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Energy transfer assays using quantum dot – gold nanoparticle complexes:
Optimizing oligonucleotide assay configuration using monovalently conjugated
quantum dots
Uvaraj Uddayasankar and Ulrich J Krull
Abstract
The energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs)
represents a popular transduction scheme in analytical assays that use nanomaterials. The
impact of the spatial arrangement of the two types of nanoparticles on analytical
performance has now been evaluated using a nucleic acid strand displacement assay. The
first spatial arrangement, Configuration 1, represents the assembly of a number of
monovalently functionalized QD-oligonucleotide conjugates around a single central
AuNP that was functionalized with complementary oligonucleotide sequences. The
assembly of these complexes, and subsequent disassembly via target oligonucleotide
mediated displacement, were used to evaluate energy transfer efficiencies. Furthermore,
the inner filter effect of AuNPs on the fluorescence intensity of the QD was studied.
AuNPs of three different diameters (6 nm, 13 nm and 30 nm) were used in these studies.
Configuration 2 was based on the placement of monovalently functionalized AuNP-
oligonucleotide conjugates around a single QD that was functionalized with a
complementary oligonucleotide. The optimal assay configuration, established by
evaluating energy transfer efficiencies and inner filter effects, was obtained by arranging
at most 15 QDs around the 13 nm AuNP (Configuration 1). These assays provided a 2.5
fold change in fluorescence intensity in the presence of target oligonucleotides. To obtain
the same response with Configuration 2 required the placement of three 6 nm AuNPs
around the QD. But the inner filter effect resulted in Configuration 2 having a 5-fold
lower fluorescence intensity when compared to Configuration 1. The use of low-cost
detection systems (digital camera) further emphasized the higher analytical performance
of Configuration 1. Response curves obtained using these detection systems
demonstrated that Configuration 1 has better sensitivities (10-fold higher) and limits of
detection (2-fold lower) when compared to Configuration 2. This study provides an
important framework for the development of sensitive assays using gold nanoparticles
and quantum dots.
Introduction
Assays based on energy transfer using quantum dots (QDs) as donors, and
metallic nanoparticles such as gold nanoparticles (AuNPs) as acceptors, have garnered
significant attention in recent years{Algar:2014cb}. While QDs have many favorable
optical properties, their physical size limits energy transfer efficiencies, {Clapp:2004kc}
thus requiring efficient energy acceptors. Metallic nanoparticles provide for competitive
energy transfer efficiencies due to their high extinction coefficients across a broad range
of wavelengths and the availability of a 2-D surface to which energy is transferred
{Singh:2010us}. While the QD-AuNP system has been utilized for the detection of
analytes such as nucleic acids {Dyadyusha:2005bp, Yeh:2010jo}, proteases
{Choi:2012ja, Kim:2008id} and inorganic ions {Li:2011ir}, the widespread adoption of
this system requires a better understanding of factors that influence its analytical
performance. Two of these factors include the inner filter effect of AuNPs and the
relative spatial arrangement of the two types of nanoparticles.
The inner filter effect refers to the attenuation of excitation source intensity and
emitted radiation due to the high extinction coefficient of AuNPs at the respective
wavelengths. Recent studies have highlighted the importance of taking this effect into
account when investigating energy transfer processes. {Han:2012iy, Zhang:2015fu}
Furthermore, it would influence the analytical performance of assays by limiting the
amount of light that may be detected. To study the impact of inner filter effect on assay
performance requires the consideration of two factors. AuNP extinction coefficient is a
function of its size, with larger AuNPs having a greater extinction coefficient.
{Haiss:2007co} However, larger AuNPs may also accommodate a greater number of
QDs on its surface. Thus, an optimal configuration may exist where the AuNP is large
enough to accommodate a sufficient number of QDs to minimize the impact of the inner
filter effect. To ensure optimal assay design, it is vital to understand and control the inner
filter effect.
Complexes of QDs and AuNPs can be created using complementary
oligonucleotide conjugates for formation of desired structures by virtue of hybridization.
{Nie:2009bp}But the large surface areas of both QDs and AuNPs necessitate stringent
control of the functionalization of these nanomaterials. The high surface area of
nanomaterials often leads to a distribution of ligand arrangements on the nanoparticle
surface. {Pons:2006kr} This increases the permutations in which the two nanoparticles
may come together, negatively impacting the performance of the assay by the reality that
the dispersity of structures provides for a dispersity of quantitative responses. Formation
of aggregates is also unavoidable as a result of the multivalency of the functionalized
nanoparticles. Due to these complications, investigations using large gold nanoparticles
have been limited. Most early studies have focused on the use of small (≤ 5 nm) AuNPs
prepared stoichiometrically to achieve monovalent conjugation (Figure 1, Configuration
2) {Pons:2007do, Choi:2012ja, Kim:2008id}. However, the conjugation of multiple
AuNPs per QD is fraught with challenges as there exists a statistical distribution of
AuNP-QD conjugate ratios, especially at low equivalences. This distribution is known to
impact energy transfer efficiencies. {Beane:2014cf} The use of monovalently
functionalized QDs may overcome some of these challenges.
