Synthesis and Characterization of Single Nanoparticle Biosensors
We synthesized nearly monodispersed (11.6 ± 3.5 nm in diameter) Ag nanoparticles as characterized by HRTEM (shown in Fig. 1S
of supporting information (SI)
) by carefully selecting experimental conditions for reduction of AgClO4
with sodium citrate and NaBH4
. Then, we functionalized these Ag nanoparticles with a mixed monolayer of MUA and MCH using the interaction of the thiol group (-SH) with the Ag nanoparticle surface to prepare AgMMUA ().
Note that the Ag nanoparticles were stabilized in solution by electrostatic repulsion of the surface adsorbed charged citrate layer.37–39
If only MUA was used to replace adsorbed citrate layer, the replacement reaction would follow a unimolecular nucleophilic substitution (SN
1) mechanism, because the citrate is a very good leaving group, and the charge-repulsion and steric hindrance of MUA makes an SN
2 mechanism nearly impossible to occur. 37–39
Thus, the presence of the MUA would accelerate the dissolving of the charged citrate layer, leading to the aggregation of nanoparticles, which is the major problem in synthesis of ω-mercaptoalkanoic acids modified Ag nanoparticles.
To solve this problem, we used a mixture of MUA and MCH with a mole ratio of 1: 9. The presence of an excess of non-charged short chain MCH allows the substitution reaction more favorable to a SN2 mechanism, which prevents the aggregation of the Ag nanoparticles. The hydroxyl group on the end of MCH makes the functionalized Ag nanoparticles more hydrophilic. Since MCH is much shorter than MUA, it will not block the carboxyl group of the MUA for further linking with IgG. The short chain of MCH is more rigid than MUA molecules; therefore, MUA surrounded by the rigid short-chain thiols of MCH is more likely to stand straight on the nanoparticle surface () rather than lay flat on the surface of the Ag nanoparticles, which reduces steric hindrance and allows the MUA to link with IgG more effectively.
With this approach, we successfully avoided aggregation of nanoparticles and prepared AgMMUA nanoparticles that are stable (size and color of single nanoparticles remain unchanged) in aqueous solution (nanopure water and buffer solution) for months. Although a similar reaction scheme had been used to functionalize the Au surface40
and nanoparticles immobilized on the surface18
, to our knowledge, it has not yet been achieved for Ag nanoparticles that freely diffuse in the solution. We found that the ratio of MUA to MCH and reaction time was crucial to preventing aggregation of nanoparticles.
NMR spectra show that MUA, MCH and citrate are attached on the surface of Ag nanoparticles with a mole ratio of 1: 3: 30 (Fig. 2S
in SI). The large and broad chemical shift at 2.5 ppm is from the –CH2
-next to thiol of both MUA and MCH,41
and citrate adsorbed on the Ag nanoparticle surface.42
Peak broadenings are observed at all chemical shifts of the NMR spectra, indicating that the MUA and MCH are on the surface of Ag nanoparticles, because nanoparticles attached to MUA and MCH slow down the rotational motion of these molecules.43–45
Furthermore, the gradient of packing density along the functional groups may also contribute to the peak broadening.
Finally, we linked the carboxyl group of AgMMUA with the amine group of IgG via a peptide bond to prepare single nanoparticle biosensors (AgMMUA-IgG), as illustrated in . By controlling the mole ratio of IgG to AgMMUA nanoparticles at less than one (0.97) during the conjugation reaction, we limit the statistical average conjugation ratio at one IgG molecule per nanoparticle. The nanoparticle biosensors prepared using covalent conjugation have several advantages over those prepared using electrostatic interaction. They are more stable and the conjugation ratio remains unchanged in buffer solution of various ionic strength and pH, allowing quantitative analysis of individual protein molecules on single living cell surface.
