Ubiquinone-quantum dot bioconjugates for in vitro and intracellular complex I sensing

Abstract

Quantum dots (QDs) have attracted increasing interest in bioimaging and sensing. Here, we report a biosensor of complex I using ubiquinone-terminated disulphides with different alkyl spacers (QnNS, n = 2, 5 and 10) as surface-capping ligands to functionalise CdSe/ZnS QDs. The enhancement or quenching of the QD bioconjugates fluorescence changes as a function of the redox state of QnNS, since QDs are highly sensitive to the electron-transfer processes. The bioconjugated QnNS-QDs emission could be modulated by complex I in the presence of NADH, which simulates an electron-transfer system part of the mitochondrial respiratory chain, providing an in vitro and intracellular complex I sensor. Epidemiological studies suggest that Parkinson's patients have the impaired activity of complex I in the electron-transfer chain of mitochondria. We have demonstrated that the QnNS-QDs system could aid in early stage Parkinson's disease diagnosis and progression monitoring by following different complex I levels in SH-SY5Y cells.

Full text

Ubiquinone-quantum dot bioconjugates
for in vitro and intracellular complex I
sensing
Wei Ma
1
*, Li-Xia Qin
1
*, Feng-Tao Liu
2
, Zhen Gu
1
, Jian Wang
2
, Zhi Gang Pan
3
, Tony D. James
4
& Yi-Tao Long
1
1
State Key Laboratory of Bioreactor Engineering & Department of Chemistry, East China University of Science and Technology, 130
Meilong Road, Shanghai, 200237, P. R. China,
2
Department of Neurology, Huashan Hospital Affiliated to Fudan University, 12
Wulumuqi Zhong Road, Shanghai, 200040, P. R. China,
3
Department of Medicine, Zhongshan Hospital Affiliated to Fudan
University, 180 Fenglin Road, Shanghai, 200032, P. R. China,
4
Department of Chemistry, University of Bath, Claverton Down, Bath,
BA2 7AY, UK.
Quantum dots (QDs) have attracted increasing interest in bioimaging and sensing. Here, we report a
biosensor of complex I using ubiquinone-terminated disulphides with different alkyl spacers (Q
n
NS, n 52,
5 and 10) as surface-capping ligands to functionalise CdSe/ZnS QDs. The enhancement or quenching of the
QD bioconjugates fluorescence changes as a function of the redox state of Q
n
NS, since QDs are highly
sensitive to the electron-transfer processes. The bioconjugated Q
n
NS-QDs emission could be modulated by
complex I in the presence of NADH, which simulates an electron-transfer system part of the mitochondrial
respiratory chain, providing an
in vitro
and intracellular complex I sensor. Epidemiological studies suggest
that Parkinson’s patients have the impaired activity of complex I in the electron-transfer chain of
mitochondria. We have demonstrated that the Q
n
NS-QDs system could aid in early stage Parkinson’s
disease diagnosis and progression monitoring by following different complex I levels in SH-SY5Y cells.
Parkinson’s disease (PD) is a complex neurodegenerative disorder affecting the elderly with many different
causes but probably then evolves via common pathways
1,2
. Currently, diagnosis of PD almost relies on
clinical acumen. There are no established laboratory tests or biosensors that can reliably and specifically
identify PD. Moreover, differential diagnosis for PD can be rather challenging due to overlapping symptoms,
particularly in its early stages
3–5
. Thus, there is an urgent clinical need to develop biosensors for the diagnosis of
PD and differentiation of disease progression. Current evidence suggests that mitochondrial NADH:ubiquinone
oxidoreductase (complex I) inhibition may be the central cause of sporadic PD and that disorders in complex I
causes the demise of dopamine neurons, which contributes to the major clinical symptoms of PD
6–8
. The
relationship between loss of complex I activity and PD progression may provide a path to early diagnosis and
monitoring of PD.
Complex I is the first enzyme of the mitochondrial respiratory chain and plays a central role in cellular energy
production, coupling electron-transfer between NADH and ubiquinone to proton translocation, helping to
provide the proton-motive force required for the synthesis of adenosine triphosphate
9
. As an essential cofactor
in the respiratory chain, ubiquinone, also known as coenzyme Q, is found at the hydrophobic core of the
phospholipid bilayer of the inner membrane of mitochondria
10
and serves as a mobile carrier transferring
electrons and protons
11,12
. The activities of reversible redox cycling between the ubiquinone and ubiquinol in
the electron transport chain allow the ubiquinone molecule to function as a valuable mediator.
Semiconductor quantum dots (QDs) have widespread applicability in areas ranging from in vivo imaging
13,14
and clinical diagnostics
15,16
in biomedicine to environmental monitoring for public health and security due to
their unique optical properties, including tunable fluorescence narrow emission, broad absorption profiles, high
signal brightness and superior photostability
17,18
. In addition, QDs are extremely sensitive to the presence of
additional charges either on their surfaces or in the surrounding environment, which can lead to a variety of
optical properties and electronic consequences
13
. The redox potential of capping molecules can be chosen to
maximize the efficiency of charge transfer to promote transfer of external electrons and holes to either the QDs’
core conduction band (CB) or the QDs’ surface states
15
. Thus, controlling charge transport across redox-active
molecules functionalised QDs has generated interest for advanced molecular and cellular imaging as well as
SUBJECT AREAS:
BIOSENSORS
PARKINSON’S DISEASE
FLUORESENT PROBES
BIOMIMETICS
Received
17 December 2012
Accepted
27 February 2013
Published
25 March 2013
Correspondence and
requests for materials
should be addressed to
Y.-T.L. (ytlong@ecust.
edu.cn)
*These authors
contributed equally to
this work.