Figure 1 (a) Schematic illustration of the assay configurations investigated. Configuration 1 arranges multiple QDs around a single AuNP while configuration 2 places multiple AuNPs around a single QD. (b) Toehold mediated strand displacement assay.
An alternative arrangement involves implementation of QDs that are
monovalently functionalized with an oligonucleotide. These monovalently conjugated
QDs are assembled on AuNPs (Configuration 1; Figure 1a). In this manuscript, the
analytical performance of multiple QDs surrounding a AuNP is compared to that of the
usual configuration where a single QD is surrounded by multiple small AuNPs
(Configuration 2; Figure 1a). For the two configurations, inner filter effects and the
quenching efficiencies were evaluated to establish optimal performance criteria. A toe-
hold mediated DNA displacement assay served as a representative assay design to assess
analytical performance (Figure 1b). In this assay, the AuNPs and QDs were each
functionalized with a different DNA strand (probe and displacer). The complementarity
of the sequences led to the formation of AuNP-QD complexes via DNA hybridization.
Introduction of a target DNA strand caused the disassembly of the complex due to the
higher stability of the probe-target hybrid (compared to the probe-displacer hybrid).
Using AuNPs of three different sizes (diameter of 6, 13 and 30 nm), the influence of the
inner filter effect and energy transfer processes were individually investigated for
Configuration 1 and Configuration 2. The ability to assemble and disassemble the QD-
AuNP conjugates (via the displacement assay) enabled the measurement of QD
fluorescence intensity when bound to and also when separated from the AuNPs. The
differences in fluorescence intensity provided insight about the energy transfer process.
For independent investigations of the inner filter effect, QDs and AuNPs that did not
interact with each other were prepared and their fluorescence intensities were monitored
at varying ratios of QD to AuNP. Finally, the response of the various configurations to
changing target concentrations was measured to understand how to optimize analytical
performance.
Experimental
A detailed list of materials, instrumentation and methods is available in the
Supporting Information.
Inner filter effect
Investigations of the inner filter effect required nanoparticles without any DNA,
but protected with mPEG ligands to mitigate non-specific interactions. All QDs were
coated with a DHLA mPEG (MW 750 g mol-1) ligand, while the AuNPs were coated with
thiol-functionalized mPEG (MW 800 g mol-1). Details of the preparation of these
nanoparticles can be found in the Supporting Information
For Configuration 1, the QDs were kept at a constant concentration of 50 nM,
with the amount of AuNPs varied to obtain the required ratios of QD to AuNP. A
solution of QDs at a concentration of 50 nM, without any AuNPs, served as a control.
For Configuration 2, the AuNP (diameter: 6 nm) was kept at a constant
concentration of 50 nM, with the amount of QDs varied to obtain the required ratios of
AuNP to QD. For the various concentrations of QDs used, a similar solution was
prepared without any AuNPs to serve as the control sample.
The luminescence of the prepared solutions were determined with a PTI
QuantaMaster spectrofluorimeter. The excitation wavelength was 405 nm, with the
fluorescence emission collected at 480 – 580 nm for QD525, 520 – 620 nm for QD575
and 580 – 680 nm for QD630. The fraction of retained fluorescence was calculated as:
Fraction of initial fluorescence intensity=I AuNP
I No AuNP 1
where IAuNP refers to the fluorescence intensity of a particular concentration of QDs in the
presence of gold nanoparticles, and INo AuNP refers to the fluorescence intensity of a
solution with the same QD concentration but without any gold nanoparticles. The QD
emission peaks were fit to a Gaussian function and the area of the Gaussian curve
represented the fluorescence intensities.
Assay assembly
Details on the preparation of the various nanoparticle – DNA conjugates is
available in the Supporting Information
Configuration 1:
QD-DNA monoconjugates ([QD] = 100 nM) were incubated with varying
amounts of DNA functionalized AuNPs in borate buffer supplemented with sodium
chloride (final concentration: 0.5 M). In addition to the QD-DNA conjugates, a quantity
of QD630 equal to QD-DNA monoconjugate was also added to the sample to serve as an
internal standard. The solutions were incubated at room temperature for 1 hour to ensure
complete hybridization. Prior to measurements, the samples were diluted such that the
QD concentration was 50 nM.
Configuration 2:
AuNP-DNA monoconjugates ([AuNP] = 50 nM) were incubated in borate buffer
that was supplemented with sodium chloride (0.5 M) with varying amounts of QDs that
had been functionalized with multiple DNA strands. The solution was spiked with a
quantity of QD630 at a concentration equal to that of the QD-DNA conjugates to serve as
an internal standard. The solutions were incubated at room temperature for 1 hour to
ensure complete hybridization. Prior to measurements, the samples were diluted such that
the AuNP concentration was 50 nM.
