrophotometric reading.40,42,43 However, ELISA suffers drawbacks of being time consuming and exhibiting a narrow dynamic range. Herein, we, for the first time, localized immunoplasmonic nanotags on a confined nanoscale region (100-nm GNIs) along the lateral direction using TIRS-SRM at the single-molecule

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level. For this, 20-nm silver nanoparticles were used as fluorescent-free metal nanoprobes (i.e., nanotags). The location and distribution of individual nanotags on GNIs were determined by applying 2D Gaussian fitting to the point spread function. After Gaussian fitting, the binding event was confirmed by the changes in the centroid positions of individual nanometals. The centroid position distance between GNIs and nanotags was resolved at subdiffraction limit resolution. In addition, this method was successfully applied to determine the exact superlocalization and interaction of individual protein molecules (i.e., antigen-antibody) as well as for the quantitative analysis of various disease-related protein molecules at the single-molecule level.

EXPERIMENTAL SECTION Reagents. 11-Mercaptoundecanoic acid (MUA, 95%), 6mercapto-1-hexanol (MCH, 97%), dimethyl sulfoxide (DMSO, 99.5%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 2-(morpholino)ethanesulfonic acid (MES), glycine, and phosphate-buffered saline (PBS) were supplied by Sigma-Aldrich (St, Louis, MO, USA). Dithiobis(succinimidyl propionate) (DSP), protein A/G, and N-hydroxysulfosuccinimide (NHSS) were purchased from Pierce (Rockford, IL, USA). Tris(base) was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA). StabilGuard was purchased from Surmodics (Eden Prairie, MN, USA). Silver nanotags approximately 20-nm in diameter (7.0 × 1010 particles/mL) were obtained from BBI Life Sciences (Cardiff, UK). Human AFP antigen and monoclonal human AFP antibody (5H7 and 4A3) were sourced from Biodesign International (ME, USA). The commercial ELISA kit was purchased from F. Hoffmann-La Roche Ltd. (Basel, Switzerland) using cobas e602 (F. Hoffmann-La Roche Ltd., Basel, Switzerland). Fabrication of GNI Arrays. The fabrication process was performed according to a previously reported procedure.44 The cleaned glass wafer with piranha solution (1:1 = H2SO4:30% H2O2) was coated with a 150 nm thick polymethylmethacrylate (PMMA) layer that served as an electron-sensitive photoresist. An electron beam (Elionix E-beam system, 100 keV/100 pA) was used to burn off the polymer in a desired pattern. Then, Au/Cr (20/5 nm thickness) was deposited by thermal evaporation to form array that consisted of 4 × 4 GNI array (100 nm in diameter and 10 μm pitch) on a 10 mm2 glass wafer (National Nanofab Center, Daejeon, Korea). Antibody Conjugation on the Silver Nanotag Surface. Antibody conjugation to prepare the immunoplasmonic nanotag was performed in two steps. First, 10 mM MUA and 30 mM MCH in ethanol were added to the water-based 20-nm silver solution. The solution was sonicated for 20 min and incubated for 2 h 30 min to form a self-assembled monolayer of MUAMCH on the 20-nm silver surface, which was then washed with deionized water. The 20-nm silver-MUA-MCH 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. Second, AFP secondary antibody (4A3) was conjugated to the 20nm silver-MUA-MCH by adding 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). After stirring at room temperature for 40 min, the 20-nm silver-MUA-MCH-NHSS was suspended in 1× PBS, and 10 μg/mL monoclonal human AFP antibody (4A3) in PBS (pH

