Chrominance to Dimension: A Real-Time Method for Measuring the

Article pubs.acs.org/ac

Chrominance to Dimension: A Real-Time Method for Measuring the Size of Single Gold Nanoparticles Chao Jing,† Zhen Gu,† Yi-Lun Ying,† Da-Wei Li,† Lei Zhang,†,‡ and Yi-Tao Long*,† †

Key Laboratory for Advanced Materials & Department of Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China ‡ School of Materials Science & Engineering, Nanjing University of Posts and Telecommunications, 9 Wenyuan Road, Nanjing 210046, P. R. China S Supporting Information *

ABSTRACT: Noble metal nanoparticles have excellent optical and chemical properties and are widely used in optics, sensors, and biomedicines. The inherent characteristics of metal nanoparticles, particularly their size, play important roles in their applications. The ability to readily measure the size of single nanomaterials on-site is crucial to the rapid development of single-particle sensors. In this study, we developed a facile and real-time method for estimating the diameter of single gold nanoparticles (GNPs) that range from 35 to 110 nm in diameter; this technique uses the chrominance of the GNP’s plasmon resonance scattering light that is captured by a dark-field microscope (DFM). The RGB (three primary colors, red, green, and blue) chrominance information from the dark-field image can be directly converted into the diameters of the GNPs using the relationship between the particle size and the scattering light peak wavelength; this conversion was carried out using Matlab program based on an RGB-To-Wavelength (RTW) process. This approach is more convenient, less time-consuming, and enables observation under arbitrary conditions compared to the scanning electron microscopy (SEM) technique. The differences between the diameters of the GNPs that were calculated using this method and those that were measured using SEM were less than 5 nm. The RTW method has also been applied in the monitoring of the refractive index of the media surrounding the GNPs, and their dynamic acting within cells in real-time.

L

infrared absorption of nanomaterials and can be tuned by adjusting the size, shape and composition of the particles.23,24 In biological sensing and detection applications, the size, shape, composition, surrounding medium, and electron density of the GNPs can impact their LSPR properties and sensing efficiencies.25,26 Especially, the particle size plays a crucial role in its LSPR characteristics and biosensor applications.27−29 Large nanoparticles (diameter d > 200 nm) exhibit strong retardation effects, which invalidate the quasi-static approximation that the plasmon resonance from the nanoparticles is negligible. In contrast, the diameter of small nanoparticles (diameter d < 20 nm) is less than the mean free path of the moving electrons; therefore, the surface electrons do not oscillate with enough energy to generate scattering light.30,31 The sensitivity of nanoparticle-based cellular sensors is also size dependent. GNPs that range from 2 to 100 nm in diameter have been used to investigate the uptake process in living cells; nanoparticles that have 40−50 nm diameters exhibited the strongest effect.32 In plasmon resonance energy transfer (PRET) detection, the efficiency of the energy transfer is relied on the overlap of the energy levels, which is determined by the sizes of the particles.33 In addition, when GNPs are used as catalysts, smaller particles are more active than larger

ocalized surface plasmon resonance (LSPR) occurs in plasmonic metallic particles (Au, Ag, Cu) which results from the interaction between incident light and surface electrons.1 This phenomenon has attracted the attention of numerous researchers.1−5 Plasmonic nanoparticles have been widely used in biology, chemistry, and photonics because of their unique optical and chemical properties.6−9 Their absorption and scattering properties can be utilized in biomolecular binding, heavy metal ions detection, and membrane transfer applications.10−12 Because of their excellent biocompatibility as well as their facile synthesis and modification, the use of gold nanoparticles (GNPs) has promoted advancements in the fields of biomedical detection, drug delivery, and disease diagnosis.13−15 For instance, proteinlabeled GNPs account for a large proportion of plasmonicbased biosensors in cancer detection and biorecognition.16 Notably, dark-field microscopy (DFM) allows plasmon resonance scattering light to be used for intracellular detection at the single-particle level.17,18 GNPs have been used as contrast agents in cell imaging and in vivo biosensing because of their nontoxicity and lack of photobleaching or blinking.2 Their scattering intensity is also stronger than the fluorescence from dyes and quantum dots.19−21 According to previous studies, the scattering light of a 50 nm GNP can exceed the fluorescence of a chromophore or a quantum dot by a factor of over 1 million.22 Another important application of plasmonic nanoparticles is photothermal therapy, which is based on the near© 2012 American Chemical Society

