U.S. patent application number 16/620690 was filed with the patent office on 2020-12-31 for method for preparing a ratiometric fluorescent sensor for phycoerythrin based on a magnetic molecularly imprinted core-shell polymer.
This patent application is currently assigned to Qingdao University. The applicant listed for this patent is Qingdao University. Invention is credited to Xiangning BU, Yongxin FU, Rijun GUI, Hui JIN.
Application Number | 20200408689 16/620690 |
Document ID | / |
Family ID | 1000005137558 |
Filed Date | 2020-12-31 |
United States Patent
Application |
20200408689 |
Kind Code |
A1 |
JIN; Hui ; et al. |
December 31, 2020 |
METHOD FOR PREPARING A RATIOMETRIC FLUORESCENT SENSOR FOR
PHYCOERYTHRIN BASED ON A MAGNETIC MOLECULARLY IMPRINTED CORE-SHELL
POLYMER
Abstract
A method for preparing a ratiometric fluorescent sensor for
phycoerythrin based on a magnetic molecularly imprinted core-shell
polymer is provided. With Fe.sub.3O.sub.4 magnetic nanoparticles as
the core, blue fluorescence-emitting carbon quantum dots (B-CDs)
are coupled on the surfaces of Fe.sub.3O.sub.4 magnetic
nanoparticles, and SiO.sub.2 shells carrying template molecules
(phycoerythrin) are grown on the surfaces of Fe.sub.3O.sub.4/B-CDs.
Then, the molecularly imprinted polymer SiO.sub.2-MIPs are obtained
by eluting the template molecules, that is,
Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained. Fluorescence
emission spectra of the dispersion of
Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs in the presence of different
concentrations of phycoerythrin are measured. By fitting the linear
relationship between the ratios I.sub.phycoerythrin/I.sub.B-CDs of
fluorescence emission peak intensities of phycoerythrin and B-CDs
and the molar concentrations of phycoerythrin, the ratiometric
fluorescent sensor for phycoerythrin is constructed.
Inventors: |
JIN; Hui; (Qingdao, CN)
; GUI; Rijun; (Qingdao, CN) ; FU; Yongxin;
(Qingdao, CN) ; BU; Xiangning; (Qingdao,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Qingdao University |
Qingdao |
|
CN |
|
|
Assignee: |
Qingdao University
Qingdao
CN
|
Family ID: |
1000005137558 |
Appl. No.: |
16/620690 |
Filed: |
March 14, 2019 |
PCT Filed: |
March 14, 2019 |
PCT NO: |
PCT/CN2019/078077 |
371 Date: |
December 9, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09K 11/06 20130101;
C09K 2211/14 20130101; C08K 2201/01 20130101; G01N 2021/6439
20130101; G01N 2600/00 20130101; C09K 11/025 20130101; C08G 77/26
20130101; G01N 21/6428 20130101; C08K 2003/2275 20130101; C08K 3/22
20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; C09K 11/02 20060101 C09K011/02; C09K 11/06 20060101
C09K011/06; C08G 77/26 20060101 C08G077/26; C08K 3/22 20060101
C08K003/22 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 8, 2018 |
CN |
201811325300.1 |
Claims
1. A method for preparing a ratiometric fluorescent sensor for
phycoerythrin based on a magnetic molecularly imprinted core-shell
polymer, comprising the following steps: (1) preparation of
aminated B-CDs: taking 0.3 mL of 1,4-dioxane and 25 mL of catechol
solution to mix well by ultrasound to obtain a first solution,
transferring the first solution to a 50 mL high-pressure reactor
with a polytetrafluoroethylene liner, heating the first solution
for a first reaction at 180.degree. C. for 12 hours to obtain a
dark brown mixture; wherein the dark brown mixture is diluted with
20 mL of double-distilled water and centrifuged at 12,000 rpm to
remove larger particles to obtain a supernatant; the supernatant is
filtered through a 0.4 micro-filtration membrane to obtain a
filtrate, and the filtrate is dialyzed through a dialysis bag with
a molecular weight cut-off of 1000 Da to remove unreacted
experimental materials to obtain a second solution; the second
solution in the dialysis bag is poured out, subjected to a rotary
evaporation to remove 90% of the liquid water, and then dried in a
vacuum to obtain the B-CDs, and the B-CDs are stored at 4.degree.