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7.4) was added. The resulting immunoplasmonic nanotags were stored at 4 °C. Nanoimmunoassay of AFP on GNI. Immunoreactions were performed at room temperature according to a previously reported procedure.43 The GNIs were reacted in a stepwise manner with 4 mg/mL of DSP in DMSO for 30 min, then rinsed with DMSO and deionized water. Next, 0.1 mg/mL of protein A/G was added to facilitate Fc binding for 1 h. Unreacted succinimide was blocked with 10 mM Tris (pH 7.5) and 1 M glycine for 30 min. The glass nanochip with GNIs was immersed in StabilGuard for 30 min and then reacted with 20 µL of 2 µg/mL primary monoclonal human AFP antibody (5H7) for 1 h. The target human AFP antigen was diluted (7.04 zM to 7.04 fM), and 20 μL of AFP antigen was loaded on the chip for 1 h. All subsequent steps were followed by washing with deionized water and briefly drying. A

B

EMCCD

λ1 λ2

EM CCD λ1 ×

OL

M M DP

CG GNI array

λ2

OL

L1 λ1 ××

DC

DP Z

λ2

P

M

EFL

Z

L2

M

Figure 1. (A) Physical setup and (B) schematic diagram of the lab-made TIRS-SRM system. Black circle: 20-nm silver nanotag on 100-nm GNI, and the inset red circle shows the super-localization image. “×” indicates the centroid of a nanometal. Abbreviations: EMCCD, electron-multiplying chargecoupled device; OL, objective lens; CG, cover glass; GNI, gold-nanoisland; EFL, evanescent field layer; DP, dove prism; L1, 405-nm laser; L2, 635-nm laser, M, mirror; P, pinhole; DC, dichroic mirror; Z, z-positioner; λ1, 405-nm illumination; λ2, 635-nm illumination.

Lab-Made TIRS-SRM System. Images of GNIs before and after the immunoreaction were collected using a lab-made TIRS-SRM system (Figure 1). The schematic representation and physical layout of the TIRS-SRM system were modified from the previously published system.36 Briefly, the TIRS-SRM imaging techniques were based on an upright Olympus BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan). For total internal reflection scattering (TIRS), two lasers with vertical polarization (p-pol) were used as follows: a 30 mW, 405-nm laser (SDL-405-LM-100T, Shanghai Dream Lasers Technology Co., Ltd., China) for illumination of the sample and a 30-mW, 635-nm laser (SDL-635-LM-400T, Shanghai Dream Lasers Technology Co., China Ltd., China) for the GNI signal. Various optical components were also used in the TIRS-SRM system to observe the scattering intensity of GNIs and the immunoplasmonic nanotags and reactions on the arrayed GNIs. Scattering signals were measured at various wavelengths with fixed prisms and were collected with a numerical aperture (NA) 0.6-1.3 objective lens (UPLANFLN, ×100). Moreover, a mirror manipulator was used to control the incident angle of light illumination to maximize scattering signals from the plasmonic nanotags. TIRS images were acquired with a QuantEM 512SC (photometrics, AZ, USA) electron-multiplying charge-coupled device (EMCCD) camera (512 × 512 pixel imaging array). The scat-


tered intensity was calculated as the difference between the intensities of selected signal regions and background regions. Images were acquired using MetaMorph (Version 7.5, Universal Imaging, Sunnyvale, CA, USA). Lab-Made Enhanced Dark Field Microscopy. Lab-made enhanced dark field microscope with an enhanced dark-field illumination device (Cytoviva Inc., Auburn, AL, USA) attached to microscope (Olympus Optical Co., Ltd., Tokyo, Japan). The device replaced the microscope's original condenser with a Cytoviva 1250 dark-field condenser (NA 1.4) attached via a fiber optic light guide to a Solarc 24 W arc lamp (WelchAllyn, Skaneateles Falls, NY, USA). Bandpass filters (406/15 nm and 620/14 nm) from Semrock (Rochester, NY, USA) were used for wavelength selection. Super-Localization of Nanotag-Antigens on GNIs. Raw diffraction-limited wide-field TIRS microscopic images of immunoplasmonic nanotags and GNIs were fit to resolve their centers with subdiffraction-limit resolution.45 The super-localization of immunoplasmonic nanotags on GNIs was achieved with the following steps. Briefly, 500 frames of TIRS images of a single immunoplasmonic nanotag and GNI at the specific scattering wavelength were acquired with the 405-nm laser and 635-nm excitation laser, respectively. The centers of the nanotag and GNI in each frame were fit with a symmetric 2D Gaussian function: 1