SCIENTIFIC REPORTS | 3 : 1537 | DOI: 10.1038/srep01537 1

ultrasensitive biosensing
14,19
. For instance, QD-dopamine bioconju-
gates stain dopamine-receptor-expressing cells in redox-sensitive
patterns
20
. Dopamine as an electron donor could sensitize QDs
through different mechanisms involving reactive oxygen species
(ROS)
20–23
. Recently, we demonstrated that coupling QDs with cyto-
chrome c is capable of fluorescence imaging of a superoxide radical
with high specificity
24
. Ubiquinone-coupled QDs could be used for
quantitative detection of ROS in living cells
25
. Cumulatively, these
results confirm a role for redox molecules, and especially quinone, in
charge-transfer interactions with QDs; however, the improvement of
QD bioconjugates compatibility in biological system and how to
exploit it as biosensors for clinical diagnostic applications is lacking.
Herein, we report the design and preparation of colloidal CdSe/
ZnS QDs utilizing three ubiquinone-terminated disulphides with
different alkyl spacer, Q
n
NS (n52, 5 and 10), appended with 1,2,3-
triazole that are synthesized as surface-capping ligands to functiona-
lise QDs (Q
n
NS-QDs). Using the Q
n
NS-QD bioconjugates, we found
that either quenching or enhancing the QDs’ emission is reversibly
tuned by the redox state of surface-capping layer, following the
transformation between oxidized ubiquinone (Q
n
NS) and reduced
ubiquinol (HQ
n
NS). There is a direct interplay between ubiquinone
and NADH in the enzymatic reaction of the electron transport chain
and it enables us to follow the activities of complex I to develop a
unique optical sensor for complex I. We have demonstrated that the
emission of Q
n
NS-QDs is enhanced with complex I in the presence
of NADH, which is attributed to the oxidized ubiquinone being
reduced to ubiquinol on the QD surface. Our strategy is aimed at
using the QD bioconjugates to follow deficient levels of complex I in
human neuroblastoma SH-SY5Y cells. We believe our approach may
hold particular promise as a powerful fluorescence biosensor target-
ing the clinical diagnosis of PD.
Results
Design of surface-capping Q
n
NS ligands.We designed QD biocon-
jugates for biosensing complex I using ubiquinone-terminated
disulphide ligands and 550-nm-emitting core-shell CdSe/ZnS QDs.
Three ligands Q
2
NS,Q
5
NS and Q
10
NS were prepared using a facile
click reaction using copper(I) tris(benzyltriazolylmethyl) amine-
catalysed 1,2,3-triazole formation
26
between alkylazide-disulphides
and ubiquinone with terminal alkynes (Fig. 1a). We introduced the
quinoid moiety in the Q
n
NS ligands to achieve the redox-switchable
fluorescence properties that could be useful for signal multiplexing.
The 1,2,3-triazole groups, similar to histidine
27
, could enhance the
compatibility of Q
n
NS-QDs in biological systems. Three alkyl linkers
confer different abilities of electron-transfer to either the QDs’ core
or the surface of QDs. Finally, the disulfide group facilitates binding
of Q
n
NS to the QDs. The synthetic procedures and structural charac-
terisation of the Q
n
NS ligands are presented in the Supplementary
Information.
Fluorescence spectra of Q
n
NS and HQ
n
NS-functionalised CdSe/
ZnS QDs.We investigated the fluorescence effects of QD biocon-
jugates by using QDs capped with oxidized Q
n
NS and reduced
HQ
n
NS because QDs are prone to exchange electrons or energy
with the attached ligands upon excitation, resulting in their fluore-
scence change. As shown in Figure 2a–c, for Q
n
NS-QD bioconju-
gates, the fluorescence intensity gradually decreased with increasing
ratios (20–100) of Q
n
NS to QDs compared to the unconjugated QDs,
at high Q
n
NS (ratio 120) levels quenching of the QD bioconjugates
was saturated giving an average coverage of 100 Q
n
NS molecules per
CdSe/ZnS QDs. As can be seen, fluorescence quenching efficiency
was dependent on the alkyl chain spacer of Q
n
NS, since a more
pronounced quenching was observed for the shorter Q
2
NS-
modified QDs. As spacer length increases, the quenching efficiency
of the three ubiquinone-functionalised CdSe/ZnS QDs (ratio 100)
decreases: ,77% (Q
2
NS-QDs), ,56% (Q
5
NS-QDs) and ,37%
(Q
10
NS-QDs), respectively. We further examined fluorescence
effects of the QDs modified with the reduced ubiquinol, HQ
n
NS.