C. away from light, or the B-CDs are dispersed in water to obtain a
B-CDs aqueous dispersion for subsequent experiments; (2)
preparation of carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles:
adding ferric chloride and ferrous chloride with a molar ratio of
2:1 into a 250 mL reaction flask to prepare a 100 mL mixed
solution, adding 10 mL of ammonia water with a mass concentration
of 25% into the 250 mL reaction flask under N.sub.2 protection to
obtain a third solution, stirring the third solution rapidly to
cause a second reaction, adjusting a pH of the third solution to
alkaline with HCl, after 10 minutes of the second reaction, adding
10 mL of trisodium citrate solution to the third solution to obtain
a fourth solution; and then placing the 250 mL reaction flask in a
water bath at 80.degree. C. continuously stirring the fourth
solution for a third reaction for 30 minutes to obtain a first
reaction production; wherein the first reaction product is
centrifuged, washed and dried to obtain the carboxylated
Fe.sub.3O.sub.4 magnetic nanoparticles; the carboxylated
Fe.sub.3O.sub.4 magnetic nanoparticles are stored at 4.degree. C.
away from light, or dispersed in water to prepare a Fe.sub.3O.sub.4
aqueous dispersion for subsequent experiments; (3) preparation of
magnetic molecularly imprinted core-shell polymers: adding 2 mL of
the B-CDs aqueous dispersion to 18 mL of aqueous dispersion
containing 0.8 mL of the Fe.sub.3O.sub.4 aqueous dispersion to
obtain a fifth solution, stirring the fifth solution for a fourth
reaction for 30 minutes, adding template molecules (the
phycoerythrin) and 20 .mu.L of 3-aminopropyltriethoxysilane to the
fifth solution to obtain a sixth solution, continuously performing
a fifth reaction on the sixth solution for 1 hour, then adding 40
.mu.L of ammonia water and 40 .mu.L of tetraethyl silicate to the
sixth solution to obtain a seventh solution, and continuously
performing a sixth reaction on the seventh solution away from light
for 12 hours to obtain a second reaction product; wherein the
second reaction product is centrifuged, and washed three times with
a solution consisting of ethanol and acetonitrile at a volume ratio
of 8:2 to remove the template molecules to obtain a product, and
then Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained from the
product by centrifugation, washing and drying; the
Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are dispersed in water to
prepare a Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs aqueous dispersion
for use; (4) at room temperature and under a magnetic stirring,
adding the phycoerythrin with a plurality of molar concentrations
to the Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs aqueous dispersion to
form a plurality of homogeneous mixtures and then incubating the
plurality of homogeneous mixtures away from light for 5 minutes;
wherein fluorescence emission spectra of the plurality of
homogeneous mixtures are measured; by fitting a linear relationship
between ratios I.sub.phycoerythrin/I.sub.B-CDs of fluorescence
emission peak intensities of the phycoerythrin and the B-CDs and
the plurality of molar concentrations of the phycoerythrin, the
ratiometric fluorescent sensor for the phycoerythrin is
constructed.
2. The method for preparing the ratiometric fluorescent sensor for
the phycoerythrin based on the magnetic molecularly imprinted
core-shell polymer according to claim 1, wherein, in step (1), a
size of each of the aminated B-CDs is 1-5 nm.
3. The method for preparing the ratiometric fluorescent sensor for
the phycoerythrin based on the magnetic molecularly imprinted
core-shell polymer according to claim 1, wherein, in step (2), a
size of each of the carboxylated Fe.sub.3O.sub.4 magnetic
nanoparticles is 10-30 nm.
4. The method for preparing the ratiometric fluorescent sensor for
the phycoerythrin based on the magnetic molecularly imprinted
core-shell polymer according to claim 1, wherein, in step (3), a
mass concentration of the B-CDs is 1-10 mg/mL, a mass concentration
of the carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles is 5-20
mg/mL, and the plurality of molar concentrations of the
phycoerythrin is 0.5-1 .mu.M.
5. The method for preparing the ratiometric fluorescent sensor for
the phycoerythrin based on the magnetic molecularly imprinted
core-shell polymer according to claim 1, wherein, in step (4), a
linear detection range of the molar concentrations of the
phycoerythrin is 1-500 nM, and a detection limit of the
phycoerythrin is 1-10 nmol/L.
