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High Sensitivity Detection of CdSe/ZnS Quantum
Dot-Labeled DNA Based on N-type Porous
Silicon Microcavities
Changwu Lv 1 , Zhenhong Jia 2, *, Jie Lv 3 , Hongyan Zhang 1 and Yanyu Li 1
1
2
3
*
School of Physical Science and Technology, Xinjiang University, Urumqi 830046, China;
[email protected] (C.L.); [email protected] (H.Z.); [email protected] (Y.L.)
College of Information Science and Engineering, Xinjiang University, Urumqi 830046, China
College of Resource and Environment sciences, Xinjiang University, Urumqi 830046, China; [email protected]
Correspondence: [email protected]; Tel.: +86-991-858-3362
Academic Editor: Alexander Star
Received: 18 October 2016; Accepted: 28 December 2016; Published: 1 January 2017
Abstract: N-type macroporous silicon microcavity structures were prepared using electrochemical
etching in an HF solution in the absence of light and oxidants. The CdSe/ZnS water-soluble quantum
dot-labeled DNA target molecules were detected by monitoring the microcavity reflectance spectrum,
which was characterized by the reflectance spectrum defect state position shift resulting from changes
to the structures’ refractive index. Quantum dots with a high refractive index and DNA coupling
can improve the detection sensitivity by amplifying the optical response signals of the target DNA.
The experimental results show that DNA combined with a quantum dot can improve the sensitivity
of DNA detection by more than five times.
Keywords: N-type porous silicon; quantum dot labeling; QD-DNA; reflectance spectrum
1. Introduction
It is advantageous to use porous silicon because of its simple preparation processes; the pore
size, pore density, and porous silicon layer thickness can be controlled by changing electrochemical
corrosion conditions (e.g., the silicon wafer type, the doping, the electrolyte ratio, and the corrosion
current density) [1–3]. A large specific surface area can adsorb large amounts of chemical and biological
molecules. These properties make porous silicon a good candidate for highly sensitive, unlabeled
optical biochemical sensors [4,5]. The combination of a biomolecules and porous silicon can result
in change of an effective refractive index. By observing the effect of this change on the reflectance
spectrum shift such as a monolayer porous silicon interference peak shift [6,7], the Bragg reflector
center position [8], or the microcavity defect state [9,10]—it is possible to detect biochemical molecules.
Quantum dots (QDs) are mostly used as high-sensitivity fluorescent molecule labels because
of their high luminescence quality, good monochromaticity, surface functionalization, and tunable
photoluminescence range [11,12]. Nanoparticles such as Au or QDs were attached to the DNA before
the DNA was hybridized with the complementary DNA for optical sensing [13,14]. Many optical
methods are used to observe the changes caused by the nanoparticle addition to the DNA such
as light scattering, SPR, fluorescence, and SERS [15]. Moreover, the high refractive index of QDs
can increase the effective refractive index of QD-labeled molecules while making them luminescent.
This causes a greater reflectance spectrum shift, improving the sensitivity of the sensor. Girija Gaur et al.
utilized reflectance spectrum shifts to detect QD-labeled small molecules of biotin in a porous silicon
monolayer [16]. The result was a six-fold increase in detection sensitivity compared to that of unlabeled
targets. We know that a porous silicon microcavity (PSM) is more sensitive to an effective refractive
index change due to a sharp dip in the reflectance spectrum for the defect state. In addition, the pore
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compared to that of unlabeled targets. We know that a porous silicon microcavity (PSM) is more
sensitive to an effective refractive index change due to a sharp dip in the reflectance spectrum for
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the defect state. In addition, the pore size is an important factor that we must consider in sensing.
The pore size of P-type porous silicon ranges from 10 nm to 30 nm [17], the size of the QD is about
4 nm,
the sizefactor
of thethat
20-base
DNAconsider
molecule
about 6The
nm pore
in length.
thatporous
the size
of the
size
is anand
important
we must
inissensing.
size ofNote
P-type
silicon
DNAfrom
molecule
quantum
dot is
labeling.
small
ratiosize
between
pore size
and
ranges
10 nmwill
to 30increase
nm [17],after
the size
of the QD
about 4 A
nm,
and the
of the 20-base
DNA
molecule
size
will
cause
the
QD-labeled
DNA
to
plug
the
pore
and
prevent
other
biological
molecule is about 6 nm in length. Note that the size of the DNA molecule will increase after quantum
molecules
from
entering
the microcavity
[18].