Received: November 23, 2011 Accepted: April 14, 2012 Published: April 14, 2012 4284

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particles.34 Therefore, selecting a size-suitable particle as a nanoprobe is critical at the time of implementing the real-time experiments. That is, single-particle detection methods require the development of a facile, real-time, and on-site method for measuring the sizes of individual nanoparticles. Many techniques are currently available for measuring the size of single nanoparticles. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) are conventional methods for determining the diameter, composition, and shape of particles. However, all these techniques could not enable the real-time monitoring of samples. Furthermore, these methods are timeconsuming and expensive. Also, they are unable to monitor the surrounding medium or other chemical changes on the nanoparticle surface. Recently, RGB (red, green, blue) has attracted the interest of researchers.35,36 In color images, there are 24 bits per pixel, which include three 8-bit integers (0−255) that indicate the intensity of the RGB colors. Each color can be deconstructed into RGB values that contain information about the chromaticity and intensity, and the chromaticity values can be converted into a spectral wavelength. The various colors of the corresponding scattering light of the GNPs with different diameters are imaged by DFM. As a consequence, the RGB information of the color images could be thus transferred into the spectral wavelength of the scattering light. In the RBG converting To Wavelength (RTW) process, the wavelength with the highest intensity is considered as the peak wavelength. The color of the scattering light reveals the dimensions of the plasmonic particle. Therefore, RGB values could be used to estimate the sizes of particles. After picking a color spot in the DFM image, the size of the single nanoparticle could be determined within 1 s using the Matlab program. In this study, we demonstrate a facile, on-site, and high-throughput method for measuring the sizes of single GNPs. The DFM capturing time for one image is less than 600 ms which is suitable for the real-time determination of fast reaction. Results show that the RTW process provided a rapid method to obtain the spectral wavelengths of more than 1000 nanoparticles within 3 min compared with the spectrograph which could only record the spectra of 1−10 particles one time. That is, the calculated diameters and peak wavelength could provide statistic data of numerous individual nanoparticles instead of the bulk properties of the sample. Moreover, RTW also enables the estimation of the surrounding medium refractive index, monitoring the interaction of biomolecules on the surface of plasmonic particles and real-time observing the nanoparticles in living cells. The method could recognize the tiny color changes of the probe nanoparticles in cell imaging.