Description
CROSS REFERENCE TO THE RELATED APPLICATIONS
[0001] This application is the national phase entry of
International Application No. PCT/CN2019/078077, filed on Mar. 14,
2019, which is based upon and claims priority to Chinese Patent
Application No. 201811325300.1, filed on Nov. 8, 2018, the entire
contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present disclosure belongs to the technical field of
preparation of nano composite material and molecularly imprinted
polymer, and more particularly to a method for preparing a
ratiometric fluorescent sensor for phycoerythrin based on a
magnetic molecularly imprinted core-shell polymer. The prepared
sensor can be used for detecting phycoerythrin with high
sensitivity and high selectivity.
BACKGROUND
[0003] Red tide that is caused by blooming red algae on the ocean
surface is a serious matter and causes increasing concerns for
human health and water resources. Phycoerythrin, one of the
important light-harvesting pigment-proteins in algae, together with
Phycocyanin and allophycocyanin, constitute the fluorescent family
of phycobiliprotein. These proteins are part of an efficient energy
transfer chain, which direct excitation energy of light-harvesting
complex to a chlorophyll-containing reaction center with a light
energy transmission efficiency of 95%. Phycoerythrin is mainly
obtained by separation and purification from red algae and has
strong fluorescence, good light absorption characteristics, high
quantum yield, extensive excitation and emission in the visible
light region and the like. The phycoerythrin can be widely used in
the technical fields of diagnosis and bioengineering, such as
fluorescence immunoassay, bicolor or multicolor fluorescence
analysis, surface antigen detection of cancer cell, flow cytometry,
antibody fluorescent labeling, bioimaging, food, cosmetics and the
like. The precise detection of phycoerythrin is an important
problem to be solved. It is very important to develop a simple and
efficient method to identify and detect phycoerythrin.
[0004] Molecular imprinting technology can capture specific
template molecules into polymer molecular cavities with high
sensitivity and selectivity in the presence of specific template
molecules. During the preparation process, removal of the template
molecules by elution, causes specific sites to remain in the formed
polymer network, and the shape and size of the specific sites match
the template molecules. The complementary relationship between the
specific sites of the molecularly imprinted polymer and the
template molecules enables high-selectivity and specific detection
of the template molecule. As a special type of receptor, the
molecularly imprinted polymer has the advantages of simple
preparation, low cost, good selectivity, high physical strength and
good thermal stability, which has been widely used in many
important fields such as chemical/biological sensor,
chromatographic separation, solid phase extraction and drug
release.
[0005] Munier et al. used a one-step purification from red edible
seaweed to obtain R-phycoerythrin (Mathilde Munier, Michele
Morancais, Justine Dumay, Pascal Jaouen, Joel Fleurence. One-step
purification of R-phycoerythrin from the red edible seaweed
Grateloupia Turuturu. Journal of Chromatography B, 992 (2015)
23-29), wherein the R-phycoerythrin was detected by high
performance liquid chromatography. Gameiro et al. performed
fluorescence characterization on phycoerythrin (Carla Gameiro,
Andrei B. Utkin, Paulo Cartaxana. Characterization of estuarine
intertidal macroalgae by laser-induced fluorescence. Estuarine,
Coastal and Shelf Science 167 (2015) 119-124). Jikui Wu et al.
reported a method for detecting DNA hybridization using
R-phycoerythrin fluorescence (Jikui Wu, Yunfei Lu, Ningna Ren,
Junling Zhang. Method for detecting DNA hybridization by using
surface cationized R-phycoerythrin. Chinese Invention Patent
Publication No. CN108037103A) Hualin Wang et al. prepared a
phycoerythrin-labeled polystyrene microsphere as a fluorescent
probe (Hualin Wang, Tao Zhang, Kedeng Zhang. Method for preparing a
fluorescent probe of a phycoerythrin-labeled polystyrene
microsphere. Chinese Invention Patent. Publication No.
CN108383936A).