𝐼𝐼�𝑥𝑥, 𝑦𝑦, 𝐼𝐼0 , 𝐴𝐴, 𝑥𝑥0 , 𝑦𝑦0 , 𝜎𝜎𝑥𝑥 , 𝜎𝜎𝑦𝑦 � = 𝐼𝐼0 + 𝐴𝐴 exp �− �� 2

𝑥𝑥−𝑥𝑥0 2 𝜎𝜎𝑥𝑥

𝑦𝑦−𝑦𝑦0

� +�

𝜎𝜎𝑦𝑦

2

� �� (1)

where I0 is a constant term representing the background noise, A is the amplitude, x0 and y0 are the coordinates of the center, and σx and σy are standard deviations of the distribution in the x and y directions, respectively. The center coordinates of the nanotag and GNI in each frame were recorded as (x, y), and the real centers of the nanotag and GNI were represented with their average value in 500 frames (𝑥𝑥̅ , 𝑦𝑦�). The center differences, dc, of the nanotag and GNI were calculated with the following equations: Δ𝑥𝑥 = �𝑥𝑥̅tag − 𝑥𝑥̅GNI �

(2)

𝑑𝑑𝑐𝑐 = ��Δ𝑥𝑥 2 + Δ𝑦𝑦 2 �

(4)

Δy = �𝑦𝑦�tag − 𝑦𝑦�GNI �

(3)

Quantification of AFP Molecules on GNIs. The relative scattering intensity (RSI) was calculated by the difference of intensity between the signal and the background noise (Figure S1). Two lasers were used to obtain the scattering signals of nanotags and GNIs. The RSI of GNIs was measured with a 635nm laser; the RSI of nanotags was measured with a 405-nm laser. The RSI signals before and after antibody immunoreactions on GNI arrays as a function of wavelength were calculated using the average corrected RSI for AFP antigen concentration. In addition, we quantitatively analyzed images from the TIRS system as follows. First, signal and background regions with the same areas were selected. Second, the sum of RSIs of occupied pixels per spot corrected by background subtraction was calculated. Calibration curves and quantified values of AFP standard were calculated as the sum of RSIs from 16 spots on a 4 × 4 GNI array. Calibration curves were fit using Excel software (Version 2016, Microsoft Co., Redmond, WA, USA). Images were acquired, and data were analyzed using MetaMorph. Spectroscopy and Electron Microscopy. UV-Vis absorbance spectra measurements were conducted with a V-670 spectrophotometer (JASCO Co., Easton, MD, USA). The maximum

wavelength of scattering signal from GNI before and after immunoreaction was decided using a hyperspectral imaging (HSI) system (CytoViva Inc., Auburn, AL, USA). The surface morphology of immunoplasmonic nanotags was characterized by transmission electron microscopy (TEM) (JEM 2100F, JEOL, Tokyo, Japan). Samples for TEM characterization were prepared by placing a drop of sample solution on a carbon-coated copper grid and drying at 37 °C. TEM measurements were conducted at an accelerating voltage of 200 kV. A scanning electron microscope (SEM) (Quanta FEG 650, FEI Co., OR, USA) with an accelerating voltage of 30 kV was used to confirm immunoreactions and the positions of nanotags on GNIs.

