applied

sciences
Article
Facile Fabrication of Micro/Nano Hierarchical SERS
Sensor via Anisotropic Etching and Electrochemical
Treatment for Malachite Green Detection
Chu-Yu Huang * and Chih-Hung Chien
Department of Mechanical Engineering, National Chung Hsing University, Taichung 402, Taiwan;
as6659871@gmail.com
* Correspondence: tomhuang@nchu.edu.tw




Received: 18 November 2019; Accepted: 29 November 2019; Published: 2 December 2019


Featured Application: The SERS active substrates developed in this study have great potential in
areas such as medicine, food safety, and biotechnology.

Abstract: We propose a facile method to produce micro/nano hierarchical surface-enhanced Raman

scattering (SERS) active substrates using simple steps and inexpensive costs. The proposed SERS
substrate is a silicon pyramid array covered by a nanostructured gold film (AuNS @ SiPA). Through
finite element method (FEM) simulation, we showed that many strong local electric field enhancements
(hot spots) were formed between the nano-gap of gold nanostructures. In addition, the micron-scale
pyramid structure not only increases the sensing surface area of the sensor, but also helps trap light.
By combining these micro and nano structures, the proposed micro/nano hierarchical SERS sensor
exhibited high sensitivity. Experimental results confirmed that the AuNS @ SiPA substrate has high
sensitivity. The SERS signal enhancement factor obtained from the Rhodamine 6G (R6G) probe
molecules was as high as 1 × 107 and the SERS substrates were found to be able to detect a very low
concentration of 0.01 nM malachite green (MG) solution. Therefore, this study provides a novel and
practical method for fabricating SERS substrates that can facilitate the use of SERS in medicine, food
safety, and biotechnology.

Keywords: SERS; surface-enhanced Raman scattering; gold nanostructure; micro/nano hierarchical;

micro-pyramid array; malachite green; MG

1. Introduction
Malachite green is often illegally used in aquaculture to prevent aquatic animals from being
infected by parasites, fungi, and protozoa [1]. According to the results of animal experiments, malachite
green can poison the liver of experimental animals, cause anemia and hyperthyroidism, and cause lung
adenomas of experimental rats [2]. In order to avoid the harm of malachite green to human health,
malachite green is prohibited from being used for aquaculture animals in the United States, and the
UK [3]. According to the “Standards for Residues of Animal Drugs” of the Department of Health
and Welfare, the residue of malachite green in aquatic products is “not allowed to be detected”. The
European Commission requires that detection methods be able to determine the sum of MG at the
minimum performance limit of 2 ng/g (approximately 5.5 nM) [4]. The detection of malachite green
residues is usually carried out at a sophisticated Laboratory using expensive equipments, such as liquid
chromatography visible detection (LC-VIS) for routine screening and liquid chromatography mass
spectrometry (LC-MS) for residue confirmation [1]. It is a time consuming and expensive process. There
is a need for high throughput and cost-effective methods for screening these aquaculture products.

Appl. Sci. 2019, 9, 5237; doi:10.3390/app9235237 www.mdpi.com/journal/applsci

Appl. Sci. 2019, 9, 5237 2 of 12

Surface-enhanced Raman spectroscopy (SERS) technology has the potential to provide a very fast and
sensitive method of detecting chemicals and biomolecules. It can be a solution for this unmet need.
The 3D microstructure of the SERS substrate has a significant impact on the SERS signal. Studies
have shown that the SERS signal can be enhanced by changing the 2D planar substrate into a
3D microstructure substrate, for example, silicon nanowire [5], and pyramid structure [6–9]. This
microstructured SERS substrate has a large surface area, therefore increases the number of effective
hot spots, and further enhances the sensitivity of the SERS signal [10]. The microstructured silicon
pyramid is particularly interesting one. Pyramid microstructure has been used in solar cells to improve
the conversion efficiency of solar cells through its light trapping effect [11–13]. Due to its low process
threshold and cost-effective performance, this is the most mature production process in the photovoltaic
industry for decades. An anisotropic wet chemical etch is typically utilized to form a micro-sized
pyramidal structure as a light-harvesting structure using different etch rates of silicon crystals at <100>
and <111> surfaces [14]. It has been shown that a randomly sized pyramid array surface can have
better light-harvesting efficiency and therefore a better absorption coefficient than planar and single
sized pyramid array surfaces [15]. In some SERS studies [7–9], It has been proven that the Si pyramids
can increase the amount of the hot spots by providing large surface area and create strong local electric
field [9]. However, substrates with only 3D microstructures are not sufficient to generate strong SERS
signals, and studies have shown that strong local surface plasmons (SERS hot spots) occur at the
nano-gaps between noble metal nanostructures [9,10]. Therefore, it is preferred that the SERS substrate
can have both micron and nanostructures, which will greatly increase the SERS signal strength.
In this study, we provide a facile method to produce SERS active substrates using simple steps
and inexpensive costs. We use wet etching to create a pyramid-shaped large structure on a silicon
wafer and then use the electrochemical process to create gold nanostructures on the pyramid surface.
In previous studies, electrochemical surface treatment was mostly used to roughening the surface
of bulk gold or bulk silver materials [16,17]. In this study, we applied the electrochemical oxidation
and reduction cycles to the gold thin film on the silicon micro-pyramid array, which leads to atomic
restructuring of the gold thin film surface. We studied the surface metal dissolution/redeposition effect
of the electrochemical treatment to the gold thin film on the micro-pyramid array silicon substrate, and
find a method to optimize the electrochemical treatment and maximized surface area of the gold thin
film on the micro-pyramid array silicon substrate. We observed the electrochemical redox cycle curves
and SERS signal of substrates, and found the relationship between electrochemical oxidation peaks
and SERS signal strength. From the relationship between electrochemical oxidation peaks and SERS
signal intensity, we obtained the best electrochemical treatment cycles for SERS substrate preparation.
In addition, by combining the micro-pyramid array and gold nanostructure on the pyramid surface,
this micro/nano hierarchical structured SERS substrate can greatly enhance the Raman signal. The
SERS enhancement effect is tested with a low concentration of Rhodamine 6G (R6G). Finally, we
applied SERS substrates prepared by optimized electrochemical parameters to the detection of R6G
and malachite green solutions. The results show that the substrate has high sensitivity and can measure
very low concentrations of R6G and malachite green.