[0006] At present, studies on Phycoerythrin focused on extraction,
isolation, purification and use as fluorescent labeling probes, and
the analysis and detection of phycoerythrin is limited to
quantitative analysis using high performance liquid chromatography
and fluorescence spectrometer. Traditional instrumental analysis
techniques generally have problems such as having cumbersome sample
pretreatment, complex operation, being time-consuming and
laboursome, having low sensitivity and poor selectivity. The
detection of phycoglobin, only relying on a single signal output,
is susceptible to factors such as background, reagents, system and
environmental conditions, resulting in fluctuations in the
measurement results. In contrast, employing the dual-signal ratio
processing to obtain the intensity ratio of the signals has a
self-calibration function, which effectively eliminates the
interference of the autologous signal and the background signal and
improves the accuracy and reliability of the detection results. In
this regard, the present disclosure reports a novel ratiometric
fluorescent sensor for phycoerythrin based on a magnetic
molecularly imprinted core-shell polymer. Using Fe.sub.3O.sub.4
magnetic nanoparticles as the cores, blue fluorescence-emitting
carbon quantum dots (B-CDs) are coupled on the surface of
Fe.sub.3O.sub.4 magnetic nanoparticles, and silica shells carrying
template molecules (phycoerythrin) are grown on the surfaces of
Fe.sub.3O.sub.4/B-CDs. Then, the molecularly imprinted polymer
SiO.sub.2-MIPs are obtained by eluting the template molecules, that
is, Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained. The magnetic
molecularly imprinted core-shell polymer can be used for
ratiometric fluorescent detection of phycoerythrin with high
sensitivity and selectivity. So far, constructing the ratiometric
fluorescent sensor based on magnetic molecularly imprinted
core-shell polymer Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs and the
ratiometric fluorescent method for detecting phycoerythrin have not
yet been reported on domestic and foreign literatures and
patents.
SUMMARY
[0007] The objective of the present disclosure is to overcome the
deficiencies of the prior art described above, and to design a
method for preparing a ratiometric fluorescent sensor for
phycoerythrin based on a magnetic molecularly imprinted core-shell
polymer, where the method is simple, has low-cost, high-sensitivity
and good-selectivity.
[0008] In order to achieve the aforementioned objective, according
to the present disclosure, a process of preparing a ratiometric
fluorescent sensor for phycoerythrin based on a magnetic
molecularly imprinted core-shell polymer includes the following
steps.
[0009] (1) Preparation of aminated B-CDs: taking 0.3 mL of
1,4-dioxane and 25 mL of catechol solution to mix well by
ultrasound, transferring to a 50 mL high-pressure reactor with a
polytetrafluoroethylene liner, heating and reacting at 180.degree.
C. for 12 hours to obtain a dark brown mixture. The prepared dark
brown mixture is diluted with 20 mL of double-distilled water and
centrifuged at 12,000 rpm to remove larger particles. The
supernatant is collected and filtered through a 0.4 .mu.m
micro-filtration membrane, and the filtrate is dialyzed through a
dialysis bag with a molecular weight cut-off of 1000 Da to remove
the unreacted experimental materials. The solution in the dialysis
bag is poured out, subjected to rotary evaporation to remove 90% of
the liquid, and then dried in a vacuum to obtain B-CDs. The B-CDs
is stored at 4.degree. C. from light or dispersed in solution to
prepare dispersing B-CDs for subsequent experiments.
[0010] (2) Preparation of carboxylated Fe.sub.3O.sub.4 magnetic
nanoparticles: adding ferric chloride and ferrous chloride with a
molar ratio of 2:1 into a 250 mL reaction flask to prepare a 100 mL
mixed solution, adding 10 mL of ammonia water with a mass
concentration of 25% into the reaction flask under N.sub.2
protection, stirring rapidly to cause a reaction, adjusting the pH
of the solution to alkaline with HCl, after 10 min of reaction,
adding 10 mL of trisodium citrate solution. Then the reaction flask
is placed in a water bath at 80.degree. C., and is continuously
stirred for reaction for 30 min. The reaction product is
centrifuged, washed and dried to obtain Fe.sub.3O.sub.4. The
Fe.sub.3O.sub.4 is stored at 4.degree. C. from light or dispersed
in solution to prepare a dispersing Fe.sub.3O.sub.4 for subsequent
experiments.
[0011] (3) Preparation of magnetic molecularly imprinted core-shell
polymers: adding 2 mL of B-CDs aqueous dispersion to 18 mL of
aqueous dispersion containing 0.8 mL of Fe.sub.3O.sub.4, stirring
and reacting for 30 min, adding template molecules (phycoerythrin)
and 20 .mu.L of 3-aminopropyltriethoxysilane, reacting continuously
for 1 hour, then adding 40 .mu.L of ammonia water and 40 .mu.L of
tetraethyl silicate, and reacting continuously away from light for
12 hours. The reaction product is centrifuged and washed three
times with a solution consisting of ethanol and acetonitrile at a
volume ratio of 8:2 to remove the template molecules, and then
Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained by
centrifugation, washing and drying. The magnetic molecularly
imprinted core-shell polymers are dispersed in solution to prepare
a dispersion for use.