Preparation of the GNPs. All glassware was immersed in an aqua regia solution (3:1 HCl/HNO3) for 12 h (Caution, aqua regia is a strong acid and is highly corrosive; it should be handled with care) and then rinsed several times with ultrapure water before use. Seed GNPs with diameters of 13 nm were prepared according to a procedure that has been described previously.37 In brief, 50 mL of 0.01 wt % HAuCl4 was added to a 100 mL round-bottom flask that was equipped with a condenser. The solution was brought to a rolling boil under vigorous stirring, and 5 mL of 38.8 mM sodium citrate was rapidly added to the vortex of the solution; the addition of sodium citrate caused the color to change from pale yellow to red. The solution was heated for 15 min and then stirred for an additional 15 min after the heating mantle had been removed. The resulting solution of seed particles was used to prepare the larger gold particles using a procedure that has been described previously.31 In brief, 25 mL of water, 1 mL of the solution of seed particles, and 100 μL of 0.2 M NH2OH·HCl were combined in a 50 mL beaker, and 3.0 mL of 0.1 wt % HAuCl4 was added dropwise under vigorous stirring. As the HAuCl4 solution was added, the color of the mixture gradually changed to dark red. The addition of the HAuCl4 was completed within 5 min. The nanoparticle solutions were stored in dark bottles at 4 °C. Preparation of the Samples. Gold nanoparticles used in the experiments were immobilized on the glass slide. The indium tin oxide (ITO) slides were selected due to their electroconductivity for the SEM experiments. The surfaces of ITO slides were cleaned in an ultrasonic bath. The slides were first sonicated in ethanol and acetone to remove oily matter and then sonicated in water to remove water-soluble matter. The samples were sonicated in each solvent for more than 1 h. The clean ITO slides (20 mm × 10 mm × 1.1 mm) were modified with thiol by incubating them in a solution of 1% v/v 3mercaptopropyl trimethoxysilane in ethanol for 12 h and then rinsed with ethanol several times and dried under a stream of nitrogen. The silanized ITO slides were then modified with GNPs by Au−S chemical adsorption by placing them in the 10 times diluted gold colloid solution for 15 min. The GNPfunctionalized ITO slides were rinsed with water and dried under a stream of nitrogen prior to the dark-field observation. Cell Culture. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented plus 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 mg/mL streptomycin and 100 U/mL penicillin) at 37 °C in the humidified atmosphere under 5% CO2. The cells were seeded in 6 cm dishes at a density of 6 × 105 cells/dish and grew for 12 h. Then cells were incubated with fresh media containing 0.15 nM GNPs (v/v, 8:1) for 24 h. As control experiments, the 10 μM taxol solution was added to the culture medium, followed by incubation at 37 °C for 5 h. Then cells were rinsed by Trisbuffered saline (TBS, 10 mM, pH = 7.3, 0.15 M NaCl). After recording the original spectra of GNPs in the cell, the 20 μM CuCl2 solution was added to the media to form the Au@Cu core−shell structure. The detailed procedure has been reported in a previous work.38 Measurement with Dark-Field Microscopy and Scattering Spectroscopy. The dark-field measurements were carried out on an inverted microscope (eclipse Ti−U, Nikon, Japan) that was equipped with a dark-field condenser (0.8 < NA < 0.95) and a 40× objective lens (NA = 0.8). The white light source was a 100 W halogen lamp. The GNPfunctionalized slides were immobilized on a platform, and the

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EXPERIMENTAL SECTION Materials. All reagents were of analytical grade. Gold chloride trihydrate (HAuCl4·3H2O, >99.0%), taxol (AR), 3mercaptopropyl trimethoxysilane (95% v/v), and the reagents for cell culture were purchased from Sigma-Aldrich Co. Ltd. Absolute ethanol (AR), acetone (AR), sodium citrate (AR), hydroxylamine hydrochloride (AR), hydrochloric acid (AR), nitric acid (AR), and copper chloride (AR) were purchased from J&K Scientific Ltd. (China). Ultrapure water with a resistivity of 18.2 MΩ cm was produced using a Milli-Q apparatus (Millipore) and used in the preparation of all the solutions. The indium tin oxide (ITO) slides were purchased from Geao Co. Ltd. (China). 4285

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white light source was used to excite the GNPs and generate plasmon resonance scattering light. A true-color digital camera (Nikon DS-fi, Japan) was used to capture the dark-field color images. The scattering light of the gold nanoparticle was split by a monochromator (Acton SP2300i, PI) that was equipped with a grating (grating density, 300 lines/mm; blazed wavelength, 500 nm) and recorded by a spectrometer CCD (CASCADE 512B, Roper Scientific, PI) to obtain the scattering spectra. Additional details of the dark-field microscopy and spectroscopy setup have been reported in a previous work.38 Figure 1 shows the DFM and spectrometer setup.

Figure 2. Dark-field images of three GNPs (1−3) with different diameters (A) and the corresponding plasmon resonance scattering spectra of the three GNPs (1−3) (B). Figure 1. Dark-field microscope and scattering light spectrometer setup. The light that scatters from GNPs is captured by the imaging CCD and the spectrometer CCD.

the particle: σsca is directly proportional to the square of V. In addition, eqs 3 and 4 present the scattering light wavelength as a function of the size of a real nanoparticle.41