The representative UV-vis absorption spectrum of bulk Ag, AgMMUA and AgMMUA-IgG nanoparticle solution shows a peak wavelength at 393, 400 and 406 nm, respectively, illustrating a red-shift as the surface of Ag nanoparticles is modified with the functional groups (). The LSPRS of representative single Ag nanoparticles from these three solutions exhibits a peak wavelength of 450, 482, and 545 nm, respectively, showing a longer wavelength and lower intensity as the surface of Ag nanoparticles is functionalized with the mixed monolayer of MUA and MCH, and linked with IgG (). The histograms of the different colors (LSPRS) of individual nanoparticles of the three solutions in shows that Ag nanoparticles in nanopure water contains blue (88%) and green (12%) nanoparticles; AgMMUA nanoparticles in nanopure water includes the light green (82%) and yellow green (19%) nanoparticles; and AgMMUA-IgG nanoparticles in PBS buffer has dark green (75%), yellow (11%), and white (14%) nanoparticles.
Figure 2 Characterization of optical properties of Ag, AgMMUA and AgMMUA-IgG nanoparticles: (A) representative UV-vis spectra of (a) 3.28 nM Ag and (b) 4.33 nM AgMMUA nanoparticles in nanopure water and (c) 5.24 nM AgMMUA-IgG nanoparticles in PBS buffer; (B) LSPR (more ...)
As described by Mie theory46
and demonstrated by our previous studies13, 15, 16
, the LSPRS and scattering intensity of single Ag nanoparticles highly depend on its size, shape, surface properties and surrounding environments. The color distribution of individual nanoparticles further illustrates that the functional groups on the surface of Ag nanoparticles lead to changes of the optical properties of individual nanoparticles, and the red shift of their LSPRS. The white color of nanoparticles observed in the AgMMUA-IgG solution is most likely attributable to the cross-linking of nanoparticles, leading to larger nanoparticles, which can be removed via centrifugation.
Characterization of Biological Activity of Single Nanoparticle Biosensors
The primary challenge of preparing effective nanoparticle biosensors, especially by covalent conjugation of protein molecules (IgG, ligand) with nanoparticles, is to retain the biological activities of those biomolecules attached on the surface of nanoparticles. To characterize the biological activity of AgMMUA-IgG, we measured their binding affinity with PrA in buffer solution, and compared their binding constant with that of IgG-PrA in solution.
The results in illustrate that the absorbance of AgMMUA-IgG nanoparticles decreases and reaches a constant value, indicating that AgMMUA-IgG nanoparticles bind with PrA and reach binding equilibrium. It is well known that PrA (a receptor) displays 3–4 binding sites for the Fc portion of rabbit IgG47–49
, which allows one PrA molecule to bind with multiple AgMMUA-IgG nanoparticles, leading to the precipitation of bound IgG-PrA.49
The peak wavelength of the spectra remains constant over time (), suggesting that AgMMUA-IgG-PrA nanoparticles did not contribute significantly to the absorbance and indicating that cross-linked AgMMUA-IgG-PrA nanoparticles precipitate from solution. Otherwise, we would observe a red shift of peak wavelength if the cross-linked larger AgMMUA-IgGPrA nanoparticles remained suspended in the solution. The extinction coefficient (molar absorptivity, ε1
) of AgMMA-IgG calculated from the absorption spectra using the Beer-Lambert law is 2.6×108
Figure 3 Characterization of biological activity of AgMMUA-IgG nanoparticles: (A) UV-vis spectra of 250 µL of 1.1 nM AgMMUA-IgG in the PBS buffer solution (a) before, at (b) 5 min and (c) 24 hours after adding 1.0 µL of 25 µM PrA, showing (more ...)
Plot of peak absorbance subtracted from baseline versus time in demonstrates a nearly first-order reaction as illustrated below:
Since the PrA molar concentration is 100 times higher than that of AgMMUA-IgG, we assume the PrA concentration remains constant over the entire reaction. Thus, a second-order (or multiple-order) reaction can be treated as a first-order reaction. Using the change of AgMMUA-IgG absorbance resulting from its binding with PrA, we calculate the amount of AgMMUA-IgG nanoparticles (0.14 nM) that bound with PrA and the amount of AgMMUA-IgG-PrA (0.14 nM) that is generated, showing equilibrium concentrations of AgMMUA-IgG (0.96 nM), PrA (99.86 nM) and AgMMUA-IgG-PrA (0.14 nM), and the equilibrium binding constant (affinity, Kb
) as 1.5×106
We further determined the binding association rate constant (ka
) and dissociation rate constant (kd
) using the slope of and by matching simulation data with the experimental data in , respectively. The detailed calculation is shown in SI. Using this approach, we can calculate the binding affinity constant (Kb
), which agrees well with the Kb
directly calculated using equilibrium concentrations. The experiments were repeated three times and the average of Kb
, (1.9 ± 0.1) ×106
, determined by both methods, agrees well with those observed in solution (3×106
) and reported in the literature50
. Thus, a primary biological activity of IgG conjugated with nanoparticles is preserved, allowing AgMMUA-IgG nanoparticles to be used to image and characterize individual receptor molecules on single living cells.