In comparison,
N-type
poresDNA
are generally
dot
labeling. A
small
ratio between
pore size and
molecule
size will cause
thesilicon
QD-labeled
to plug
much
(50 nm–120
[19,20]. molecules from entering the microcavity [18]. In comparison,
the
porelarger
and prevent
otherm)
biological
this pores
paper,are
N-type
porous
microcavity
structures
N-typeIn
silicon
generally
muchsilicon
larger (50
nm–120 nm)
[19,20]. are used. Hydroxyl-modified
CdSe/ZnS
are coupled
with DNA molecules;
the QDs
used to amplify the
In this water-soluble
paper, N-typeQDs
porous
silicon microcavity
structures are
used.areHydroxyl-modified
effective
refractive
index
change.
CdSe/ZnS water-soluble QDs are coupled with DNA molecules; the QDs are used to amplify the
The
schematic
image
of the sensing principle is shown in Figure 1. After the hybridization
effective
refractive
index
change.
between
the complementary
and
the probe
DNA in
in Figure
the PSM
structure,
the effectivebetween
refractive
The schematic
image of the DNA
sensing
principle
is shown
1. After
the hybridization
index
of
the
structure
changed,
and
the
red
shift
of
the
spectrum
can
be
observed
in
the
reflection
the complementary DNA and the probe DNA in the PSM structure, the effective refractive index
of
spectrum.
are marked
by ashift
series
of spectrum
chemical can
couplings
to theintarget
DNA, the
effective
the
structureQDs
changed,
and the red
of the
be observed
the reflection
spectrum.
refractive
indexby
ofaDNA-QD
significantly
increased
due
to the
highthe
refractive
index
of theindex
quantum
QDs
are marked
series of chemical
couplings
to the
target
DNA,
effective
refractive
of
dots.
The
red
shift
in
the
reflectance
spectrum
increased
compared
to
the
unmodified
DNA.
QDs
DNA-QD significantly increased due to the high refractive index of the quantum dots. The red shift in
play
a role in
increasing
the spectral
response
signal. This DNA.
method
increases
theinsensitivity
the
reflectance
spectrum
increased
compared
to the unmodified
QDs
play a role
increasingof
detecting
DNA
hybridization.
the spectral response signal. This method increases the sensitivity of detecting DNA hybridization.
Figure
1. 1.
Schematic
diagram
ofof
thethe
sensing
principle.
Figure
Schematic
diagram
sensing
principle.
Experimental
2. 2.
Experimental
2.1.
Materials
and
Instruments
2.1.
Materials
and
Instruments
Ethanolamine
Ethanolaminehydrochloride
hydrochloride(EA),
(EA),2-[4-(2-hydroxyethyl)-1-piperazinyl]
2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic
ethanesulfonicacid
acid
(HEPES)
buffer
(pH 9.0),
glutaraldehyde
(GA, 50%), and
aminopropyl
triethoxysilane
(APTES,
99%) were
(HEPES)
buffer
(pH
9.0), glutaraldehyde
(GA,
50%), and
aminopropyl
triethoxysilane
purchased
Aladdin Reagent
Co., Ltd. (Shanghai,
China), CdSe/ZnS
(APTES, from
99%)Shanghai
were purchased
from Shanghai
Aladdin Reagent
Co., Ltd. carboxyl-modified
(Shanghai, China),
water-soluble
quantum dots were water-soluble
purchased fromquantum
Wuhan Jiayuan
Dots Biotech
Ltd.
CdSe/ZnS carboxyl-modified
dots Quantum
were purchased
fromCo.,Wuhan
(Wuhan,
China),
along
with
a
phosphate
buffer
(PBS,
pH
7.4),
1-(3-dimethylaminopropyl)-3-ethyl
Jiayuan Quantum Dots Biotech Co. Ltd. (Wuhan, China), along with a phosphate buffer
carbodiimide
(EDC), and N-hydroxysulfosuccinimide
All (EDC),
purchased
(PBS, pH hydrochloride
7.4), 1-(3-dimethylaminopropyl)-3-ethyl
carbodiimide (Sul-NHS).
hydrochloride
and
reagents
were analytical grade.
N-hydroxysulfosuccinimide
(Sul-NHS). All purchased reagents were analytical grade.
0 -NH2,
Target
DNA:
5′-CGCGGCCTATCAGCTTGTTG-3′-NH2,
Target
DNA:
50 -CGCGGCCTATCAGCTTGTTG-3
Probe
DNA:
5′-CAACAAGCTGATAGGCCGCG-3′-NH2.