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αsphere =

RESULTS AND DISCUSSION Localized Surface Plasmon Resonance Scattering Spectra of GNPs. As displayed in Figure 2A, the scattering light of GNP is recorded as a color spot in the DFM image. Each GNP is represented by a spot, and the color of the spot indicates its size. Figure 2B shows the scattering spectra of three GNPs with different colors (1−3) that were collected by the spectrometer CCD. It can be seen that the scattering wavelength peaks (λmax) of the three GNPs are 540, 580, and 620 nm, respectively. Clearly, the corresponding intensity of the GNP spectrum increases from 1200 to 2000 au. These results indicate that both the λmax and the intensity of the scattering light increase as the size of the GNPs becomes larger and agreed with the previous report.39 On the basis of the above discussion, the size of a GNP has a significant impact on the intensity and wavelength of its scattering spectrum. Mie theory calculated the intensity of the scattering light of a GNP from the scattering cross section σsca (eq 1).40 8π 3 2 α 3λ 4 ε − εm α = 3V ε + 2εm σsca =

( 101 )(ε + εm)χ 2 + O(χ 4 )

1

(

1 3

+

εm εεm

)

−

1 (ε 30

+ 10εm)χ 2 − i

4π 2εm 3/2V 3λ 3

+ O(χ 4 )

V (3)

χ=

πa λ

(4)

In eqs 3 and 4, αsphere is the polarizability of the nanosphere, χ is the size parameter, O(χ4) is infinitely small and is on the same order as χ4, the quadratic term in the numerator corresponds to the retardation of the excitation field and the quadratic term in the denominator corresponds to the retardation of the depolarization field that is inside of the particle. These two retardation effects reduce the resonance energy of the restoring force between oppositely charged particles that are separated. RGB Converted to Spectral Wavelength (RTW). A Matlab program was used to convert the RGB information (VR, VG, VB) that is contained in the dark-field image into information about the scattering light λmax (eqs 5 and 6). In these equations, x, y, and z are the coordinates of the chromaticity diagram (Figure S-1 in the Supporting Information).

(1)

(2)

where α is the polarizability, λ is the wavelength of the light, V is the volume of the particle, εm is the dielectric constant of the surrounding environment, and ε is the dielectric constant of the GNP. Equations 1 and 2 show the relationship between the intensity of the plasmon resonance scattering and the volume of

( X Y Z ) = ( VR VG

⎞ ⎛ 0.490 0.177 0 ⎜ VB ) × ⎜ 0.310 0.812 0.010 ⎟⎟ ⎟ ⎜ ⎝ 0.200 0.011 0.990 ⎠ (5)

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Figure 3. Dark-field images of the 15 GNPs whose scattering light was recorded by the spectrometer CCD for comparison with the calculated peak wavelengths. The GNPs are approximately 52 nm in diameter and are immersed in water.

Table 1. Comparison of the Calculated (λcal) and the Experimental Peak Wavelengths (λexp) of the GNPs That Are Indicated in Figure 3 no.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

λexpa (nm) λcalb (nm)

559 560

551 556

554 555

560 559

550 554

560 559

559 559

555 557

557 556

556 558

557 556

553 555

557 558

556 554

557 556

a

The experimental peak wavelengths were determined using a spectrometer CCD. bThe peak wavelengths of the GNPs were calculated by converting the RGB information of the scattering light into a measurement of the diameter.

⎛x⎞ ⎛ X ⎞ ⎜ y ⎟ = ⎜ Y ⎟ /(X + Y + Z) ⎜ ⎟ ⎜ ⎟ ⎝z ⎠ ⎝Z ⎠

plasmon resonance of gold colloid in water has been expressed as a function of the diameter d of the particles (eq 7).43,44 (6)

