Luminescent semiconductor quantum dots (QDs) have become popular for biological imaging, labeling, and sensing due to their bright photoluminescence (PL).1 These QDs are characterized by an excited state—reached after absorbing energy from a source at a given wavelength—and a ground state, achieved when the energy absorbed in the form of a photoluminescent emission is released. Such PL emissions can be tuned according to the size of QDs by what is known as quantum confinement effects. Luminescent QDs show higher performance than other light-emitting sensors: they are excited with high efficiency over a broad range of wavelengths and produce narrow PL emission profiles on returning to their ground state.
Thus, when QDs are mixed with materials, such as biological cells, they can be selectively excited by choosing a wavelength that other materials do not absorb. In addition, QDs are more resistant to photobleaching—they are not destroyed by exposure to light—than organic dyes traditionally used in biological analysis. Consequently, they remain emissive longer under continuous excitation.
In applications where QDs are used as sensors, a change in QD PL signals the presence of target species or analytes. Currently, detection of analytes in biological systems using QDs is commonly performed using Förster resonance energy transfer (FRET).2 This technique measures the degree of energy transfer from QDs to proximal organic dyes—known as acceptor dyes due to their ability to absorb energy—which changes according to the analyte. Here, we report the creation of biosensors that exploit QD optical properties, which are inherently sensitive to charge-transfer (CT) processes.3 Until now, CT-based sensing with QDs has received far less attention than FRET-based methods because it is more complex.
CT processes involving oxidation and reduction (redox) are ubiquitous in biological systems. An important class of molecules that undergo redox processes in such systems are catechols. The catecholamine neurotransmitter dopamine is the most prominent among this class. Its chemical state changes, via oxidation, from a reduced state as a hydroquinone in acidic media—i.e., with pH below 7—to an oxidized state as a quinone in basic media—pH above 7—at a rate that increases with pH. This redox process also exhibits a linear change of its formal potential (Ef) versus pH.
Numerous studies investigating the interaction of these catechol species with QDs have provided mixed results and interpretations vis-à-vis their mechanism of action, including whether dopamine acts as an electron acceptor, donor, or even a FRET donor. We therefore designed a series of highly controlled experiments involving water-soluble QDs and dopamine to elucidate the fundamental nature of these interactions and ultimately to exploit them for intracellular pH sensing.4
To prepare well-defined cadmium selenide/zinc sulfide core/shell QD-dopamine assemblies for analysis, we used a peptide as a physical bridge between the nanocrystal surface of the QD and the catechol ring (see Figure 1). We have shown that peptides containing polyhistidine sequences self-assemble to the surface of QDs via metal-affinity coordination of the imidazolium side chains of the polyhistidine to the zinc ions of the QD shell. We therefore prepared a dopamine-labeled peptide for self-assembly to the surface of the QDs. This method allowed us, first, to control the number of dopamine-peptide compounds assembled per QD and, second, to control whether the dopamine was in a hydroquinone or a quinone state via solution pH (see Figure 1).
Figure 1. Photoluminescence (PL) mechanism of quantum dot (QD)-dopamine conjugates in acidic and basic conditions. hv: Photon. His6: Hexahistidine. e−, h+: Charge carriers. ET: Electron transfer. CB: Conduction band. VB: Valence band.
Steady-state PL data collected from 550nm-emitting QDs self-assembled with increasing ratios of dopamine-peptide compounds at pH 4.8 and 9.3 is shown in Figure 2. Increased QD PL quenching occurred consistently with increasing ratios of dopamine-peptide/QD and also with increasing pH. These results suggest that the quinone, an oxidized form of dopamine, was responsible for quenching. This was confirmed by exposing QDs to dopamine-peptide constructs that were pre-reduced or pre-oxidized to hydroquinone or quinone states, respectively. We found that substantial QD PL quenching occurred with the dopamine-peptide constructs in the quinone form while minimal quenching was observed with the hydroquinone species.
Figure 2. Representative PL spectra in arbitrary units (AU) from 550nm-emitting QDs assembled with increasing ratios of dopamine-peptide construct at pH 4.8 (A) and 9.3 (B). The QD emission is centered at 553nm, but is referred to as 550nm for simplicity.
