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On-chip sample pretreatment using a porous polymer monolithic column forsolid-phase microextraction and chemiluminescence determination ofcatechins in green tea
Ling Lin,ab Hui Chen,b Huibin Wei,b Feng Wang*a and Jin-Ming Lin*b
Received 26th June 2011, Accepted 23rd July 2011
DOI: 10.1039/c1an15530j
A porous polymer monolithic column for solid-phase microextraction and chemiluminescence
detection was integrated into a simple microfluidic chip for the extraction and determination of
catechins in green tea. The porous polymer was prepared by poly(glycidyl methacrylate-co-ethylene
dimethacrylate) and modified with ethylenediamine. Catechins can be concentrated in the porous
polymer monolithic column and react with potassium permanganate to give chemiluminescence. The
microfluidic chip is reusable with high sensitivity and very low reagent consumption. The on-line
preconcentration and detection can be realized without an elution step. The enrichment factor was
calculated to be about 20 for catechins. The relative chemiluminescence intensity increased linearly with
concentration of catechin from 5.0 � 10�9 to 1.0 � 10�6 M and the limit of detection was 1.0 � 10�9 M.
The proposed method was applied to determine catechin in green tea. The recoveries are from 90% to
110% which benefits the actual application for green tea samples.
Introduction
Rapid development in biotechnology has led to advances in the
fields of pharmacy, materials and environmental sciences, while
current separation technology struggles to meet the demands of
a fast, efficient, high flux method of separation. In recent years,
sample pre-treatment technology was focused on solvent-free
and miniaturized methods.1–3 Solid-phase microextraction
(SPME) with its advantages of solvent-free extraction, low
sample consumption, simple and flexible operation has been
widely used.4,5 Since being introduced by Pawliszyn in the early
1990s,6 the technology and application of SPME have advanced
rapidly.7–10 Polymer monoliths as a new type of structural
material used in sample preparation have several merits of simple
preparation, high permeability, and good biocompatibility.11
Extraction materials currently include magnetic beads,12
membranes,13 monolithic columns,14 etc., of which the mono-
lithic column with its good permeability, high gathering effi-
ciency, and convenience has been widely used as a solid phase
extraction material. The glycidyl methacrylate (GMA) mono-
lithic column has epoxy groups that are easily modified to
various functional groups, and has been used in sample
concentrating.14
aState Key Laboratory of Chemical Resource Engineering, BeijingUniversity of Chemical Technology, Beijing, 100029, China. E-mail:[email protected]; Fax: +86-10-62792343; Tel: +86-10-62792343bBeijing Key Laboratory of Microanalysis and Instrumentation,Department of Chemistry, Tsinghua University, Beijing, 100084, China.E-mail: [email protected]
4260 | Analyst, 2011, 136, 4260–4267
The microfluidic system, which features analytical automa-
tion, integration and micromation, has considerable advantages
in reducing reagent consumption and shortening the analysis
time. In order to achieve an on-line sample pretreatment method
in a microfluidic chip, an integrated microfluidic system was
desired. The monolithic column method is suitable for complex
sample pretreatment in a microfluidic analysis system. In recent
years, several groups have designed many microfluidic devices
that integrated the monolithic column in chips as a sample
preparation technique.15–19
Chemiluminescence (CL) is the phenomenon of light emission
from the reaction between two or more than two compounds. An
external light source is unnecessary for CL, which eliminates
stray light and the instability caused by fluctuations from the
light source. This reduces noise and thus improves the signal to
noise ratio. Therefore, there are many reports concerning the
application of CL as a detection system in microchips, e.g., CL
resonance energy transfer-based detection for microchip elec-
trophoresis,20 microfluidic CD4+ T-cell counting,21 CL lensless
imaging for personalized diagnostics through multiplex bio-
analysis22 and CL flow-through DNA microarray analysis.23
These successful applications of CL as a highly sensitive detec-
tion method in microchips encourage us to develop a more
simple microfluidic device which can not only concentrate the
target compounds on the chip channel, but also detect at the
same channel using CL. However, in many cases, the CL
detection suffers from the effect of reagents and low selectivity. A
method which can concentrate the target compounds in a solid
phase and then react with CL reagents directly is desirable.