0 -NH2.
Probe
DNA:
50 -CAACAAGCTGATAGGCCGCG-3
DNA
primers
were
purchased
from
INVITROGEN
TRADING Co., Ltd. (Shanghai, China).
DNA primers were purchased from INVITROGEN TRADING Co., Ltd. (Shanghai, China).
Field emission scanning electron microscopy (FESEM, ZEISS SUPRA 55VP) was used to
Field emission
scanning
electron
microscopy
ZEISS
SUPRA 55VP)
wasmicroscopy
used to
characterize
the porous
silicon
surface
and cross (FESEM,
section. The
transmission
electron
characterize
the
porous
silicon
surface
and
cross
section.
The
transmission
electron
microscopy
(TEM) image was acquired using a 200 kV field emission transmission electron microscope
(TEM)
image was
usingspectrum
a 200 kVis field
emission
transmission
microscope
(JEM-2100F).
Theacquired
absorption
measured
using
a Nano electron
Drop 2000
UV-Vis
(JEM-2100F).
The
absorption
spectrum
is
measured
using
a
Nano
Drop
2000
UV-Vis
spectrophotometer
spectrophotometer (Thermo Scientific, Waltham, MA, USA). The fluorescence emission spectrum
(Thermo Scientific, Waltham, MA, USA). The fluorescence emission spectrum was measured using
a Hitachi UV-Vis spectrofluorometer (Hitachi F-4600, Tokyo, Japan). A Hitachi UV-Vis spectrophotometer
(Hitachi U-4100, Tokyo, Japan) was used to measure the sample reflectance spectrum.
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was measured using a Hitachi UV-Vis spectrofluorometer (Hitachi F-4600, Tokyo, Japan). A Hitachi
3 of 8
(Hitachi U-4100, Tokyo, Japan) was used to measure the sample
reflectance spectrum.
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2017,
17, 80
UV-Vis
spectrophotometer
2.2. Porous Silicon Microcavity Preparation
N-type heavily doped silicon wafers with 0.01 ohm
·cm to 0.02 ohm
·cm and (100) crystal
ohm·
ohm·
orientation were diced into 1 cm
These pieces
pieces were
were subsequently
subsequently cleaned
cleaned using
using acetone,
acetone,
cm22 pieces. These
ethanol, and deionized water for 10 min each, and then dried in air. The samples were etched with
a single cell under constant current, using a mixture of hydrofluoric acid and water; the HF acid
concentration
HF:
200200
mLmL
water).
TheThe
corrosion
current
density
cyclecycle
was
concentration was
wasroughly
roughly5%
5%(25
(25mL
mL40%
40%
HF:
water).
corrosion
current
density
14
mA
mA28for
4 s,for
with
interval
of 5 s. The
etched
current
was
14 for
mA4.5
fors and
4.5 s28
and
mA
4 s,an
with
an interval
of 5 defect
s. The layer
defectwas
layer
was using
etcheda using
a
density
14 mAoffor
s. Note
the N-type
silicon
poresilicon
size is pore
relatively
large;
the pores
etched
using
current of
density
149 mA
for that
9 s. Note
that the
N-type
size is
relatively
large;
the pores
corrosion
currents
of 14 mA
and 28ofmA
Figure
respectively.
corresponding
etched using
corrosion
currents
14 are
mAshown
and 28inmA
are2a,b,
shown
in Figure The
2a,b,
respectively. pore
The
sizes
are roughly
30–40
nm
and
40–50 30–40
nm. The
for are
the large
biomolecules
corresponding
pore
sizes
are
roughly
nmpore
and diameters
40–50 nm.are
Thelarge
poreenough
diameters
enough
and
thebiomolecules
quantum dots
to enter
the porous
The
are silicon.
round, The
withpores
relatively
thick walls,
for the
and
the quantum
dotssilicon.
to enter
thepores
porous
are round,
with
as
shown in
Figure
2a.asInshown
Figurein
2b,Figure
the pore
are2b,
irregular
with
thin walls.
Figure with
2c shows
relatively
thick
walls,
2a. shapes
In Figure
the pore
shapes
are irregular
thin
awalls.
cross-sectional
of athe
PSM. The defect
layer
is inPSM.
the middle,
withlayer
eight is
upper
and
eight lower
Figure 2c view
shows
cross-sectional
view
of the
The defect
in the
middle,
with
layers.
Figure
2deight
shows
an enlarged
view of2dthe
crossan
section.
eight upper
and
lower
layers. Figure
shows
enlarged view of the cross section.