Surprisingly, the fluorescence intensity of reduced HQ
n
NS-QD
bioconjugates gradually enhanced at given ratio of HQ
n
NS to QDs
from 20 to 100. At a HQ
n
NS/QDs ratio of 120, no additional increase
of fluorescence was observed which indicated that on average 100
HQ
n
NS ligands assembled to these CdSe/ZnS QDs (Fig. 2a–c).
The alkyl chain spacer-dependent trend for fluorescence enhance-
ment of HQ
n
NS-QDs (ratio 100) was ,44% (HQ
2
NS-QDs), ,33%
(HQ
5
NS-QDs) and ,23% (HQ
10
NS-QDs), respectively. The insets
in Figure 2a–c schematically depict the linear correspondence of QD
bioconjugates fluorescence changes as a function of either ubiquinol
or ubiquinone ratio and reaches saturation.
Electrochemical switching fluorescence of Q
n
NS modified CdSe/
ZnS QDs.In this work, we investigated the redox properties of the
surface-capping ligands effect on the fluorescence of QD biocon-
jugates following the transformation between Q
n
NS and HQ
n
NS
state when applying constant potential. As depicted in Figure 3a, b
and c, at the applied reduction potential of 20.30 V vs. SCE, the
fluorescence intensity of Q
2
NS,Q
5
NS and Q
10
NS-modified CdSe/
ZnS QDs increased during electrolysis. It should be noted that the
Q
n
NS ligands are reduced to generate the ubiquinol, which resulted
in an enhancement of QDs’ fluorescence. When applying a constant
potential of 0.10 V vs. SCE to Q
2
NS-QDs (50 s), Q
5
NS-QDs (55 s)
and Q
10
NS-QDs (60 s), a decrease in fluorescence intensity was ob-
served indicating that the capping layer of HQ
n
NS was reoxidized to
the Q
n
NS layer. We then looked to confirming that the redox couple
ubiquinon/ubiquinol is responsible for the QDs’ fluorescence quen-
ching/enhancement. Control experiments indicated that no effect
was observed on the fluorescence intensity when unmodified CdSe/
ZnS QDs were used at a potential of 20.30 V or 0.10 V vs. SCE under
the same conditions (data not shown). Moreover, in situ UV-vis
spectroelectrochemical experiments have also confirmed that
transformation of surface-capping layer between ubiquinone and
ubiquinol could be modulated effectively by electrochemistry (see
Fig. S1, Supplementary Information).
Figure 3d, e and f present the time-dependent changes in fluor-
escence intensity for ubiquinone/ubiquinol upon repeated reduction
and oxidation cycles of the Q
n
NS-QDs with applied potential.
Clearly, the fluorescence intensity of Q
2
NS,Q
5
NS and Q
10
NS-
modified QDs are enhanced significantly with an increase of elec-
trolysis time (0,50 s, 0,55 s and 0,60 s, respectively) at the
potential of 20.30 V vs. SCE. Subsequently, QDs’ fluorescence
intensity gradually decreased at the potential of 0.10 V vs. SCE with
electrolysis time (0,50 s, 0,55 s and 0,60 s, respectively). Clearly
demonstrating that the oxidized capping layer (ubiquinone) converts
to its reduced form (ubiquinol) and is reoxidized again (ubiquinone)
on the QD surface. These results show excellent reversibility over
three cycles with only small losses of fluorescence intensity in the
subsequent cycles and rapid transformation between redox states of
surface-capping ligand, Q
n
NS and HQ
n
NS,circa 2 min. Overall,
these results indicate the feasibility of using such QD bioconjugates
as switchable fluorescence sensors.
The biocatalytic reduction of Q
n
NS-modified CdSe/ZnS QDs
utilizing complex I and NADH.To further investigate the viabi-
lity of Q
n
NS-CdSe/ZnS QDs’ compatibility in biological systems, we
coupled the biocatalytic reduction to complex I enzymatic reaction
using NADH cofactors to mimic the well-known ‘Q cycle’ model in
electron-transfer process of the respiratory chain (Fig. 1b)
28
.As
shown in Figure 4a, b, c, the time-dependent fluorescence intensity
of Q
n
NS-QD bioconjugates increase upon the interaction with
complex I (4.9 U) in the presence of 10 mM NADH. The results
indicate that the biocatalytic two-electron, two-proton reduction of
Q
n
NS utilising complex I and NADH cofactor results in HQ
n
NS-
functionalised QDs and switches on the QDs’ fluorescence. The
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 3 : 1537 | DOI: 10.1038/srep01537 2

Figure 1
|
Schematic of ubiquinone-CdSe/ZnS QDs as redox fluorescence biosensor for Parkinson’s disease diagnosis. (a), Ubiquinone-terminated
disulphides (Q
n
NS) synthesis and self-assembly of Q
n
NS on to CdSe/ZnS QDs. (b), Conceptual visualisation of Q
n
NS-QDs as complex I sensor in vitro.