RESULTS AND DISCUSSION AFP Antibody Conjugation on the Nanotag Surface. Plasmonic nanometals act as transducers that convert small changes in the local refractive index (RI) into a spectral shift. Nanometals possess strong absorption and scattering in the visible region, and surface modification of these nanometals leads to changes in their extinction in the UV-Vis region. The dependence of the extinction maximum wavelength upon surface modification of plasmonic nanometals can be expressed as follows.46,47 Δ𝜆𝜆𝑚𝑚𝑚𝑚𝑚𝑚 = 𝑚𝑚Δ𝑛𝑛 �1 − 𝑒𝑒𝑒𝑒𝑒𝑒 �−

2𝑑𝑑 𝑙𝑙𝑑𝑑

��

(5)

Here, λmax is the extinction maximum wavelength; m and n are the refractive indexes of the bulk nanometal and the absorbed surface layer, respectively; d is the effective thickness of the adsorbed layer, and ld is the characteristic electromagnetic decay length. The extinction maximum (λmax) of the silver nanotags was observed by means of UV-Vis spectroscopy both before and after conjugation with the secondary antibody. Therefore, successful antibody conjugation on the 20-nm silver nanometal (i.e., nanotag) surface was indicated by a spectral shift.47 The UV-Vis spectrum of bare silver showed a peak located at 401 nm, which after secondary AFP antibody conjugation, was located at 409 nm (Figure 2A). Conjugation of the antibody on the nanotag surface was confirmed by the 8-nm redshifted spectrum. A 0.025

Absorbance (a. u.)

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Nanotag Nanotag-4A3

8 nm

NH CO

NH OC

HN CO

O

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C HN

300

350

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450

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600

Wavelength (nm)

B

Nanotag-4A3

Nanotag

1 nm

1 nm

Figure 2. (A) UV-Vis spectra of 20-nm silver nanometal ensemble (solid line, λmax = 401 nm) and 20-nm silver nanotag4A3 dispersion (dotted line, λmax = 409 nm). 4A3 is the AFP secondary antibody. (B) TEM images of the 20-nm silver nanotag surface.

The nanotag static structure was obtained using TEM (Figure 2B). TEM images clearly showed formation of the conjugate.



400

ⅲ

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5.0 ×102

2.5 × 103

RSI

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Scattering intensity

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900 500 nm

0

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Figure 3. The scattering intensity spectra of (A(i)) 100-nm GNI and (B(i)) 20-nm silver nanotags on 100-nm GNI using HSI. TIRS images of 4×4 GNI arrays (A(ii)) before and (B(ii)) after immunoreaction. Relative scattering intensity (RSI) of 100-nm GNIs (A(iii)) before and (B(iii)) after immunoreaction using TIRS-SRM with a 30-mW, 405-nm laser.

Optimization of Optical Control in the Evanescent Field Layer. The localization precision depended on achieving the maximum signal-to-noise ratio (SNR) in the image. To maximize the RSI and SNR, we adjusted various optical controls such as the NA of the objective lens, laser power, and exposure time. The NA was associated with resolving power. A higher NA value produced high resolution, collected more light, and generally produced brighter images.48 Increasing the NA values led to an increase in the background noise intensity and decrease in SNR48. The scattering intensity that includes the background noise, increased rapidly in the range of NA value 0.6-0.8 (Figure S2). In this range the background noise increased slowly on compared to the scattering intensity of the signal that led to the

increase in SNR value. However, there was no significant increase in signal intensity above NA 0.8, although there was a gradual increase in background noise. Thus, the SNR got decreased after NA 0.8 due to increase in the background noise intensity. Therefore, considering the correlation of emission intensity and background signal, an NA value of 0.8 was selected (right in Figure 4A). 20

10

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SNR

RSI ( ×103 )

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8 4

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0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