Figure 2.
2. Scanning
and
Figure
Scanning electron
electron microscopy
microscopy image
image (a)
(a) subjected
subjected to
to aa corrosion
corrosion current
current 14
14 mA
mA and
(b) subjected
2828
mA;
(c) (c)
cross-sectional
view
of the
(d) an
enlarged
view
(b)
subjected to
toaacorrosion
corrosioncurrent
current
mA;
cross-sectional
view
of PSM;
the PSM;
(d)
an enlarged
of
the
cross
section.
view of the cross section.
2.3. Quantum
2.3.
Quantum Dot
Dot Coupled
Coupled with
with DNA
DNA
A 5050uL uL
CdSe/ZnS
carboxyl-modified
water-soluble
dot(8 µM
solution
(8 µ M
A
CdSe/ZnS
carboxyl-modified
water-soluble
quantumquantum
dot solution
concentration)
concentration)
was
diluted
with
PBS
to
1
µ
M.
Next,
40
µ
L
of
EDC
(0.01
M
concentration)
and
40 µ L
was diluted with PBS to 1 µM. Next, 40 µL of EDC (0.01 M concentration) and 40 µL of Sul-NHS
of Sul-NHS
(0.01 M concentration)
added
to the
reactionAfter
mixture.
Afterfor
reacting
for5010µL
min,
(0.01
M concentration)
were addedwere
to the
reaction
mixture.
reacting
10 min,
of
50
µ
L
of
amino-modified
DNA
(40
µ
M
concentration)
was
added
and
the
reaction
vessel
amino-modified DNA (40 µM concentration) was added and the reaction vessel was shaken gentlywas
for
shaken
gently
h in
the dark at Centrifugation
room temperature.
Centrifugation
can remove
excess
DNA
10
h in the
darkfor
at 10
room
temperature.
can remove
excess DNA
due to the
solubility
due
to
the
solubility
differences
between
DNA
and
carboxyl
moiety
on
the
QD.
The
QD-modified
differences between DNA and carboxyl moiety on the QD. The QD-modified DNA separates because
DNA separates
because many
unlinked
thesolubility
surface of
QDDNA.
have The
less QD-DNA
solubility
many
unlinked carboxyl
moieties
on the carboxyl
surface ofmoieties
QD haveon
less
than
than DNA.
The than
QD-DNA
weight
is larger
than the
DNA,
while
the solution.
unlinkedAfter
DNAcentrifugation
is still in the
weight
is larger
the DNA,
while
the unlinked
DNA
is still
in the
solution.
After
centrifugation
at
10,000
rpm
for
10
min,
the
supernatant
was
removed
and PBS
the
at 10,000 rpm for 10 min, the supernatant was removed and the precipitate was diluted with
precipitate
was
diluted
with
PBS
into
a
different
concentration
of
DNA
solution
and
stored
at
4
°C.
into a different concentration of DNA solution and stored at 4 ◦ C. The fluorescence emission spectra
of the quantum dot and the quantum dot-DNA solution were measured from a 100 µL sample
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The fluorescence emission spectra of the quantum dot and the quantum dot-DNA solution were
measured from a 100 µ L sample using an excitation wavelength of 370 nm, an excitation voltage of
400
v, and
a slit width
of 10 nm.ofThe
and absorption
of QDs
after
using
an excitation
wavelength
370fluorescence
nm, an excitation
voltage ofspectra
400 v, and
a slitbefore
widthand
of 10
nm.
DNA
coupling are
in spectra
Figure of
3.QDs
Thebefore
emission
peakDNA
of carboxyl-modified
The fluorescence
and shown
absorption
and after
coupling are shown CdSe/ZnS
in Figure 3.
water-soluble
quantum
dots is located atCdSe/ZnS
528 nm. Note
that the quantum
peak shifted
nm at
after
The emission peak
of carboxyl-modified
water-soluble
dotstois 530
located
528 the
nm.
quantum
with DNA.
spectrum
shows
thatwith
thereDNA.
is an The
added
260 nm
Note thatdot
thecoupled
peak shifted
to 530 The
nm absorption
after the quantum
dot
coupled
absorption
absorption
peak after
the QD
combined
with
Thus,peak
bothafter
the fluorescence
and absorption
spectrum shows
that there
is an
added 260
nmDNA.
absorption
the QD combined
with DNA.
spectra
indicate
that
the
quantum
dot
and
DNA
conjunction
was
successful.