Imaging and Sensing of Individual Receptor Molecules on Single Living Cells
We anchored the T-ZZ onto the membrane of living cells to create the desired low coverage of individual ZZ molecules (0.21–0.37 molecules/µm2
) on living cells28–30
, which were detected by single AgMMUA-IgG nanoparticles and imaged in real-time using SNOMS (). Since the mole ratio of IgG to AgMMUA nanoparticles is 0.97, a single IgG molecule is statistically conjugated with a single AgMMUA nanoparticle. Although multiple IgG molecules may be present in a single AgMMU-IgG nanoparticle, the low coverage of T-ZZ molecules on the surface of single living cells, and the low concentration of AgMMUA-IgG nanoparticles greatly reduces the possibility of having more than one T-ZZ molecule bound with a single AgMMUA-IgG nanoparticle, because, at such low coverage (0.21–0.37 molecules/µm2
), it is extremely unlikely to have two neighboring T-ZZ molecules within the proximity of a single AgMMUA-IgG nanoparticle (~15 nm in diameter). If two T-ZZ molecules were located within a cross sectional area of a single nanoparticle (2.3×10−4
), the coverage of T-ZZ on a single living cell surface would be 8.7}104
. For those nanoparticles that are not conjugated with IgG, they will not bind with T-ZZ, which will not be detected on cell surface. Thus, no washing step is needed for this assay, and the possible small variation of conjugation ratio of nanoparticles with IgG will not affect SMD of individual receptors on single living cells.
Figure 4 Imaging and sensing of single T-ZZ molecules on single living cells using single AgMMUA-IgG nanoparticle biosensors. The living cells attached with T-ZZ (0.37 molecules/µm2) were incubated with 5.56 nM AgMMUA-IgG nanoparticles and imaged using (more ...)
Thus, by controlling a low conjugation ratio of IgG with AgMMUA nanoparticles and low coverage of T-ZZ on living cell surface, we ensure that a SMD scheme is appropriately applied for the reaction described below:
Fibroblast cells are highly adhesive cells and were directly cultured on coverslips. Thus, the cells would not move during the experiments. Diffusion of the attached T-ZZ molecules is confined within the cell membrane, which is orders of magnitudes slower than the Brownian motion of single nanoparticles in solution.16, 17, 51
Therefore, once AgMMUA-IgG nanoparticles bind with T-ZZ on the cell surface, the diffusion of the nanoparticles is negligible in comparison with the free Brownian motion of individual nanoparticles in the solution, showing that the bound AgMMUA-IgG nanoparticles become bright spots shrunk to the resolution limit of the CCD with a 2-pixel diameter. This unique feature allows us to avoid the washing step and directly image the number of individual T-ZZ molecules bound with AgMMUA-IgG nanoparticles on the surface of cells. We also acquired sequence images of living cells incubated with AgMMUA-IgG over time, and did not observe significant diffusion of T-ZZ bound with AgMMUA-IgG on living cells. We also used the scattering intensity of individual nanoparticles to determine whether the clusters of T-ZZ (more than a single nanoparticle) were present within the spatial resolution of CCD camera, showing no evidence of aggregation of T-ZZ.
Interestingly, we observed a red shift of the LSPRS of AgMMUA-IgG nanoparticles as they bound with T-ZZ on living cells, showing more orange and red nanoparticles (&), which may be attributable to the change of the surface properties and dielectric constant of embedded medium of nanoparticles. This interesting phenomenon offers an additional feature, allowing us to distinguish bound and unbound AgMMUA-IgG nanoparticles.