2. Methodology and Experiments

2.1. Fabrication of the Silicon Micro-Pyramid Structures

To fabricate silicon micro-pyramid structures, we first cleaned the silicon wafer with ethanol and
acetone. The silicon wafer was then etched at 85 ◦ C for 40 min in a mixed solution of NaOH (1.8 wt%),
water and isopropyl alcohol (IPA, 5 vol%). During the NaOH etching, the etching solution was stirred
using a magnetic stirrer, and the rotational speed of the magnetic stirring was set to 200 rpm. After
etching, the wafer was rinsed with deionized water and dried with nitrogen.
 Appl.Sci.
Appl. Sci.2019,
2019,9,9,5237
x FOR PEER REVIEW 33 of
of 12
12

2.2. Fabrication of the Hierarchical Micro/Nanostructure

2.2. Fabrication of the Hierarchical Micro/Nanostructure
To create a hierarchical micro/nanostructure, a gold thin film with a thickness of 100 nm was
first To
deposited
create a on micro-pyramids
hierarchical using a dc magnetron
micro/nanostructure, sputtering
a gold thin film withsystem (Cressington
a thickness of 100 nm 108was
Sputter
first
Coater). The
deposited wafer with theusing
on micro-pyramids golda dc
film deposited
magnetron was diced
sputtering into(Cressington
system 15 × 15 mm 108chips before
Sputter the
Coater).
electrochemical
The wafer with thetreatment.
gold film The chip waswas
deposited then attached
diced to ×
into 15 a glass
15 mm slide and
chips wire connected
before as shown
the electrochemical
in Figure 1c.
treatment. The
The conductive
chip was then wire was to
attached covered
a glasswithslidesilicone
and wire to connected
prevent reaction
as shown with inelectrolyte. The
Figure 1c. The
chip (working
conductive wireelectrode)
was covered waswiththensilicone
immersed in the KCL
to prevent solution
reaction along with aThe
with electrolyte. counter electrode
chip (working
(platinum)was
electrode) andthen
a reference
immersed electrode (Ag/AgCl).
in the KCL solutionThe sample
along withwas then electrochemically
a counter electrode (platinum) treated
andby a
applied oxidation
reference electrodeand reduction
(Ag/AgCl). cycles
The to form
sample was thethennanostructure on thetreated
electrochemically surfaceby ofapplied
the goldoxidation
thin film.
Thereduction
and electrochemical
cycles totreatment
form the was performed
nanostructure onby theusing a VidaBio
surface VS1thin
of the gold electrochemical potentiostat
film. The electrochemical
with a voltage
treatment sweep range
was performed of −800
by using mV to VS1
a VidaBio 800 electrochemical
mV, voltage sweep rate: 50 with
potentiostat mV/s, oxidation
a voltage and
sweep
Reduction
range cycles
of −800 mV tofrom
8001mV,
to 15 cyclessweep
voltage in a KCLrate:solution
50 mV/s, with a concentration
oxidation and Reductionof 0.2cycles
mol/L.from
The 1micro-
to 15
pyramids
cycles combined
in a KCL solution with
with a the nanostructured
concentration gold The
of 0.2 mol/L. thin film formedcombined
micro-pyramids the hierarchical
with the
micro/nanostructure.
nanostructured gold thinThefilm
fabrication
formed the process of the micro/nanostructure.
hierarchical described micro/nanoThe hierarchical
fabricationstructure
process ofis
illustrated
the describedin micro/nano
Figure 1. hierarchical structure is illustrated in Figure 1.

Figure 1. The fabrication process of the described micro/nano hierarchical structure. (a) The silicon
Figure 1. The fabrication process of the described micro/nano hierarchical structure. (a) The silicon
wafer was wet etched in a mixed solution of NaOH, water, and isopropyl alcohol. (b) Depositing a gold
wafer was wet etched in a mixed solution of NaOH, water, and isopropyl alcohol. (b) Depositing a
thin film on the micro-pyramid array using a sputtering system. (c) Attach the chip to a slide before
gold thin film on the micro-pyramid array using a sputtering system. (c) Attach the chip to a slide
electrochemical treatment. (d) The sample was treated with electrochemical oxidation and reduction
before electrochemical treatment. (d) The sample was treated with electrochemical oxidation and
cycles to create nanostructures on the surface of the gold thin film.
reduction cycles to create nanostructures on the surface of the gold thin film.
2.3. Experiments
2.3. Experiments
The surface morphology of the fabricated SERS substrate was investigated by using a scanning
Themicroscope
electron surface morphology of the fabricated
(SEM, JSM-7800F, SERSTokyo,
JEOL Ltd., substrate wasand
Japan) investigated
an atomic byforce
usingmicroscope
a scanning
electron microscope (SEM, JSM-7800F, JEOL Ltd., Tokyo, Japan) and an atomic
(AFM, Bruker Dimension Icon, Bruker Corporation, Billerica, USA). The SERS performances of force microscope
(AFM, Bruker
fabricated Dimension
substrates Icon,with
were tested Bruker
R6GCorporation,
and malachiteBillerica, USA). The
green solutions. Five SERS performances
microliters of differentof
fabricated substrates
concentrations were tested
of R6G aqueous withand
solution R6G and malachite
malachite green were
green solution solutions. Five dropped
separately microliters
ontoof
different concentrations of R6G aqueous solution and malachite green solution
the prepared SERS substrates and allowed to dry at room temperature for Raman spectroscopy were separately
dropped onto the
measurements. SERSprepared
spectra SERS substratesby
were measured and allowed to Raman
a microscope dry at room temperature
spectrometer for Raman
(Nanofinder 30,
spectroscopy
Tokyo measurements.
Instruments, SERSunder
Tokyo, Japan) spectra were measured
excitation of a 632.8bynma HeNe
microscope Raman spectrometer
laser, wherein the effective
(Nanofinder
power 30, Tokyo
of the laser source Instruments,
was set to 0.1 Tokyo,
mW. The Japan) under
diameter excitation
of the focusedof a 632.8
laser nmthe
spot on HeNe laser,
surface of
wherein the effective power of the laser source was set to 0.1 mW. The diameter of the focused laser
spot on the surface of the sample was approximately 2 μm, and the integration time per measurement
 pyramid array surface can have better light collection efficiency than a planar and single-sized
pyramid array surface over a wide wavelength range. The average spacing between the micro-
pyramids is about 2.8 μm, and the average width of the micro-pyramids at the base is about 1.4 μm.
The AFM image in Figure 2b indicates that the average height of these micro-pyramids is about 2 μm.
A gold film with a thickness of 100 μm was then deposited on the micro-pyramid array by
Appl. Sci. 2019, 9, 5237 4 of 12
sputtering and then electrochemical treated to form nanoparticles on the gold film surface. The size
of these nanoparticles was controlled by electrochemical redox cycles. Figure 3a–d show SEM images
of these
the sample gold
was nanoparticles
approximately with2 different
µm, and theelectrochemical
integration time redox cycles
per of 0, 5, 10, was
measurement and 15, respectively.
10 s. The SERS
As can
substrate be seen from
performance wasthese
then figures, the morphology
characterized by calculatingof the
thegold
SERSfilm on the surface
enhancement of the micro-
factor.
pyramid structure after electrochemical treatment clearly shows the formation of many tiny nano-
3.grain
Results and Discussion
structures. It can be seen that the longer the duration, the larger the diameter of these
nanoparticles. In the first 10 redox cycles, the diameter of these nanoparticles has relatively faster
3.1. Characterization
increasement, andof the
the Developed Substrates
nanoparticles become larger and larger. After 10 redox cycles, the
nanoparticles’
The SEM image changes are not
showed in obvious.
Figure 2aHowever, if too many
is the anisotropic wet redox
etchedcycles
siliconare performed, thearray
micro-pyramid gold
film will become
structure. From the tooimage,
thin and
oneexpose
can alsothesee
silicon
that pyramid
the produced(as shown in Figure 4).
micro-pyramid array is a randomly
Figure 3c,d
sized pyramid shows
array that the
surface. Thisdiameter of the
feature may benanoparticles
an advantage,isasabout 20 nm in
mentioned when
[15],the redox cycle is
a random-sized
10–15 cycles.
pyramid array The gapcan
surface between the nanoparticles
have better light collection is efficiency
in the rangethanofaaplanar
few nanometers and ispyramid
and single-sized suitable
for producing
array surface over many intense
a wide hot spots. range.
wavelength Therefore,
Theitaverage
is very helpful
spacingtobetween
enhancethe themicro-pyramids
SERS effect. Figureis
4 shows that when the redox cycle was set to 30 cycles, the gold film became
about 2.8 µm, and the average width of the micro-pyramids at the base is about 1.4 µm. The AFM too thin and exposed
the underlying
image in Figure 2b pyramid.
indicates that the average height of these micro-pyramids is about 2 µm.