Quantum
dot-labeled
Thus, both the fluorescence and absorption spectra indicate that the quantum dot and DNA conjunction
DNA
has little effect
on thedot-labeled
overall photoluminescence
intensity.
was successful.
Quantum
DNA has little effect
on the overall photoluminescence intensity.
(a)
(b)
Figure
Figure3.3. Fluorescence
Fluorescenceand
andabsorption
absorptionspectra
spectraofofQD
QDand
andthe
theQD-labeled
QD-labeledDNA:
DNA:QD
QDrepresents
representsthe
the
fluorescence
spectrum
of
the
quantum
dots,
QD
+
DNA
represents
the
fluorescence
spectrum
of
the
fluorescence spectrum of the quantum dots, QD + DNA represents the fluorescence spectrum of the
quantum
quantumdot-labeled
dot-labeledDNA.
DNA.(a)
(a)fluorescence
fluorescence spectra;
spectra; (b)
(b) absorption
absorption spectra.
spectra.
2.4.
2.4.PSM
PSMFunctionalization
Functionalization
To
immobilize
silicon
surface,
thethe
PSM
requires
a
To immobilizethe
thetarget
targetbiological
biologicalmolecules
moleculestotothe
theporous
porous
silicon
surface,
PSM
requires
series
of of
functional
processes
to to
stabilize
a series
functional
processes
stabilizethe
theporous
poroussilicon
siliconsurface
surfaceand
andmodify
modifythe
thepore
poresurface
surface
functional
groups.
First,
the
freshly-prepared
PSM
was
oxidized;
the
samples
were
heated
◦C
functional groups. First, the freshly-prepared PSM was oxidized; the samples were heatedto
to500
500°C
for
30
min
in
an
oxidizing
furnace.
Next,
they
were
soaked
in
5%
APTES
for
1
h,
rinsed
with
for 30 min in an oxidizing furnace. Next, they were soaked in 5% APTES for 1 h, rinsed with deionized
deionized
water,
dried
air, and at
incubated
at 10
100
°C for
10silanization,
min. After silanization,
the amino
water, dried
in air,
and in
incubated
100 ◦ C for
min.
After
the amino groups
were
groups
were
immobilized
to
the
pore
surface.
The
samples
were
immersed
in
a 2.5%
immobilized to the pore surface. The samples were immersed in a 2.5% glutaraldehyde solution
for
glutaraldehyde
solution
for
1
h,
then
rinsed
with
PBS
(pH
7.4)
and
dried
in
air
to
modify
the
surface
1 h, then rinsed with PBS (pH 7.4) and dried in air to modify the surface of the porous silicon with
of
porous
silicon
an aldehyde
Thewith
aldehyde
group can
be coupled with
an the
aldehyde
group.
Thewith
aldehyde
group can group.
be coupled
amino-modified
DNA.
amino-modified DNA.
2.5. DNA Probe Connected to Porous Silicon Microcavity
2.5. DNA Probe Connected to Porous Silicon Microcavity
A 50 µL DNA probe (10 µM concentration) was dropped into the functionalized PSM sample
◦ C, the samples
A a50pipette.
µ L DNAAfter
probe2 (10
waswere
dropped
into
thePBS
functionalized
sample
using
h atµ M
37 concentration)
rinsed
with
to remove PSM
excess
DNA.
using
a pipette. After 2aldehyde
h at 37 °C,
the samples
were
rinsed
PBSwith
to remove
DNA.buffer,
The
The excess/unreacted
functional
groups
were
thenwith
closed
EA (3Mexcess
in HEPES
◦
excess/unreacted
aldehyde
functional
groups
were
then
closed
with
EA
(3M
in
HEPES
buffer,
pH
pH 9.0) incubated at 37 C for 1 h. Finally, the samples were rinsed with PBS and dried in air. 9.0)
incubated at 37 °C for 1 h. Finally, the samples were rinsed with PBS and dried in air.
2.6. Target DNA Detection
2.6. Target DNA Detection
Fifty microliters of target DNA labeled with QDs at different concentrations were attached to the
microliters
of target
DNA
labeled at
with
were attached
to
PSMFifty
samples.
The samples
were
incubated
37 ◦QDs
C forat2 different
h to allowconcentrations
DNA to hybridize.
Afterwards,
the
samples.
