Thermosensitive Metal Chelation Dual-Template Epitope Imprinting

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Thermo-sensitive Metal Chelation Dual-template Epitope Imprinting Polymer using Distillation-precipitation Polymerization for Simultaneous Recognition of Human Serum Albumin and Transferrin Ya-Ping Qin, Chao Jia, Xi-Wen He, Wen-You Li, and YuKui Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00327 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 20, 2018

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ACS Applied Materials & Interfaces

Thermo-sensitive Metal Chelation Dual-template Epitope Imprinting Polymer using Distillation-precipitation Polymerization for Simultaneous Recognition of Human Serum Albumin and Transferrin

Ya-Ping Qina, Chao Jiaa, Xi-Wen Hea, Wen-You Li*,a,b, Yu-Kui Zhanga,c

a

College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory

of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, China b

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),

Tianjin 300071, China c

National Chromatographic Research and Analysis Center, Dalian Institute of

Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

ABSTRACT A new type of thermo-sensitive dual-template epitope molecular imprinting polymer was prepared coated on magnetic carbon nanotubes (MCNTs@D-EMIP) for simultaneous recognition of human serum albumin (HSA) and transferrin (Trf) via the strategies of dual-template epitope imprinting, metal chelation imprinting and distillation-precipitation polymerization (DPP). C-terminal peptides of HSA and C-terminal peptides of Trf were selected as templates, zinc acrylate and N-isopropylacrylamide (NIPAM) were

used

as functional monomers,

and

MCNTs@D-EMIP was prepared by the method of DPP. The two types of template epitopes were immobilized by metal chelation and six-membered ring formed with zinc acylate. MCNTs@D-EMIP was prepared only in 30 min which was much shorter than other polymerization methods. The resultant MCNTs@D-EMIP showed excellent specific recognition ability towards HSA and Trf. The adsorption amounts of MCNTs@D-EMIP for HSA and Trf were 103.67 mg g-1 and 68.48 mg g-1 and imprinting factors were 2.57 and 2.17, respectively. In addition, MCNTs@D-EMIP displayed thermo-sensitive property to realize temperature controlled recognition and 1

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release of target proteins. Furthermore, the results of HPLC analysis proved that MCNTs@D-EMIP could be applied to specifically recognize two types of targets simultaneously in bio-sample. The proposed strategy provided a preparation method for thermo-sensitive dual-template epitope imprinting polymer via dual-template imprinting, metal chelation imprinting and DPP.

KEYWORDS: dual-template epitope imprinting, molecular imprinting polymer, thermo-sensitive recognition, metal chelation imprinting; distillation-precipitation polymerization.

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INTRUDUCTION Molecular imprinting technology (MIT)1,2 has experienced the development stages of bulk imprinting3,4, surface imprinting5-7 and epitope imprinting8-10 and it has been applied in various areas, such as bio-separation11, drug delivery12 and bio-imaging13,14. Epitope molecular imprinting technology15,16 (EMIT) is a new molecular imprinting strategy for recognition towards bio-macromolecule, which was proposed by Rachkov and Minoura17,18 and has caused wide-spreading attention. In the process of epitope imprinting, an exposed domain (epitope) of target protein is used as template to imprint the recognition site and the target protein is recognized by capturing the epitope of the protein. Since EMIT has been put forward, a great diversity of epitope imprinting polymers were generated with various functions, such as fluorescent epitope imprinting polymer19, magnetic epitope imprinting polymer20,21 and quartz crystal microbalance imprinting sensor 22. Capturing and recognizing various proteins simultaneously plays an important role in precise diagnose and high-through analysis23,24. As an artificial antibody, molecular imprinting polymer has great potential to meet this demand because of its high selectivity and plasticity. Recently, dual-template molecular imprinting technology (DMIT) has been proposed and applied for specific recognition towards two types of targets simultaneously25-27. During the process of preparing dual-template molecular imprinting polymer (DMIP), two types of molecules were used as templates, so that corresponding imprinted sites were produced for recognition towards two types of targets. According to reports, DMIT has been used to prepare DMIP for simultaneous recognition for two types of small molecules28-30 and proteins31. However, imprinting polymerization methods were limited for dual-template protein imprinting because proteins were sensitive to harsh experimental conditions, such as high temperature, organic solvent and high/low pH. Dual-template epitope imprinting could solve this problem well because epitope was not as sensitive as protein so that the polymerization method was not limited. In dual-template epitope imprinting, the terminal peptides of the two target proteins were selected as templates to prepare imprinting polymer and two types of corresponding imprinted sites would be 3