(a) (b)
Figure2.2.(a)
Figure (a)The
TheSEM
SEMimage
imageofofpyramid
pyramidmicrostructure
microstructureformed
formedby byanisotropic
anisotropicwet
wetetching.
etching.(b)
(b)The
The
pyramidsize
pyramid sizemeasured
measuredbybyananatomic
atomic force
force microscope
microscope (AFM),
(AFM), thethe overall
overall pyramid
pyramid average
average height
height is 2is
2 μm,
µm, andand
thethe average
average spacing
spacing is 2.8
is 2.8 µm.μm.

A gold film with a thickness of 100 µm was then deposited on the micro-pyramid array by
sputtering and then electrochemical treated to form nanoparticles on the gold film surface. The size of
these nanoparticles was controlled by electrochemical redox cycles. Figure 3a–d show SEM images of
these gold nanoparticles with different electrochemical redox cycles of 0, 5, 10, and 15, respectively.
As can be seen from these figures, the morphology of the gold film on the surface of the
micro-pyramid structure after electrochemical treatment clearly shows the formation of many tiny
nano-grain structures. It can be seen that the longer the duration, the larger the diameter of these
nanoparticles. In the first 10 redox cycles, the diameter of these nanoparticles has relatively faster
increasement, and the nanoparticles become larger and larger. After 10 redox cycles, the nanoparticles’
changes are not obvious. However, if too many redox cycles are performed, the gold film will become
too thin and expose the silicon pyramid (as shown in Figure 4).
Appl. Sci.
Appl. Sci. 2019,
2019, 9,
9, 5237
x FOR PEER REVIEW 55 of 12
of 12
Appl. Sci. 2019, 9, x FOR PEER REVIEW 5 of 12

Figure 3. (a) The gold film morphology on the surface of the micro-pyramid structure before
Figure 3. (a) The gold film morphology on the surface of the micro-pyramid structure before
electrochemical
Figure 3. (a) Thetreatment,
gold film themorphology
surface is relatively
on the smoothof(compared
surface with the electrochemically
the micro-pyramid structure treated
before
electrochemical treatment, the surface is relatively smooth (compared with the electrochemically
treated surface).
electrochemical (b) The
treatment,gold
the film morphology
surface is after
relatively 5 redox
smooth cycles.
(compared (c) The
with gold
the film morphology
electrochemically
surface). (b) The gold film morphology after 5 redox cycles. (c) The gold film morphology after 10
after 10
treated
redox redox(d)
cycles. cycles.
surface). The (d) The
(b) The
gold filmgold
gold film film morphology
morphology
morphology after
after after 15
5 redox
15 redox redox cycles.
cycles.
cycles. (c) The gold film morphology
after 10 redox cycles. (d) The gold film morphology after 15 redox cycles.