This journal is ª The Royal Society of Chemistry 2011
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In this work, we try to use a monolithic column for the solid-
phase microextration of our selected sample. The CL reagent
KMnO4 was used to react with the target compounds in the
monolithic column. Both solid-phase microextraction and CL
detection were integrated into a microfluidic chip. The analysis of
tea polyphenols was used as an application example in the
present work. Based on the reported KMnO4-catechins CL
reaction,24–26 their CL signals can be detected when a KMnO4
solution is pumped into the chip channel. A reusable catechins
analysis system was achieved with high sensitivity and low
reagent consumption. The on-line preconcentration and detec-
tion can be realized without an elution step.
Experimental
Chemicals and materials
Glycidyl methacrylate (GMA, 97% pure), ethylene dimethacry-
late (EDMA, 98% pure), ethylenediamine (99% pure), cyclo-
hexanol, 1-dodecanol, 3-(trimethoxysilyl) propyl methacrylate
(g-MAPS, 95% pure), and epicatechin (EC), catechin (CCN),
epicatechin-3-gallate (ECG), epigallocatechin (EGC), epi-
gallocatechin-3-gallate (EGCG) were obtained from J&K
Scientific Ltd. (Beijing, China). Standard solutions (C, EC, ECG,
EGC, EGCG, were initially prepared at 1� 10�7 M by dissolving
the appropriate amount of solid in deionized water before use
and diluting to the required concentration. All water was freshly
deionized using an ultraviolet ultrapurewater system (18.3 MU
cm, Barnstead, IO, USA). 2,20-Azobis (2-methylpropionitrile)
(AIBN) was bought from Shanghai Chemical Co. Ltd.
(Shanghai, China) and was of analytical reagent grade. Potas-
sium permanganate was from Beijing Chemical Reagent
Company (Beijing, China). The CL solution of KMnO4 was
prepared in 2.0 M sulfuric acid solution. The diluted solutions
were made just prior to use. Silicon wafers were from Xilika
Crystal polishing Material Co., Ltd. (Tianjin, China). Negative
photoresist (SU-8 2050), and developer were purchased from
Microchem Corp. (Newton, MA, USA). Poly(dimethylsiloxane)
(PDMS) and curing agent were obtained from Dow Corning
(Midland, MI, USA). Glass slides were obtained from Fisher
Scientific (Pittsburgh, PA, USA). All of the other reagents not
mentioned above were of analytical grade.
Apparatus
The batch CL experiment using a 3 mL glass cuvette and the flow
CL experiment generated on the chip were performed with an
BPCL ultraweak chemiluminescence analyzer (Insitute of
Biophysics, Chinese Academy of Science, Beijing, China).
Fourier transform infrared (FT-IR) measurements were carried
out with a Perkin Elmer 100 FT-IR spectrometer (Massachu-
setts, MA, USA). The transmission electron microscope was
operated at 100 kV, JEOL 6301F (Japan).
Fabrication of microfluidic chip
The microfluidic chip for chemiluminescence detection of tea
polyphenols was fabricated from poly (dimethylsiloxane)
(PDMS) produced by standard soft lithography techniques.17,27
As shown in Fig. 1a and 1b, the microchannel designed was
This journal is ª The Royal Society of Chemistry 2011
20 mm in length, 250 mm in width and 80 mm in depth. The radius
of the circle in the center of the channel for placing the monolithic
column was 5 mm. The layout of the chip was fabricated through
coating negative photoresists SU-8 2050 on a silicon wafer
cleaned by piranha solution. After photolithographic patterning
of a photoresist coated silicon wafer, a mold that carried a relief of
the desired microstructure was generated. A 10 : 1 premixed
PDMS prepolymer was prepared according to the manufacturer
instructions, degassed in a vacuum chamber for 1 h and then
poured on the mold and cured in a 70 �C oven for 2 h. The PDMS
was cut from the mold with a surgical scalpel and then the mold
was carefully peeled off. The channel inlet and outlet were
punched by a flat-tip syringe needle. The channels were sealed
with a glass cover slip after oxygen plasma treatment for 90 s.
Preparation of generic poly(glycidyl methacrylate-co-ethylene
dimethacrylate) monolithic column
A custom quartz glass tube (3 cm� 5 mm, i.d.) was first activated
by 1.0 M NaOH and 1.0 M HCl for 30 min, respectively. A
3-(triethoxysilyl) propyl methacrylate ethanol solution (2 : 8, v/v)
was used to fill the activated quartz glass tube. After sealing the
two ends of the quartz glass tube with silicon rubber, the reaction
was allowed to perform at 40 �C for 6 h. Then, the residual
solution was driven out and the quartz glass tube was washed
thoroughly with methanol. Nitrogen gas was driven through the
capillary to dry the inner surface before further use.