Fluorescence intensity measurements
The fluorescence intensities of all solutions were measured using an
epifluorescence microscope (described in the Supporting Information). The
epifluorescence microscope made it possible to collect emission data from small solution
volumes (≤10 μL). The fluorescence spectrum for the different samples were obtained,
and then normalized to the peak intensity corresponding to QD630. The normalized
QD525 spectrum was fit to a Gaussian function to enable calculation of peak area.
Response to target hybridization was obtained by measuring the fluorescence
intensity of solutions where the QD-AuNP complexes were incubated with the specified
amount of target for at least 1 hour. Care was taken to ensure that the monovalently
functionalized nanoparticles were always at 50 nM prior to measuring the fluorescence
intensity.
The contrast ratio was obtained as per the following equation,
Contrast ratio=I TGT
I 02
where ITGT corresponds to the area of the emission envelop for a solution with target, and
I0 refers to the area of the emission spectrum for a solution without any target.
For most measurements, the contrast ratios were measured by incubating the QD-
AuNP complexes with an excess of target (>100 equiv.) for at least 1 hour. For the
calibration curve measurements, the QD-AuNP complexes were incubated with a series
of target amounts for at least 1 hour.
Fluorescence intensity determination from an iPad camera
The plastic tubes containing QD-AuNP conjugates that were incubated with
varying amounts of target were placed at a distance of 5 cm from a UV lamp (365 nm;
UVGL-58, LW/SW, 6W; The Science Company, Denver, CO, USA), with the lamp
coplanar to the tubes. The fluorescent image was collected 20 cm above the samples (90o
with respect to the light source) using a camera that was integral to an Apple iPad 4
(Cupertino, CA). The distance of the camera from the samples was chosen to ensure all
tubes were captured in a single image frame. Measurements were taken in a dark
environment to reduce background due to ambient light. The images were obtained under
the default settings without any special precautions.
The image was processed using ImageJ, where the RGB color splitting function
was used to generate the images of the green channel (QD525) and the red channel
(QD630). The intensities of the green channel were then normalized to that of the red
channel, and this served as the response signal for the assay. The contrast ratios were
calculated as described above.
Results and discussion
Assay design
A toe-hold mediated DNA strand displacement assay served as a model system to
optimize assay configurations that utilized quantum dots (QDs) as energy donors and
gold nanoparticles (AuNPs) as acceptors. The displacement assay required initial
formation of a DNA hybrid between a probe strand and a shorter displacer strand. Each
strand was immobilized onto a different nanoparticle, and upon DNA hybridization the
nanoparticles were brought in close proximity to facilitate efficient energy transfer. For
this system of nanoparticles, the displacer strand had to be at least 16 bases long
(Supporting Information, Figure S3). Shorter displacer strands resulted in lower
hybridization efficiencies (higher background), while longer DNA strands did not lead to
further improvements in hybridization efficiency. The target DNA was complementary to
the probe strand and was longer than the displacer strand. The probe-target hybrid had 30
complementary base pairs, compared to a maximum of 16-20 base pairs for the probe-
displacer hybrid. This ensured that the probe-target hybrid was more thermodynamically
stable than the probe-displacer hybrid, favoring its formation in the presence of target
DNA strands. Hybridization of the target DNA strand with the probe strand caused the
disassembly of the AuNP-QD complex, thus decreasing the energy transfer efficiency.
Two different configurations were investigated in these experiments (Figure 1).
Configuration 1 refers to an assay in which QDs were arranged around a single AuNP by
hybridization of complementary DNA strands. Configuration 2 made use of a single QD
that was surrounded by multiple AuNPs. For both configurations, the DNA sequences
were designed to ensure minimal separation distance between the two nanoparticles. This
required the placement of both nanoparticles (QDs and AuNPs) on the same end of the
DNA hybrid. Depending on the configuration, monovalent functionalization of the QD
(configuration 1) or the AuNP (configuration 2) with the probe DNA strand was required.
The monovalent functionalized nanoparticles were selected to provide for reduced steric
and electrostatic hindrance towards the binding of long target DNA strands. Monovalent
conjugates also prevented the formation of crosslinked aggregates.
The QDs were functionalized with a bidentate poly(ethylene glycol) methyl ether
ligand (DHLA – mPEG), which is known to provide QDs with excellent colloidal
stability under a variety of solution conditions (pH and ionic strength), and also to
prevent non-specific interactions with biomolecules and other interfaces.
{Susumu:2007fy} DNA conjugation onto the QDs was achieved using the well-known
interactions of a hexahistidine moiety with the inorganic surface of the QDs.
{Medintz:2007ch} AuNPs were functionalized with thiol terminated DNA strands using
the “salt aging” protocol discussed by Hurst et al. {Hurst:2006ea} The AuNPs were
further stabilized using a thiol functionalized poly(ethylene glycol) methyl ether ligand,
for the same reason QDs were functionalized with mPEG ligands (see above). When
required, monovalent conjugates of both QDs and AuNPs were prepared using the
protocol developed by Uddayasankar et al. {Uddayasankar:2014fi} (See supporting
information, Figure S2).
