- Email: [email protected]
Quantitative nanoimmunosensor based on dark-field illumination with enhanced sensitivity and on–off switching using scattering signals
Biosensors and Bioelectronics 79 (2016) 709–714
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Quantitative nanoimmunosensor based on dark-field illumination with enhanced sensitivity and on–off switching using scattering signals Seungah Lee a,1, He Nan b,1, Hyunung Yu c, Seong Ho Kang a,b,n a Department of Applied Chemistry and Institute of Natural Sciences, College of Applied Science, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea b Department of Chemistry, Graduate School, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea c Nanobio Fusion Research Center, Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 29 August 2015 Received in revised form 11 December 2015 Accepted 2 January 2016 Available online 4 January 2016
A nanoimmunosensor based on wavelength-dependent dark-field illumination with enhanced sensitivity was used to detect a disease-related protein molecule at zeptomolar (zM) concentrations. The assay platform of 100-nm gold nanospots could be selectively acquired using the wavelength-dependence of enhanced scattering signals from antibody-conjugated plasmonic silver nanoparticles (NPs) with on–off switching using optical filters. Detection of human thyroid-stimulating hormone (hTSH) at a sensitivity of 100 zM, which corresponds to 1–2 molecules per gold spot, was possible within a linear range of 100 zM–100 fM (R ¼0.9968). A significantly enhanced sensitivity ( 4-fold) was achieved with enhanced dark-field illumination compared to using a total internal reflection fluorescence immunosensor. Immunoreactions were confirmed via optical axial-slicing based on the spectral characteristics of two plasmonic NPs. This method of using wavelength-dependent dark-field illumination had an enhanced sensitivity and a wide, linear dynamic range of 100 zM–100 fM, and was an effective tool for quantitatively detecting a single molecule on a nanobiochip for molecular diagnostics. & 2016 Elsevier B.V. All rights reserved.
Keywords: Quantification Enhanced dark-field illumination Ultra-high sensitivity Switchable nanoimmunosensor
1. Introduction Methods for quantitatively detecting targets such as proteins, viruses, food poisoning-causing bacteria and nucleic acids at a high speed and with high sensitivity are important for disease diagnosis. Target molecular quantitative detection methods combine a molecule-specific detection system, an example being an immunoassay with a generating enzyme such as horseradish peroxidase. However, obtaining a single-molecule detection limit for ultra-trace amounts of a sample for conventional immunoassays is difficult. Developing analytical techniques with highly sensitive detection for disease diagnosis is important. Scattering imaging methods offer quantitative analysis for a broad class of immunoassays and are favored over fluorescence imaging methods, as scattering signals are at least 50 times more intense than fluorescence signals (Francois et al., 2003). Enhanced darkfield microscopy provides the light economy and increases the n Corresponding author at: Department of Applied Chemistry and Institute of Natural Sciences, College of Applied Science, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea. E-mail address: [email protected] (S.H. Kang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2016.01.003 0956-5663/& 2016 Elsevier B.V. All rights reserved.
resolution by employing a collimating light adapter, three collimating lenses, and a convex mirror (Vodyanoy et al., 2009a, 2009b; Vainrub et al., 2006; Vodyanoy and Pustovyy, 2009). These results in an enhanced sensitivity compared with conventional dark-field microscopy. In particular, the images from the enhanced dark-field showed 10-times higher signal-to-noise (S/N) ratios than those of the conventional dark-field (Zhang et al., 2015a). Furthermore, our enhanced dark-field illumination methods with wavelength selectivity have exceptional sensitivity for nanometaltagged proteins on nanoarray chips in sandwich immunoassays (Lee et al., 2013). Recently, Zhang et al. obtained super-resolution images of plasmonic nanometals by using an enhanced dark-field illumination system. They resolved the coordinates (x, y) of plasmonic nanometals by fitting their point spread function (PSF) with a two-dimensional Gaussian via a least-squares criterion algorithm (Zhang et al., 2015b). Plasmonic nanoparticles (NPs) are an important class of nonfluorescent probes that have large cross-sections, surface reaction under mild conditions, optical stability and resistance to photobleaching or photoblinking under continuous illumination (Wang and Tao, 2013; Stender et al., 2013; Crut et al., 2014). Plasmonic noble metal NPs have been used extensively to investigate NPbased catalysis (Zhang et al., 2011), disease diagnosis (Stoeva et al.,
710
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
2006; Georganopoulou et al., 2005; Chen et al., 2011), drug delivery (Paciotti et al., 2006; Cheng et al., 2008), sensor (Kreno et al., 2010; Lee and Mirkin, 2008), and photonic devices (Wurtz et al., 2007). The wavelength difference between silver and gold plasmonic NPs is used for immunoassays on nanobiochips for disease diagnosis (Lee et al., 2013; Lee and Kang, 2014; Lee et al., 2015a). Nanoarray chip immunoassays have been developed for detection using total internal reflection scattering based on the reconstruction of three-dimensional positions; these assays have nanoscale accuracy for lateral resolution (Lee et al., 2015b). However, nonfluorescence immunoassays of an infinitesimal sample concentration up to 10 nm axial resolution using enhanced dark-field microscopy with a z-nanopositioner have not yet been reported. Herein, we used high-sensitivity immunoassay chips and enhanced dark-field illumination to investigate the quantification, localization and interactions of antibodies and antigens. The detection limit was at the single-molecule level (1–2 molecules per gold spot) for ultrasensitive, quantitative analyses of images of the scattering signals. Based on the structural configuration of goldnanopatterned chips and antibody-conjugated silver NPs, the axial resolution ( 10 nm) of a sandwich immunoassay complex was determined using enhanced dark-field illumination with an axial nanopositioner.
for 4 h at room temperature and then stored for 12 h at 4 °C. Excess hydroxylamine was quenched with 10 mM Tris (pH 7.5) and 1 M glycine with incubation for 15 min. The final silver NP-antibody suspension was centrifuged (17,000 rpm, 90 min, 4 °C) to remove the unbound antibody. Silver NP antibodies were resuspended in a PBS buffer and stored at 4 °C. 2.3. hTSH sandwich nanoimmunoassays on chips Sandwich immunoassays of hTSH on gold-nanopatterned chips were fabricated as previously reported (Lee and Kang, 2013) (Supplementary Fig. S1B). Gold-nanopatterned chips (Supplementary Fig. S2) were immersed for 30 min in 4 mg/mL DSP in DMSO and rinsed with distilled water. Protein A/G was added at 0.1 mg/mL for 1 h to facilitate Fc binding. Unreacted succinimide groups were blocked with 10 mM Tris (pH 7.5) and 1 M glycine for 30 min. Chips were incubated with StabilGuard for 30 min and rinsed briefly with distilled water. Chips were incubated in 20 μL of 2 μg/mL TSH-antibody in PBS with a pH of 7.4 for 1 h. After washing, TSH standard protein was diluted and incubated on the chip for 1–4 h. For 100 zM assays, 500 μL of TSH were loaded for 1–2 molecules/spot. Monoclonal mouse TSH-antibody-silver NPs were reacted for 2 h for dark-field scattering images. Because of their size, the antibodies conjugated to the silver NPs were incubated for longer than the molecular antigens.