Figure 4. The gold film on the surface

4. The surface of
of the
the pyramid
pyramid is is oxidized
oxidized and
and corroded.
corroded. After too many
oxidization-reduction
oxidization-reduction
Figure cycles,
cycles,
4. The gold film the gold
gold film
thesurface
on the ofbecame
film became too
too thin
the pyramid is and
thin and exposed
andthe
exposed
oxidized the pyramid.
pyramid.
corroded. After too many
oxidization-reduction cycles, the gold film became too thin and exposed the pyramid.
When the gold film on the surface of the pyramid is subjected to cyclic voltammetry redox
scanning,
Whenthe thegold
goldonfilm
the surface
on the is first oxidized
surface into the solution
of the pyramid and then
is subjected reduced
to cyclic to the surface
voltammetry of
redox
scanning, the gold on the surface is first oxidized into the solution and then reduced to the surface of
oxidation peak occurs, which is about 530 mV. The oxidation peak currents were further plotted
against the scanning cycles in Figure 6. As can be seen from Figures 5 and 6, when the number of
scanning cycles increased, the peak current of the oxidation also increased. When it reaches a certain
amount of cycles (in this case around 10 cycles), the oxidation peaks reach a plateau state. The change
inAppl.
the Sci.
microscopic
2019, 9, 5237
surface area of the electrode produced by the oxidation-reduction cycles6 of is 12
proportional to the oxidation peak, as Daubinger and Netzahualcóyotl mentioned in [18,19].
Therefore, a higher oxidation peak indicates that more nanostructures were produced on the
electrodeFigure 3c,dduring
surface showsthe thatoxidation-reduction
the diameter of the scanning
nanoparticles is about 20 nm when the redox cycle is
process.
10–15
SERS measurements of substrates electrochemically treatedofwith
cycles. The gap between the nanoparticles is in the range a few nanometers
different and is suitable for
oxidation-reduction
producing
cycles many intense
were conducted hot spots. Therefore,
for comparison. From theitSERSis very helpful
spectra ofto1 enhance
μM R6G the SERS measured
solution effect. Figureby 4
shows that
substrates withwhen the redox
different cycle was set to cycle
oxidation-reduction 30 cycles,
timesthe (0, gold film
2, 4, 6, became
8, 10, 12, and too14,
thin and exposed
respectively), thethe
underlying
SERS pyramid.
characteristic peak intensity of R6G at 1510 cm−1 increase rapidly in the first few cycles and
reach aWhen
plateau the gold
after 10film
cycleson(as
theshown
surface of the pyramid
in Figure 7). It has theis subjected
same tendency to cyclic voltammetry
as the oxidation peakredox
ofscanning,
the redoxthe gold
curve on theinsurface
shown Figureis6.first
Thisoxidized
is because into the solution
higher oxidation andpeaks
then reduced to the reaction
indicate more surface of
the gold
surface film.
area, During
which means thismore
process, the gold surface
nanostructures is granulated
were created. More to form many gold
nanostructures not nano-gaps.
only have aIn
the experiment,
larger reaction area webut
repeated the cyclichigher
also produce voltammetry
density redox
SERS scanning
hot spotscycle andmultiple
lead to times.
higher Figure
SERS 5
shows theTherefore,
intensities. cyclic voltammetry
the optimalcurves from the
number of 1st scan to the 15th scan
oxidation-reduction cycle can
cycles in a be0.2 M KCl solution
determined by at
a scan ratethe of 50 −1
mV s . The position of the blue
monitoring oxidation-reduction plots. Once it arrow
reachesinathe figure state,
plateau showsitthe position
means the where
optimal the
oxidation peak occurs,
oxidation-reduction cycleswhich is about
for SERS 530 mV. Thehas
enhancement oxidation peak currents were further plotted against
been reached.
theAnother
scanningnote cycles in Figure
worth 6. As can
mentioning be seen
is that fromcurrent
the peak Figuresof5 and 6, when the
the reduction number
is less thanofthescanning
peak
cycles increased,
oxidation the peak
current, which current
means that of
thethe oxidation
redox process also increased. When
is irreversible. it reaches
Therefore, a certain
the gold amount
film on the
of cycles
surface (in this
of the case around
pyramid 10 cycles),
is gradually the oxidation
oxidized peaks reach
and dissolved. a plateau
This will causestate. The film
the gold changeon inthethe
microscopic
surface surface area
of the pyramid of the electrode
to gradually decrease.produced
This is bywhy thetoooxidation-reduction
many redox scan cycles cyclescan
is proportional
cause the
to the
gold filmoxidation
to become peak, as Daubinger
too thin and expose andtheNetzahualcóyotl
underlying pyramid mentioned
(as shownin [18,19].
in FigureTherefore, a higher
4). Therefore,
weoxidation
need topeak indicates
carefully that more
monitor nanostructures were produced
the oxidation-reduction curves and on stop
the electrode surface during the
the oxidation-reduction
oxidation-reduction
process once the oxidation scanning
peakprocess.
reaches a plateau.

1st cycle
40 2nd cycle
15th
10th
3rd cycle
30 4th cycle
5th cycle
20 1st 6th cycle
7th cycle
10
8th cycle
I(uA)

9th cycle
0
10th cycle
11th cycle
-10
12th cycle
13th cycle
-20
14th cycle
15th cycle
-30

-40
-1000 -800 -600 -400 -200 0 200 400 600 800 1000
E(mV)
Figure 5. The cyclic voltammetry curves from the 1st scan to the 15th scan cycle at a sweeping rate of
50 mV s−1 in the 0.2 M KCl solution. Each cycle curve was plotted in a different line style for clarity.
The position of the blue arrow in the figure shows the position where the oxidation peak occurs, which
is about 530 mV. We can see from the figure that the oxidation peak current increases with the number
of scanning cycles. However, the increment gradually reduces, and its increment was quite limited
after the number of scanning cycles exceeded 10.
 The position of the blue arrow in the figure shows the position where the oxidation peak occurs,
which is about 530 mV. We can see from the figure that the oxidation peak current increases with the
number of scanning cycles. However, the increment gradually reduces, and its increment was quite
limited after the number of scanning cycles exceeded 10.
Appl. Sci. 2019, 9, 5237 7 of 12

25 REVIEW
Appl. Sci. 2019, 9, x FOR PEER 7 of 12

Figure 5. The cyclic voltammetry curves from the 1st scan to the 15th scan cycle at a sweeping rate of
50 mV s−1 in the 0.220M KCl solution. Each cycle curve was plotted in a different line style for clarity.
The position of the blue arrow in the figure shows the position where the oxidation peak occurs,

Oxidation peak (uA)

which is about 530 mV. We can see from the figure that the oxidation peak current increases with the
15
number of scanning cycles. However, the increment gradually reduces, and its increment was quite
limited after the number of scanning cycles exceeded 10.
10