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Compared to the bare 20-nm silver nanotag, a shadow coating was observed surrounding the metal after conjugation with antibody molecules. These results confirmed that the silver nanotag was successfully conjugated with secondary AFP antibody molecules (4A3). The nanotagged-AFP antibody was now ready to capture target antigens, indicating the successful attachment of the target protein to the nanotag surface. Characterization of Scattering Spectrum on GNIs. The scattering signal from bare GNIs occurred at a wavelength of approximately 610 nm (Figure 3A(i)). Primary antibody (5H7) immobilized on the GNIs was used to capture AFP molecules (antigen). The 20-nm silver nanotagged-antibody (4A3) was employed as a detection antibody. After immunoreaction, the scattering peak was red-shifted to approximately 650 nm (Figure 3B(i)). The scattering signal from GNI got decreased after immunoreaction, as it was covered by silver nanotag. A pronounced shoulder on the left flank of the spectrum occurred at approximately 525 nm (white box in Figure 3B(i)) after the immunoreaction, which indicated the representative properties of immunoplasmonic nanotags, as distinguished from 100-nm GNIs (Figure 3B). Also, RSI changes before and after the immunoreaction using 405-nm laser illumination demonstrated the successful immunoreaction. The GNI images in the TIRS were observed to be brighter (Figure 3A(ii) and B(ii)), with the intensity increasing ~14 fold (Figure 3A(iii) and B(iii)). In addition, the scattering signals remained stable without photobleaching (Movie S1). Through differences in the RSI signal, the antigen-antibody binding event (immunoreactions) on GNIs was confirmed.


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15 10

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Figure 4. The RSI of 20-nm silver nanotagged-AFP on 100-nm GNIs and SNR plots with respect to the change in (A) NA, (B) laser power, and (C) EMCCD exposure time on illumination at 405 nm wavelength. RSI = Relative scattering intensity. Black arrows represent optimum conditions.

Adjustment of the laser power was carried out to increase the emission intensity within the range of 0 to 40 mW. The emission intensity is proportional to the incident irradiance, as I = kP0c, where I is the emission intensity; k is a constant; P0 is the incident irradiance (i.e., laser power); and c is the concentration of analyte. Therefore, increasing the laser power also increased the RSI. A higher laser power led to an increase in the RSI (left in Figure 4B); however, considering the SNR (right in Figure 4B), all experiments were carried out with a laser power of 30 mW. The exposure time of the EMCCD was associated with the signal intensity and streaking.49 The correlation of signal intensity and exposure time is shown in Figure 4C. A longer exposure time of the EMCCD led to an increase in the RSI without photobleaching compared to fluorescence (left in Figure 4C).47 However, considering the SNR (right in Figure 4C), 140 ms was selected for the experiments. Location of Antigen-Antibody Binding. Light sources with wavelengths of 405 nm and 635 nm illuminated the interface of the two mediums, combining to create an incident angle (θ) chosen such that it was larger than the critical angle of 62.9°. We constructed a trapezoidal prism where the incident angle of light illumination could be adjusted within the range of 67.6° to 71.0° using a mirror manipulator. The angular effect of the incident light was observed by detecting the immunoplasmonic nanotags with laser illumination at 405 nm and GNIs at 635 nm. The scattering intensity peak exhibited some flat space at the top (red-dotted boxes in Figure 5A). This phenomenon showed


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that the signal from immunoplasmonic nanotags was neither one point nor fixed, because the location of nanotags on GNIs changed even though they were in confined nanoscale regions. Through SEM images, we could confirm the various positions of dispersed individual nanotags on GNIs (white arrows in Figure 5B). A λ1, 405 nm

λ2, 635 nm

5.0×102

RSI

5.0×103

RSI

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


67

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71

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50 nm

50 nm

50 nm

Figure 5. (A) The RSI plots of 20-nm silver nanotagged-AFP on 100-nm GNIs, with respect to the change in incident angle (θ) on illumination with 405 nm laser (left) and 635 nm laser (right), respectively. (B) SEM images of 20-nm silver nanotag on a 100-nm GNI after immunoreaction. Right corresponds to SEM data cited in Ref. 37. RSI = Relative scattering intensity.