2. Materials and methods 2.1. Reagents 11-Mercaptoundecanoic acid (MUA, 95%), 6-mercapto-1-hexanol (MCH, 97%), 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO, 99.5%), 2(morpholino)ethanesulfonic acid (MES), glycine, and phosphatebuffered saline (PBS) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Dithiobis(succinimidyl propionate) (DSP) and Protein A/ G 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 NPs approximately 80 nm in diameter (1.83 pM, 1.10 109 particles/mL) were obtained from BBI Life Sciences (Cardiff, UK). Monoclonal mouse anti-human thyroid stimulating hormone (TSH-antibody, 2TS11-10C7 and 2TS11-5E8) was purchased from HyTest (Turku, Finland) and human thyroid stimulating hormone protein (hTSH-antigen, 30-AT09) was purchased from Fitzgerald (North Acton, MA, USA). Samples were spiked with standard protein in human serum. 2.2. Antibody conjugation on silver NP surfaces Antibody conjugation to silver NPs was performed in two steps (Supplementary Fig. S1A). First, 10 mM MUA and 30 mM MCH in ethanol were added to a water-based NP solution. The solution was sonicated for 10 min and incubated for 2.5 h to form a selfassembled monolayer of MUA-MCH on the silver NP surface, which was washed with ultrapure water and centrifuged (12,000 rpm, 90 min, 4 °C). Silver NP-MUA-MCH was suspended in 50 mM MES and 0.1 M NaCl (pH 6.0) to generate a carboxylic acidterminated alkanethiol monolayer on the surface. Second, antibody was conjugated to silver NP-MUA-MCH by adding 40 mg EDC (2 mg/mL in 50 mM MES pH 6.0) and 196 μg Nhydroxysulfosuccinimide (NHSS, Pierce, IL, USA; 2 mg/mL in 1 PBS). After stirring at room temperature for 40 min, silver NPMUA-MCH-NHSS was washed by centrifugation (17,000 rpm, 30 min, 4 °C) and dissolved in 1 PBS, and then 10 μg/mL monoclonal mouse anti-human TSH-antibody in PBS (pH 7.4) was added. Reactions in the solution occurred while on a rotary shaker
2.4. Wavelength-dependent enhanced dark-field illumination detection Silver NP-TSH antibodies on chips were studied by resonant light scattering using a lab-built wavelength-dependent enhanced dark-field microscope with an enhanced dark-field illumination device (Cytoviva Inc., Auburn, AL, USA) attached to an upright Olympus BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) (Fig. 1). 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 metal halide light source (Welch Allyn, Skaneateles Falls, NY, USA) (Supplementary Fig. S3). A 100 oil iris objective (UPLANFLN, adjustable numerical aperture, from 0.6 to 1.3) was used for dark-field light scattering imaging. The smallest NA value (i.e., 0.6) was used in all of the experiments due to the low noise and background scattering (Taylor and Bowen, 2013; Ha et al., 2012). Bandpass filters (473/10 nm and 605/10 nm) from Semrock (Rochester, NY, USA) were used for wavelength selection. Color dark-field light scattering photographs and high-sensitivity dark-field light scattering images of silver NP-TSH antibodies and gold nanospots were captured with a Nikon D3S digital camera (Tokyo, Japan) for qualitative analysis as well as with an electron-multiplying charge-coupled device camera (QuantEM 512SC, Photometrics, AZ, USA) for quantitative analysis. Z-axis sectioning at 10-nm intervals was achieved using a z-nanopositioner (LEP MAC 5000, LUDL Electronic Products Ltd. NY, USA). 2.5. Quantitative analysis of hTSH Relative plasmon resonance scattering (PRS) signals were corrected for the background and filtered for nonvalid spots. Images were acquired by changing bandpass filters (473/10 nm and 605/ 10 nm) to switch the scattering signals of silver NPs and gold nanospots on and off. The relative scattering intensity (RSI) of gold spots was measured with a 605/10 nm bandpass filter; the RSI of the sliver NPs was measured with a 473/10 nm bandpass filter. The relative intensity of PRS signals before and after silver NP-TSH antibody immune reactions on gold-nanopatterned chips as a function of wavelength was calculated using the average corrected
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
(A)
EMCCD
(B)
DSLR
711
(C)
EM CCD
Silver NPs
Linkers Au
DSLR BF BF
OL
OL
GNC
Cr
Z
EDFC Z
EDFC
Fig. 1. Physical layout (A) and schematic diagram (B) of the wavelength-dependent dark-field illumination system. Schematic diagram (C) of the sandwich nanoimmunoassay on gold-nanopatterned chips. Z, z-nanopositioner; NPs, nanoparticles; GNC, gold-nanopatterned chip; OL, objective lens; BF, bandpass filter; DSLR, digital single-lens reflex; EMCCD, electron-multiplying charge-coupled device; EDFC, enhanced dark-field condenser.
(A) AFM
120
Height (nm)
100 80 60 40
~120 nm
20 0 0
200 nm
0.1 0.2 0.3 0.4 0.5
Distance (µm)
(B) SEM
100 nm
100 nm
Fig. 2. (A) AFM height profiles after immunoreaction with antibody-labeled silver NPs on 100-nm gold-nanopatterned chips. (B) SEM images of 80-nm silver NP-TSH antibody (right, arrows) on a 100-nm gold spot (left). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
PRS intensity for antigen concentration. In addition, we quantitatively analyzed images from the wavelength-dependent enhanced dark-field illumination system as follows. First, signal and background regions with the same area were selected. Second, we calculated the sum of the PRS intensities of occupied pixels per spot, corrected by background subtraction. Calibration curves and quantified values of standard TSH were calculated as the sum of the PRS intensities from 20 spots on a 4 5 gold-nanopatterned chip. Calibration curves were fitted using the Excel software (Version 2010, Microsoft Co., Redmond, WA, USA). Images were acquired and data were analyzed using MetaMorph (Version 7.0, Universal Imaging, Sunnyvale, CA, USA).
2.6. SEM and AFM images A scanning electron microscope (SEM, Quanta FEG 650, FEI Co., OR, USA) with an accelerating voltage of 30 kV was used to confirm immunoreactions. A commercial atomic force microscope (AFM, n-Tracer, Nanofocus Inc., Oberhausen, Germany) was used for the measurement, which has a separate piezoelectric response between the XY-motion and the Z-approach. For measurement of the surface topography, AFM was used in a tapping mode with a resonant frequency of the probe set to 61.2 kHz and a scan rate of 0.5 Hz.