Under oxidized state (Q
n
NS), ubiquinone functions as a favorable electron acceptor, this results in effective QDs’ fluorescence quenching. Addition of
complex I to Q
n
NS-QDs solution in the presence of NADH, ubiquinone coupled electron transfer and proton translocation from NADH, producing
reduced ubiquinol (HQ
n
NS) form on the surface of QDs to mimic the initial stages of the respiratory chain. Ubiquinol when in close proximity to the QDs
produces fluorescence enhancement. (c), Energetic diagram of the QDs bioconjugates between QDs and Q
n
NS/HQ
n
NS. (d), Fluorescence spectra of
ubiquinone/ubiquinol- functionalised CdSe/ZnS QDs. (e), Cyclic voltammetry of Q
n
NS-CdSe/ZnS QDs. (f), Visualisation of Q
n
NS-CdSe/ZnS QDs as an
intracellular complex I sensor. The mitochondrial-specific neurotoxin, rotenone, inhibits complex I and leads to Parkinson’s-like pathogenesis.
Parkinson’s disease is characterised by impaired activity of complex I in mitochondria.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 3 : 1537 | DOI: 10.1038/srep01537 3

insets in Figure 4a, b, c illustrate that QDs’ fluorescence intensity
increased as a function of incubation time, and reaches saturation
after ,4.5, ,5.0 and ,5.5 min for the Q
2
NS,Q
5
NS and Q
10
NS-QDs
systems, respectively. Notably, we incorporated a 1,2,3-triazole
group in these ligands that could be useful for enzyme affinity.
Therefore in control experiments linking groups without triazoles
Q
n
S(see Fig. S2, Supplementary Information) were prepared. Figure
S2 shows similar time-dependent fluorescence enhancements from
QDs assembled with Q
n
Sexcept a significant increase in incubation
time compared to triazole linked Q
n
NS-QDs. The fluorescence
Figure 2
|
Effects of ubiquinone and ubiquinol on fluorescence spectra of QDs. (a–c), Representative fluorescence spectra collected from
550-nm-emitting hydrophilic CdSe/ZnS QDs (0.2 M PBS buffer; pH 8.0) recorded before and after self-assembly with an increasing ratio of ubiquinol
and ubiquinone added to PBS buffer at Q
2
NS (a), Q
5
NS (b) and Q
10
NS (c). Spectra were collected on a Shimadzu Cary Eclipse (Varian) fluorometer with
350 nm excitation. Inset: Plot of QD bioconjugates fluorescence at 550 nm versus HQ
n
NS and Q
n
NS to QDs ratio. Standard deviations are calculated
from at least three replicate samples are shown. (d), Normailized cyclic voltammetry of Q
n
NS modified QDs on glassy carbon electrode in PBS buffer of
pH 58.0 at a 100 mV?s
21
scan rate. SCE is saturated calomel electrode.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 3 : 1537 | DOI: 10.1038/srep01537 4

intensity reaches saturation after ,7.0, ,8.0 and ,10.5 min for Q
1
S,
Q
5
Sand Q
10
S-QDs system, respectively. This is because the triazole
groups behave similar to the histidine ligand could be used to cap
enzyme through proteins or peptides-affinity coordination of
triazole residues for simultaneously improving QD bioconjugates’
aqueous dispersion and biocompatibility
27
; Which results in the
triazole Q
n
NS ligands having better binding affinity with complex I.
As shown in Figure 4d, as the concentrations of complex I
increase, the fluorescence intensity of Q
n
NS-QDs in the presence
of NADH becomes more intense with respect to the initial fluor-
escence intensity (F
0
), which agrees with the functionalisation of
higher HQ
n
NS concentrations on the QDs’ surface. However, con-
trol experiments revealed that the Q
n
NS-QDs’ fluorescence was
insensitive to high concentrations of complex I when NADH was
excluded from the system (data not shown). The changes in the
fluorescence intensities of three QD systems were obtained with
NADH for complex I in the range of 0.02 U to 4.9 U, 0.03 U to
4.1 U and 0.04 U to 3.1 U for Q
2
NS,Q
5
NS and Q
10
NS, respectively.
The Q
2
NS-QDs showed a lower detection limit and wider response
range compared to both Q
5
NS and Q
10
NS-QDs.
Moreover, the corresponding spectral changes in absorption by
UV–vis spectroscopy were monitored upon addition of complex I
in the presence of NADH. As shown in Figure 4e, in the absence of
complex I, the solution displays a band at 275 nm, attributed to the p-
p* absorption spectrum of ubiquinone and NADH. The remaining
peak at 340 nm, is characteristic of NADH. Clearly, as the concen-
tration of complex I increases, the absorbance at 275 nm decreases
and the NADH absorption peak at 340 nm gradually disappears, with
subsequent buildup of the absorbance at 290 nm (Fig. 4e, 1.5 U com-
plex I and 3.1 U complex I), and then the band is shifted to longer
wavelengths (from 290 to 305 nm) as the concentration of complex I
is increased (Fig. 4e, 4.1 U complex I), which indicates that ubiqui-
none could be reduced to ubiquinol
29
. This absorption profile demon-
strates that ubiquinone accepts two-electrons and two-protons from
Figure 3
|
The fluorescence spectra changes with applied potential. (a–c), The fluorescence emission spectra of Q
2
NS (a), Q
5
NS (b) and Q
10
NS (c)
functionalised CdSe/ZnS QDs changes with applied potential. At the applied potential of 20.30 V vs. SCE, the fluorescence intensity of QD bioconjugates
gradually increased. At a constant applied potential of 0.10 V vs. SCE, the fluorescence intensity decreased over time. (d–f), Time-dependent fluorescence
intensity changes upon the redox cycle of Q
2
NS (d), Q
5
NS (e) and Q
10
NS (f) functionalised CdSe/ZnS QDs with applied potential. The fluorescence cyclic
change follows with a cyclic change of the electrolysis time.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 3 : 1537 | DOI: 10.1038/srep01537 5

NADH to convert to ubiquinol on the QDs’ surface under biocatalysis
of complex I to mimic the initial stage of respiration.