To confirm the accuracy of the RTW method, several GNPs were selected and their scattering spectra were recorded with the spectrometer CCD and then the recorded data were compared with the data that were calculated using the Matlab program. Figure 3 shows the dark-field images of the selected 15 GNPs which are approximately 52 nm in diameter and are immersed in water. The experimental and calculated peak wavelengths of the GNPs were presented in Table 1. Obviously, the differences between the experimental (λexp) and the calculated (λcal) data do not exceed 5 nm. Also, the average values of λexp and λcal are 556.1 and 556.8 nm, respectively, and the average difference between λexp and λcal is only 1.670 nm. It can be concluded that the peak wavelengths that were calculated using the RTW method are in good agreement with the experimental results. Therefore, this method of converting the RGB information from a dark-field image into information about the scattering light could be used to estimate the λmax of single particles. Notably, the RTW program could obtain the scattering light peak wavelength of more than 1000 GNPs in 3 min using an ordinary laptop. Figures S-3 and S-4 in the Supporting Information show the spectral data of 1766 GNPs in one dark-field image calculated by Matlab. Because of the small nanoparticles whose diameter were less than 35 nm could not be measured by our CCD, the distribution histogram for the peak wavelengths of 1766 GNPs was not symmetric. Thus, the RTW program has much more efficiency and is readily avaliable than a spectrograph which records the spectra of only 1−10 GNP at one time. Estimation of GNPs’ Diameter. On the basis of the success of the RTW approach, the relationship between the size and the plasmon resonance scattering wavelength λmax of the GNPs has been determined, and this method has been used to estimate the diameters of the particles. In recent decades, great effort has been put into determining the relationship between the sizes of GNPs and the wavelengths of their scattering light.28,39,41,42 Notably, the peak position of the surface

λ water = λ 0 + L1 exp(L 2d)

(7)

λmax = mnmed + b

(8)

λmax = (m + m0)nmed + b

(9)

Δλmax = (m + m0)Δnmed

(10)

In eqs 7−10, λ0, L1, and L2 are the fit parameters that were determined from the theoretical values: λ0 = 512 nm, L1 = 6.53, and L2 = 0.0216. This formula was used to estimate the λmax of the GNPs in water. In the dark-field experiment, single nanoparticles are immobilized on a glass slide and exposed to air. Because of the intense effect of surrounding medium on LSPR, the calculation should take the change in the local refractive index into account.13 According to a previous report, the λmax of the plasmon resonance is proportional to the refractive index of the surroundings nmed (eq 8). In eq 8, m is the refractive index sensitivity of the nanoparticles, which is approximately 44 nm/refractive index unit (RIU) for gold nanospheres.45 Because the value of m is dependent on the particles’ shape, a constant parameter m0 (m0 = 76) has been introduced in eq 9 to account for synthetic nanoparticles that are not regular spheres. Therefore, when the diameter d is constant, λmax can be expressed as a function of Δnmed (eq 11). λmax = λ water + Δλmax = (m + m0)Δnmed + λ 0 + L1 exp(L 2d)

nmed = αn2 + (1 − α)n1

(11) (12)

In eq 11, λmax is the peak wavelength of the light that is scattered from a GNP in an arbitrary medium whose refractive index is nmed, and λwater is the peak wavelength of the plasmon resonance of gold colloid in aqueous solution. The nanoparticles that are immobilized on a glass slide are exposed to two media: the substrate and air (Figure 4). Equation 12 expresses the refractive index of the surroundings as a function of n1 and n2,46 where n1 and n2 are the refractive indices of the air and the substrate, respectively, and α is a weight factor. 4287