712
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
3. Results and discussion 3.1. Sandwich nanoimmunoreactions on gold-nanopatterned chips The structure of sandwich nanoimmunoassays on chips was a gold nanospot, antibody-conjugated silver NPs, and biomolecules such as DSP linker, protein A/G, antibody, or antigen. According to a previous study (Lee and Kang, 2014), the average height of the 100-nm gold nanospots was approximately 14.5 nm. Other lengths for molecules oriented on the gold nanospot were 1.2 nm for DSP, 1.4–5.0 nm for protein A/G, 14.5 nm for the primary antibody, 4.0– 6.5 nm for the antigen and 14.5 nm for the second antibody (Bjork et al., 1972; Lee et al., 2007; Lynch et al., 2004). The diameter of the silver NP was 80 nm. Theoretically, the total height of the sandwich immunoreacted molecules on gold nanospots on glass substrates was approximately 130–136 nm. AFM was used to confirm immunoreactions. The height of 100-nm gold spots increased by 120 nm after immunoreaction with TSH protein and 80-nm silver NPs (Fig. 2A). SEM images detected 80-nm silver NPs on 100-nm gold spots (Fig. 2B). Although the gold spots did not have exactly the same aspect ratios, target TSHs with silver NP-detection probes might have been produced during sandwich nanoimmunoreactions on the chips. 3.2. Plasmonic metal dependence on wavelength Primary antibody immobilized on gold-nanopatterned chips was employed to capture the target antigens. Silver NP-labeled secondary antibodies were used to form a sandwich immune complex on the chips. The plasmon resonance of silver NPs was typically in the blue–green wavelength range at a lower wavelength than gold. The intrinsic scattering color of a single 100-nm gold spot was yellow (Absmax ¼650 nm), while the resonant scattering peak of the chip after immunoreaction with the silver NPlabeled secondary antibody (Absmax ¼456 nm, Supplementary Fig. S4) was blue-shifted (Absmax ¼ 600 nm) (Lee et al., 2013). The
(B) After reaction
3.3. Axial resolution based on optical sectioning Gold and silver NPs have different plasmon resonance values corresponding to their wavelengths. Gold nanospots have strong PRS after treatment with approximately 600 nm light. The PRS of silver NPs is in the 460-nm region (Lee et al., 2013). Images of gold nanospots on the bottom and the silver NPs on the top were acquired based on z-slices in 10-nm steps in the sandwich immunoassay. Relative PRS intensities were corrected as the sum of PRS intensities after background subtraction of 20 gold spots on 4 5 nanopatterned chips. Gold-nanopatterned chips were optically sectioned at 10 nm with an axial nanopositioner, and the intensity distribution was plotted from the sliver NP to the gold spot for the z-orientation of biomolecules on the gold nanospots (Fig. 3C). In the axial intensity (Iz) distribution of the PSF, the locations of the maximum RSI before and after the immunoreaction show a clear distinction in scattering intensity profiles. In an aberration-corrected wide-field microscope, resolution is most often approximated to have an isotropic Gaussian profile in the lateral (x, y)-dimensions, whose full-width-at-half-maximum
(C) Profiles of scattering intensity
Relative scattering intensity
(A) Before reaction
intrinsic scattering color changed from yellow to blue after immunoreaction with the silver NP-labeled secondary antibody (Fig. 3A and B). These results indicated that silver NP-labeled secondary antibodies with an emission maximum around 456 nm were linked at the top of the gold nanospot and had a resonant scattering peak around 600 nm, according to the sandwich immunoreaction. In addition, we carried out sandwich immunoassay without TSH antigen and detected the scattering image as a negative control to demonstrate the specificity of the chip using the Nikon D3S digital camera (Supplementary Fig. S5). The scattering image of the chip after immunoreaction without antigen did not show up as blue because the color is a characteristic of the intrinsic scattering color of silver NP (Lee et al., 2013). The result demonstrates that the non-specific binding of silver NP-antibodies has not occurred on the chip.
0
2
4
6
Z-position (nm, ×103)
8
10
Fig. 3. Color camera images before (A, orange) and after (B, blue) immunoreaction. Profiles of the scattering intensity before (red dot) and after (blue dot) immunoreaction (C). The standard TSH concentration was 1 fM. Profiles of scattering intensity were acquired with sequential z-axis imaging at 10-nm intervals from silver NPs to gold spot using a high-sensitivity electron-multiplying charge-coupled device camera. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
(theoretical concentration¼8.0 fM) and was quantified using the chip (experiment concentration ¼7.7 fM). The fluorescent-free detection of biomolecules on nanobiochips using a combination of wavelength-dependent enhanced dark-field illumination and a plasmonic NP probe might be used for clinical diagnosis with ultrahigh sensitivity.
Relative scattering intensity
300 250 y = 35.629x - 56.043 R = 0.9968 (100 zM-100 fM)
200
713
150 4. Conclusions
100 50 0
1
2
3
4
5
6
7
8
9
Log [TSH (zM)] Fig. 4. Quantitative PRS response curves for target protein molecules (standard TSH) in the linear dynamic concentrations range of 100 zM–100 fM using a highsensitivity electron-multiplying charge-coupled device camera. Relative scattering intensities were corrected via background subtraction.
(FWHM) value is expressed by Eq. (1) (Brauchle et al., 2010):
FWHMx, y =
λ , 2NA
(1)
where λ is the emission wavelength and NA is the numerical aperture of the microscope objective. The FWHM value of the PSF in the axial (z)-dimension is expressed as Eq. (2):
FWHMz =
2λ . NA2
In summary, an ultrahigh-sensitivity, quantitative nanoimmunoassay using antibody-conjugated plasmonic NPs was investigated using simple switchable signals of an optical bandpass filter coupled with enhanced dark-field illumination. A silver NP probe and a single gold spot were selectively localized using an axial scattering intensity profile acquired with sequential z-axis images at 10-nm intervals. Nanoimmunoreactions were confirmed by measuring heights through the axial orientation of biomolecules using z-axis sectioning of intensities. Depending on the wavelength, the quantification of human TSH had a wide dynamic linear range from 100 zM to 100 fM with a strong correlation coefficient (R ¼0.9968) by suppressing the scattering intensity of the gold spot. Therefore, this method can be suggested as an effective immunoassay tool based on non-fluorescence three-dimensional super-resolution imaging of biomolecules on nanobiochips at single molecule level (1–2 molecules/spot).