525

020
Oxidation peak (uA)

0 2 4 6 8 10 12 14
15
oxidation-reduction cycles

Figure 6. The oxidation peak

10 currents at 530 mV were plotted against the scanning cycles. It can be
seen that the oxidation peak current
Figure 6. The oxidation peak currentsincreases
at 530 with the number
mV were plotted of scanning
against cycles. The
the scanning increment
cycles. It can be
gradually reduces, and when the voltammetry redox scan reaches a certain amount of cycles
seen that the oxidation5peak current increases with the number of scanning cycles. The increment(in this
case around 10 cycles), the oxidation peaks reach a plateau state.
gradually reduces, and when the voltammetry redox scan reaches a certain amount of cycles (in this
casemeasurements
SERS around 10 cycles),ofthe oxidationelectrochemically
substrates peaks reach a plateau state. with different oxidation-reduction
treated 0
cycles were conducted for comparison. From the SERS spectra of 1 µM R6G solution measured by
substrates with different oxidation-reduction
0 2 4 cycle6 times8 (0, 2,10
4, 6, 8,1210, 12,
14and 14, respectively), the
at 1510 cm−1 increase
SERS characteristic peak intensity of R6G oxidation-reduction cycles rapidly in the first few cycles and
35000
reach a plateau after 10 cycles (as shown in Figure 7). It has the same tendency as the oxidation peak
SERS peak intensity @ 1510 cm-1

of the redox curve shown30000 in Figure 6. This is because higher oxidation peaks indicate more reaction
Figure
surface area, 6. The
which oxidation
means more peak currents at 530 mV
nanostructures werewere plottedMore
created. against the scanning cycles.
nanostructures not It can have
only be a
seen that the oxidation
25000
peak current increases with the number of scanning cycles. The increment
larger reaction area but also produce higher density SERS hot spots and lead to higher SERS intensities.
gradually reduces, and when the voltammetry redox scan reaches a certain amount of cycles (in this
Therefore, the optimal number of oxidation-reduction cycles can be determined by monitoring the
case around 10 cycles),
20000 the oxidation peaks reach a plateau state.
oxidation-reduction plots. Once it reaches a plateau state, it means the optimal oxidation-reduction
cycles for SERS enhancement 15000 has been reached.

10000
35000
SERS peak intensity @ 1510 cm-1

5000
30000

0
25000
0 2 4 6 8 10 12 14

20000
oxidation-reduction cycles

15000
Figure 7. It shows the surface-enhanced Raman scattering (SERS) experiments results of 1 μM
Rhodamine 6 (R6G) solution
10000 measured by SERS chips with different oxidation-reduction cycles (0, 2,
4, 6, 8, 10, 12, and 14, respectively). It can be seen that in the first few cycles, the intensity of the SERS
5000
characteristic peak of R6G at 1510 cm−1 increased rapidly and reached a plateau after 10 cycles.
0
0 2 4 6 8 10 12 14
oxidation-reduction cycles

Figure 7. It shows the surface-enhanced Raman scattering (SERS) experiments results of 1 µM

Figure 67.(R6G)
Rhodamine It shows the measured
solution surface-enhanced
by SERSRaman scattering
chips with (SERS)
different experiments results
oxidation-reduction cyclesof(0,1 2,μM
4,
Rhodamine 6 (R6G) solution measured by SERS chips with different oxidation-reduction cycles (0, 2,
6, 8, 10, 12, and 14, respectively). It can be seen that in the first few cycles, the intensity of the SERS
4, 6, 8, 10, 12, and 14, respectively). It can be seen that in the first few cycles, the intensity of the SERS
characteristic peak of R6G at 1510 cm−1 increased rapidly and reached a plateau after 10 cycles.
characteristic peak of R6G at 1510 cm−1 increased rapidly and reached a plateau after 10 cycles.
Appl. Sci. 2019, 9, 5237 8 of 12

Another note worth mentioning is that the peak current of the reduction is less than the peak
oxidation current, which means that the redox process is irreversible. Therefore, the gold film on the
surface of the pyramid is gradually oxidized and dissolved. This will cause the gold film on the surface
of the pyramid to gradually decrease. This is why too many redox scan cycles can cause the gold film
to become too thin and expose the underlying pyramid (as shown in Figure 4). Therefore, we need to
carefully monitor the oxidation-reduction curves and stop the oxidation-reduction process once the
oxidation peak
Appl. Sci. 2019, 9, xreaches a REVIEW
FOR PEER plateau. 8 of 12

3.2. Simulations
3.2. Simulations
Simulations were performed to further understand the effects of gold nanoparticles decorated
Simulations were performed to further understand the effects of gold nanoparticles decorated
micro-pyramid
micro-pyramidarray arraysubstrate. WeWe
substrate. calculated the local
calculated the electric field of the
local electric fieldgold
of nanoparticles decorated
the gold nanoparticles
micro-pyramid structure using
decorated micro-pyramid a commercial
structure the finite element
using a commercial method
the finite element(FEM) software
method (FEM)(COMSOL
software
Multiphysics). We set the diameter of gold nanoparticles to 20 nm and pitch
(COMSOL Multiphysics). We set the diameter of gold nanoparticles to 20 nm and pitch between between nanoparticles to 22
nm. Figure 8 shows a cross-sectional view of the electric field distribution between two
nanoparticles to 22 nm. Figure 8 shows a cross-sectional view of the electric field distribution between micro-pyramids
with
two an incident light wavelength
micro-pyramids of 633 nm.
with an incident lightInwavelength
this figure, ofthe633
normalized
nm. In thiselectric fieldthe
figure, represents
normalizedthe
ratio between the electric field excited by the local surface plasmon resonance
electric field represents the ratio between the electric field excited by the local surface plasmon and the background
electric field.
resonance andWe thecan clearly see
background that afield.
electric strongWeelectric field see
can clearly is formed around
that a strong gold field
electric nanoparticles,
is formed
especially
around gold nanoparticles, especially between nanoparticles. V-shaped caves formed also
between nanoparticles. V-shaped caves formed between the pyramids can help the
between to
enhance the electric field by concentrating the input of light energy. This micro/nano
pyramids can also help to enhance the electric field by concentrating the input of light energy. This hierarchical
structured
micro/nanosubstrate helps
hierarchical to form many
structured strong
substrate hottospots.
helps Since strong
form many the SERS hotenhancement
spots. Since the effect
SERSis
proportional to the fourth power of the local electric field intensity [20], these hot
enhancement effect is proportional to the fourth power of the local electric field intensity [20], these spots have a very
good enhancement
hot spots have a veryeffect
goodon enhancement
the SERS signal.effect on the SERS signal.