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of the GNPs were also calculated from the RGB information of the scattering light using Matlab, and the results are illustrated in Figure 6D. The distributions indicate that the estimated diameters of the GNPs are approximately 52 ± 10 (D1), 59 ± 10 (D2), and 70 ± 10 nm (D3). It could be concluded that the average difference between the calculated diameters and the SEM diameters is less than 5 nm. The reason is that the increased heterogeneity in the calculated sizes may be due to the inhomogeneous shapes of the particles. As the SEM images displayed, the shapes of the GNPs are not regular spheres and the inhomogeneity of the shapes affects the scattering of the GNPs.47 Therefore, this method of calculating the dimension from the chrominance is appropriate for nanospheres with diameters from 35 to 110 nm that could be detected by DFM, which is rapid for monitoring the particles in bulk and in realtime. Also, the entire process takes less than 5 min and the acquisition time for one DFM image is less than 1 s, which makes it more convenient than the expensive SEM characterization. Besides, our method provides a high-throughput measurement for calculating the diameters for numerous nanoparticles. Figure S-5 in the Supporting Information displays the diameter distribution of 1766 GNPs calculated by Matlab within 3 min. Calculation of Surrounding Medium Refractive Index. The LSPR of the plasmonic particles is dependent on the surrounding medium.1 In recent years, many biosensors have been developed based on changes in the surrounding medium by detecting shifts in the LSPR peak wavelength.11 Therefore, for a gold nanoparticle with a known diameter, the RTW method could be used to calculate the refractive index of the surrounding medium and characterize the binding interactions on the surfaces of the particles. For example, Figure 3 shows GNPs with diameters of approximately 52 nm that are immersed in water. Using a weight factor of α = 0.82, the average n1 that is calculated from eq 10 is 1.34, which is close to the refractive index of 1.33 of water. Thus, the calculated refractive index could be used to monitor biomolecular interactions, changes in the molecular structure, and specific binding interactions. Also, this approach is more convenient than commercial SPR instruments and allows the system to be monitored at the single-particle level. Furthermore, this method could monitor the rapid reactions of dark-field biosensors by eliminating the need to collect spectra so that the entire image could be captured within 1 s. Analysis of GNPs in a Living Cell. The RTW method was also used for intracellular detection and biosensing. Following the development of a high-resolution microscope, GNPs became widely used in cell imaging and biorecognition because of their biocompatibility, stability, and nontoxicity.48,49 In a previous work, we used the scattering properties of gold and copper nanoparticles to detect NADH in living cells using DFM.38 The GNPs were used as single nanoprobes and incubated in living cells for 24 h. The NADH in cells reduces copper ions to copper atoms, which could adsorb onto the surface of gold nanoparticles to form the Au@Cu core−shell structure. Results show that the formation of the core−shell structure induces a red shift in the scattering wavelength of the nanoparticles. The addition of the cancer drug taxol limits the generation of NADH, and the decrease in the amount of NADH prevents the copper ions from being reduced. The structure of the GNPs in the cells did not change, which indicates that the cancer drug was effective. Therefore, this method could be applied to screen cancer drugs in living cells.

Figure 4. Illustration of the surrounding medium for GNPs. Under the dark-field condition, GNPs are surrounded by a glass substrate and by air. In this illustration, n1 and n2 are the refractive indices of the air and the glass substrate, respectively.

Because of the rough surface of the glass slide, the refractive index of the substrate accounts for a larger proportion of the total surroundings than the air. Thus, the relationship between the diameter d and the λmax can be obtained (eq 11). To determine the value of α, the scattering spectra of the 52 and 59 nm GNPs were recorded by the spectrometer CCD, and the corresponding average peak wavelengths were 543 and 546 nm, respectively. As shown in Figure 5, the peak wavelengths of the

Figure 5. The peak wavelengths of 52 (black solid line) and 59 nm (red solid line) GNPs that were calculated using different values of α. The dotted lines represent the average peak wavelengths of the 52 (black) and 59 nm (red) GNPs that were measured by the spectrometer CCD.

GNPs were calculated using different values of α. For n1 = 1.003 and n2 = 1.520, results indicate that the calculated diameter is in good agreement with the experimental data when α = 0.82. The peak wavelengths of the GNPs that were calculated from the RGB values can be used to estimate the diameter of the GNPs using the relationship between the diameter d and λmax. Gold nanoparticles with diameters of approximately 52, 59, and 70 nm were prepared to verify the accuracy of this measurement method. Figure 6A shows the SEM images of the 52 (A1), 59 (A2), and 70 nm (A3) particles, and the distributions of the GNP diameters determined from the SEM data that were presented in Figure 6B1, B2, and B3. Clearly, the three sizes of GNPs are distributed around 52 ± 5, 59 ± 5, and 70 ± 5 nm. It should be noted that the heterogeneity in the size distribution of the GNPs could be caused by the limitations of our synthesis method, that is, the homogeneity of the particle sizes may decrease when the diameter increases. Figure 6C1, C2, and C3 show the dark-field images of the three sizes of GNPs, and results indicate that the color of the scattering light changes from green to yellow (C1−C3) as the diameters of the GNPs increased from 52 to 70 nm. In addition, the diameters 4288