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generated to capture corresponding target proteins simultaneously. Therefore, dual-template epitope imprinting polymer could be applied to recognize two types of proteins in bio-samples. However, dual-template epitope imprinting is still in its infancy. N-isopropylacrylamide (NIPAM) is a type of thermo-sensitive material, and the poly N-isopropylacrylamide (pNIPAM) hydrogels has temperature controlled property. pNIPAM has a low critical solution temperature (LCST). When the temperature is lower than LCST, the pNIPAM hydrogel is highly swollen, and when the temperature is above LCST, the hydrogel is at a state of shrinking32,33. NIPAM has been widely used in molecular imprinting to realize temperature-controlled recognition and release for target molecule such as organic small molecules34 and proteins35,36. While NIPAM has been rarely used in dual-template epitope imprinting. Distillation-precipitation

polymerization

(DPP)37

is

a

simple

and

rapid

polymerization approach for preparing monodisperse nano/microparticles. In the process of DPP, monomer, cross-linker and initiator are added into the organic solvent with low boiling point to form a mixture. The solvent will be evaporated out with the temperature increases and the concentration of the reactants gradually increases, which would result in that the reaction rate becomes faster and faster. Therefore, the polymerization could be completed in a very short time. Based on the merit of fast reaction rate, DPP has been used in molecular imprinting, especially for organic small molecules38,39. DPP could not be used in protein imprinting due to the sensitivity of proteins to the condition of DPP including organic solvent and high temperature. Interestingly, DPP has the potential to be applied in epitope imprinting for final recognition of target protein because epitope is not as sensitive as protein to the harsh conditions. However, the application of DPP in epitope imprinting for specific recognition of proteins has rarely been reported. Magnetic carbon nanotubes (MCNTs) are a new type of nanomaterial, which have been widely researched in material science40. Compared with traditional magnetic material and carbon nanotubes, MCNTs contain the properties of magnetism and high surface area. Because of their merits, MCNTs have been applied in so many areas41, 4


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such as treatment of environment, separation of biomolecules and drug delivery. In recent years, MCNTs have been used as supporting carrier in molecular imprinting polymer42 to increase adsorption capacity and make the treatment of sample brief. HSA and Trf existed in human serum albumin sample, which have a close relationship to clinical diseases. HSA is the most abundant protein in human serum albumin real sample and it is a carrier of fatty acid and drug, which could reflect the physical state. Compared with HSA, the content of Trf is much much lower in human serum albumin sample. The unnormal content of Trf is associated with these diseases, such as ischemic anemia, oxyhepatitis and malignant tumor. Therefore, it is necessary and important to detect the contents of HSA and Trf simultaneously from human serum albumin sample to diagnose some diseases. In this work, the thermo-sensitive dual-template epitope imprinting polymer coated on MCNTs (MCNTs@D-EMIP) was synthesized for simultaneous recognition of HSA and Trf via the methods of dual-template epitope imprinting, metal chelation imprinting and DPP. C-terminal peptides of HSA and C-terminal peptides of Trf were used as templates, zinc acrylate and NIPAM were selected as functional monomers, ethylene glycol dimethacrylate (EGDMA), azobisisobutyronitrile (AIBN) and acetonitrile (ACN) played the roles of cross-linker, initiator and solvent, respectively. The two types of template epitopes were immobilized by metal chelation and six-membered ring formed with zinc acylate. Because of the application of DPP, MCNTs@D-EMIP could be prepared only in 30 min which was much shorter than other polymerization methods19-21,28,43,44. The prepared MCNTs@D-EMIP displayed specific recognition ability towards HSA and Trf. The adsorption amounts of MCNTs@D-EMIP for HSA and Trf were 103.67 mg g-1 and 68.48 mg g-1 and their imprinting factors were 2.57 and 2.17, respectively. Besides, MCNTs@D-EMIP showed excellent theromo-sensitive property towards two types of target proteins. The proposed strategy revealed the feasibility of fabrication of a thermo-sensitive dual-template epitope imprinting polymer coated on MCNTs via dual-template imprinting, metal chelation imprinting and DPP.