Discussion
The fluorescence enhancement and quenching of QD bioconjugates
could be reversibly switched with the redox state of surface-capping
ligands, between Q
n
NS and HQ
n
NS. Following photoexcitation of
Q
n
NS-QD bioconjugates, QDs’ CB electron is transferred to the low-
est unoccupied molecular orbital (LUMO) of the ubiquinone accep-
tor and the electron is then shuttled back to the QDs’ valence band
(VB) through non-radiative pathways, releasing heat (Fig. 1c). Thus,
ubiquinones exhibit surface-related trap states acting as fast non-
radiative de-excitation routes for photoinduced electron carriers,
leading to fluorescence quenching
24
. It is worth noting that assem-
bling HQ
n
Sligands on the CdSe/ZnS QDs produced a significant
fluorescence enhancement. Here, the photoexcited HQ
n
NS-QD bio-
conjugates decay radiatively to the ground state because the HQ
n
NS
ligands could act as poor electron accepter/donors. This in turn will
result in a recovery of high luminescence compared to unmodified
QDs. Moreover, the ubiquinol also provides an efficient passivation
of the surface trap states to overcome the potential surface defects,
giving rise to a significantly enhanced fluorescence in such QD bio-
conjugates. As can be shown in Figure 1c, the bandgap of surface-
capping ligand ubiquinol is larger than that of CdSe/ZnS QDs and
the hole trapping is negligible. Upon excitation, the resulting elec-
trons and holes are confined in the regions of the ubiquinol func-
tionlised CdSe/ZnS QDs and thereby enhance the fluorescence. In
Figure 4
|
Complex I sensing
in vitro
.(a–c), Time-dependent fluorescence changes upon the interaction of Q
2
NS (a), Q
5
NS (b) and Q
10
NS (c)
functionalised CdSe/ZnS QDs with complex I (4.9 U) in the presence of 10 mM NADH. The insets illustrate the increase of fluorescence (DF) as a
function of incubation time. (d) Calibration curve corresponding to the fluorescence analysis of variable concentrations of complex I of Q
n
NS-QDs in the
presence of NADH. (e) Absorption spectra changes observed upon addition of complex I to a deaerated PBS solution of Q
5
NS-functionalised CdSe/ZnS
QDs in the presence of 10 mM NADH. All measurements were performed in a 0.2 M deaerated PBS solution of pH 8.0.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 3 : 1537 | DOI: 10.1038/srep01537 6

addition, the fluorescence efficiency and stability of HQ
n
NS-QD
bioconjugates against photo-oxidation has shown significant im-
provement due to the antioxidation effect of ubiquinol. Therefore,
there is the remarkable fluorescence enhancement for HQ
n
NS-
functionalised QDs.
We show fluorescence change efficiency to be dependent on alkyl
spacer, as more pronounced change was observed for shorter spacer
in Figure 2a–c. This arises as decreasing spacer distance allows for
faster electron-transfer rate, enhancing the ability of electron transfer
between photoexcited QDs and Q
n
NS. To confirm that the spacer-
dependent Q
n
NS ligands in the QD bioconjugates determine the
magnitude of electron-transfer ability and concomitant QDs’ fluor-
escence difference, we exploited the electrochemistry of Q
n
NS-QDs
in PBS buffer. Only a single pair of oxidation/reduction peaks were
observed from Q
n
NS-QDs, the voltammetric responses range from
reversible for Q
2
NS-QDs to irreversible for Q
5
NS-QDs and Q
10
NS-
QDs based on the peak to peak separation (DE
p
) as shown in
Figure 2d. This is a typical cyclic voltammogram of surface-confined
quinone monolayers measured in buffer where two-electron, two-
proton transfer processes occur
30–32
. As the alkyl spacer increases, the
DE
p
increases because the electron transfer from photoexcited QDs
to the redox active ubiquinone moiety is forced to proceed at a larger
distance, slowing the overall electron-transfer rate and redox kin-
etics. Moreover, we retain the same electrochemistry as free Q
n
NS
(unmodified to QDs) in PBS buffer. As observed, the peak potentials
of free Q
n
NS are slightly different and redox shape is almost ident-
ical. As the spacer length increases from Q
2
NS to Q
10
NS, their cyclic
voltammograms undergo a consistent shift in anodic and cathodic
peak to lower potential (see Fig. S3, Supplementary Information).