A polymerization mixture containing 24% (w/w) GMA
(functional monomer), 16% (w/w) EDMA (crosslinker), 15%
(w/w) cyclohexanol (porogens), 45% (w/w) 1-dodecanol (poro-
gens), and 1% AIBN initiator (w/w with respect to the total
monomer content) was sonicated to obtain a homogeneous
solution, and then purged with nitrogen for 15 min. The quartz
glass tube was filled with the polymerization mixture using
a syringe and sealed with rubber septa at both ends. The poly-
merization was carried out in a water bath at 60 �C for 12 h. The
column was then rinsed with acetonitrile to remove the porogens.
Amine-modified monolithic column
The monolithic capillary obtained was modified with diethyl-
amine to introduce weak amino-exchange groups at the surface
of the polymeric skeleton. The reaction scheme is presented in
Fig. 2. The column material was prepared by soaking in ethyl-
enediamine solution and placed in a 70 �C water bath for 4 h.
This completed the preparation of the diethylamine-modified
poly(GMA-co-EDMA) monolithic material used for SPME.
CL measurements
The configuration of the on-chip SPME-photomultiplier tube
(PMT) used for the study is shown in Fig. 1c and 1d. Because
potassium permanganate has a strong oxidability, in acidic
medium it is capable of redox reacting with a majority of organic
compounds, in the course of which the energy transfer is often
accompanied by a chemiluminescence reaction. Therefore, in the
experiment we used concentrated sulfuric acid to control the pH
of the potassium permanganate solution, rather than other
buffer solutions, since other components in the buffer solutions
will interfere with the chemiluminescence reaction. We placed
Analyst, 2011, 136, 4260–4267 | 4261
Fig. 1 Integration of capillary monolithic column and CL of KMnO4 system into a microfluidic chip. (a) Schematic diagram of setup, design of
microfluidic channel (20 mm long, 250 mmwide and 80 mm deep). The radius of the circle in the center of channel for putting the monolithic column was
5 mm. The blue light is detected by a silicon photodetector and is then converted to a photocurrent; (b) Polyphenols extraction onto the monolithic
column. The CL signal could be detected when a KMnO4 solution was pumped into the microfluidic channel; (c) Microfluidic chip on the photodetector;
(d) Pictures of a whole CL reaction device.
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a photomultiplier tube which connected to a BPCL ultra-weak
luminescence analyzer directly under the PDMS microfluidic
channel for CL detection. The CL profile and intensity were
displayed and integrated for a 0.1 s interval while the voltage of
PMTwas set at 1.3 kV. The sample solution was connected to the
SPME column by a black tube. A solution of KMnO4 (pH 2.0),
which was prepared fresh before each experiment, was then
flowed through the microfluidic channel after finishing the
extraction.
Pre-treatment of tea sample
Commercial green tea leaves harvested in China were obtained
from a nearby supermarket. The dried tea leaves (1.0 g) were
extracted with 100 mL of hot water at 80 �C for 10 min and the
leaves were filtered out by filter paper. The solution was diluted
to an appropriate concentration (10 times) with water before
detection and the sample pre-treatment was carried out just
before analysis.
Results and discussion
Preparation of poly(GMA-co-EDMA) monolithic column
The preparation of the poly(glycidyl methacrylate-co-ethylene
dimethacrylate) (GMA-EDMA) monolith is shown in Fig. 2.
The principle was based on the report from Svec’s group.28 It was
polymerized in situ from GMA and EDMA by heating in the
4262 | Analyst, 2011, 136, 4260–4267
presence of porogenic solvent and initiator. The epoxide groups
of the polymerized glycidyl methacrylate were allowed to react
with diethylamine according to the reaction. The setup of the
SPME in the microfluidic channel is shown in Fig. 1a and 1b.
Monoliths were prepared at the desired location in the quartz
glass tube which was integrated into the microfluidic channel. A
cross-linker provides the material with high mechanical stability
and contributes to the formation of the macroporous structure.