The samples
were
incubated
at 37
°C for 2 htarget
to allow
to hybridize.
the PSM
samples
were rinsed
with PBS
to wash
away the
unconnected
DNADNA
and QDs,
and then
Afterwards,
dried in air. the samples were rinsed with PBS to wash away the unconnected target DNA and
QDs, The
and then
dried
in air.
sample
reflectance
spectra were measured after each step of sample functionalization
The sample
reflectance
spectra
after
each
of sampleoffunctionalization
(oxidization,
silanization,
GA)
after were
probemeasured
combination
and
the step
hybridization
the DNA probe
(oxidization,
silanization, GA) after probe combination and the hybridization of the DNA probe
with QD-DNA.
with QD-DNA.
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3. Results and Discussion
3. Results and Discussion
3. Results and Discussion
The defect state in the high reflection region of the PSM reflectance spectrum is near 650 nm.
The
defect
state
inoxidized,
the high reflection and
region
of the PSM reflectance
spectrum is near
650
nm.
WhenThe
thedefect
sample
wasin
functionalized
with glutaraldehyde,
the650
probe
state
the high silanized,
reflection region
of the PSM reflectance
spectrum isand
near
nm.
When the hybridized
sample waswith
oxidized,
silanized, and functionalized
with
glutaraldehyde,
and the
DNA
complementary
QD-DNA,
the
reflectance
spectrum
the probe
PSM
When was
the sample was oxidized,
silanized, andtarget
functionalized
with
glutaraldehyde,
and the of
probe
DNA
DNA
was
hybridized
with
complementary
target
QD-DNA,
the
reflectance
spectrum
of
the PSM
showed
a
regular
red
shift.
Figure
4
shows
the
reflectance
spectrum
shifts.
The
reflectance
spectrum
was hybridized with complementary target QD-DNA, the reflectance spectrum of the PSM showed
showed
a regular
red shift.
Figureindex
4 shows
the reflectance
spectrum
shifts.
The reflectanceand
spectrum
shift
reflects
PSM
refractive
changes
that
result
from
functionalization
DNA
a regular
red the
shift.
Figure
4 shows
the reflectance
spectrum
shifts.the
The
reflectance spectrum
shift
shift
reflects
the
PSM
refractive
index
changes
that
result
from
the
functionalization
and
DNA
connection.
When
a small molecule
was grafted
to the
porous
silicon surface, the
effective
refractive
reflects the PSM
refractive
index changes
that result
from
the functionalization
and
DNA connection.
connection.
a small
molecule
was
grafted
the reflectance
porous silicon
surface, the effective
refractive
index
theWhen
porous
silicon
becomes
large,
andtosilicon
the
spectrum
to the
red.ofThe
When aofsmall
molecule
was grafted
to the
porous
surface, the
effectiveshifts
refractive
index
the
index ofofthe
silicon
becomes index
large, shift
and has
the areflectance
spectruminshifts
to the
red. The
The
amount
redporous
shift and
the refractive
linear
a amount
certain
range.
porous silicon
becomes
large,
and the reflectance
spectrum
shiftsrelationship
to the red. The
of red shift
amount of red
shift and
the
refractive index
shift has a linear concentrations
relationship in and
a certain
range. The
relationship
between
target
thebetween
reflectance
and the refractive
indexthe
shift
has aDNA
linear molecules
relationshipatindifferent
a certain range. The relationship
the
relationship
between
the
target
DNA
molecules
at
different
concentrations
and
the
reflectance
spectrum
redmolecules
shift is shown
in Figure
5.
target DNA
at different
concentrations
and the reflectance spectrum red shift is shown in
spectrum red shift is shown in Figure 5.
Figure 5.
Figure 4. PSM reflectance spectrum following (a) oxidation; (b) silanization; (c) glutaraldehyde;
Figure
4.4. PSM
reflectance
spectrum following
(a) oxidation; (b) silanization; (c) glutaraldehyde;
Figure
PSM (e)
reflectance
(d)
DNA
probe;
QD-DNAspectrum
target. following (a) oxidation; (b) silanization; (c) glutaraldehyde;
(d)
DNA
probe;
(e)
QD-DNA
target.
(d) DNA probe; (e) QD-DNA target.
Figure 5. Relationship between the target DNA at different concentrations and the reflectance spectrum
Figure 5. Relationship between the target DNA at different concentrations and the reflectance
shift.
The5.black
squares represent
the QD-DNA
and the
dots represent
the control
DNA
Figure
Relationship
target DNA
atred
different
and
the(unlabeled).
reflectance
spectrum
shift.
The blackbetween
squaresthe
represent
the QD-DNA
and concentrations
the red dots represent
the
control
spectrum
shift. The black squares represent the QD-DNA and the red dots represent the control
DNA
(unlabeled).