Acknowledgments
(2)
In single-molecule experiments, high-NA objectives were used to collect the maximum number of photons. Physical limitations restrained the maximum collection of NA to about 1.3 for oil immersion objectives. In this case, the extent of the PSF axial profile typically approaches about 716 nm (at 605-nm wavelength) and 560 nm (at 473-nm wavelength). This measurement is roughly two times the lateral profile. The axial position is not as readily discernible because: (1) the PSF shape is symmetrical around the optical axis and the focal plane in the absence of aberrations in the optical system; and (2) the lateral width of the PSF varies little near the focal plane (Thompson et al., 2002; Mortensen et al., 2010; Hajj et al., 2014). By this method, deciding the accurate axial location of two nanometals after immunoreaction is difficult. However, two mountaintop gradations were observed in the scattering intensity profile (Fig. 3C). This finding was attributed to antibody-conjugated silver NPs immunoreacting on the 100-nm gold spots. 3.4. Quantification of the TSH protein on gold-nanopatterned chips TSH protein quantitatively analyzed by labeling plasmonic NPs and using wavelength-dependent dark-field illumination had enhanced sensitivity. A strong relationship was observed between the relative increase in the enhanced dark-field illumination intensity of the immune-targeted silver NP-antibody and the standard TSH concentration on a 100-nm gold-patterned chip. The linear dynamic range was seven orders, from 100 zM to 100 fM (Fig. 4). The method achieved a limit of detection (LOD) of 100 zM, corresponding to 1–2 molecules/gold spot. This value was approximately 4 times lower than the LOD obtained by a previous fluorescent labeling method (Lee and Kang, 2012). The value was, however, superior to the guideline value of the National Academy of Clinical Biochemistry (0.02 mI U/L¼ 118.7 fM) (Baloch et al., 2003). A clinical sample spiked with normal human serum
This research was 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) and by a Nano-Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (2009-0082580).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.01.003.
References Baloch, Z., Carayon, P., Conte-Devolx, B., Demers, L.M., Feldt-Rasmussen, U., Henry, J. F., LiVosli, V.A., Niccoli-Sire, P., John, R., Ruf, J., Smyth, P.P., Spencer, C.A., Stockigt, J.R., 2003. Thyroid 13, 3–126. Bjork, I., Petersson, B.A., Sjöquist, J., 1972. Eur. J. Biochem. 29, 579–584. Brauchle C., Lamb D.C. and Michaelis J., 2010. Single particle tracking and single molecule energy transfer, Wiley VCH, pp. 5. Crut, A., Maioli, P., Del Fatti, N., Vallee, F., 2014. Chem. Soc. Rev. 43, 3921–3956. Chen, S., Svedendahl, M., Duyne, R.P., Käll, M., 2011. Nano Lett. 11, 1826–1830. Cheng, Y., Samia, A.C., Meyers, J.D., Panagopoulos, I., Fei, B., Burda, C., 2008. J. Am. Chem. Soc. 130, 10643–10647. Francois, P., Bento, M., Vaudaux, P., Schrenzel, J., 2003. J. Microbiol. Methods 55, 755–762. Georganopoulou, D.G., Chang, L., Nam, J.M., Thaxton, C.S., Mufson, E.J., Klein, W.L., Mirkin, C.A., 2005. Proc. Natl. Acad. Sci. USA 102, 2273–2776. Hajj, B., Beheiry, M.E., Izeddin, I., Darzacq, X., Dahan, M., 2014. Phys. Chem. Chem. Phys. 16, 16340–16348. Ha, J.W., Marchuk, K., Fang, N., 2012. Nano Lett. 12, 4282–4288. Kreno, L.E., Hupp, J.T., Van Duyne, R.P., 2010. Anal. Chem. 82, 8042–8046. Lee, J.M., Park, H.K., Jung, Y., Kim, J.K., Jung, S.O., Chung, B.H., 2007. Anal. Chem. 79, 2680–2687. Lee, J.S., Mirkin, C.A., 2008. Anal. Chem. 80, 6805–6808. Lee, S., Kang, S.H., 2014. Biosens. Bioelectron. 60, 45–51. Lee, S., Kang, S.H., 2013. Analyst 138, 3478–3482. Lee, S., Kang, S.H., 2012. Talanta 99, 1030–1034.
714
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
Lee, S., Park, G., Chakkarapani, S.K., Kang, S.H., 2015a. Biosens. Bioelectron. 63, 444–449. Lee, S., Yu, H., Kang, S.H., 2013. Chem. Commun. 49, 8335–8337. Lee, S., Yu, H., Kang, S.H., 2015b. Chem. Commun. 51, 945–947. Lynch, M., Mosher, C., Huff, J., Nettikadan, S., Johnson, J., Henderson, E., 2004. Proteomics 4, 1695–1702. Mortensen, K.I., Churchman, L.S., Spudich, J.A., Flyvbjerg, H., 2010. Nat. Methods 7, 377–381. Paciotti, G.F., Kingston, D.G.I., Tamarkin, L., 2006. DrugDev. Res. 67, 47–54. Stender, A.S., Marchuk, K., Liu, C., Sander, S., Meyer, M.W., Smith, E.A., Neupane, B., Wang, G., Li, J., Cheng, J.X., Huang, B., Fang, N., 2013. Chem. Rev. 113, 2469–2527. Stoeva, S.I., Lee, J.S., Smith, J.E., Rosen, S.T., Mirkin, C.A., 2006. J. Am. Chem. Soc. 128, 8378–8379. Taylor, M.A., Bowen, W.P., 2013. Appl.Optics 52, 5718–5723.