Figure8.8.A
Figure Across-sectional
cross-sectionalview
viewofofthe
theelectric
electricfield
fielddistribution
distributionbetween
betweentwo
twomicro-pyramids
micro-pyramidswith with
anincident
an incident light wavelength
wavelengthof of633
633nm.
nm.InIn
this figure,
this thethe
figure, normalized electric
normalized fieldfield
electric represents the ratio
represents the
between
ratio the electric
between Field Field
the electric excited by theby
excited local
thesurface plasmon
local surface resonance
plasmon and theand
resonance background electric
the background
field. field.
electric

3.3.
3.3. SERS
SERS Measurements
Measurements
Figure
Figure99 shows
shows the
the SERS
SERS spectra
spectra of
of R6G
R6G solutions with concentrations
solutions with concentrations of 10−6−6M,
of 11 ××10 M,1 1× ×
1010
−7
−7 M,

M, ×−810 −8 1 −9× M, −9
1 ×110 M, and
M, and 1 × 10 10 respectively,
M, respectively,
usingusing our SERS
our SERS substrate
substrate with
with 10 10 oxidation-reduction
oxidation-reduction cycles.
cycles. The Raman band assignments of the main peaks for R6G
The Raman band assignments of the main peaks for R6G were given in Figurewere given in Figure9.9. Even
Even atat aa low
low
concentration −1
concentration of of11nM,
nM,characteristic
characteristicpeaks
peaksof
ofR6G
R6Gatat607,
607,765,
765,1176,
1176,1304,
1304,1355,
1355,1503,
1503,andand1640
1640cm cm−1
were
wereclearly
clearlyobserved
observedfrom fromthese
thesespectra.
spectra.These
Thesespectra
spectraindicate
indicatethat
thatour
ourSERS
SERSsubstrates
substrateshavehaveaagoodgood
enhancement factor.
enhancement factor.
We used the following well-established formula [21] to estimate the enhancement factor (EF)
value of R6G:
E F = ( I S E R S / I b u lk ) / ( N b u lk / N S E R S ) (1)

where I SERS is the vibrational intensity of the SERS of the R6G molecule at 1510 cm−1, and I bulk is
Appl. Sci. 2019, 9, x FOR PEER REVIEW 9 of 12

the SERS substrate is densely packed with R6G molecules after immersed in 10−6 M R6G solution for
12 h.Sci.
Appl. However, the R6G is likely to cover the substrate at a lower density. Therefore, the SERS
2019, 9, 5237 9 of 12
enhancement factor calculated here is the lower limits.

C-H C-H C-O-C arom arom arom

C-C-C
ring ip out of plane ip str C-C str C-C str C-C str

bend bend bend

1362

1510
35000 1 uM R6G
0.1 uM R6G
10 nM R6G

612
30000
1nM R6G
Intensity (a.u.)

1311

1650
25000

1186
20000
773

1601
1575
15000

10000

5000
x2
0 x3
600 800 1000 1200 1400 1600 1800
-1
Raman shift (cm )

Figure 9. Shows the SERS spectra of R6G solutions with concentrations of 1 × 10−6 M, 1 × 10−7 M, 1 ×
10−8 M, and 1 × 10−9 M, respectively, using our SERS substrate with 10 oxidation-reduction cycles.
Figure 9. Shows the SERS spectra of R6G solutions with concentrations of 1 × 10−6 M, 1 × 10−7 M, 1 ×
10−8 M, and 1 × 10−9 M, respectively, using our SERS substrate with 10 oxidation-reduction cycles.
We used the following well-established formula [21] to estimate the enhancement factor (EF)
value of R6G:
The developed SERS substrates were also tested with low concentration malachite green
EF = (ISERS /Ibulk )/(Nbulk /NSERS ) (1)
solution. Figure 10 shows measured SERS spectra for malachite green concentrations ranging from
0.01
where nMISERS
to 10isnMtheon SERS substrates.
vibrational intensity The
of Raman
the SERS characteristic peaks of malachite
of the R6G molecule at 1510 cmgreen−1 , and
at 1620 cmthe
Ibulk is −1,

1370 cm−1, 1175

vibrational cm−1of
intensity , 916
the cm −1, and 802 cm−1 are in-plane C–C stretching vibration of the ring,
normal Raman spectrum of the R6G molecule at 1510 cm . NSERS is the −1

overlapping effect of irradiated

number of molecules in-plane N–phenyl stretching
by laser spots under vibration
SERS, andand in-plane
Nbulk C–C stretching
is the number of molecules vibration,
of the
in-plane
bulk sample aromatic C–H vibration,
irradiated by laser spots ring-breathing,
under normal andRaman
out-of-plane phenyl–H
condition. The EF bending, respectively.
was calculated to be
These × 107 , whichpeaks
about 1characteristic is quiteof good
malachitefor a green are clearly
gold-based SERS observed
substrate from theThis
[21,22]. spectra.
resultAccording
is assuming to the
the
result, the SERSissubstrates
SERS substrate densely packedwere foundwith R6Gto bemolecules
able to detect
after immersed −6
a low concentration
in 10 M of R6G 0.01 nM malachite
solution for 12 h.
green
However, solution,
the R6Gwhich is very
is likely to close
coverto the
the finding at
substrate in aprevious studies[23,24].
lower density. The
Therefore, theenhancement
SERS enhancement factors
for different concentrations of
factor calculated here is the lower limits. malachite green were also calculated. The Raman characteristic peak
values Theofdeveloped
malachiteSERS green at 1370were
substrates cm also
−1 acquired
tested withfromlow Figure 10 weremalachite
concentration used to green calculate the
solution.
enhancement
Figure 10 shows factors for different
measured SERS spectra concentrations
for malachite of malachite green. The ranging
green concentrations resulting from enhancement
0.01 nM to
factors
10 nM on forSERS
concentrations
substrates. of The10Raman
nM, 1 nM, 0.1 nM, and
characteristic peaks0.01ofnM malachite
malachite green
green are cm
at 1620 1.21 −1×, 1370 cm−1×,
107, 1.49
10 7
1175, 7.81 −1
cm ×, 10 916, cm
7 −1
and ,4.05
and×802 10 cm
8 −1
, respectively.
are in-planeIt C–C
can be seen that
stretching when the
vibration concentration
of the ring, overlapping of theeffect
MG
solution
of in-plane to be measured
N–phenyl is lower vibration
stretching than 1 nM, andas in-plane
the concentration decreases,
C–C stretching the enhancement
vibration, in-plane aromatic factor
(or
C–H sensitivity)
vibration, of the developedand
ring-breathing, chip becomes larger,
out-of-plane ratherbending,
phenyl–H than a constant value.
respectively. These characteristic
peaks of malachite green are clearly observed from the spectra. According to the result, the SERS
substrates were found to be able to detect a low concentration of 0.01 nM malachite green solution,
which is very close to the finding in previous studies [23,24]. The enhancement factors for different
concentrations of malachite green were also calculated. The Raman characteristic peak values of
malachite green at 1370 cm−1 acquired from Figure 10 were used to calculate the enhancement factors
for different concentrations of malachite green. The resulting enhancement factors for concentrations
of 10 nM, 1 nM, 0.1 nM, and 0.01 nM malachite green are 1.21 × 107 , 1.49 × 107 , 7.81 × 107 , and 4.05 ×
108 , respectively. It can be seen that when the concentration of the MG solution to be measured is lower
than 1 nM, as the concentration decreases, the enhancement factor (or sensitivity) of the developed
chip becomes larger, rather than a constant value.
 Appl.
Appl. Sci.Sci.
2019,2019, 9, x FOR PEER REVIEW
9, 5237 10 10 of 12
of 12