In the subsequent discussion, the QDs are classified using their emission
wavelength. For example, QDs that have an emission wavelength of 525 nm are referred
to as QD525. The AuNPs are classified using their diameter. For example, an AuNP with
a diameter of 6 nm would be referred to as 6AuNP.
Configuration 1: One AuNP – multiple QD system
Forming QD-AuNP complexes in solution results in a decrease of the
fluorescence intensity from QDs. This occurs for two reasons: the inner filter effect and
non-radiative energy transfer.
Assessing the influence of an inner filter effect requires QDs and AuNPs that co-
exist in solution, but with no physical contact between the two nanoparticles. Both the
QD525s and the AuNPs of three different diameters (6, 13 and 30 nm) were coated with
mPEG based ligands to minimize any non-specific interactions. The concentration of
AuNPs was varied while keeping the QD concentration constant at 50 nM. This provided
for solutions with varying ratios of QDs to AuNPs and since QD concentration was held
constant, an increase ratio is indicative of decreasing AuNP concentration. An idealized
physical model based on a packing arrangement of spheres around a central sphere was
taken into consideration when determining the appropriate ratios of QD525 to AuNP for
the different sizes of AuNPs (Supporting information, Table S1). The different sizes of
AuNPs were expected to accommodate varying numbers of QDs, necessitating the
investigation of multiple ratios of QDs per AuNP.
The presence of any inner filter effect was assessed by measuring the fluorescence
intensity of the series of solutions relative to a solution of QDs with no AuNPs. The data
from these experiments are presented in Figure 2 for three different sizes of AuNP (a – 6
nm, c – 13 nm and e – 30 nm). For all sizes of AuNPs, the general trend indicates a
decrease in fluorescence intensity for higher concentrations of gold nanoparticle (lower
ratios of QD to AuNP). Depending on the spectral characteristics of the chromophore, the
decrease in fluorescence intensity due to an inner filter effect is a combination of both a
decrease in penetration depth of excitation intensity, and a decrease in the emitted
intensity from the fluorophores. The absorbance spectra of the AuNPs and the emission
spectrum of QD525 are available in Figure S4. AuNPs have a significant extinction
coefficient at 405 nm (wavelength used to excite QDs), resulting in the attenuation of the
excitation source intensity. In fact, even the smallest AuNPs used in these experiments
(6AuNP) have an extinction coefficient at 405 nm (1.3x107 M-1cm-1) that is almost 20
times greater than the extinction coefficient of QD525 (6.8x105 M-1cm-1) at that
wavelength. In addition, the high spectral overlap between the emission of QD525 and
the plasmon absorption peaks of the AuNPs further attenuate the light intensity emitted
by the QDs (for spectral overlap integrals, see Table S2 in the Supporting information).
The 30AuNPs have the most significant impact with regards to decreasing the observed
photoluminescence of QDs due to the inner filter effect. Even for the highest ratios of QD
to AuNP (i.e. lowest AuNP concentration) that were investigated, only 30 % of the
fluorescence intensity was observable. For the 6AuNP and 13AuNP systems, up to 80 %
of the fluorescence intensity was retained at the highest ratios of QD to AuNP. Since the
attenuated fluorescence intensity is due to a reduction in excitation radiation and emitted
radiation, the product of the transmittance at the excitation wavelength and emission
wavelength (of a solution of AuNPs) should provide an estimate of the inner filter effect.
Figure S5 in the Supporting Information serves to compare the fluorescence intensities
observed to those predicted by the transmittance of AuNP solutions. While an exact
quantitative agreement between the theoretical predictions and experimental observations
was not expected, the trends that were experimentally observed closely followed the
theoretical predictions. Although the mPEG coatings are expected to reduce the
interactions between the nanoparticles, {Choi:2012ja} deviations between theory and
experimental data could result from some non-specific interactions between the
nanoparticles. Furthermore, colloidal AuNPs often have a distribution in the size of
nanoparticles. A distribution in the size of AuNPs results in a distribution of extinction
coefficients of the nanoparticles, adding more uncertainty to the prediction of the inner
filter effect. The most accurate method to assess the influence of an inner filter effect is
by experimental quantification.{Zhang:2015fu}
Figure 2 Effect of gold nanoparticle size on the performance of displacement assays involving quantum dots as donors and gold nanoparticles as acceptors. The trends in the inner filter effect (left axis) and contrast ratio (right axis) as a function of number of QDs per AuNP for three different sizes (diameter) of AuNPs; (a) 6 nm (c) 13 nm and (e) 30 nm. The TEM images provide confirmation of QD binding onto AuNPs of the three different sizes: (b) 6 nm (d) 13 nm and (f) 30 nm.