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EXPERIMENTAL SECTION Reagents and Instruments. The reagents and instruments used in this work are shown in Supporting Information. Synthesis of Vinyl-modified MCNTs (MCNTS@MPS). The MCNTs@MPS was fabricated via three steps which have been shown in our previous works45. MCNTs were prepared via hydrothermal method and then silica layer coated MCNTs (MCNTs@SiO2) was synthesized via the hydrolysis of tetraethoxysilicane

(TEOS).

Finally,

MCNTs@MPS was

prepared

via

the

modification of 3-methacryloyloxypropyltrimethoxysilane (MPS) on the surface of MCNTs@SiO2. Synthesis of MCNTs@D-EMIP. The MCNTs@D-EMIP was fabricated by the method of DPP as follows: 200 mg MCNTs@MPS, 36 mg C-terminal peptides of HSA, 4 mg C-terminal peptides of Trf, 100 mg zinc acrylate, 50 mg NIPAM and 40 mL ACN were dispersed to a 100 mL one-necked bottle by ultrasound for 30 min. Then 550 µL EGDMA and 60 mg AIBN were dispersed to the solution by ultrasound. Finally, the bottle was installed into distillation-precipitation polymerization device and the device was preheated to 80℃ and the target temperature was set at 95℃. As the temperature increased, the solvent evaporated gradually. The reaction was stopped until approximately 25 mL ACN was evaporated and collected, which only needed 30 min. The resultant imprinting polymers were collected by a magnet and washed with a mixed solution of methanol and acetic acid (9 : 1, v/v) to remove templates until no templates were detected by HPLC. Finally, the MCNTs@D-EMIP was washed with ethanol and distilled water three times and dried at 50℃. Non-imprinting polymer (MCNTs@D-ENIP) was synthesized using the same process but without the addition of templates. Synthesis of Dual-template Epitope Molecular Imprinting Polymer on MCNTs with acrylic acid

(AA)

as Functional Monomer (MCNTs@D-EMIP-AA).

In order to investigate the effect of metal chelation and six-membered ring, MCNTs@D-EMIP-AA was prepared by the same method as MCNTs@D-EMIP. The only difference was that AA and NIPAM were used as functional monomers which 6


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had only hydrogen bond interaction with the two types of template epitopes instead of metal chelation and six-membered ring. MCNTs@D-ENIP-AA was fabricated using the same procedure but without the addition of templates. Rebinding Experiment. The imprinting polymers were used to adsorb HSA and Trf respectively to investigate the specific recognition ability for the two target proteins. 4.0 mg MCNTs@D-EMIP or MCNTs@D-ENIP was dispersed into 4 mL protein solution at the concentration of 0.5 mg mL-1 for adsorption on a rotator. After saturated, the concentrations of HSA and Trf in supernatant were measured by UV-vis at the wavelength of 278 nm to evaluate their adsorption amounts. Thermo-sensitive Adsorption Experiment. 4.0 mg MCNTs@D-EMIP or MCNTs@D-ENIP was dispersed in 4 mL target proteins solution at the concentration of 0.5 mg mL-1 and incubated on a rotator over the temperature change between 20 ℃ and 45 ℃ . The adsorption amounts of imprinting polymer for HSA and Trf at different temperature were evaluated to estimate its thermo-sensitive property. Selective and Competitive Experiment. In order to investigate the selectivity of MCNTs@D-EMIP, selective experiment and competitive experiment were performed. In selectivity experiment, 4.0 mg MCNTs@D-EMIP or MCNTs@D-ENIP was dispersed into 4 mL competitive protein solution (BSA , Mb or Cyt C) with a concentration of 0.5 mg mL-1 for adsorption on a rotator. After saturation, the concentrations of supernatants were measured by UV-vis spectrometry at the wavelength of 278 nm, 410 nm and 410 nm for BSA, Mb and Cyt C respectively to evaluate their adsorption amounts. In competitive experiment, 4.0 mg MCNTs@D-EMIP or MCNTs@D-ENIP was dispersed into a protein mixture solution of HSA, Trf, Mb and Cyt C at the concentration of 0.5 mg mL-1 for each protein and incubated on a rotator. After saturation, the concentrations of the four proteins in the supernatant were detected by HPLC at the wavelength of 214 nm to evaluate the adsorption amounts of proteins. Real Sample Analysis. 7