Special care was taken to eliminate oxygen (a typical inhibitor in
free radical polymerization) during preparation. Therefore, the
prepared prepolymer solution was degassed by sonication to
eliminate the dissolved oxygen. To remove the cross-linker,
porogens and initiator in the SPME, the prepared SPME was
rinsed thoroughly with acetonitrile and water.
Porogenic solvents are another important part of the poly-
merization mixtures. Their function is to dissolve all monomers
and initiator to form a homogeneous solution and to control the
phase separation process during polymerization in order to
achieve the desired pore structure. A porogenic mixture con-
sisting of methanol and hexane was considered to be suitable for
the preparation of the monolithic concentrators.
This porogenic solvent was developed via an extensive study of
a large number which will be published elsewhere. They are
characterized by very large pores that provide the monoliths with
a low flow resistance and allow the use of a high flow rate. Flow
rates of up to 50 mL min�1 can be achieved without causing
mechanical damage to the monoliths.
This journal is ª The Royal Society of Chemistry 2011
Fig. 2 Preparation of polymer monolithic material. Reaction of poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic material with
ethylenediamine solution with amine modification.
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Since the tea polyphenols are hydrophobic and aromatic with
a benzene ring and a phenolic hydroxyl, the poly(glycidyl
methacrylate-co-ethylene dimethacrylate) was modified on the
hydrophobic skeleton to form a strong bonding between the
amino functional group and the analyte. The role of hydrophobic
function, p–p interactions and hydrogen bonding, can increase
the adsorption of the monolithic column.
Fig. 3a is the appearance of the scanning electron micrograph
prepared from the poly(GMA-co-EDMA) monolithic column.
We can see from this figure, the overall size of the column has
a regular and uniform shape. The surface of the whole column
Fig. 3 SEM micrograph of the internal structure of the poly (GMA-co-E
magnification; (b) FT-IR spectra of monolithic column before (line 1) and af
This journal is ª The Royal Society of Chemistry 2011
shows the structure of particle clusters. Microsphere particles
interconnected into a continuous skeleton and between the skel-
eton there were interconnected macroporous structures. These
micron-scale macropores provide good permeability. It is condu-
cive to increase the extraction capacity andaccelerate the exchange
of analytes in the material. From Fig. 3a we can also see that there
is no obvious cleft on the surface of the monolithic material.
The chemical state of the modified amino monolithic column
surface was characterized by FT-IR spectroscopy. As the
comparison shows in Fig. 3b, the modification of the monolithic
column surface by the ethylenediamine solution gives a newly
DMA) monolithic columns. (a) GMA-co-EDMA monolith at 50 mm
ter amine modification (line 2).
Analyst, 2011, 136, 4260–4267 | 4263
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developing absorption peak at 3300 cm�1 and 3570 cm�1, which is
assigned to –NH2 groups and –OH groups respectively. The shift
of the peak was caused by the –NH2 group being modified onto
the monolithic column. It suggests that the –NH2 groups have
been successfully grafted onto the monolithic column after
modification. And the vibrations of C]O and C–O (1730 cm�1
and 1150 cm�1 respectively) were observed. They were covalently
bonded to the monolithic column frameworks enhancing the
stability and hydrophobicity of the monolithic column in
aqueous systems.
Fig. 4 The CL profile for the detection of 1.0 � 10�7 M catechins by on-
chip polymer monolithic column SPME (a) and by injection without
preconcentration (b).
Sample pretreatment using the prepared material for SPME
The extraction capacity of the polymer monolithic column was
tested with series of standardized catechin solutions, the
concentration range of which varied from 1.0 � 10�9 to 1.0 �10�3 M. The results indicated that the extraction capacity
increased with increasing concentration of the analyte. It
demonstrated that the polymer monolithic column exhibited
high extraction potential for the target analytes. The large
extraction capacity was due to the extraction mechanism, which
was mainly nonspecific adsorption derived from the interaction
of polar groups between the analytes and functional monomers
in the polymeric sorbent.
The monolithic column was synthesized inside a quartz glass
tube (3 cm � 4 mm i.d.). An activation step of the polymer
monolithic column involves methanol and water (1 : 1, v/v)
flowing through the extraction column with a speed of 20 ml
min�1. Then a rinse step with acetonitrile and water (1 : 1, v/v)
was added to the polymer monolithic column with a speed of
20 ml min�1 after the CL reaction step in order to wash off salts
and/or superfluous catechins remaining on the surface of the
column. These rinse solutions were subsequently analyzed for the
presence of catechins. Catechin was not detected in any of these
solutions indicating that no detectable amount of catechins
remained on the column after the CL reaction step.