Thompson, R.E., Larson, D.R., Webb, W.W., 2002. Biophys. J. 82, 2775–2783. Vainrub, A., Pustovyy, O., Vodyanoy, V., 2006. Opt. Lett. 31, 2855–2857. Vodyanoy, V., Pustovyy, O., 2009. Patent No. US 7,564,623 B2, Jul 21. Vodyanoy, V., Vainrub, A., Pustovyy, O., 2009a. Patent No. US 7,551,349 B2, Jun 23. Vodyanoy, V., Pustovyy, O., Vainrub, A., 2009b. Patent No. US 7,542,203 B2, Jun 2. Wang, W., Tao, N., 2013. Anal. Chem. 86, 2–14. Wurtz, G.A., Evans, P.R., Hendren, W., Atkinson, R., Dickson, W., Pollard, R.J., Zayats, A.V., Harrison, W., Bower, C., 2007. Nano Lett. 7, 1297–1303. Zhang, L., Li, Y., Li, D.W., Jing, C., Chen, X., Lv, M., Huang, Q., Long, Y.T., Willner, I., 2011. Angew. Chem. Int. Ed. 123, 6921–6924. Zhang, P., Park, S., Kang, S.H., 2015a. Bull. Korean Chem. Soc. 36, 1172–1177. Zhang, P., Lee, S., Yu, H., Fang, N., Kang, S.H., 2015b. Sci. Rep. 5, 11447. http://dx.doi. org/10.1038/srep11447.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Quantitative nanoimmunosensor based on dark-field illumination with enhanced sensitivity and on–off switching using scattering signals Seungah Lee a,1, He Nan b,1, Hyunung Yu c, Seong Ho Kang a,b,n a Department of Applied Chemistry and Institute of Natural Sciences, College of Applied Science, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea b Department of Chemistry, Graduate School, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea c Nanobio Fusion Research Center, Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 29 August 2015 Received in revised form 11 December 2015 Accepted 2 January 2016 Available online 4 January 2016
A nanoimmunosensor based on wavelength-dependent dark-field illumination with enhanced sensitivity was used to detect a disease-related protein molecule at zeptomolar (zM) concentrations. The assay platform of 100-nm gold nanospots could be selectively acquired using the wavelength-dependence of enhanced scattering signals from antibody-conjugated plasmonic silver nanoparticles (NPs) with on–off switching using optical filters. Detection of human thyroid-stimulating hormone (hTSH) at a sensitivity of 100 zM, which corresponds to 1–2 molecules per gold spot, was possible within a linear range of 100 zM–100 fM (R ¼0.9968). A significantly enhanced sensitivity ( 4-fold) was achieved with enhanced dark-field illumination compared to using a total internal reflection fluorescence immunosensor. Immunoreactions were confirmed via optical axial-slicing based on the spectral characteristics of two plasmonic NPs. This method of using wavelength-dependent dark-field illumination had an enhanced sensitivity and a wide, linear dynamic range of 100 zM–100 fM, and was an effective tool for quantitatively detecting a single molecule on a nanobiochip for molecular diagnostics. & 2016 Elsevier B.V. All rights reserved.
Keywords: Quantification Enhanced dark-field illumination Ultra-high sensitivity Switchable nanoimmunosensor
1. Introduction Methods for quantitatively detecting targets such as proteins, viruses, food poisoning-causing bacteria and nucleic acids at a high speed and with high sensitivity are important for disease diagnosis. Target molecular quantitative detection methods combine a molecule-specific detection system, an example being an immunoassay with a generating enzyme such as horseradish peroxidase. However, obtaining a single-molecule detection limit for ultra-trace amounts of a sample for conventional immunoassays is difficult. Developing analytical techniques with highly sensitive detection for disease diagnosis is important. Scattering imaging methods offer quantitative analysis for a broad class of immunoassays and are favored over fluorescence imaging methods, as scattering signals are at least 50 times more intense than fluorescence signals (Francois et al., 2003). Enhanced darkfield microscopy provides the light economy and increases the n Corresponding author at: Department of Applied Chemistry and Institute of Natural Sciences, College of Applied Science, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea. E-mail address: [email protected] (S.H. Kang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2016.01.003 0956-5663/& 2016 Elsevier B.V. All rights reserved.
resolution by employing a collimating light adapter, three collimating lenses, and a convex mirror (Vodyanoy et al., 2009a, 2009b; Vainrub et al., 2006; Vodyanoy and Pustovyy, 2009). These results in an enhanced sensitivity compared with conventional dark-field microscopy. In particular, the images from the enhanced dark-field showed 10-times higher signal-to-noise (S/N) ratios than those of the conventional dark-field (Zhang et al., 2015a). Furthermore, our enhanced dark-field illumination methods with wavelength selectivity have exceptional sensitivity for nanometaltagged proteins on nanoarray chips in sandwich immunoassays (Lee et al., 2013). Recently, Zhang et al. obtained super-resolution images of plasmonic nanometals by using an enhanced dark-field illumination system. They resolved the coordinates (x, y) of plasmonic nanometals by fitting their point spread function (PSF) with a two-dimensional Gaussian via a least-squares criterion algorithm (Zhang et al., 2015b). Plasmonic nanoparticles (NPs) are an important class of nonfluorescent probes that have large cross-sections, surface reaction under mild conditions, optical stability and resistance to photobleaching or photoblinking under continuous illumination (Wang and Tao, 2013; Stender et al., 2013; Crut et al., 2014). Plasmonic noble metal NPs have been used extensively to investigate NPbased catalysis (Zhang et al., 2011), disease diagnosis (Stoeva et al.,
710
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
2006; Georganopoulou et al., 2005; Chen et al., 2011), drug delivery (Paciotti et al., 2006; Cheng et al., 2008), sensor (Kreno et al., 2010; Lee and Mirkin, 2008), and photonic devices (Wurtz et al., 2007). The wavelength difference between silver and gold plasmonic NPs is used for immunoassays on nanobiochips for disease diagnosis (Lee et al., 2013; Lee and Kang, 2014; Lee et al., 2015a). Nanoarray chip immunoassays have been developed for detection using total internal reflection scattering based on the reconstruction of three-dimensional positions; these assays have nanoscale accuracy for lateral resolution (Lee et al., 2015b). However, nonfluorescence immunoassays of an infinitesimal sample concentration up to 10 nm axial resolution using enhanced dark-field microscopy with a z-nanopositioner have not yet been reported. Herein, we used high-sensitivity immunoassay chips and enhanced dark-field illumination to investigate the quantification, localization and interactions of antibodies and antigens. The detection limit was at the single-molecule level (1–2 molecules per gold spot) for ultrasensitive, quantitative analyses of images of the scattering signals. Based on the structural configuration of goldnanopatterned chips and antibody-conjugated silver NPs, the axial resolution ( 10 nm) of a sandwich immunoassay complex was determined using enhanced dark-field illumination with an axial nanopositioner.
for 4 h at room temperature and then stored for 12 h at 4 °C. Excess hydroxylamine was quenched with 10 mM Tris (pH 7.5) and 1 M glycine with incubation for 15 min. The final silver NP-antibody suspension was centrifuged (17,000 rpm, 90 min, 4 °C) to remove the unbound antibody. Silver NP antibodies were resuspended in a PBS buffer and stored at 4 °C. 2.3. hTSH sandwich nanoimmunoassays on chips Sandwich immunoassays of hTSH on gold-nanopatterned chips were fabricated as previously reported (Lee and Kang, 2013) (Supplementary Fig. S1B). Gold-nanopatterned chips (Supplementary Fig. S2) were immersed for 30 min in 4 mg/mL DSP in DMSO and rinsed with distilled water. Protein A/G was added at 0.1 mg/mL for 1 h to facilitate Fc binding. Unreacted succinimide groups were blocked with 10 mM Tris (pH 7.5) and 1 M glycine for 30 min. Chips were incubated with StabilGuard for 30 min and rinsed briefly with distilled water. Chips were incubated in 20 μL of 2 μg/mL TSH-antibody in PBS with a pH of 7.4 for 1 h. After washing, TSH standard protein was diluted and incubated on the chip for 1–4 h. For 100 zM assays, 500 μL of TSH were loaded for 1–2 molecules/spot. Monoclonal mouse TSH-antibody-silver NPs were reacted for 2 h for dark-field scattering images. Because of their size, the antibodies conjugated to the silver NPs were incubated for longer than the molecular antigens.