[0012] (4) At room temperature and stirring magnetically, a certain
dosage of phycoerythrin is added to the polymer dispersion to form
a homogeneous mixture and then incubated away from light for 5
minutes. Fluorescence emission spectra of the homogeneous mixture
in the presence of different concentrations of phycoerythrin are
measured. By fitting a linear relationship between the ratios
(I.sub.phycoerythrin/I.sub.B-CDs) of fluorescence emission peak
intensities of phycoerythrin and B-CDs and the molar concentrations
of phycoerythrin, the ratiometric fluorescent sensor for
phycoerythrin is constructed.
[0013] In step (1), the size of the aminated B-CDs is 1-5 nm.
[0014] In step (2), the size of the carboxylated Fe.sub.3O.sub.4
magnetic nanoparticles is 10-30 nm.
[0015] In step (3), the mass concentration of the B-CDs is 1-10
mg/mL, the mass concentration of the Fe.sub.3O.sub.4 magnetic
nanoparticles is 5-20 mg/mL, and the dosage of phycoerythrin is
0.5-1 .mu.M.
[0016] In step (4), the linear detection range of the molar
concentration of phycoerythrin is 1-500 nM, and the detection limit
is 1-10 nmol/L.
[0017] The advantages of the present disclosure are as follows. The
present application discloses a novel ratiometric fluorescent
sensor for phycoerythrin based on a magnetic molecularly imprinted
core-shell polymer. With Fe.sub.3O.sub.4 magnetic nanoparticles as
the core, blue fluorescence-emitting carbon quantum dots (B-CDs)
are coupled on the surfaces of Fe.sub.3O.sub.4 magnetic
nanoparticles, and silica shells carrying template molecules
(phycoerythrin) are grown on the surfaces of Fe.sub.3O.sub.4/B-CDs.
Then, the molecularly imprinted polymer SiO.sub.2-MIPs are obtained
by eluting the template molecules, that is,
Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained. Fluorescence
emission spectra of the homogeneous mixture of
Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs in the presence of different
concentrations of phycoerythrin are measured. By fitting the linear
relationship between the ratios (I.sub.phycoerythrin/I.sub.B-CDs)
of fluorescence emission peak intensities of phycoerythrin and
B-CDs and the molar concentrations of phycoerythrin, the
ratiometric fluorescent sensor for phycoerythrin is constructed,
which can be used for ratiometric fluorescent detection of
phycoerythrin with high sensitivity and selectivity. Compared with
the prior art, according to the present disclosure, the method has
the advantages of having simple operation, low cost, extensive
resources of raw materials, strong anti-interference ability of the
ratiometric signal, good accuracy, high sensitivity and high
selectivity, which can be developed into a novel ratiometric
fluorescent sensor for the efficient detection of
phycoerythrin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic diagram showing the preparation for
the ratiometric fluorescent sensor for phycoerythrin based on a
magnetic molecularly imprinted core-shell polymer and the detection
for phycoerythrin;
[0019] FIG. 2 shows the fluorescence emission spectra of sensor
system measured by the ratiometric fluorescent sensor of the
present disclosure, corresponding to different molar concentrations
of phycoerythrin;
[0020] FIG. 3 shows the linear relationship between the ratios
(I.sub.phycoerythrin/I.sub.B-CDs) of fluorescence emission peak
intensities of phycoerythrin and B-CDs and the different molar
concentrations of phycoerythrin.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0021] The present disclosure will be further described below in
conjunction with the drawings and specific embodiments.
Embodiment 1
[0022] This embodiment relates to a method for preparing a
ratiometric fluorescent sensor for phycoerythrin based on a
magnetic molecularly imprinted core-shell polymer. A process of
preparing the ratiometric fluorescent sensor and the principle of
the ratiometric fluorescent detection of phycoerythrin are shown in
FIG. 1, and the specific process steps are as follows.