In order to investigate the potential application of MCNTs@D-EMIP in real bio-sample, MCNTs@D-EMIP was used to recognize target proteins in human serum albumin real sample. Compared with HSA, the content of Trf in human serum albumin real sample is very low. In order to get better adsorption efficiency, we added Trf into commercial human serum albumin real sample to adjust the contents of HSA and Trf. The commercial human serum albumin real sample was diluted 200-fold with PBS buffer solution (20 mM, pH 7.0) and 1.0 mg Trf was added into 50 mL diluted real sample. The above mixture real sample containing HSA and Trf was used for adsorption. MCNTs@D-EMIP or MCNTs@D-ENIP was dispersed into above mentioned sample and incubated to saturation. After saturation, the imprinting polymers were collected by a magnet and the collected polymers was eluted with a solution of SDS (5%, m/v) and acetic acid (5%, v/v) to remove the specifically adsorbed proteins. MCNTs@D-ENIP needed to pass through the same operation. Finally, the original real sample, the supernatant after adsorption and the eluent were detected by HPLC at the wavelength of 214 nm to analyze the specific recognition ability in real sample towards two types of target proteins.

RESULTS AND DISCISSION Preparation and Characterization of MCNTs@D-EMIP. The general procedure is shown in Scheme 1A from which we could find out that the MCNTs@D-EMIP was prepared via three steps including the preparation of MCNTs by hydrothermal method, modification of MCNTs by silylation modification and preparation of MCNTs@D-EMIP by DPP. In this study, C-terminal peptides of HSA and C-terminal peptides of Trf were used as templates simultaneously, zinc acrylate and NIPAM were used as functional monomers to prepare thermo-sensitive MCNTs@D-EMIP via DPP. As main functional monomer, zinc acrylate could form metal chelation and six-membered ring to immobilize more templates to generate more imprinted sites, which is shown in Scheme 1B. The prepared MCNTs@D-EMIP containing two types of corresponding imprinted sites could recognize HSA and Trf simultaneously. In addition, the application of DPP allowed the preparation process to 8


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be completed in a very short time. The addition of NIPAM assisted the MCNTs@D-EMIP to realize temperature controlled adsorption and release of target proteins. In a word, this method provides a new thought to prepare imprinting polymer for recognition of two types of targets in complex real samples.

Scheme 1. (A) Schematic representation of the synthesis of the MCNTs@D-EMIP. (B) Metal-chelation and six-membered ring formed between two types of template epitopes and zinc acrylate.

The size and shape of the samples were examined by TEM images (Figure 1) and the size distributions of samples were shown in Figure S1. The diameters of MCNTs,

9



MCNTs@SiO2, MCNTs@MPS and MCNTs@D-EMIP samples were 148±24 nm, 206±21 nm, 227±22 nm and 339±24 nm, respectively, which indicated that the synthetic process was performed successfully. Besides, the peaks at 1620 and 3420 cm−1 were ascribed to CNTs and the peak at 577 cm−1 to Fe−O, which illustrated that the MCNTs were comprised of CNTs and Fe3O4 (Figure S2A). The peaks at 1090 cm−1 and 1736 cm−1 were ascribed to the asymmetric stretching peak of Si−O−Si (Figure S2B-b) and the stretching vibration of C=O in the functional monomer respectively (Figure S2B-d). In addition, the magnetic property of samples were characterized and the results were shown in Figure S3 which proved that the samples had enough magnetic property to be separated by a magnet quickly and the modification and imprinting processes were performed successfully.