2. Materials and methods 2.1. Reagents 11-Mercaptoundecanoic acid (MUA, 95%), 6-mercapto-1-hexanol (MCH, 97%), 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO, 99.5%), 2(morpholino)ethanesulfonic acid (MES), glycine, and phosphatebuffered saline (PBS) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Dithiobis(succinimidyl propionate) (DSP) and Protein A/ G 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 NPs approximately 80 nm in diameter (1.83 pM, 1.10 109 particles/mL) were obtained from BBI Life Sciences (Cardiff, UK). Monoclonal mouse anti-human thyroid stimulating hormone (TSH-antibody, 2TS11-10C7 and 2TS11-5E8) was purchased from HyTest (Turku, Finland) and human thyroid stimulating hormone protein (hTSH-antigen, 30-AT09) was purchased from Fitzgerald (North Acton, MA, USA). Samples were spiked with standard protein in human serum. 2.2. Antibody conjugation on silver NP surfaces Antibody conjugation to silver NPs was performed in two steps (Supplementary Fig. S1A). First, 10 mM MUA and 30 mM MCH in ethanol were added to a water-based NP solution. The solution was sonicated for 10 min and incubated for 2.5 h to form a selfassembled monolayer of MUA-MCH on the silver NP surface, which was washed with ultrapure water and centrifuged (12,000 rpm, 90 min, 4 °C). Silver NP-MUA-MCH was suspended in 50 mM MES and 0.1 M NaCl (pH 6.0) to generate a carboxylic acidterminated alkanethiol monolayer on the surface. Second, antibody was conjugated to silver NP-MUA-MCH by adding 40 mg EDC (2 mg/mL in 50 mM MES pH 6.0) and 196 μg Nhydroxysulfosuccinimide (NHSS, Pierce, IL, USA; 2 mg/mL in 1 PBS). After stirring at room temperature for 40 min, silver NPMUA-MCH-NHSS was washed by centrifugation (17,000 rpm, 30 min, 4 °C) and dissolved in 1 PBS, and then 10 μg/mL monoclonal mouse anti-human TSH-antibody in PBS (pH 7.4) was added. Reactions in the solution occurred while on a rotary shaker
2.4. Wavelength-dependent enhanced dark-field illumination detection Silver NP-TSH antibodies on chips were studied by resonant light scattering using a lab-built wavelength-dependent enhanced dark-field microscope with an enhanced dark-field illumination device (Cytoviva Inc., Auburn, AL, USA) attached to an upright Olympus BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) (Fig. 1). 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 metal halide light source (Welch Allyn, Skaneateles Falls, NY, USA) (Supplementary Fig. S3). A 100 oil iris objective (UPLANFLN, adjustable numerical aperture, from 0.6 to 1.3) was used for dark-field light scattering imaging. The smallest NA value (i.e., 0.6) was used in all of the experiments due to the low noise and background scattering (Taylor and Bowen, 2013; Ha et al., 2012). Bandpass filters (473/10 nm and 605/10 nm) from Semrock (Rochester, NY, USA) were used for wavelength selection. Color dark-field light scattering photographs and high-sensitivity dark-field light scattering images of silver NP-TSH antibodies and gold nanospots were captured with a Nikon D3S digital camera (Tokyo, Japan) for qualitative analysis as well as with an electron-multiplying charge-coupled device camera (QuantEM 512SC, Photometrics, AZ, USA) for quantitative analysis. Z-axis sectioning at 10-nm intervals was achieved using a z-nanopositioner (LEP MAC 5000, LUDL Electronic Products Ltd. NY, USA). 2.5. Quantitative analysis of hTSH Relative plasmon resonance scattering (PRS) signals were corrected for the background and filtered for nonvalid spots. Images were acquired by changing bandpass filters (473/10 nm and 605/ 10 nm) to switch the scattering signals of silver NPs and gold nanospots on and off. The relative scattering intensity (RSI) of gold spots was measured with a 605/10 nm bandpass filter; the RSI of the sliver NPs was measured with a 473/10 nm bandpass filter. The relative intensity of PRS signals before and after silver NP-TSH antibody immune reactions on gold-nanopatterned chips as a function of wavelength was calculated using the average corrected
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
(A)
EMCCD
(B)
DSLR
711
(C)
EM CCD
Silver NPs
Linkers Au
DSLR BF BF
OL
OL
GNC
Cr
Z
EDFC Z
EDFC
Fig. 1. Physical layout (A) and schematic diagram (B) of the wavelength-dependent dark-field illumination system. Schematic diagram (C) of the sandwich nanoimmunoassay on gold-nanopatterned chips. Z, z-nanopositioner; NPs, nanoparticles; GNC, gold-nanopatterned chip; OL, objective lens; BF, bandpass filter; DSLR, digital single-lens reflex; EMCCD, electron-multiplying charge-coupled device; EDFC, enhanced dark-field condenser.
(A) AFM
120
Height (nm)
100 80 60 40
~120 nm
20 0 0
200 nm
0.1 0.2 0.3 0.4 0.5
Distance (µm)
(B) SEM
100 nm
100 nm
Fig. 2. (A) AFM height profiles after immunoreaction with antibody-labeled silver NPs on 100-nm gold-nanopatterned chips. (B) SEM images of 80-nm silver NP-TSH antibody (right, arrows) on a 100-nm gold spot (left). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
PRS intensity for antigen concentration. In addition, we quantitatively analyzed images from the wavelength-dependent enhanced dark-field illumination system as follows. First, signal and background regions with the same area were selected. Second, we calculated the sum of the PRS intensities of occupied pixels per spot, corrected by background subtraction. Calibration curves and quantified values of standard TSH were calculated as the sum of the PRS intensities from 20 spots on a 4 5 gold-nanopatterned chip. Calibration curves were fitted using the Excel software (Version 2010, Microsoft Co., Redmond, WA, USA). Images were acquired and data were analyzed using MetaMorph (Version 7.0, Universal Imaging, Sunnyvale, CA, USA).
2.6. SEM and AFM images A scanning electron microscope (SEM, Quanta FEG 650, FEI Co., OR, USA) with an accelerating voltage of 30 kV was used to confirm immunoreactions. A commercial atomic force microscope (AFM, n-Tracer, Nanofocus Inc., Oberhausen, Germany) was used for the measurement, which has a separate piezoelectric response between the XY-motion and the Z-approach. For measurement of the surface topography, AFM was used in a tapping mode with a resonant frequency of the probe set to 61.2 kHz and a scan rate of 0.5 Hz.