DNA (unlabeled).
Figure
5 shows both the QD-DNA red shift and the control DNA at different concentrations.
WhenFigure
the concentrations
QD-DNA
were
µM,and
0.5 µM,
1 µM, 2.5
µM,atand
5.0 µM,concentrations.
the reflectance
5 shows bothofthe
QD-DNA
red0.1shift
the control
DNA
different
Figure
5 shows
the
QD-DNA
red
and
the
concentrations.
spectra
red concentrations
shifts
wereboth
11 nm,
nm, 23.5 were
nm, shift
280.1
nm,
and
31control
nm,
When
the
control
When
the
of 18
QD-DNA
µ M,
0.5
µ M,respectively.
1 DNA
µ M, at
2.5different
µ
M, and
5.0 µ M,DNA
the
When
the
concentrations
of
QD-DNA
were
0.1
µ
M,
0.5
µ
M,
1
µ
M,
2.5
µ
M,
and
5.0
µ
M,
the
concentrations
were
0.5shifts
µM, 1were
µM,11
2.5nm,
µM,18and
µM,
the28reflectance
spectra
red shifts were
10 nm,
reflectance
spectra
red
nm,5.0
23.5
nm,
nm, and 31
nm, respectively.
When
the
reflectance
spectra
red
shifts
were
11
nm,
18
nm,
23.5
nm,
28
nm,
and
31
nm,
respectively.
When
the
control DNA concentrations were 0.5 µ M, 1 µ M, 2.5 µ M, and 5.0 µ M, the reflectance spectra red
control DNA concentrations were 0.5 µ M, 1 µ M, 2.5 µ M, and 5.0 µ M, the reflectance spectra red
Sensors 2017, 17, 80
6 of 8
14 nm, 18 nm, and 23 nm, respectively. It can be seen from the figure that the QD-DNA red shift is
larger than that of the control DNA at the same concentration. As shown in Figure 4, the red shift
of the 0.1 µM QD-DNA at point a is equivalent to the red shift of the control DNA concentration at
0.5 µM (point a0 ). The red shift of the 0.5 uM QD-DNA at point b is similar to the red shift of the 2.5 µM
control DNA (point b0 ). Finally, the red shift of the 1 µM QD-DNA at point c is a bit larger than that of
the 5 µM control DNA concentration at point c0 . That is to say, the detection sensitivity of QD-DNA
is roughly five times that of conventional DNA detection. As can be seen from Figure 4, at DNA
concentrations below 0.5 µM, the slope of the QD-DNA (14.34 nm/µM) data points is greater than the
slope of controlled DNA (2.67 nm/µM) at the concentration range from 0.5 µM to 5 µM. Therefore,
the QD-DNA detection sensitivity is at least five times higher than conventional DNA detection.
When QD-DNA is chosen as the target molecule for the reflectance spectrum shift, the sensitivity
is improved due to the high refractive index of QDs. The QDs and DNA molecules are bonded together
to increase the effective refractive index of DNA molecules, and then hybridized with a DNA probe in
the PSM. This increases the PSM effective refractive index shift. To further improve the DNA detection
sensitivity using QD as a marker, one should optimize the ratio of QDs to DNA. Table 1 shows the red
shifts for two different ratios of QDs and DNA: 1:20 and 1:5 at different concentrations. The 1:20 ratio
exhibits a 7 nm and a 12 nm red shift at 0.1 µM and 0.5 µM DNA concentrations, respectively. The red
shift for the ratio of 1:5 is clearly larger than 1:20. However, the red shift in the 0.5 uM control DNA
is 10 nm, so the detection sensitivity for 1:20 is not significantly improved. However, the detection
sensitivity increased appreciably for the 1:5 ratio, indicating that the ratio of quantum dots and DNA
has an impact on the detection sensitivity.
Table 1. Red shifts for two different concentration ratios (1:20 and 1:5) of quantum dots and DNA at
a DNA concentration of 0.1 µM and 0.5 µM. Results for control DNA are also given.
QD:DNA
1:20
1:5
ck DNA
0.1 µM
0.5 µM
7 ± 0.8
12 ± 1
11 ± 1
18 ± 1.5
–
10 ± 0.9
The TEM image (Figure 6a) shows that the QD is about 4 nm, which is appropriate for our
porous silicon holes. The refractive index of a QD is related to its size. Generally, the larger the
QD is, the larger the refractive index. A QD with a large refractive index has a stronger spectral
response, so the sensitivities scale with the QD size. However, the particle size will affect the particles
entering into the porous silicon hole. The PSM structure detection sensitivity will be greatly reduced
when the QDs cannot enter the pores, so the appropriate size is an important factor for increasing
the sensitivity. The absorption spectroscopy image (Figure 6b) shows that the peak is located at
525 nm and the emission spectroscopy image (Figure 6b) shows that the peak is located at 500 nm.