The DE
p
of Q
2
NS-QDs, Q
5
NS-QDs and Q
10
NS-QDs are 33, 107 and
223 mV, respectively, while the DE
p
value of free Q
2
NS,Q
5
NS and
Q
10
NS were typically larger. The great differences of voltammetric
response between free Q
n
NS and Q
n
NS-QDs under the same con-
ditions indicate that electron-transfer ability is affected when Q
n
NS
is covalently bound to the QDs’ surface. We show fluorescence
change efficiency to be dependent on alkyl spacer, as decreasing
spacer distance enhances the ability of electron-transfer between
the photoexcited QDs and the Q
n
NS.
It is important to note that the capping layer of Q
n
NS on QDs can
be reduced to the ubiquinol form and effectively produce the high
fluorescence of HQ
n
NS-modified QDs, even at low concentrations of
complex I. Similar to what occurred in initial stage of natural respir-
atory chain, changes in absorption were noted under our constructed
QDs system as ubiquinol formed during the incubation process. This
is also consistent with the electrochemical reduction of the ubiqui-
none capping layer to the reduced state ubiquinol that yields QDs of
enhanced fluorescence. Exploiting these results suggest that Q
n
NS-
QDs could be a biocompatible fluorescence biosensor for tracking of
complex I. Our goal was to establish a model to monitor intracellular
complex I level using human neuroblastoma SH-SY5Y cells labelled
with Q
n
NS-QDs. The mitochondrial-specific neurotoxin such as
rotenone was shown to inhibit complex I in the electron-transfer
chain of mitochondria and induce PD, and PD patients were found
to have the reduced levels of complex I activity
33
. Several lines of
evidence suggest that complex I deficiency of mitochondria could
represent an early critical evaluation in the pathogenesis of sporadic
PD (Fig. 1f)
5–8
. We selected Q
2
NS-CdSe/ZnS QDs with the best
detection limit for complex I to investigate whether there are signifi-
cant differences in fluorescence imaging of QDs as a function of
complex I. Complex I levels decrease in human SH-SY5Y cells after
24 hr of exposure to 100 nM (Damage I), 500 nM (Damage II) and
1mM (Damage III) rotenone. In Figure 5, Q
2
NS-CdSe/ZnS QDs
label SH-SY5Y living cells with different complex I levels: with no-
damage (Normal), QDs’ fluorescence is striking bright at cellular
region. As the cell becomes more damaged (Damage I and
Damage II), QD labelling produces a moderate fluorescence. With
the most-damaged cellular conditions (Damage III), QD labelling
throughout the cell shows only faint fluorescence under identical
conditions. A steady decrease in QDs’ fluorescence correlated to
complex I deficiency, whereas bright-field measurements clearly
show that the cells are viable and overall cellular morphology
appeared unperturbed during the process of these experiments
(Fig. 5). The cell fluorescent micrographs exhibit excellent agreement
with the in vitro data from Figure 4. These results suggest methods
for our constructed Q
n
NS-QDs system could trace complex I defi-
cient levels in SH-SY5Y cells and have raised exciting possibilities in
fluorescence biosensor targeting for PD diagnosis. Moreover, we also
investigated the cytotoxicity of Q
2
NS-QD labelled SH-SY5Y cells by
the MTT assay. It is worthy to note that the cell viability after addi-
tion of Q
2
NS-QDs was found to be above 80% at concentrations
ranging from 3.75 to 200 mg?ml
21
for different rotenone damaged
SH-SY5Y cells (see Fig. S4, Supplementary Information). Thus,
Q
2
NS modified CdSe/ZnS QDs are suitable for use in some potential
biomedical applications.
In summary, we report the development of a novel biosensing
approach using surface-attached CdSe/ZnS QDs exploiting three
ubiquinone-terminated disulphides (Q
n
NS). The fluorescence
enhancement of reduced HQ
n
NS-modified QDs and quenching of
oxidized Q
n
NS-modified QDs could be reversibly tuned with the
transformation between Q
n
NS and HQ
n
NS state. In the presence
of NADH and complex I, the surface attached layer Q
n
NS-QDs
was reduced to HQ
n
NS by proton coupled electron-transfer from
NADH to ubiquinone, which in turn enabled us to probe the com-
plex I level by modulating the QDs’ fluorescence intensity. Impor-
tantly, the utility of the system is demonstrated by monitoring the
fluorescence change to trace complex I levels in human neuroblas-
toma SH-SY5Y cells. Our results demonstrate that the Q
n
NS-QDs
biosensor could be useful for early detection of PD and monitoring
disease progression. We believe that our biosensing approach is a
significant step forward toward molecular diagnosis of PD.
Methods
Fluorescence spectra of functionalised CdSe/ZnS QDs.550 nm-emitting CdSe/ZnS
core-shell QDs were hydrophilic with carboxylic acid ligands. 1 mM Q
n
NS (oxidized
Figure 5
|
Intracellular complex I level sensing. Bright field image and
fluorescent micrographs collected from human neuroblastoma SH-SY5Y
cells with 550-nm-emitting Q
2
NS-functionalised QDs. Complex I
deficient level increased in human SH-SY5Y cells after 24 hr of exposure to
100 nM (Damage I), 500 nM (Damage II) and 1 mM (Damage III)
rotenone. A steady fluorescence decrease in Q
2
NS-QDs label SH-SY5Y cell
fluorescent micrographs as increase of complex I deficiency exhibits
excellent concordance with in vitro result data. Merged images are shown
in the bottom row.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 3 : 1537 | DOI: 10.1038/srep01537 7

form, ubiquinone) and HQ
n
NS (reduced form, ubiquinol) stock solutions were
resolubilised in Millipore H
2
O/dimethylsulphoxide (90:10) and self-assembled to
CdSe/ZnS QDs in 0.2 M PBS at pH 8.0 for 30 min (for detailed procedures, see
Supplementary Information). The Q
n
NS and HQ
n
NS-functionalised QDs stock
solutions (0.2 mM) were purged with N
2
for 5 min before fluorescence analysis.