712
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
3. Results and discussion 3.1. Sandwich nanoimmunoreactions on gold-nanopatterned chips The structure of sandwich nanoimmunoassays on chips was a gold nanospot, antibody-conjugated silver NPs, and biomolecules such as DSP linker, protein A/G, antibody, or antigen. According to a previous study (Lee and Kang, 2014), the average height of the 100-nm gold nanospots was approximately 14.5 nm. Other lengths for molecules oriented on the gold nanospot were 1.2 nm for DSP, 1.4–5.0 nm for protein A/G, 14.5 nm for the primary antibody, 4.0– 6.5 nm for the antigen and 14.5 nm for the second antibody (Bjork et al., 1972; Lee et al., 2007; Lynch et al., 2004). The diameter of the silver NP was 80 nm. Theoretically, the total height of the sandwich immunoreacted molecules on gold nanospots on glass substrates was approximately 130–136 nm. AFM was used to confirm immunoreactions. The height of 100-nm gold spots increased by 120 nm after immunoreaction with TSH protein and 80-nm silver NPs (Fig. 2A). SEM images detected 80-nm silver NPs on 100-nm gold spots (Fig. 2B). Although the gold spots did not have exactly the same aspect ratios, target TSHs with silver NP-detection probes might have been produced during sandwich nanoimmunoreactions on the chips. 3.2. Plasmonic metal dependence on wavelength Primary antibody immobilized on gold-nanopatterned chips was employed to capture the target antigens. Silver NP-labeled secondary antibodies were used to form a sandwich immune complex on the chips. The plasmon resonance of silver NPs was typically in the blue–green wavelength range at a lower wavelength than gold. The intrinsic scattering color of a single 100-nm gold spot was yellow (Absmax ¼650 nm), while the resonant scattering peak of the chip after immunoreaction with the silver NPlabeled secondary antibody (Absmax ¼456 nm, Supplementary Fig. S4) was blue-shifted (Absmax ¼ 600 nm) (Lee et al., 2013). The
(B) After reaction
3.3. Axial resolution based on optical sectioning Gold and silver NPs have different plasmon resonance values corresponding to their wavelengths. Gold nanospots have strong PRS after treatment with approximately 600 nm light. The PRS of silver NPs is in the 460-nm region (Lee et al., 2013). Images of gold nanospots on the bottom and the silver NPs on the top were acquired based on z-slices in 10-nm steps in the sandwich immunoassay. Relative PRS intensities were corrected as the sum of PRS intensities after background subtraction of 20 gold spots on 4 5 nanopatterned chips. Gold-nanopatterned chips were optically sectioned at 10 nm with an axial nanopositioner, and the intensity distribution was plotted from the sliver NP to the gold spot for the z-orientation of biomolecules on the gold nanospots (Fig. 3C). In the axial intensity (Iz) distribution of the PSF, the locations of the maximum RSI before and after the immunoreaction show a clear distinction in scattering intensity profiles. In an aberration-corrected wide-field microscope, resolution is most often approximated to have an isotropic Gaussian profile in the lateral (x, y)-dimensions, whose full-width-at-half-maximum
(C) Profiles of scattering intensity
Relative scattering intensity
(A) Before reaction
intrinsic scattering color changed from yellow to blue after immunoreaction with the silver NP-labeled secondary antibody (Fig. 3A and B). These results indicated that silver NP-labeled secondary antibodies with an emission maximum around 456 nm were linked at the top of the gold nanospot and had a resonant scattering peak around 600 nm, according to the sandwich immunoreaction. In addition, we carried out sandwich immunoassay without TSH antigen and detected the scattering image as a negative control to demonstrate the specificity of the chip using the Nikon D3S digital camera (Supplementary Fig. S5). The scattering image of the chip after immunoreaction without antigen did not show up as blue because the color is a characteristic of the intrinsic scattering color of silver NP (Lee et al., 2013). The result demonstrates that the non-specific binding of silver NP-antibodies has not occurred on the chip.
0
2
4
6
Z-position (nm, ×103)
8
10
Fig. 3. Color camera images before (A, orange) and after (B, blue) immunoreaction. Profiles of the scattering intensity before (red dot) and after (blue dot) immunoreaction (C). The standard TSH concentration was 1 fM. Profiles of scattering intensity were acquired with sequential z-axis imaging at 10-nm intervals from silver NPs to gold spot using a high-sensitivity electron-multiplying charge-coupled device camera. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
(theoretical concentration¼8.0 fM) and was quantified using the chip (experiment concentration ¼7.7 fM). The fluorescent-free detection of biomolecules on nanobiochips using a combination of wavelength-dependent enhanced dark-field illumination and a plasmonic NP probe might be used for clinical diagnosis with ultrahigh sensitivity.
Relative scattering intensity
300 250 y = 35.629x - 56.043 R = 0.9968 (100 zM-100 fM)
200
713
150 4. Conclusions
100 50 0
1
2
3
4
5
6
7
8
9
Log [TSH (zM)] Fig. 4. Quantitative PRS response curves for target protein molecules (standard TSH) in the linear dynamic concentrations range of 100 zM–100 fM using a highsensitivity electron-multiplying charge-coupled device camera. Relative scattering intensities were corrected via background subtraction.
(FWHM) value is expressed by Eq. (1) (Brauchle et al., 2010):
FWHMx, y =
λ , 2NA
(1)
where λ is the emission wavelength and NA is the numerical aperture of the microscope objective. The FWHM value of the PSF in the axial (z)-dimension is expressed as Eq. (2):
FWHMz =
2λ . NA2
In summary, an ultrahigh-sensitivity, quantitative nanoimmunoassay using antibody-conjugated plasmonic NPs was investigated using simple switchable signals of an optical bandpass filter coupled with enhanced dark-field illumination. A silver NP probe and a single gold spot were selectively localized using an axial scattering intensity profile acquired with sequential z-axis images at 10-nm intervals. Nanoimmunoreactions were confirmed by measuring heights through the axial orientation of biomolecules using z-axis sectioning of intensities. Depending on the wavelength, the quantification of human TSH had a wide dynamic linear range from 100 zM to 100 fM with a strong correlation coefficient (R ¼0.9968) by suppressing the scattering intensity of the gold spot. Therefore, this method can be suggested as an effective immunoassay tool based on non-fluorescence three-dimensional super-resolution imaging of biomolecules on nanobiochips at single molecule level (1–2 molecules/spot).