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Figure 6. (A) SEM images of the 52 (A1), 59 (A2), and 70 nm (A3) GNPs. (B) The size distributions of the 52 (B1), 59 (B2), and 70 nm (B3) GNPs that were determined from the SEM images in part A. (C) The dark-field images of the 52 (C1), 59 (C2), and 70 nm (C3) GNPs. (D) The size distributions of the 52 (D1), 59 (D2), and 70 nm (D3) GNPs that were calculated using Matlab. Each size distribution diagram includes data from 100 nanoparticles.

based on the RGB information that is contained in the darkfield images of the plasmon resonance scattering from GNPs. Using Matlab, the RGB information from the light that is scattered from the GNPs is directly converted into a

The shifts in the scattering spectra of the nanoparticles in the cell were detected by the spectrometer CCD, but the process was time-consuming and the spectra were not intense enough for the influence of adiaphanous organelles in cell. Hence, the RTW method facilitates the monitoring of NADH by readily determining the peak scattering wavelengths of the GNPs and the Au@Cu NPs in a cell (Figure 7). GNPs with diameters of approximately 50 nm were incubated in HeLa cells. After the addition of copper chloride, the color of the nanoparticles changed from green to red. Figure 7A,B shows the HeLa cells incubated with gold nanoparticles with (A) and without (B) the treatment of the cancer drug taxol. The wavelengths of the particles 1−4 that are labeled in Figure 7D are 585, 580, 585, and 593 nm, respectively, which indicates that the NADH reduced the copper ions to copper atoms and that the copper atoms were then adsorbed onto the GNPs. With the treatment of the cancer drug, the nanoparticles did not undergo a red shift. The calculated peak wavelengths of the particles in Figure 7C are 569, 570, 565, and 563 nm, respectively. The detailed information of nanoparticles in the cell in the presence and absence of taxol were displayed in Figure S-6 in the Supporting Information. These results indicated that GNPs could be used as biosensors to screen the effectiveness of cancer drugs and map the NADH in a cell. Thus, the RTW method makes this technique easier and less time-consuming in comparison to our previous recording approach.

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Figure 7. (A) DFM images of GNPs that are contained within HeLa cells; the HeLa cells have been treated with taxol (10 μM) and then incubated in a TBS solution that contains 20 μM CuCl2 for 3 h. (B) DFM images of GNPs that are contained within HeLa cells that have not undergone treatment with taxol. Parts C and D are the detailed views of the DFM images in parts A and B, respectively.

CONCLUSIONS In summary, we developed a chrominance-to-dimension method for the on-site estimation of the diameters of single GNPs that range from 35 to 110 nm in size. This technique is 4289

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measurement of their diameter using the relationship between the particle size and the peak wavelength of the scattering light. This approach is more convenient and less costly than conventional techniques, such as SEM. It is important to note that the differences between the GNP diameters that were calculated using this method and those that were measured via SEM technique were less than 5 nm. This method could be applied in various conditions without vacuum limitations which enables the on-site determination. The ability to measure the size of GNPs is useful for real-time biosensing applications because the measurement time for one image takes less than 600 ms. Moreover, this method could be used to estimate the refractive index of the local medium and monitor the changes in the particle’s surroundings, which makes the use of plasmon resonance based biosensors more direct and facile. Furthermore, the RGB-To-Wavelength method was successful in determining the peak wavelengths of the nanoparticles in a living cell and bypassing the complicated process of collecting the spectra by a spectrograph. These advancements support cell imaging techniques and other color-coded biosensors and provide more specific information about the intracellular interactions. This method also enables the biomolecular interactions in cells including protein degeneration and antigen−antibody binding to be continuously monitored by detecting changes in the particle’s surrounding medium. We believe that the approach could have wide-ranging applications in sample characterization, biosensing, and cell imaging.

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ASSOCIATED CONTENT

S Supporting Information *

The Matlab program which was used to calculate the diameter of GNPs and the chromaticity diagram. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Science Fund for Distinguished Young Scholars (21125522), the National Natural Science Foundation of China (91027035, 21007015), the Fundamental Research Funds for the Central Universities (WK1013002) and the Open Project Program of the State Key Laboratory of Chemical Engineering (ECUST, SKL-ChE11C01). We especially thank Li Ni, Jin Huang and Tao Xie from ECUST for the Matlab programming.

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