Cell Culture, cellular imaging and cytotoxicity.SH-SY5Y neuroblastoma cells were
grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco BRL, Paisley, UK)
supplemented with 10% fetal bovine serum (Gibco BRL), 100 U?ml
21
penicillin and
100 mg?ml
21
streptomycin. Cells were maintained in a humidified 5% CO
2
atmosphere at 37uC. Environmental toxins such as the complex I inhibitor rotenone
induce selective death of dopaminergic neurons through inhibition of the
electron-transfer chain complex I activity. Rotenone was added directly to the media
at appropriate concentrations. Complex I deficiency levels increased in human
SH-SY5Y cells after 24 hr of exposure to 100 nM, 500 nM and 1 mM rotenone.
Under these conditions, cell viability decreased by about 13%, 15% and 18% after
24 hr, respectively. SH-SY5Y cells with different complex I levels were plated into a
24-well culture plate (200-300 cells/well), and allowed to adhere for 10 hr before
treatment. Culture medium containing 200 mg?ml
21
Q
n
NS-functionalised CdSe/ZnS
QDs were added and incubated for 10 hr. Next, the growth medium was removed,
and the cells were fixed with 4% methanal solution at room temperature for 20 min,
followed by washing three times with PBS solution. The cover glass was then mounted
on a microscopic glass slide and was studied under a microscope. The images were
taken by using an inverted fluorescence scanning microscope with an objective lens
(360). All background parameters (the laser intensity, exposure time, objective lens)
were kept constant when the different fluorescence images were captured.
The cytotoxicity assays were performed by the MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) assay. SH-SY5Y cells were placed in 96-well
culture plates (10
4
cells/well), and allowed to attach for 24 hr before treatment. The
cells were treated with Q
n
NS-functionalised CdSe/ZnS QDs ranging from 3.75 to
200 mg?ml
21
. The cell viability was evaluated by the MTT assay for different rotenone
damaged SH-SY5Y cells after 24 hr treatment. The optical density in control and
sample-treated wells was measured in an automated microplate reader (Multiskan Ex,
Lab systems, Finland) at a test wavelength of 470 nm. The cytotoxicity of modified
QDs was expressed as IC
50
(concentration of 50% cytotoxicity, which was
extrapolated from linear regression analysis of the experimental data).
1. Siderowf, A. & Stern, M. Update on Parkinson disease. Ann. Intern. Med. 138,
651–658 (2003).
2. Dawson, T. M. & Dawson, V. L. Neuroprotective and neurorestorative strategies
for Parkinson’s disease. Nat. Neurosci. 5, 1058–1061 (2002).
3. Jellinger, K. A. Neuropathological spectrum of synucleinopathies. Mov. Disord.
18, S2–S12 (2003).
4. Litvan, I. et al. The etiopathogenesis of Parkinson disease and suggestions for
future research. Part I. J. Neuropathol. Exp. Neurol. 66, 251–257 (2007).
5. Shi, M. et al. Cerebrospinal fluid biomarkers for Parkinson disease diagnosis and
progression. Ann. Neurol. 69, 570–580 (2011).
6. Dawson, T. M. & Dawson, V. L. Molecular pathways of neurodegeneration in
Parkinson’s disease. Science 302, 819–822 (2003).
7. Valente, E. M. et al. Hereditary early-onset Parkinson’s diseasecaused by
mutations in PINK1. Science 304, 1158–1160 (2004).
8. Betarbet, R. et al. Chronic systemic pesticide exposure reproduces features of
Parkinson’s disease. Nat. Neurosci. 3, 1301–1306 (2000).
9. Sazanov, L. A. & Hinchliffe, P. Structure of the hydrophilic domain of respiratory
complex I from Thermus thermophilus. Science 311, 1430–1436 (2006).
10. Jeya, M., Moon, H. J., Lee, J. L., Kim, I. W. & Lee, J. K. Current state of coenzyme
Q
10
production and its applications. Appl. Microbiol. Biot. 85, 1653–1663 (2010).
11. Matthews, R. T., Yang, L., Browne, S., Baik, M. & Beal, M. F. Coenzyme Q
10
administration increases brain mitochondrial concentrations and exerts
neuroprotective effects. Proc. Natl. Acad. Sci. U. S. A. 95, 8892–8897 (1998).
12. Do, T. Q., Schultz, J. R. & Clarke, C. F. Enhanced sensitivity of ubiquinone-
deficient mutants of Saccharomyces cerevisiae to products of autoxidized
polyunsaturated fatty acids. Proc. Natl. Acad. Sci. U. S. A. 93, 7534–7539 (1996).