To test the long-term stability and reuse of the polymer
monolithic column, a large number of the measurements
described above were carried out using a single microfluidic chip
containing the same polymer monolithic column. The monolithic
column used over a period of 2 months with 30 injections of
different concentrations of catechins showed no evident oxida-
tion due to KMnO4, indicating the reusability of the column.
The extraction efficiency of the polymer monolithic column for
catechins was studied. The extraction efficiency can be repre-
sented by the enrichment factor,29 which is the ratio of the peak
area obtained with on-chip polymer monolithic column SPME
(Fig. 4a) to that of the corresponding analyte in the extraction
solution without preconcentration, i.e. direct injection (Fig. 4b).
According to the extraction results as shown in Fig. 4, the
enrichment factor was calculated to be 20 for catechins. Appar-
ently, the enrichment ability of on-chip polymer monolithic
column SPME was sufficient to concentrate trace catechins in
a tea sample for analysis by CL.
Optimization of the CL detection conditions
Parameters were investigated to establish the optimal conditions
for the KMnO4-hydroxyl CL detection system as shown in
4264 | Analyst, 2011, 136, 4260–4267
Fig. 5. The CL intensities in the figures were generated by sub-
tracting the background from the peak value of the original
intensities.
The effect of concentration on the CL intensity was studied. In
Fig. 5a, the CL intensity increased steadily with the increasing
KMnO4 concentration from 0 to 1.0 � 10�6 M and then the CL
intensity began to decline from 1.0 � 10�6 M to 1.0 � 10�4 M in
the flow-injection system. Therefore, 1.0 � 10�6 M of KMnO4
was selected in the CL system.
As shown in Fig. 5b the CL intensity was increased with
increasing the pH value and reached a maximum at pH 2.08 in
the flow-injection system. There was a much weaker CL signal
when the pH was above 4.30. The pH of the KMnO4 solution
was demonstrated to have a significant effect on the CL
intensity, and pH 2.08 was selected for the following CL
reaction.
As shown in Fig. 5c, the flow rate of sample solution from 0 mL
min�1 to 50 mL min�1 was tested in our experiment and the peak
value of the CL intensity was the largest in the 30 mLmin�1 infuse
rate experiment. But the noise was also increased with increasing
rates. Considering analytical precision and solution consump-
tion, the optimal flow rate was 30 mL min�1.
Time control of monolithic column sample extraction is
necessary. The effects of extraction time were determined in the
range from 1 to 30 min. The results shown in Fig. 5d indicated
that 5 min was effective for our experiments.
Effects of catechin type on CL intensity
Under the optimized experimental conditions, the CL behavior
of CCN, EG, EGC, ECG and EGCG single standard solutions
at the concentration of 1 � 10�7 M with the proposed method
was studied, respectively. As shown in Fig. 6, the variability of
CL intensity from different catechins was small, indicating that
the CL doesn’t depend on the catechin type. This study
demonstrated that the total catechin amount can be measured by
this CL reaction method.
This journal is ª The Royal Society of Chemistry 2011
Fig. 5 Effects of several parameters on the CL intensity. Conditions: (a) pH 2.10, 1 � 10�7 M catechins and flow rate 30 mL min�1. (b) 1 � 10�6 M
KMnO4, 1 � 10�7 M catechins and flow rate 30 mL min�1. (c) pH 1.90, 1 � 10�6 M KMnO4 and 1 � 10�7 M catechins. (d) pH 2.05, 1 � 10�6 M KMnO4,
1 � 10�7 M catechins and flow rate 30 mL min�1.
Fig. 6 Effects of catechin type on CL intensity.
This journal is ª The Royal Society of Chemistry 2011
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Analytical performance of on-chip CL method
Under the optimum experimental conditions, the CL intensity
increased linearly in the concentration range from 5.0 � 10�9 M
to 1.0 � 10�6 M for catechin (Fig. 7). The detection limit of this
method is 1.0 � 10�9 M catechin at S/N ratio of 3. The relative
standard deviation (RSD) of ten parallel measurements (for
single column) at 1.0 � 10�8 M catechins in the CL system was
4.8%. The column-to-column RSD was found to be 5.7% (n¼ 5).