A

ⅰ

ⅱ

ⅲ

ⅳ

ⅴ

ⅵ

ⅶ

B

GNI

∆xa ∆yb dcc

ⅷ

ⅰ

ⅱ

ⅲ

Table 1. Comparison of Dynamic Linear Ranges and LODs for Various AFP Detection Methods

ⅸ

ⅳ

ⅴ

ⅵ

nanotags on GNIs were blurred because of the diffraction limit of light (Figure S3A and C). It was difficult to distinguish the exact positions of nanotags on GNIs. Fortunately, however, due to the specific characterization of plasmonic effects of silver nanometals and GNIs, their maximum scattering wavelengths were different (Figure 3), which enabled them to be separated into spectral domains. After the silver nanotags and GNIs were detected, their exact centers were resolved by 2D Gaussian fitting (Figure S3B and D). Compared with the wide-field diffraction limit TIRS images, the super-localized images of TIRSSRM provided much higher spatial resolution of individual nanotags on GNIs at the single-molecule level. This result showed that the antibody-antigen binding reaction could be observed directly. The super-localization result of TIRS-SRM before the reaction showed no center changes (Figure S3B, dc = ∆x = ∆y = 0), which implies that only GNIs exist in the system without any immunoreactions. However, the super-localized TIRS-SRM images after reaction (Figure S3D) showed the clear presence of nanotags on GNIs (∆x = 19.7 nm, ∆y = 4.5 nm, and dc = 20.2 nm), providing direct evidence of the successful immunoreactions, and showed the possibility of direct quantification of AFP molecules due to the protein-protein interaction on nanoscale substrate regions. More importantly, the centers distance between the silver nanotag and GNI could be measured exactly on less than 10 nm subdiffraction-limited images by the developed TIRS-SRM method (Figure 6). In addition, since immunoreactions of the silver nanotags on GNIs were stochastic, the distribution of nanotags on GNIs should be random. The super-localization data of nine spots (Figure 6) and the RSI measurements of AFP-immunoplasmonic nanotags on different selected GNIs over time after immunoreactions (Movie S2) provided the most intuitive data of this speculation and matched the results acquired with the SEM data (Figure 5B).

ⅶ

ⅷ

ⅸ

28.11 8.40 1.92 6.10 1.79 0.02 1.71 25.25 28.61 12.65 17.10 8.86 16.77 9.83 0.01 11.32 5.24 4.41 30.83 19.05 9.07 17.84 9.99 0.02 11.45 25.79 28.95

Figure 6. (A) Super-localization of silver nanotags at nine spots on GNIs. (B) The x, y coordinates of 20-nm silver nanotags on GNIs. When the coordinates of the GNI center were fixed at (0, 0), the silver nanotag coordinates were determined as relative coordinates. aThe relative coordinate difference of silver nanotag along the x direction. bThe relative coordinate difference of silver nanotag along the y direction. cDistance between the centroid of GNI and silver nanotag (nm). Scale bar, 10 nm.

Super-Localization of Antigen Molecules on GNIs by TIRS-SRM. Wide-field TIRS images of immunoplasmonic

method

dynamic linear range (M) LOD (M)

ref

UCL

1.40 × 10-13–8.40 × 10-10

2.80 × 10-13

53

-12

54

5.60 × 10-14

55

-13

56

5.60 × 10-13

57

-18

50

7.00 × 10-14

51

1.36 × 10

-11

52

7.20 × 10

-19

58

-11

FPLS

5.54 × 10 –1.12 × 10

SPFS

– -13

-9

-9

PEC

7.00 × 10 –7.00 × 10

ECI

2.80 × 10-12–1.40 × 10-9 -16

-13

ECL

7.20 × 10 –7.00 × 10

CLI

7.00 × 10-12–1.40 × 10-9

RRS

-11

-10

-19

-8

1.36 × 10 –4.36 × 10

TIRFM

7.20 × 10 –1.00 × 10

ELISA

8.52 × 10-12–1.70 × 10-8

TIRS-SRM

-20

1.41 × 10 –7.04 × 10

-15

5.88 × 10

5.32 × 10

2.88 × 10

8.52 × 10-12

this work

-21

this work

7.04 × 10

Abbreviations: UCL, upconverting luminescence; FPLS, fluorescence photoluminescence spectroscopy; SPFS, surface plasmon field-enhanced fluorescence spectroscopy; PEC, photoelectrochemical; ECI, electrochemical immunoassay; ECL, electrochemiluminescence; CLI, chemiluminescence immunoassay; RRS, resonance Rayleigh scattering; TIRFM, total internal reflection fluorescence microscopy; ELISA, enzyme-linked immunosorbent assay; LOD, limit of detection.