By establishing the trends associated with the inner filter effect, it was then
possible to evaluate the influence of AuNP size on the contrast ratio. Contrast ratio was
used as a parameter to evaluate energy transfer efficiencies, and is defined as the ratio of
the fluorescence intensity after target binding to that prior to introduction of the
oligonucleotide targets. Incubation of the AuNP-DNA conjugates with monovalently
functionalized QD-DNA conjugates resulted in the formation of “satellite structures” as
confirmed with TEM imaging (Figure 2(b), (d) and (f)). The monovalency of the QDs
prevented the crosslinking of multiple AuNPs. Introduction of the target oligonucleotides
resulted in the disassembly of the AuNP-QD satellite structures. To minimize the
influence of variable hybridization efficiencies, a high equivalence of target (>100 equiv.
with respect to the QD-DNA monoconjugates) was used. The plots represented in Figure
2 demonstrate the trends in contrast ratio for different ratios of QDs per AuNP, with each
of the panels corresponding to a different AuNP diameter. The highest contrast ratios
were observed for the 13 nm AuNP system, and even the lowest contrast ratio observed
with this system was higher than the largest ratios observed for the other two sizes of
AuNPs.
To investigate the quenching efficiencies of the AuNP – QD conjugates, the
fluorescence lifetime of QDs hybridized to AuNPs of the different diameters were
compared to QDs in the absence of AuNPs. The lowest QD to AuNP ratio presented in
Figure 2 was used for all sizes of AuNPs to ensure minimal contribution from QDs that
were not bound to AuNPs. The fluorescence lifetime, and associated quenching
efficiencies are presented in Table 1.
Table 1 Fluorescence lifetimes and quenching efficiency of QD525 with three different gold nanoparticle sizes.
Sample Fluorescence lifetime (ns)
Quenching efficiencya (%)
QD525 (No AuNP) 5.3 ± 0.3 -
QD525 – 6AuNP 3.2 ± 0.2 40 ± 1
QD525 – 13AuNP 1.7 ± 0.2 68 ± 1
QD525 – 30AuNP 3.6 ± 0.4 32 ± 1
aQuenching efficiency=(1−( τ AuNP
τ0 ))×100; where τAuNP refers to the fluorescence lifetime in the
presence of AuNPs and τ0 refers to that in the absence of AuNPs
Compared to QDs alone, the QDs incubated with AuNPs have a statistically
significant decrease in fluorescence lifetime, as is expected in scenarios involving non-
radiative energy transfer.{Lakowicz:tx} Comparing the fluorescence lifetimes in the
presence and absence of quencher allows the calculation of energy transfer efficiency (or
quenching efficiency). The 13AuNPs had a higher energy transfer efficiency compared to
the 6AuNP. This was expected given that the spectral overlap integral of the 13AuNP is
approximately an order of magnitude greater than that of the 6AuNP (Supporting
information, Table S2). These results are consistent with previous studies where larger
AuNPs provide for a higher quenching efficiency {Griffin:2009wr}, and is also
supported by the higher contrast ratio observed for the 13AuNP – QD system.
The 30AuNP – QD system had the lowest energy transfer efficiency of 32 %,
even though it has a spectral overlap integral that is 24 times greater than that of the
13AuNP (Table S2). This lower energy transfer efficiency is also consistent with the
lower contrast ratio observed for the 30AuNP – QD system. This issue was further
investigated by analyzing the hybridization efficiency between the AuNPs and QDs. This
was done only for the 13AuNP - QD and 30AuNP – QD system to resolve the
inconsistencies seen in the quenching efficiency measurements. Taking into account the
theoretical maximum of the number of QDs that may be assembled onto the AuNPs (See
Supporting Information, Table S1), the 13AuNP and 30AuNP were first incubated with
varying equivalents of QD525-DNA monoconjugates. The samples containing the two
nanoparticles were centrifuged at a sufficient centrifugal force to precipitate the AuNPs
while keeping the QDs in solution. Comparison of the quantity of QDs remaining in
solution to the initial amount of QDs provided an estimate of the number of QDs bound
to the AuNPs (Figure 3).
Figure 3 Hybridization efficiency of QD525 – probe DNA monoconjugates onto gold nanoparticles functionalized with displacer DNA. (a) 13 nm AuNP (b) 30 nm AuNP.
As the number of QDs incubated with 13AuNP increased, a decrease in the
hybridization efficiency was observed (Figure 3a). A lower hybridization efficiency at
QD to 13AuNP ratios greater than 10 is consistent with the trend of decreasing contrast
ratio as the ratio of QD to AuNP increases (Figure 2c). The QDs not bound onto the
13AuNP contributed to the fluorescence intensity measured. This provides higher
background fluorescence regardless of the presence of target, leading to a smaller
contrast ratio. The lack of quantitative binding, even for the smallest ratios of QD525 to
13AuNP, can be attributed to the equilibrium that exists between the bound and free form
of the QD-DNA monoconjugates. Decreasing the concentration of 13AuNP (i.e.
increasing ratio of QD to AuNP) results in a shift in the equilibrium to the unbound state,
consistent with the lower hybridization efficiencies observed at higher ratios of QD to
13AuNP. The fixed geometric volume around the AuNPs also limits the total number of
QDs that may be immobilized per AuNP.