The results indicate that the proposed CL method has good
linearity and high sensitivity and precision.
Interference studies
Compounds of similar structures to catechins which could
interfere in the detection were evaluated. The interferences were
added into 1.0 � 10�8 M catechins and examined by the
proposed method. Most ions did not interfere with the deter-
mination of high concentration levels of catechins solution. The
Analyst, 2011, 136, 4260–4267 | 4265
Fig. 7 (a) Relationship between the CL intensity and the concentration of catechins. (b) CL signals for the determination of catechins. Concentration of
catechins (M) (1) 1.0 � 10�9; (2) 1.0 � 10�8; (3) 1.0 � 10�7; (4) 1.0 � 10�6; (5) tea sample 1; (6) tea sample 2.
Table 2 Result of catechins determination and recoveries in three greentea samples
Sample No.Added(�10�7 M)
Observed(�10�7 M) Recovery (%) RSD (%, n ¼ 3)
1 0 0.22 — —0.40 0.63 103 3.20.20 0.44 110 5.80.10 0.31 90 2.3
2 0 0.20 — —
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effects of some components of green tea, such as glucose and
sucrose, caffeine, albumin, glutamic acid were also investigated
and their interference could be excluded. Glutamic acid with the
concentration beyond 2.0 � 10�4 M could affect and quench the
CL. But their interferences could be neglected due to the low
concentration in samples and the appropriate dilution. The
results are summarized in Table 1, which demonstrated the high
selectivity of the present method for the determination of green
tea.
0.40 0.57 93 4.10.20 0.39 95 2.50.10 0.29 90 6.23 0 0.37 — —0.80 1.22 106 1.70.40 0.76 98 3.40.20 0.58 105 7.2
Application
The proposed method was applied to the determination of green
tea (Fig. 7). Different kinds of green tea were purchased from
a market. Samples were prepared as described above. The results
showed that the coexisting substances in the samples did not
interfere with the determination. Recovery tests were performed
to evaluate the accuracy of this method. The results for the
contents and recoveries are summarized in Table 2. The recov-
eries from 90% to 110% indicate that the method outlined in this
experiment is a direct and simple way to analyze the catechins in
green tea.
Aside from the inherent advantage of CL detection, the high
sensitivity was also due to the existence of large numbers of
Table 1 Assessment of the interference by metal ions and organiccompounds in the CL method for green tea
Species addedMole ratio(Cspecies/CAA)
Variation of the CLpeak height (%, n ¼ 5)
K+ 1000 0.12PO4
3� 1000 1.02SO4
2� 1000 0.69Ca2+ 1000 1.12Glucose 200 �1.48Sucrose 200 2.32Caffeine 200 4.12Albumin 150 2.62Glutamic acid 2 �4.61
4266 | Analyst, 2011, 136, 4260–4267
recognition sites for catechins in the column. Hence, all the
catechins adhered on the monolithic column and can react with
KMnO4 solution. In addition, this strategy can be also utilized in
the detection of other tea samples or analytes, since any aptamer-
target binding event can be translated to an adsorption modifi-
cation on the monolithic column principally. Using this method,
only 5 min was required to finish the sample extraction, while the
CL detection can also be accomplished in less than 5 min.
Consequently, due to the features of microfluidic devices, the
time and sample volume consumption were significantly
decreased compared to other methods for catechin detection,30,31
and it was exactly in conformity with the desire of sample
detection.
Conclusions
In conclusion, a simple and highly efficient microfluidic chip was
designed by the combination of solid phase micro-extraction and
chemiluminescence. As to demonstrate the applications of the
microfluidic chip, polyphenols were extracted from green tea and
detected via on-line chemiluminescence using it. A reusable
catechins analysis system was achieved with high sensitivity and
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very low reagent consumption. The online preconcentration and
detection can be realized without an elution step. Good stability
and reusability of SPME was achieved which benefits the actual
application for green tea samples. And the high sensitivity of the
CL was successfully introduced into the detection of catechins in
samples, which can be a cheap and effective method for the
quality detection of green tea.
Acknowledgements
This work was supported by National Natural Science Foun-
dation of China (Nos. 20935002, 90813015).
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