Analytical Chemistry Quantitative Analysis of AFP Molecules in Human Blood Samples. AFP was quantified using various detection methods including with an electrochemiluminescence immunosensor (2.81 aM),50 a chemiluminescence immunosensor (70 fM),51 and resonance scattering spectroscopy (13.6 pM)52 (Table 1). In particular, total internal reflection fluorescence (TIRF) microscopy had a limit of detection (LOD) of ~700 zM AFP.58 The LOD from commercial kit (i.e., ELISA) as a clinical immunoassay was 8.52 pM. Compared to previous methods, the developed TIRS-SRM method showed excellent detection sensitivity with a wide linear dynamic range of 14.08 zM and 7.04 fM (correlation coefficient, R = 0.9946) under 405-nm laser illumination (Figure 7A). The intensity was at least 15.5 times higher than the other fluorescence-free detection (i.e., enhanced dark field microscopy) (Figure S4). In addition, the LOD (SNR = 3) was 7.04 zM, a value at least 100 times lower than previous LODs using TIRF (700 zM).58 The AFP standard solution (1 fg/mL = 14.08 aM), normal (nonpathologic) human serum (top in Figure 7B, 0.08 fg/mL = 1.17 aM), and human serum spiked with AFP (bottom in Figure 7B, 1.18 fg/mL = 16.65 aM) were analyzed using the GNI arrays. The standard calibration curve could be used to detect AFP in human blood samples with high accuracy and super-sensitivity. These results also indicate that TIRS-SRM is an effective assay method for quantification of various disease-related protein molecules in human blood samples with super-sensitive and high reliability at the single-molecule level. A

B 18

0.25

14 10

Log [RSI]

6

y = 0.09x + 5.64 (R = 0.9946)

SI (×103)

2

LOD = 7.04 zM (S/N = 3)

14 10 6 2

-3

-2

-1

0

1

our recordings registered the spatial coordinates of each molecule with nanometer precision. In addition, this method was successfully applied to determine AFP in human blood samples. The TIRS-SRM method was demonstrated to be an effective tool for super-localizing nanoparticles on defined nanoscale regions at subdiffraction limit resolution and a reliable method for the quantitative detection of a disease-related protein molecule at the single-molecule level.

ASSOCIATED CONTENT Supporting Information Figure S1, TIRS imaging and scattering intensity value of the GNI without/with AFP antigen as negative control (PDF). Figure S2. the scattering intensity of signal and background noise of 20-nm silver nanotagged-AFP on 100-nm GNIs with respect to the change in NA. (PDF) Figure S3, wide-field TIRS images and super-localization of immunoplasmonic nanotags on GNIs arrays before and after immunoreactions (PDF). Figure S4, enhanced dark field imaging under 406 nm and 620 nm bandpass filter, and spectrum of the arc lamp (PDF). Movie S1, RSI of immunoplasmoic nanotag-AFP before and after immunoreaction on 100-nm GNIs under illumination of a 405-nm laser. (AVI). Movie S2, RSI measurements of immunoplasmonic nanotag-AFP on different selected GNIs shown in Figure 6B over time after immunoreaction under illumination at 405 nm and 635 nm (AVI). The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *

18

SI (×103)

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

Page 6 of 15

2

3

4

Log [AFP (aM)]

Figure 7. (A) Standard calibration curve of AFP molecules with a linear dynamic range of 14.08 zM–7.04 fM. (B) SI values of AFP molecules in only a human serum sample (top) and a serum sample spiked with AFP standard (bottom). Scale bar: 500 nm. RSI: relative scattering intensity. LOD: limit of detection. SNR: signal-tonoise ratio (S/N).