Figure 5 Imaging and characterization of the concentration dependence of binding kinetics of AgMMUA-IgG with T-ZZ on single living cells at the single-molecule resolution. Sequence optical images of representative cells selected from at least 120 cells at each (more ...)
To measure their binding affinity and determine the dependence of binding kinetics on concentration of AgMMUA-IgG nanoparticles, we prepared one surface coverage of T-ZZ molecules (0.37 molecules/µm2
) on living cells and used four concentrations (1.39, 2.78, 5.56, and 11.12 nM) of AgMMU-IgG nanoparticles (Table I
in SI). Although the binding dissociation constant (KD
) of IgG with T-ZZ on the living cells has not yet been reported, the KD
of T-ZZ with IgG on phospholipids (not on living cell membrane) was estimated as 43 nM by previous bulk measurement.28, 52
Thus, the selected concentrations of AgMMUA-IgG nanoparticles mimic the ligand (IgG) concentrations well below its KD
Using CCD microarray, we were able to image the distribution and binding reaction of T-ZZ molecules on several living cells simultaneously (). Representative cells selected from the full-frame images similar to those in are illustrated in , showing the concentration and time dependence of AgMMUA-IgG nanoparticles binding with T-ZZ on living cells. At the same concentration of AgMMUA-IgG, more AgMMUA-IgG nanoparticles bind with T-ZZ molecules on living cells as incubation time increases from 0 to 180 min (the vertical column of ), demonstrating time-dependence and real-time monitoring of the binding reaction on living cells. At a given incubation time, as AgMMUA-IgG concentration increases, more AgMMUA-IgG nanoparticles bind with T-ZZ molecules on the cells (the horizontal row of ), showing concentration dependence.
Using the same approach, we investigated the dependence of binding kinetics on the coverage of T-ZZ on living cells. We used a single concentration of AgMMUA-IgG nanoparticles (2.78 nM) to detect individual T-ZZ molecules on living cells, which were present with two different coverages of T-ZZ (0.21 and 0.37 molecules/µm2). We observed more AgMMUA-IgG nanoparticles bound with T-ZZ on living cells as incubation time increased (vertical column of ). At the given incubation time, more AgMMUA-IgG nanoparticles bound with T-ZZ on living cells with the higher coverage of T-ZZ (horizontal row of ), showing the dependence of binding reaction rate on the coverage of T-ZZ on cell surface.
To determine possible non-specific interactions of AgMMUA-IgG nanoparticles with living cell membrane, three blank control experiments were performed, by incubating 11.12 nM AgMMUA (not yet conjugated with IgG) with living cells attached with T-ZZ prepared as described in , and by incubating 11.12 nM AgMMUA-IgG nanoparticles (the highest concentration used in ) with living cells that were not attached with T-ZZ molecules, showing no significant number of bound AgMMUA-IgG nanoparticles on living cells ().
The viability of the cells during each experiment was monitored using a Trypan Blue cell viability assay, showing more than 90% of cells remained alive for the entire experimental duration from treatment with T-ZZ at pH 4.8 for 45 min, rinsing with the buffer at pH 7.4, and imaging in the microchannel for 1.5 hr.
Plots of the number of bound AgMMUA-IgG nanoparticles with T-ZZ on living cells versus reaction time for the study of dependence of AgMMUA-IgG concentration (), T-ZZ coverage on cell surface (), and blank control experiments (), are presented in , respectively. The results further illustrate that binding kinetics depend on ligand concentrations (AgMMUA-IgG) and the coverage of model receptors (T-ZZ) on the cell surface. The control experiments show insignificant non-specific interaction of AgMMUA-IgG nanoparticles with cell membrane.
Figure 6 Characterization of binding affinity and kinetics of AgMMUA-IgG with T-ZZ on living cells and determination of dynamic range of single nanoparticle biosensors. Plots of number of single T-ZZ molecules bound with AgMMUA-IgG nanoparticles on single living (more ...)