Because the degree of QD PL quenching was pH-dependent in these assemblies, we examined whether the conjugates could quantitatively measure changes in pH. First, we measured changes in the PL of QD-dopamine conjugates with increasing pH against those of a standard fluorescent nanoparticle, a 20nm-diameter nanosphere (Fluorophorex, FLX): see Figure 3(A). The QD PL decreased by ∼70% as the pH increased from 6.5 to 11.5, while the FLX PL remained unperturbed. Importantly, when we plotted the QD/FLX PL and the dopamine-peptide construct's formal potential Ef versus pH, they were both linear and superimposable, see Figure 3(B). This shows that QD PL quenching is directly related to the pH-dependent redox properties of the dopamine. Moreover, the plot of QD/FLX PL versus pH provides a calibration curve for determining solution pH from QD-dopamine/FLX PL data.
Figure 3. (A) PL spectra from QD-dopamine conjugates and fluorescent nanosphere (FLX) against pH. Inset: QD and FLX PL normalized to pH 6.5. (B) Plots of dopamine-peptide formal potential (Ef) versus normal hydrogen electrode (NHE) and the ratio of QD/FLX PL, both plotted against pH.
To evaluate the potential of using these QD-dopamine assemblies as intracellular pH sensors, we measured pH changes in the cytosol (matrix inside the cell). We microinjected COS-1 cells, typically used in research, with a mixture of QD-dopamine conjugates and FLX. We then turned the cytosolic pH alkaline through drug-induced intracellular alkalosis. Time-resolved fluorescent micrographs of COS-1 cells showed that QD PL was quenched as cytosolic pH increased: see Figure 4(A).
We used the ratio of QD/FLX PL and the calibration curve in Figure 3(B) to determine the cytosolic pH at each time point shown in Figure 4(B). Single-cell pH measurements were consistent with data averaged from all the cells together.
(A) Fluorescent micrographs of COS-1 cells co-injected with QD-dopamine conjugates and FLX during drug-induced alkalosis. (B) Average and single-cell pH values derived from intracellular fluorescence in Figure 4
Understanding CT between QDs and redox species such as dopamine is important for expanding and improving QD-based nanoscale sensors. We determined that the quinone species behave as potent electron acceptors and quench QD PL. Since the rate of quinone formation and concomitant QD PL quenching is pH-dependent, the QD-dopamine assemblies were used to detect intracellular pH changes. In the future, we plan to improve the pH sensitivity of our current QD-assemblies in biological systems and to explore the interaction of other redox materials with QDs.
Michael H. Stewart, Kimihiro Susumu
Optical Sciences Division
US Naval Research Laboratory
Michael Stewart received his PhD (2007) from the University of Michigan in synthetic organometallic chemistry. He then completed a National Research Council postdoctoral fellowship at the US Naval Research Laboratory, where he is now a research chemist.
Kimihiro Susumu received his PhD in chemistry from Kyoto University, Japan (1997). He then carried out postdoctoral research at University of Pennsylvania. Since 2006 he has been at the US Naval Research Laboratory, where he is working on developing biocompatible QDs for sensing and imaging.
Center for Bio/Molecular Science and Engineering
US Naval Research Laboratory
Igor Medintz received a PhD in molecular biology from the City University of New York (1998), after which he completed a post-doctoral fellowship at the College of Chemistry, University of California, Berkeley (2001). He has been at the US Naval Research Laboratory since 2002, where he is a research biologist. He is on the program committee of two SPIE symposia.
1. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, S. Weiss, Quantum dots for live cells, in vivo imaging, and diagnostics, Science
307, pp. 538-544, 2005. doi:10.1126/science.1104274
2. I. L. Medintz, H. Mattoussi, Quantum dot-based resonance energy transfer and its growing application in biology, Phys. Chem. Chem. Phys
. 11, pp. 17-45, 2009. doi:10.1039/b813919a
3. F. M. Raymo, I. Yildiz, Luminescent chemosensors based on semiconductor quantum dots, Phys. Chem. Chem. Phys.
9, pp. 2036-2043, 2007. doi:10.1039/b616017d
4. I. L. Medintz, M. H. Stewart, S. A. Trammell, K. Susumu, J. B. Delehanty, B. C. Mei, J. S. Melinger, J. B. Blanco-Canosa, P. E. Dawson, H. Mattoussi, Quantum-dot/dopamine bioconjugates function as redox coupled assemblies for in vitro and intracellular pH sensing, Nat. Mater
. 9, pp. 676-684, 2010. doi:10.1038/nmat2811