CONCLUSION The super-localization of individual immunoplasmonic nanotags on nanoscale substrate regions (GNIs) was coordinated by 2D Gaussian fitting using TIRS-SRM at the singleparticle level. Individual antigen-antibody interactions and binding events were confirmed by measuring changes of the centroid position distribution region of individual particles at the single-molecule level. The dispersed super-location of immunoplasmonic nanotags on GNIs was also identified at subdiffraction limit resolution. The centroid positions of GNIs and nanotags were calculated at less than 10 nm resolution. The quantification of AFP target molecules showed a wide linear dynamic range of 14.08 zM–7.04 fM (R = 0.9946). The LOD (SNR = 3) was 7.04 zM, which was 100–5 × 109 times lower than previous LODs. As well as demonstrating the ultimate biosensor performance of detecting individual AFP molecules,

E-mail: [email protected]. Tel.: +82 31-201-3349. Fax: +82 31201-2340.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work as supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (No. 2015R1A2A2A01003839).

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For TOC only

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

×

λ1 TIRS-SRM

2D Gaussian fitting

λ1 λ2

λ2

×

8


Page 9 of 15

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


A

B

EMCCD

λ1 λ2

EM CCD λ1 ×

OL

M M DP

CG GNI array

λ2

OL

L1 λ1 ××

M

EFL

DC

DP Z

Z


λ2

P

M

L2


A Absorbance (a. u.)

0.025

Nanotag Nanotag-4A3

8 nm

NH CO

NH OC

HN CO

O

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

Page 10 of 15

C HN

300

350

400

450

500

550

Wavelength (nm)

B

Nanotag-4A3

Nanotag

1 nm ACS Paragon Plus Environment

1 nm

600

Page 11 of 15

400

5.0

ⅲ

ⅱ

×102

2.5 × 103

RSI

ⅰ


A

500

600

700

800

900

Wavelength (nm)

0

500 nm

30

60

90

120

90

120

Distance

B

400

ⅱ

2.0 ×10

ⅲ

2.5 × 103

RSI

ⅰ


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


500

600

700

800

900

Wavelength (nm)

500 nm


0

30

60

Distance


20

10

16

8

12

6

SNR

RSI ( ×103 )

A

8 4

4 2

0

0

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

NA

NA

16

8

12

6

SNR

RSI ( ×103 )

B

8

4

4

2

0

0 0

10

20

30

40

0

10

Laser power (mW)

20

30

40

Laser power (mW)

C 30

10

25

8

20

SNR

RSI ( ×103 )

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

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4 2

5 0

6

0

50

100

150

200

250

300

0

0

Exposure time (ms)


50

100

150

200

Exposure time (ms)

250

300

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A λ1, 405 nm

λ2, 635 nm

5.0×102

67

RSI

5.0×103

RSI

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


68

69

70

Incident angle (θ)

71

67

68

69

70

Incident angle (θ)

71

B

50 nm

50 nm


50 nm


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

A

B

Page 14 of 15

ⅰ

ⅱ

ⅲ

ⅳ

ⅴ

ⅵ

ⅶ

ⅷ

ⅸ

GNI

∆xa ∆yb dcc

ⅰ

ⅱ

ⅲ

ⅳ

ⅴ

ⅵ

ⅶ

ⅷ

ⅸ

28.11 8.40 1.92 6.10 1.79 0.02 1.71 25.25 28.61 12.65 17.10 8.86 16.77 9.83 0.01 11.32 5.24 4.41 30.83 19.05 9.07 17.84 9.99 0.02 11.45 25.79 28.95 ACS Paragon Plus Environment

Page 15 of 15

A

B 18

0.25

14 10

Log [RSI]

6

y = 0.09x + 5.64 (R = 0.9946)

SI (×103)

2

18

LOD = 7.04 zM (S/N = 3)

14 10 6 2

-3

-2

-1

0

1

2

3

4

Log [AFP (aM)]


SI (×103)

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


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