malachite green 10nM

45000malachite green 1nM

1620
malachite green 0.1nM
40000malachite green 0.01nM

1175

1370
35000

Raman Intensity (a.u.)

1394
802
30000

1596
1221
916
25000

1297
20000

15000

10000

5000

0
600 800 1000 1200 1400 1600 1800
-1
Wavenumber (cm )

Figure 10. The figure shows measured SERS spectra for malachite green concentrations ranging from
0.01Figure
nM to10.
10 The figure
nM on SERSshows measured SERS spectra for malachite green concentrations ranging from
substrates.
0.01 nM to 10 nM on SERS substrates.
4. Conclusions
4. In
Conclusions
conclusion, we have presented a facile method for synthesizing hierarchical
micro/nanostructured
In conclusion, we active
SERS havesubstrates
presented using
a simple
facile steps
method and inexpensive costs. The
for synthesizing SERS
hierarchical
active substrates were fabricated
micro/nanostructured through
SERS active wet etching
substrates to simple
using create asteps
pyramid-shaped large structure
and inexpensive costs. TheonSERS
a
silicon wafer and then used electrochemical treatments to create gold nanostructure
active substrates were fabricated through wet etching to create a pyramid-shaped large structure onon the pyramid
surface. From
a silicon wafertheand
relationship
then usedbetween electrochemical
electrochemical treatments oxidation
to createpeaks and SERS signal
gold nanostructure intensity,
on the pyramid
wesurface.
optimizedFromthethe
electrochemical treatment
relationship between for SERS substrate
electrochemical preparation.
oxidation peaks and Simulation has shown
SERS signal intensity,
thatwemany strong the
optimized hot electrochemical
spots can be formed between
treatment the nano-gap
for SERS substrate ofpreparation.
gold nanostructures.
Simulation According
has shown
to the
that many strong hot spots can be formed between the nano-gap of gold nanostructures.aAccording
SERS experiments, the micro/nano hierarchical structured SERS substrate showed strong
SERS enhancement factor up the 7
× 10 . The developed
to 1 micro/nano SERS substrates were further showed
applied to the
to the SERS experiments, hierarchical structured SERS substrate a strong
detection of malachite green solutions. The results showed that the substrate has high
SERS enhancement factor up to 1 × 10 . The developed SERS substrates were further applied to the
7 sensitivity and
candetection
measure of very low concentrations
malachite of malachite
green solutions. green
The results (0.01 nM),
showed indicating
that the substrateitshas
practical applicationand
high sensitivity
potential
can measure very low concentrations of malachite green (0.01 nM), indicating its practical is
in food contamination detection. Therefore, the developed SERS active substrate highly
application
promising
potential in food contamination detection. Therefore, the developed SERS active substrate isfood
for use in rapid chemical and biomolecular detection applications, including medicine, highly
safety, and biotechnology.
promising for use in rapid chemical and biomolecular detection applications, including medicine,
food safety, and biotechnology.
Author Contributions: Conceptualization, C.-Y.H.; methodology, C.-Y.H.; investigation, C.-H.C.; data curation,
C.-H.C.;
Authorwriting—original
Contributions: draft preparation, C.-Y.H.;
conceptualization, C.-Y.H.;writing—review and editing,
methodology, C.-Y.H.; C.-Y.H.; supervision,
investigation, C.-H.C.; dataC.-Y.H.;
curation,
project administration,
C.-H.C.; C.-Y.H.;draft
writing—original funding acquisition,
preparation, C.-Y.H.writing—review and editing, C.-Y.H.; supervision, C.-
C.-Y.H.;
Funding: Thisadministration,
Y.H.; project research was C.-Y.H.;
funded funding
by Ministry of Science
acquisition, C.-Y.H.and Technology, Taiwan, grant number
MOST 107-2221-E-005-045.
Funding: This research was funded by Ministry of Science and Technology, Taiwan, grant number MOST 107-
2221-E-005-045. The authors would like to thank the Ministry of Science and Technology of the Republic of
Acknowledgments:
China, Taiwan, for financially supporting this research under Contract No. Grant MOST 107-2221-E-005-045.
Acknowledgments:
Conflicts The
of Interest: The authors
authors wouldnolike
declare to thank
conflict the Ministry of Science and Technology of the Republic of
of interest.
China, Taiwan, for financially supporting this research under Contract No. Grant MOST 107-2221-E-005-045.
References
Conflicts of Interest: The authors declare no conflict of interest.

1. Andersen, W.C.; Turnipseed, S.B.; Roybal, J.E. Quantitative and confirmatory analyses of malachite green
References
and leucomalachite green residues in fish and shrimp. J. Agric. Food Chem. 2006, 54, 4517–4523. [CrossRef]
1. [PubMed]
Andersen, W.C.; Turnipseed, S.B.; Roybal, J.E. Quantitative and confirmatory analyses of malachite green
2. andY.;
Deng, leucomalachite greenproperties
Chu, D. Coherence residues inoffish and shrimp.
different J. Agric.
light sources andFood Chem.
their effect2006, 54,image
on the 4517–4523.
sharpness and
2. speckle
Deng,ofY.;
holographic Sci. Rep. 2017,
displays. properties
Chu, D. Coherence 7, 1–12. [CrossRef]
of different light sources[PubMed]
and their effect on the image sharpness
and speckle of holographic displays. Sci. Rep. 2017, 7, 1–12.
Appl. Sci. 2019, 9, 5237 11 of 12