Acknowledgments
(2)
In single-molecule experiments, high-NA objectives were used to collect the maximum number of photons. Physical limitations restrained the maximum collection of NA to about 1.3 for oil immersion objectives. In this case, the extent of the PSF axial profile typically approaches about 716 nm (at 605-nm wavelength) and 560 nm (at 473-nm wavelength). This measurement is roughly two times the lateral profile. The axial position is not as readily discernible because: (1) the PSF shape is symmetrical around the optical axis and the focal plane in the absence of aberrations in the optical system; and (2) the lateral width of the PSF varies little near the focal plane (Thompson et al., 2002; Mortensen et al., 2010; Hajj et al., 2014). By this method, deciding the accurate axial location of two nanometals after immunoreaction is difficult. However, two mountaintop gradations were observed in the scattering intensity profile (Fig. 3C). This finding was attributed to antibody-conjugated silver NPs immunoreacting on the 100-nm gold spots. 3.4. Quantification of the TSH protein on gold-nanopatterned chips TSH protein quantitatively analyzed by labeling plasmonic NPs and using wavelength-dependent dark-field illumination had enhanced sensitivity. A strong relationship was observed between the relative increase in the enhanced dark-field illumination intensity of the immune-targeted silver NP-antibody and the standard TSH concentration on a 100-nm gold-patterned chip. The linear dynamic range was seven orders, from 100 zM to 100 fM (Fig. 4). The method achieved a limit of detection (LOD) of 100 zM, corresponding to 1–2 molecules/gold spot. This value was approximately 4 times lower than the LOD obtained by a previous fluorescent labeling method (Lee and Kang, 2012). The value was, however, superior to the guideline value of the National Academy of Clinical Biochemistry (0.02 mI U/L¼ 118.7 fM) (Baloch et al., 2003). A clinical sample spiked with normal human serum
This research was 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) and by a Nano-Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (2009-0082580).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.01.003.
References Baloch, Z., Carayon, P., Conte-Devolx, B., Demers, L.M., Feldt-Rasmussen, U., Henry, J. F., LiVosli, V.A., Niccoli-Sire, P., John, R., Ruf, J., Smyth, P.P., Spencer, C.A., Stockigt, J.R., 2003. Thyroid 13, 3–126. Bjork, I., Petersson, B.A., Sjöquist, J., 1972. Eur. J. Biochem. 29, 579–584. Brauchle C., Lamb D.C. and Michaelis J., 2010. Single particle tracking and single molecule energy transfer, Wiley VCH, pp. 5. Crut, A., Maioli, P., Del Fatti, N., Vallee, F., 2014. Chem. Soc. Rev. 43, 3921–3956. Chen, S., Svedendahl, M., Duyne, R.P., Käll, M., 2011. Nano Lett. 11, 1826–1830. Cheng, Y., Samia, A.C., Meyers, J.D., Panagopoulos, I., Fei, B., Burda, C., 2008. J. Am. Chem. Soc. 130, 10643–10647. Francois, P., Bento, M., Vaudaux, P., Schrenzel, J., 2003. J. Microbiol. Methods 55, 755–762. Georganopoulou, D.G., Chang, L., Nam, J.M., Thaxton, C.S., Mufson, E.J., Klein, W.L., Mirkin, C.A., 2005. Proc. Natl. Acad. Sci. USA 102, 2273–2776. Hajj, B., Beheiry, M.E., Izeddin, I., Darzacq, X., Dahan, M., 2014. Phys. Chem. Chem. Phys. 16, 16340–16348. Ha, J.W., Marchuk, K., Fang, N., 2012. Nano Lett. 12, 4282–4288. Kreno, L.E., Hupp, J.T., Van Duyne, R.P., 2010. Anal. Chem. 82, 8042–8046. Lee, J.M., Park, H.K., Jung, Y., Kim, J.K., Jung, S.O., Chung, B.H., 2007. Anal. Chem. 79, 2680–2687. Lee, J.S., Mirkin, C.A., 2008. Anal. Chem. 80, 6805–6808. Lee, S., Kang, S.H., 2014. Biosens. Bioelectron. 60, 45–51. Lee, S., Kang, S.H., 2013. Analyst 138, 3478–3482. Lee, S., Kang, S.H., 2012. Talanta 99, 1030–1034.
714
S. Lee et al. / Biosensors and Bioelectronics 79 (2016) 709–714
Lee, S., Park, G., Chakkarapani, S.K., Kang, S.H., 2015a. Biosens. Bioelectron. 63, 444–449. Lee, S., Yu, H., Kang, S.H., 2013. Chem. Commun. 49, 8335–8337. Lee, S., Yu, H., Kang, S.H., 2015b. Chem. Commun. 51, 945–947. Lynch, M., Mosher, C., Huff, J., Nettikadan, S., Johnson, J., Henderson, E., 2004. Proteomics 4, 1695–1702. Mortensen, K.I., Churchman, L.S., Spudich, J.A., Flyvbjerg, H., 2010. Nat. Methods 7, 377–381. Paciotti, G.F., Kingston, D.G.I., Tamarkin, L., 2006. DrugDev. Res. 67, 47–54. Stender, A.S., Marchuk, K., Liu, C., Sander, S., Meyer, M.W., Smith, E.A., Neupane, B., Wang, G., Li, J., Cheng, J.X., Huang, B., Fang, N., 2013. Chem. Rev. 113, 2469–2527. Stoeva, S.I., Lee, J.S., Smith, J.E., Rosen, S.T., Mirkin, C.A., 2006. J. Am. Chem. Soc. 128, 8378–8379. Taylor, M.A., Bowen, W.P., 2013. Appl.Optics 52, 5718–5723.
Thompson, R.E., Larson, D.R., Webb, W.W., 2002. Biophys. J. 82, 2775–2783. Vainrub, A., Pustovyy, O., Vodyanoy, V., 2006. Opt. Lett. 31, 2855–2857. Vodyanoy, V., Pustovyy, O., 2009. Patent No. US 7,564,623 B2, Jul 21. Vodyanoy, V., Vainrub, A., Pustovyy, O., 2009a. Patent No. US 7,551,349 B2, Jun 23. Vodyanoy, V., Pustovyy, O., Vainrub, A., 2009b. Patent No. US 7,542,203 B2, Jun 2. Wang, W., Tao, N., 2013. Anal. Chem. 86, 2–14. Wurtz, G.A., Evans, P.R., Hendren, W., Atkinson, R., Dickson, W., Pollard, R.J., Zayats, A.V., Harrison, W., Bower, C., 2007. Nano Lett. 7, 1297–1303. Zhang, L., Li, Y., Li, D.W., Jing, C., Chen, X., Lv, M., Huang, Q., Long, Y.T., Willner, I., 2011. Angew. Chem. Int. Ed. 123, 6921–6924. Zhang, P., Park, S., Kang, S.H., 2015a. Bull. Korean Chem. Soc. 36, 1172–1177. Zhang, P., Lee, S., Yu, H., Fang, N., Kang, S.H., 2015b. Sci. Rep. 5, 11447. http://dx.doi. org/10.1038/srep11447.