Figure 3b illustrates the hybridization efficiencies of monovalent QD-DNA
conjugates on 30AuNPs. A decrease in the hybridization efficiency was noted as the ratio
of QDs to 30AuNP was increased as also observed for the 13AuNP-QD assemblies, but
the reduction of hybridization efficiency was more pronounced for the 30AuNPs. As the
size of an AuNP increases, the curvature of the surface decreases. A direct consequence
of surface curvature is the divergence angle between immobilized oligonucleotides.
Using the divergence angles estimated by Hill et al {Hill:2009vr}, 25-mer
oligonucleotides on 30AuNPs should have a divergence angle of 140, while those on the
13AuNPs have a divergence angle of approximately 240. The reduced divergence angle
on the 30AuNPs could lead to greater steric interactions between neighboring
biomolecules, shifting the equilibrium to the unbound state of QD-DNA monoconjugates.
Steric limitations are particularly important considerations in this system due to proximal
positioning of the QD and AuNP, and could explain the low hybridization efficiencies on
the 30AuNPs. The lower hybridization efficiency of the 30AuNPs results in a higher
background signal from QDs that remain in bulk solution (prior to target introduction).
This has the consequence of lowering the contrast ratio, even though larger nanoparticles
are known to have a higher quenching efficiency. These observations reflect the trends
observed in the contrast ratio as a function of AuNP size (Figure 2), and are consistent
with the fluorescence lifetime data (Table 1), for which the smaller decrease in lifetime
was attributed to the presence of QDs not hybridized onto the AuNPs.
It should be noted that purification of AuNP-QD complexes from unhybridized
QD-DNA conjugates was attempted by centrifugation. However, centrifugation
negatively impacted the colloidal stability of the AuNP-QD complexes, and significant
sedimentation was observed. The response of the purified complexes to the presence of
target DNA strands was very low (results not shown).
The DNA sequences were designed to ensure that the QDs and AuNPs were on
the same end of the DNA hybrid. Investigation of the impact of proximal versus distal
positioning of QDs was completed to evaluate any impact of steric hindrance on surface
loading (Figure 4).
Figure 4 (a) Schematic illustration of the proximal and distal configuration of the nanoparticle complex for nucleic acid displacement assays (b) Contrast ratio of distal and proximal configurations for AuNPs of three different sizes.
The contrast ratios obtained for the three different sizes of AuNPs for both the
proximal and distal configurations are listed in Figure 4b. For all sizes of AuNPs, the
contrast ratio was higher for the proximal than the distal configuration. The proximal
design positions the QD closer to the AuNP surface, improving non-radiative energy
transfer efficiency. Figure S6 in the supporting information illustrates the absolute
fluorescence intensities for the QD-AuNP conjugates before and after the introduction of
target. For the distal and proximal configurations, the differences observed in the
fluorescence intensities after the target DNA strand displaces the QDs from the AuNPs is
minimal (<5 %), while the differences in fluorescence intensity prior to target
introduction are greater. The difference is greatest for the 13AuNP system, where the
distal configuration has a 28 % greater fluorescence intensity when compared to the
proximal configuration. These observations suggest that the variations in contrast ratios
between proximal and distal configurations arise predominantly from the lower
quenching efficiency in the distal configuration. The differences are not as significant for
the 6AuNP and 30AuNP system due to their initial low contrast ratios. These
observations demonstrate the lack of any detrimental effects to placing the nanoparticles
in a proximal arrangement.
Contrast ratio and inner filter effects were also determined for QDs that emit at
575 nm and 630 nm (Figure 5). Monovalent conjugates of QDs with emission
wavelengths of 575 nm and 630 nm were prepared by the same protocol used for the
QD525. Complexes of QDs and AuNPs were prepared using only 13AuNP so they may
be evaluated with the optimal performance previously noted for QD525. Emission spectra
overlaid onto the absorption spectra of 13AuNPs are provided in the supporting
information (Figure S7)
Figure 5 Effect of QD emission wavelength on displacement assay performance where QDs serve as the donor and 13AuNPs serve as the acceptor, with multiple QDs arranged around the AuNP (Configuration 1). The trends in inner filter effect (left axis) and contrast ratio (right axis) as a function of number of QDs per AuNP for AuNPs of two different emission wavelengths (a) 575 nm (b) 630 nm.
A greater fraction of fluorescence intensity is preserved for the QD575 and
QD630 compared to the QD525 (Figure 2c) when considering the inner filter effect. This
may be rationalized by the smaller spectral overlap integral between the emission
intensities of the QDs and the 13AuNP (Table S3). The higher fluorescence intensities
were accompanied by a lower contrast ratio, and the results are consistent with the
decrease in the spectral overlap between the emission of the QDs and the absorbance of
13AuNPs.