Our reflectance spectrum (Figure 4) shows that the dip is located at 650 nm; far away from the emission
and the absorption peaks. Furthermore, the reflection light intensity is much stronger than the QD
emission and absorption. Therefore, the QD absorption and emission have almost no impact on the
sensing sensitivity.
At present, the detection limit of the latest porous silicon optical biosensor is about 40 nM [21,22].
In this paper, the sensor detection limit is also at this level when using the PSM to detect the control
DNA without a quantum dot. However, the QD-labeled DNA detection sensitivity is five times higher
than that of the conventional method. Using the QD-labeled DNA strand as an analyte has its own
drawbacks; the method is more complicated and interferes with the target DNA more than the label-free
detection method. As a result, the detection limit of this method is lower (0.1 nm/14.34 = 6.97 nM),
and the sensitivity of this method is much higher.
Sensors 2017, 17, 80
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Figure6.6. (a)
(a)The
TheTEM
TEMimage
imageand
and(b)
(b)the
theabsorption
absorptionand
andemission
emissionspectra
spectraof
ofQD.
QD
Figure
4. Conclusions
Conclusions
4.
In this
thiswork,
work,
we fabricated
macro-porous
silicon microcavities
andQD-labeled
prepared
In
we fabricated
N-typeN-type
macro-porous
silicon microcavities
and prepared
QD-labeled
DNA with carboxyl-CdSe/ZnS
QDs. Bythe
increasing
pore
size, more
quantum
DNA
with carboxyl-CdSe/ZnS
soluble QDs.soluble
By increasing
pore size,the
more
quantum
dots-labeled
dots-labeled
DNA
molecules
can
be
bonded
to
the
microcavity
pores.
The
effective
refractive
index
DNA molecules can be bonded to the microcavity pores. The effective refractive index is increased
by
is increasedthe
bytarget
combining
the target
DNA with the high-refractive-index
QDs; thus,
PSM spectral
combining
DNA with
the high-refractive-index
QDs; thus, PSM spectral
responses
to the
responses
the analytes
increased.between
The relationship
between
the QD-DNA
concentration
analytes
aretoincreased.
Theare
relationship
the QD-DNA
concentration
and the
red shifts of and
the
the
red
shifts
of
the
PSM
reflectance
spectrum
was
measured
using
a
detection
method
on
PSM reflectance spectrum was measured using a detection method based on refractive indexbased
changes.
refractive
changes.
Thedemonstrated
results showed
that the
demonstrated
method
of QD-labeled
DNA
The
resultsindex
showed
that the
method
of QD-labeled
DNA
increased
the reflectance
increased
the
reflectance
spectrum
red
shift,
improving
sensitivity
by
a
factor
of
five
times
spectrum red shift, improving sensitivity by a factor of five times compared to conventional DNA
compared to detection
conventional
DNAbased
hybridization
methods
on porous
Asbea
hybridization
methods
on porousdetection
silicon. As
a result,based
DNA labeled
withsilicon.
QDs can
result,
withDNA
QDssensing.
can be used for high sensitivity DNA sensing.
used
forDNA
highlabeled
sensitivity
Acknowledgments: This
This work
work was
was supported
supported by
by the
the National
National Science
Science Foundation
Foundation of
of China
China (No.
(No. 61575168,
Acknowledgments:
61665012
61665012 and
and 11504313),
11504313), Xinjiang
Xinjiang Science
Scienceand
andTechnology
TechnologyProject
Project(No.
(No.201412112).
201412112).
Author
Author Contributions:
Contributions: Z.J.
Z.J. and
and C.L.
C.L. conceived
conceived and
and designed
designed the
the experiments;
experiments; C.L.
C.L. performed
performed the
the experiments;
experiments;
C.L., H.Z. and Y.L. analyzed the data; J.L. contributed reagents/materials/analysis tools; C.L. and Z.J. wrote
C.L., H.Z. and Y.L. analyzed the data; J.L. contributed reagents/materials/analysis tools; C.L. and Z.J. wrote
the paper.
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Conflicts of Interest: The authors declare no conflict of interest.
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