3. Chemicals in Food: Safety Controls—GOV.UK. Available online: https://www.gov.uk/guidance/chemicals-

in-food-safety-controls (accessed on 1 November 2019).
4. Laboratory Information Bulletin (LIB) 4363: Malachite Green and Leucomalachite Green in Fish
and Shrimp|FDA. Available online: https://www.fda.gov/food/laboratory-methods-food/laboratory-
information-bulletin-lib-4363-malachite-green-and-leucomalachite-green-fish-and-shrimp (accessed on 26
September 2019).
5. Akin, M.S.; Yilmaz, M.; Babur, E.; Ozdemir, B.; Erdogan, H.; Tamer, U.; Demirel, G. Large area uniform
deposition of silver nanoparticles through bio-inspired polydopamine coating on silicon nanowire arrays for
practical SERS applications. J. Mater. Chem. B 2014, 2, 4894–4900. [CrossRef]
6. Wang, Y.; Lu, N.; Wang, W.; Liu, L.; Feng, L.; Zeng, Z.; Li, H.; Xu, W.; Wu, Z.; Hu, W.; et al. Highly effective
and reproducible surface-enhanced Raman scattering substrates based on Ag pyramidal arrays. Nano Res.
2013, 6, 159–166. [CrossRef]
7. Xiu, X.; Guo, Y.; Li, C.; Li, Z.; Li, D.; Zang, C.; Jiang, S.; Liu, A.; Man, B.; Zhang, C. High-performance 3D
flexible SERS substrate based on graphene oxide/silver nanoparticles/pyramid PMMA. Opt. Mater. Express
2018, 8, 844. [CrossRef]
8. Chen, S.; Liu, B.; Zhang, X.; Mo, Y.; Chen, F.; Shi, H.; Zhang, W.; Hu, C.; Chen, J. Electrochemical fabrication
of pyramid-shape silver microstructure as effective and reusable SERS substrate. Electrochim. Acta 2018, 274,
242–249. [CrossRef]
9. Li, Z.; Xu, S.C.; Zhang, C.; Liu, X.Y.; Gao, S.S.; Hu, L.T.; Guo, J.; Ma, Y.; Jiang, S.Z.; Si, H.P. High-performance
SERS substrate based on hybrid structure of graphene oxide/AgNPs/Cu film@pyramid Si. Sci. Rep. 2016, 6,
38539. [CrossRef]
10. Xu, J.; Li, C.; Si, H.; Zhao, X.; Wang, L.; Jiang, S.; Wei, D.; Yu, J.; Xiu, X.; Zhang, C. 3D SERS substrate based on
Au-Ag bi-metal nanoparticles/MoS 2 hybrid with pyramid structure. Opt. Express 2018, 26, 21546. [CrossRef]
11. Campbell, P.; Green, M.A. Light trapping properties of pyramidally textured surfaces. J. Appl. Phys. 1987, 62,
243–249. [CrossRef]
12. Mahmoud Al, A.; Lahlouh, B. Silicon Pyramid Structure as a Reflectivity Reduction Mechanism. J. Appl. Sci.
2017, 17, 374–383. [CrossRef]
13. Zhang, C.; Chen, L.; Zhu, Y.; Guan, Z. Fabrication of 20.19% Efficient Single-Crystalline Silicon Solar Cell
with Inverted Pyramid Microstructure. Nanoscale Res. Lett. 2018, 13, 91. [CrossRef] [PubMed]
14. Wang, X.; Yang, Z.; Gao, P.; Yang, X.; Zhou, S.; Wang, D.; Liao, M.; Liu, P.; Liu, Z.; Wu, S.; et al. Improved
optical absorption in visible wavelength range for silicon solar cells via texturing with nanopyramid arrays.
Opt. Express 2017, 25, 10464. [CrossRef] [PubMed]
15. Zhong, S.; Wang, W.; Zhuang, Y.; Huang, Z.; Shen, W. All-Solution-Processed Random Si Nanopyramids for
Excellent Light Trapping in Ultrathin Solar Cells. Adv. Funct. Mater. 2016, 26, 4768–4777. [CrossRef]
16. Wain, A.J.; O’connell, M.A. Advances in surface-enhanced vibrational spectroscopy at electrochemical
interfaces. Adv. Phys. X 2017, 2, 188–209. [CrossRef]
17. Wang, W.; Huang, Y.F.; Liu, D.Y.; Wang, F.F.; Tian, Z.Q.; Zhan, D. Electrochemically roughened gold
microelectrode for surface-enhanced Raman spectroscopy. J. Electroanal. Chem. 2016, 779, 126–130. [CrossRef]
18. Arroyo-Currás, N.; Scida, K.; Ploense, K.L.; Kippin, T.E.; Plaxco, K.W. High Surface Area Electrodes Generated
via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based Biosensors. Anal.
Chem. 2017, 89, 12185–12191. [CrossRef] [PubMed]
19. Daubinger, P.; Kieninger, J.; Unmüssig, T.; Urban, G.A. Electrochemical characteristics of nanostructured
platinum electrodes-A cyclic voltammetry study. Phys. Chem. Chem. Phys. 2014, 16, 8392–8399. [CrossRef]
20. Moskovits, M. Surface-enhanced Raman spectroscopy: A brief retrospective. J. Raman Spectrosc. 2005, 36,
485–496. [CrossRef]
21. Litti, L.; Meneghetti, M. Predictions on the SERS enhancement factor of gold nanosphere aggregate samples.
Phys. Chem. Chem. Phys. 2019, 21, 15515–15522. [CrossRef]
22. He, Y.; Song, C.; Que, L. Nanoforest-based SERS sensor fabricated using a maskless process for detecting
chemical and pathogen. Microsyst. Technol. 2019, 25, 4349–4356. [CrossRef]
Appl. Sci. 2019, 9, 5237 12 of 12

23. Song, J.; Huang, Y.; Fan, Y.; Zhao, Z.; Yu, W.; Rasco, B.; Lai, K. Detection of Prohibited Fish Drugs Using
Silver Nanowires as Substrate for Surface-Enhanced Raman Scattering. Nanomaterials 2016, 6, 175. [CrossRef]
[PubMed]
24. Polavarapu, L.; Porta, A.L.; Novikov, S.M.; Coronado-Puchau, M.; Liz-Marzán, L.M. Pen-on-Paper Approach
toward the Design of Universal Surface Enhanced Raman Scattering Substrates. Small 2014, 10, 3065–3071.
[CrossRef] [PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).