Configuration 2: One QD – multiple AuNP system
The configuration most commonly used in assay development makes use of a
single QD surrounded by multiple AuNPs that serve as acceptors. Such a configuration
takes advantage of higher energy transfer efficiencies obtained by surrounding a single
donor (QD) with multiple acceptors (AuNPs). QD525 was functionalized with multiple
DNA strands, with successful conjugation confirmed using agarose gel electrophoretic
analysis and UV-vis spectroscopy (Figure S8). 6AuNPs were monofunctionalized with
DNA using the same protocol developed for QDs (see Supporting Information, Figure
S2). Assembly of the conjugates was accomplished by incubating the DNA modified
QDs and AuNPs at specific ratios. 6AuNP, rather than smaller AuNPs, was chosen as the
acceptor to provide for improved analytical performance.{Choi:2012ja} In these
experiments the AuNP concentration was kept constant at 50 nM, with the amount of
QDs varied to obtain the specified AuNP to QD ratios. It is important to note that in
Configuration 1, it was the QDs that were kept at a constant concentration, with the
concentration of AuNP varied to obtain the desired ratios. In Configuration 1, it was the
QDs that hybridized to the target. In Configuration 2, the target hybridized to the 6AuNP.
To make a valid comparison between the two configurations, the concentration of
nanoparticles that responds to the target DNA should be the same. Contrast ratios were
obtained by taking fluorescence measurements before and after incubation of the
complexes with an excess of target DNA (>100 equiv.). Figure 6 depicts the contrast
ratios for varying ratios of AuNP to QD.
Figure 6 Inner filter effect (left axis) and contrast ratio (right axis) as dependent on the number of AuNPs per QD, for a displacement assay system involving multiple AuNPs interacting with a single QD (Configuration 2).
Increasing the ratio of AuNPs to QD resulted in an increase in the contrast ratio.
For assays based on energy transfer, a greater number of acceptors per donor improves
energy transfer efficiency. {Medintz:2003fr} This provides for a greater quenching
efficiency of the QDs, leading to a larger contrast ratio. For equivalences <2, a contrast
ratio of less than 1.5 is obtained. Equivalences <2 should be avoided due to the presence
of QDs that are not associated with an acceptor AuNP. The high surface area of the QDs
results in a statistical distribution of QD-AuNP conjugates that can impact assay
performance, especially at low equivalences. {Scott:2014iv} Assuming the distribution
follows Poisson statistics, an AuNP to QD equivalence of 1 leaves 37 % of the QDs
without an acceptor, and this fraction decreases to 14 % for an equivalence of 2. QDs that
have no acceptor would contribute to the background signal without taking part in the
assay. Above an equivalence of two, contrast ratios greater than 2 are obtained with 5
AuNPs per QD providing a contrast ratio of approximately 3. Equivalences above 5 were
not attempted because the spectrofluorimeter could not detect the fluorescence intensity
of the QDs in solutions for AuNP to QD ratios greater than 5.
The inner filter effect was also investigated (Figure 6). Both QDs and AuNPs
were functionalized with mPEG ligands to reduce non-specific interactions. Increasing
the ratio of AuNP to QD leads to a decrease in the observed fluorescence intensity, yet
the correlation between the decrease in fluorescence and the ratio of AuNPs per QD is
not as pronounced as that observed for Configuration 1 (Figure 2). This is attributed to
the fact that the concentration of AuNPs was kept constant for the different solutions. The
constant concentration of AuNPs attenuated the excitation and emitted radiation to the
same extent in solutions with varying ratios of AuNP to QD. For the ratios of AuNP to
QD investigated, only 15 - 26 % of the fluorescence was observed (when compared to a
situation with no AuNPs).
Comparing Configuration 1 and Configuration 2
The analytical performance of the two configurations was investigated by looking
at the response of the systems to increasing target concentrations (Figure 7).
Figure 7 Comparing the analytical performance of the two configurations of displacement assay. (a) The contrast ratio as a function of target concentration for the two different assay configurations, each performed at two different ratios of the QDs and AuNPs. (b) Absolute fluorescence intensities of the sample solutions corresponding to the different assay configurations.
Figure 7a provides the response curves for four different nanoparticle
configurations (two representative examples of Configuration 1, two of Configuration 2).
All arrangements had the same number of monovalent nanoparticles, and this is reflected
in the response curves where the response saturated using the same amount of target.
Presented in Figure 7b are the absolute fluorescence intensities of the solutions for the
four different arrangements. The variations in the observed fluorescence intensity arise
from the differential inner filter effects of the various nanoparticle arrangements. To
determine the optimal arrangement, both fluorescence intensity and any inner filter effect
need to be considered concurrently.
The response curves with the lowest and highest responses to changing quantity
of target correspond to Configuration 2 with two 6AuNP per QD525 and Configuration 1
with 5 QD525 per 13AuNP, respectively. This particular arrangement of Configuration 1
had a greater fluorescence intensity than that of the Configuration 2, which is a
consequence of the lower inner filter effect observed when multiple QDs are associated
with a single AuNP, rather than multiple AuNPs being associated with a single QD.
The other two response curves presented in Figure 7a correspond to Configuration
1 with 15 QD525 per 13AuNP and Configuration 2 with three 6AuNP per QD525. Both
these curves show similar sensitivities to changing amounts of target, but the observed
fluorescence intensities are significantly different between these configurations. The
intensities of the Configuration 1 arrangement were approximately five times greater than
for the construct based on multiple 6AuNPs arranged around a single QD.