[0023] Preparation of aminated B-CDs: 0.3 mL of 1,4-dioxane and 25
mL of catechol solution are chosen to be mixed well by ultrasound,
and transferred to a 50 mL high-pressure reactor with a
polytetrafluoroethylene liner. Heating and reacting is performed at
180.degree. C. for 12 hours to obtain a dark brown mixture. The
prepared dark brown mixture is diluted with 20 mL of
double-distilled water and centrifuged at 12,000 rpm to remove
larger particles. The supernatant is collected and filtered through
a 0.4 .mu.m micro-filtration membrane, and the filtrate is dialyzed
through a dialysis bag with a molecular weight cut-off of 1000 Da
to remove the unreacted experimental materials. The solution in the
dialysis bag is poured out, subjected to rotary evaporation to
remove 90% of the liquid, and then dried in a vacuum to obtain
B-CDs. The B-CDs is stored at 4.degree. C. from light or dispersed
in solution to prepare dispersing B-CDs for subsequent experiments,
wherein the average size of the B-CDs is 2 nm.
[0024] Preparation of carboxylated Fe.sub.3O.sub.4 magnetic
nanoparticles: ferric chloride and ferrous chloride with a molar
ratio of 2:1 are added into a 250 mL reaction flask to prepare a
100 mL mixed solution, 10 mL of ammonia water with a mass
concentration of 25% is added into the reaction flask under N.sub.2
protection, stirring rapidly for reaction, the pH of the solution
is adjusted to alkaline with HCl, after 10 min of reaction, 10 mL
of trisodium citrate solution is added. The reaction flask is
placed in a water bath at 80.degree. C., and is continuously
stirred for reaction for 30 min. The reaction product is
centrifuged, washed and dried to obtain Fe.sub.3O.sub.4. The
Fe.sub.3O.sub.4 is stored at 4.degree. C. from light or dispersed
in solution to prepare a dispersing Fe.sub.3O.sub.4 for subsequent
experiments, wherein the average size of Fe.sub.3O.sub.4 is 15
nm.
[0025] Preparation of magnetic molecularly imprinted core-shell
polymers: 2 mL of B-CDs aqueous dispersion is added to 18 mL of
aqueous dispersion containing 0.8 mL of Fe.sub.3O.sub.4, wherein
the mass concentration of B-CDs is 2 mg/mL and the mass
concentration of Fe.sub.3O.sub.4 magnetic nanoparticles is 10
mg/mL, after stirring and reacting for 30 min, template molecules
(phycoerythrin) and 20 .mu.L of 3-aminopropyltriethoxysilane are
added, wherein the dosage of phycoerythrin is 0.5 .mu.M, the
reaction is continued for 1 hour, then 40 .mu.L of ammonia water
and 40 .mu.L of tetraethyl silicate are added, and the reaction is
carried out away from light for 12 hours. The reaction product is
centrifuged and washed three times with a solution consisting of
ethanol and acetonitrile at a volume ratio of 8:2 to remove the
template molecules, and then Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs
are obtained by centrifugation, washing and drying. The magnetic
molecularly imprinted core-shell polymers are dispersed in solution
to prepare a dispersion for use.
[0026] At room temperature and stirring magnetically, a certain
dosage of phycoerythrin is added to the polymer dispersion to form
a homogeneous mixture and then incubated away from light for 5
minutes. Fluorescence emission spectra of the homogeneous mixture
in the presence of different concentrations of phycoerythrin are
measured. By fitting a linear relationship between the ratios
(I.sub.phycoerythrin/I.sub.B-CDs) of fluorescence emission peak
intensities of phycoerythrin and B-CDs and the molar concentrations
of phycoerythrin, the ratiometric fluorescent sensor for
phycoerythrin is constructed. FIG. 2 shows the fluorescence
emission spectra of sensor system measured by the ratiometric
fluorescent sensor of the present disclosure, corresponding to
different molar concentrations of phycoerythrin. As shown in FIG.
3, the linear detection range of molar concentration of
phycoerythrin obtained from the ratiometric fluorescent sensor of
the present disclosure, is 5-250 nM, and the detection limit is 2
nM.
Embodiment 2
[0027] in this embodiment, the schematic diagram of the preparation
process of the ratiometric fluorescent sensor and the principle of
the ratiometric fluorescent detection of phycoerythrin are the same
as embodiment 1, and the process steps for preparing aminated B-CDs
and carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles are also
the same as embodiment 1, wherein the average size of B-CDs is 3 nm
and the average size of Fe.sub.3O.sub.4 is 20 nm. Other specific
process steps are as follows.