We determined the binding association (k1
) and dissociation (k−1
) rate constants, and binding affinity constant (KB
) at (9.0 ± 2.6) × 103
, (3.0 ± 0.4) × 10−4
, and (3.0 ± 0.6) × 107
, respectively. The KB
agrees well with those reported from bulk measurement.28, 52
We also determined the number of T-ZZ molecules on living cells prepared as described in , showing 4640 and 2687 molecules of T-ZZ on the surface of 40 living cells, respectively. Thus, on average, there are 119 and 67 molecules per cell, which creates coverage of 0.37 and 0.21 molecules/µm2
, respectively. The calculation details are described in Table I
At higher AgMMUA-IgG nanoparticle concentrations (2.78, 5.56 and 11.12 nM), we observed a decrease in the number of bound nanoparticles after the binding reaction reached equilibrium at 80–100 min. This interesting phenomenon was not observed at the lowest concentration (1.39 nM) of AgMMUA-IgG (: a), nor at the lower coverage of T-ZZ (: a), suggesting that, as more AgMMUA-IgG nanoparticles bound with T-ZZ on living cell surface, it may become harder to distinguish two nearby nanoparticles within the spatial resolution of the CCD camera (single pixel, 65 nm) using optical microscopy, or that the higher concentrations of bound T-ZZ may lead to dissociation. The results also suggest that a high coverage of bound nanoparticles on living cells may promote their tendency to aggregate. Effort has been made to distinguish multiple nanoparticles from a single nanoparticle within the spatial resolution of CCD camera (1–2 pixel) using the scattering intensity of single nanoparticles. However, we found no evidence that multiple nanoparticles were present within the spatial limit of the CCD camera. Thus, the observation may be attributable to the dissociation of AgMMUA-IgG from T-ZZ. Studies are under way to further characterize the mechanism.
The binding rates of AgMMUA-IgG nanoparticles (1.39, 2.78, 5.56 and 11.12 nM) with T-ZZ on living cells were calculated from the slopes of (a–d) within the first 60 min before reaching equilibrium, showing 3.0, 5.3, 20 and 27 molecules/min, respectively. A plot of the binding rates versus concentration of AgMMUA-IgG shows the high dependence of binding rates on concentration (). Using the same approach, we found that the binding rate for 2.78 nM of AgMMUA-IgG nanoparticles with two different coverages of T-ZZ (0.21 and 0.37 molecules/µm2) in is 2.3 and 5.3 molecule/min, respectively, showing the dependence of binding rate with the coverage of T-ZZ on living cells.
Figure 7 Dependence of binding rates and fraction (f) of T-ZZ molecules bound with AgMMUA-IgG on living cells upon the concentration of AgMMUA-IgG: (A) Plot of binding rates calculated from during 0–60 min reaction time (prior to equilibrium) versus (more ...)
We further analyzed the bound AgMMUA-IgG nanoparticles versus the concentration at binding equilibrium (80–120 min in ). Plots of fraction (f
) of bound T-ZZ on living cells versus concentration of AgMMUA-IgG (isotherms in ) further indicate that we detected T-ZZ molecules on living cells below their binding dissociation constant. Due to the limit of spatial resolution, higher concentrations of AgMMUA-IgG were not used to detect higher coverage of bound T-ZZ on living cells. Nevertheless, we are able to calculate KB
as (4.8 ± 2.0) ×107
1 from the isotherms in , which is similar to those calculated from . The isotherms show a sigmoidal shape, suggesting that the binding reaction is cooperative, which means that T-ZZ molecules on the surface of living cells are not completely independent. In other words, the binding reaction of AgMMUA-IgG nanoparticles with individual T-ZZ molecules becomes more favorable in the presence of surrounding bound T-ZZ molecules. A plausible explanation would be that the presence of T-ZZ molecules bound with AgMMUA-IgG nanoparticles on cell surface made it easier for other nanoparticles to dock onto the cell surface. Similar phenomena where bound nanoparticles on the cell surface serve as nucleation sites to help more nanoparticles to dock on the surface were also observed in our previous study.15
Taken together, our results show that single nanoparticle biosensors can be used to map low-coverage single receptor molecules (less than ~50 receptors per cell) on single living cells in real-time, which is what we aim for. Using optical microscopy, single nanoparticle biosensors are not suitable for detecting high coverage of protein molecules on single living cells, because of limitation of optical resolution. Using TEM, one can expect to map high coverage of protein molecules on cell surface using single nanoparticle biosensors, but TEM cannot offer real-time kinetic measurements on living cells.