An optimal configuration should provide high absolute fluorescence intensities
(i.e. low inner filter effect) while at the same time offering a high contrast ratio to allow
for sensitive measurements. This is certainly desirable when assays require the use of
simplified readout platforms such as consumer cellphone/tablet cameras. Using these
criteria, the configuration based on 15 QD525s around a single 13AuNP is optimal
(Figure 8).
Figure 8 Fluorescence images collected using an iPad camera for QD-AuNP complexes incubated with varying amounts of target (pmol) (i) 0 (ii) 0.20, (iii) 0.30, (iv) 0.40, (v) 0.50, (vi) 0.60, (vii) 0.70, (viii) 0.80, (ix) 1.00, (x) 2. (a) 2 6AuNP per QD525 (b) 3 6AuNP per QD525 (c) 15 QD525 per 13AuNP (d) 5 QD525 per 13AuNP (e) Response curves obtained from images in panel (a) and (b). (f) Response curves obtained from images in panel (c) and (d).
Fluorescence photographs (using UV lamp excitation) of the solutions used to
obtain the response curves depicted in Figure 7 are shown in Figure 8. Visual inspection
of the images shows the significant improvement in assay performance that may be
obtained from Configuration 1 (Figure 8c and d), as opposed to Configuration 2 (Figure
8a and b). Fluorescence is difficult to visually discern in panels (a) and (b), while the
fluorescence intensities are obvious in panels (c) and (d). Intensity measurements
determined using a spectrofluorimeter (Figure 7) demonstrated that Configuration 2
provided similar sensitivities when compared to Configuration 1 (Table 2). However, the
use of a tablet camera to collect a fluorescence image using Configuration 2 provided
data that was of low quality for construction of a response curve (Figure 8e). The use of
Configuration 1 provided images that yielded a response curve that were better suited for
quantitative analysis (Figure 8f).
Table 2 Response curve sensitivity for Configuration 1 and Configuration 2
Configuration Slope ( x 10-13 )aSpectrofluorimeter iPad Camera
5 QD525 per 13AuNP 2.8 ± 0.7 2.1 ± 0.815 QD525 per 13AuNP 2.7 ± 0.4 2.3 ± 0.63 6AuNP per QD525 2.1 ± 0.5 0.1 ± 1632 6AuNP per QD525 1.8 ± 0.4 0.2 ± 0.7
aThe slope was determined from the equation of the sigmoidal curve for the different
dose-response plots: Contrast ratio=base+ max
1+e( xhalf −x
slope )
This is further supported by sensitivity measurements of the various response
curves, which is presented in Table 2. Using the spectrofluorimeter to measure the
samples provided similar sensitivities for all configurations, with Configuration 1 having
slightly higher sensitivities (at most 25 % better). But looking at the data collected using
the iPad camera, Configuration 2 had sensitivities that were an order of magnitude lower
than those for Configuration 1. In addition, the high uncertainties associated with the
slope of the response curves obtained by the iPad camera is indicative of poor fit of the
data to the expected sigmoidal curve shape. Comparing the two configurations,
Configuration 1 provides higher signal intensities while still maintaining the assay
response to target hybridization (contrast ratio).
Conclusion
The relative arrangement of QDs and AuNPs has a significant impact on the
analytical performance of assays that rely on energy transfer between the two
nanoparticles. Using monovalently functionalized nanoparticles, QD-AuNP complexes
were prepared in two different configurations. Configuration 1 assembled monovalent
QD-DNA conjugates around a central AuNP and made use of three different sizes of
AuNPs (6 nm, 13 nm and 30 nm). Configuration 2 placed monovalent 6AuNP-DNA
conjugates around a central QD. Both these configurations were assembled using DNA
hybridization. Energy transfer processes were investigated by looking at fluorescence
intensity changes as the complexes were disassembled using DNA strand displacement
reactions. Additionally, the strong optical absorption characteristics of AuNPs led to
significant reductions of fluorescence intensity due to the inner filter effect. Both these
factors were concurrently evaluated to establish the optimal configuration. Optimum
performance was obtained using Configuration 1 with at most 15 QDs assembled around
a 13AuNP. This system offered a 2.5 fold increase in fluorescence signal upon target
recognition. Similar increases in signal intensity using Configuration 2 required at least
three 6AuNP around the QD. However, this resulted in Configuration 2 having a 5 fold
lower signal intensity (compared to Configuration 1) due to inner filter effects. Dose-
response curves were also constructed for the two configurations, the sensitivities of
which indicated that Configuration 1 provided better analytical performance. In fact,
when low cost detectors were used (digital camera), Configuration 2 had sensitivities that
were an order of magnitude lower than those obtained using Configuration 1. These
results serve to provide important guidelines for the design of assays that use gold
nanoparticles and quantum dots as part of an energy transfer assay.
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