13. Michalet, X. et al. Quantum dots for live cells, in vivo imaging, and diagnostics.
Science 307, 538–544 (2005).
14. Melinger, J. S., Blanco-Canosa, J. B., Dawson, P. E. & Mattoussi, H. Quantum-dot/
dopamine bioconjugates function as redox coupled assemblies for in vitro and
intracellular pH sensing. Nat. Mater. 9, 676–684 (2010).
15. Gao, X., Cui, Y., Levenson, R. M., Chung, L. W. K. & Nie, S. In vivo cancer targeting
and imaging with semiconductor quantum dots. Nat. Biotechnol. 22, 969–976
(2004).
16. Gao, J. et al. In vivo tumor-targeted fluorescence imaging using near-infrared
non-cadmium quantum dots. Bioconjugate Chem. 21, 604–609 (2010).
17. Gill, R., Zayats, M. & Willner, I. Semiconductor quantum dots for bioanalysis.
Angew. Chem. Int. Ed. 47, 7602–7625 (2008).
18. Medintz, I. L., Tetsuouyeda, H., Goldman, E. R. & Mattoussi, H. Quantum dot
bioconjugates for imaging, labelling and sensing. Nat. Mater. 4, 435–446 (2005).
19. Chan, W. C. W. & Nie, S. Quantum dot bioconjugates for ultrasensitive
nonisotopic detection. Science 281, 2016–2018 (1998).
20. Medintz, I. L., Tetsuouyeda, H., Goldman, E. R. & Mattoussi, H. Quantum dot
bioconjugates for imaging, labelling and sensing. Nat. Mater. 4, 435–446 (2005).
21. Clarke, S. J. et al. Photophysics of dopamine-modified quantum dots and effects
on biological systems. Nat. Mater. 5, 409–417 (2006).
22. Cooper, D. R. et al. Photoenhancement of lifetimes in CdSe/ZnS and CdTe
quantum dot-dopamine conjugates. Phys. Chem. Chem. Phys. 11, 4298–4310
(2009).
23. Cooper, D. R., Dimitrijevic, N. M. & Nadeau, J. L. Photosensitization of CdSe/ZnS
QDs and reliability of assays for reactive oxygen species production. Nanoscale 2,
114–121 (2010).
24. Qin, L. X. et al. Coenzyme Q functionalised CdTe/ZnS quantum dots for reactive
oxygen species (ROS) imaging. Chem.-Eur. J. 19, 5262–5271 (2011).
25. Li, D.-W. et al. CdSe/ZnS quantum dot-Cytochrome c bioconjugates for selective
intracellular O
2
?
-
sensing. Chem. Commun. 47, 8539–8541 (2011).
26. Chan, T. R., Hilgraf, R., Sharpless, K. B. & Fokin, V. V. Polytriazoles as copper (I)-
stabilizing ligands in catalysis. Org. Lett. 6, 2853–2855 (2004).
27. Shen, R. et al. Multifunctional conjugates to prepare nucleolar-targeting CdS
quantum dots. J. Am. Chem. Soc. 132, 8627–8634 (2010).
28. Hirst, J. Towards the molecular mechanism of respiratory complex I. Biochem. J.
425, 327–339 (2010).
29. Ma, W. et al. In situ spectroeletrochemistry and cytotoxic activities of natural
ubiquinone analogues. Tetrahedron 67, 5990–6000 (2011).
30. Costentin, C. Electrochemical approach to the mechanistic study of proton-
coupled electron transfer. Chem. Rev. 108, 2145–2179 (2008).
31. Hong, H. G. & Park, W. Electrochemical characteristics of hydroquinone-
terminated self-assembled monolayers on gold. Langmuir 17, 2485–2492 (2001).
32. Abhayawardhana, A. D. & Sutherland, T. C. Heterogeneous proton-coupled
electron-transfer of a hydroxy-anthraquinone self-assembled monolayer.
J. Electroanal. Chem. 653, 50–55 (2011).
33. Sherer, T. B., Betarbet, R., Kim, J. H. & Greenamyre, J. T. Selective microglial
activation in the rat rotenone model of Parkinson’s disease. Neurosci. Lett. 341,
87–90 (2003).
Acknowledgements
This research was supported by the 973 Program (2013CB733700) and the National Science
Fund for Distinguished Young Scholars (21125522).
Author contributions
W.M. and L.-X.Q. contributed equally to this work. W. M. synthesized Q
Q
n
NS
compounds.
W.M. and L.-X.Q. designed and performed all the experiments, and wrote the manuscript.
F.-T.L. and J.W. grew cell cultures and assisted with cellular experiments. G.Z. drew and
summarized the figures. Y.-T.L., Z.G.P. and T.D.J. finalized the preparation of the
manuscript. Y.-T.L. designed and managed the project. All the authors discuss the results
and commented on the manuscript.
Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
License: This work is licensed under a Creative Commons
Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this
license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
How to cite this article: Ma, W. et al. Ubiquinone-quantum dot bioconjugates for in vitro
and intracellular complex I sensing. Sci. Rep. 3, 1537; DOI:10.1038/srep01537 (2013).
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 3 : 1537 | DOI: 10.1038/srep01537 8