[0028] Preparation of magnetic molecularly imprinted core-shell
polymers: 2 mL of B-CDs aqueous dispersion is added to 18 mL of
aqueous dispersion containing 0.8 mL of Fe.sub.3O.sub.4, wherein
the mass concentration of B-CDs is 5 mg/mL, and the mass
concentration of Fe.sub.3O.sub.4 magnetic nanoparticles is 15
mg/mL, after stirring and reacting for 30 minutes, template
molecules (phycoerythrin) and 20 .mu.L of
3-aminopropyltriethoxysilane are added, wherein the dosage of
phycoerythrin is 0.8 .mu.M, the reaction is continued for 1 hour,
then 40 .mu.L of ammonia water and 40 .mu.L of tetraethyl silicate
are added, and the reaction is carried out away from light for 12
hours. The reaction product is centrifuged and washed three times
with a solution consisting of ethanol and acetonitrile at a volume
ratio of 8:2 to remove the template molecules, and then
Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained by
centrifugation, washing and drying. The magnetic molecularly
imprinted core-shell polymers are dispersed in solution to prepare
a dispersion for use.
[0029] At room temperature and stirring magnetically, a certain
dosage of phycoerythrin is added to the polymer dispersion to form
a homogeneous mixture and then incubated away from light for 5
minutes. Fluorescence emission spectra of the homogeneous mixture
in the presence of different concentrations of phycoerythrin are
measured. By fitting a linear relationship between the ratios
(I.sub.phycoerythrin/I.sub.B-CDs) of fluorescence emission peak
intensities of phycoerythrin and B-CDs and the molar concentrations
of phycoerythrin, the ratiometric fluorescent sensor for
phycoerythrin is constructed. The linear detection range of molar
concentration of phycoerythrin is 5-500 nM, and the detection limit
is 5 nM.
Embodiment 3
[0030] in this embodiment, the schematic diagram of the preparation
process of the ratiometric fluorescent sensor and the principle of
the ratiometric fluorescent detection of phycoerythrin, and the
process steps for preparing aminated B-CDs and carboxylated
Fe.sub.3O.sub.4 magnetic nanoparticles are all the same as
embodiment 1, wherein the average size of B-CDs is 5 nm and the
average size of Fe.sub.3O.sub.4 is 25 nm. Other specific process
steps are as follows.
[0031] Preparation of magnetic molecularly imprinted core-shell
polymers: 2 mL of B-CDs aqueous dispersion is added to 18 mL of
aqueous dispersion containing 0.8 mL of Fe.sub.3O.sub.4, wherein
the mass concentration of B-CDs is 8 mg/mL, and the mass
concentration of Fe.sub.3O.sub.4 magnetic nanoparticles is 20
mg/mL, after stirring and reacting for 30 min, template molecules
(phycoerythrin) and 20 .mu.L of 3-aminopropyltriethoxysilane are
added, wherein the dosage of phycoerythrin is 1 .mu.M, the reaction
is continued for 1 hour, then 40 .mu.L of ammonia water and 40
.mu.L of tetraethyl silicate are added, and the reaction is carried
out away from light for 12 hours. The reaction product is
centrifuged and washed three times with a solution consisting of
ethanol and acetonitrile at a volume ratio of 8:2 to remove the
template molecules, and then Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs
are obtained by centrifugation, washing and drying. The magnetic
molecularly imprinted core-shell polymers are dispersed in solution
to prepare a dispersion for use.
[0032] At room temperature and stirring magnetically, a certain
dosage of phycoerythrin is added to the polymer dispersion to form
a homogeneous mixture and then incubated away from light for 5
minutes. Fluorescence emission spectra of the homogeneous mixture
in the presence of different concentrations of phycoerythrin are
measured. By fitting a linear relationship between the ratios
(I.sub.phycoerythrin/I.sub.B-CDs) of fluorescence emission peak
intensities of phycoerythrin and B-CDs and the molar concentrations
of phycoerythrin, the ratiometric fluorescent sensor for
phycoerythrin is constructed. The linear detection range of molar
concentration of phycoerythrin is 10-500 nM, and the detection
limit is 8 nM.
[0033] The above disclosures are only described as some preferred
embodiments of the present invention. It should be noted that those
skilled in the art can also make several improvements and
modifications without departing from the principles of the present
invention, and these improvements and modifications shall still
fall within the protection scope of the present invention.
* * * * *