Figure 1. TEM images of MCNTs (A), MCNTs@SiO2 (B), MCNTs@MPS (C) and MCNTs@D-EMIP (D).

Detection of Template Peptides to Track the Process of Imprinting. MCNTs@D-EMIP could recognize the two types of target proteins by the corresponding imprinted sites, which means that more imprinted sites could get better recognition ability. In the process of imprinting, the two types of template epitopes were immobilized by the force formed with functional monomer and then removed by eluent and corresponding imprinted sites were generated. In order to evaluate the imprinting process and investigate the mass and ratio of template peptides immobilized, the eluent was detected by HPLC and the utilization of epitope (UE,

10


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Equation S1) was calculated. As shown in Figure S4, the two types of template epitope were removed completely after washed by the eluent for eight times and UE of them were 64.46 % and 30.01%, respectively. Optimization of Mass of Functional Monomers and the Volume of Cross-linker. In the process of imprinting, zinc acrylate and NIPAM were selected as functional monomers and the mass of them affected the final recognition efficiency including adsorption amounts and imprinting factors for two types of target proteins. In order to increase the specific recognition ability and insure the thermo-sensitivity of MCNTs@D-EMIP, the total mass of functional monomers was optimized and mass ratio of zinc acrylate and NIPAM was kept at 2:1 (Figure 2A). Zinc acrylate played the role of immobilizing peptides of HSA and Trf via metal chelation and six-memebered ring to generate imprinted sites, and NIPAM was responsible for endowing thermo-sensitive property for MCNTs@D-EMIP. Under the condition of less mass, zinc acrylate could not immobilize enough templates to generate enough imprinted sites to capture target proteins. Less NIPAM would lead to the absence of thermo-sensitive property of MCNTs@D-EMIP. While in the case of much more functional monomers, the net structure of imprinting polymer would be loose due to a low cross-link degree (CLD, Equation S2) so that the recognition ability and selectivity would decrease. As shown in Figure 2A, when the total mass of functional monomer was 150 mg (the mass of zinc acrylate and NIPAM were 100 mg and 50 mg, respectively), the final imprinting efficiency got to be the best. Besides, cross-link degree also influenced the final imprinting results (Figure 2B), which could be calculated based on Equation S2. The influence could be described as follows: When the cross-link degree is lower, the imprinting polymer matrix would be too loose to have precise recognition sites and non-specific adsorption would increase in some degree; On the other hand, when the cross-link degree is much more, it is more difficult for target protein to transfer into the polymer matrix. The results shown in Figure 2B indicated that MCNTs@D-EMIP got the best recognition ability for two types of target proteins when the volume was 550 µL and the cross-link degree was calculated to be 75.94%. 11



Figure 2. (A) Influence of the total mass of the monomers on adsorption capacity and imprinting factor. (B) Influence of the volume of cross-linker on adsorption capacity and imprinting factor. The error bars represent the standard deviation of three parallel experiments.

Binding Properties of MCNTs@D-EMIP and MCNTs@D-ENIP. The binding isotherms of HSA and Trf onto the MCNTs@D-EMIP and MCNTs@D-ENIP were investigated in the concentration range from 0.1 to 0.5 mg mL-1. The results were shown in Figure 3A which indicated that the adsorption amounts of HSA and Trf increased with the increase of protein concentration and reached to saturation at 0.5 mg mL-1 and the maximum adsorption amounts (Q, Equation S3) for HSA and Trf were 103.67 and 68.48 mg g-1 and their corresponding imprinting factors (IF, Equation S4) were 2.57 and 2.17, respectively. Figure 3B showed the kinetics adsorption process of MCNTs@D-EMIP and MCNTs@D-ENIP towards two types of target proteins. From the results we could find out that MCNTs@D-EMIP exhibited specific recognition ability for HSA and Trf while MCNTs@D-ENIP didn’t show specific selective ability. The adsorption curves indicated that the MCNTs@D-EMIP reached to equilibrium at 2 h and 1 h for HSA and Trf, respectively, while MCNTs@D-ENIP costed less time due to the absence of imprinted sites. Finally, the imprinting factors got up to 2.57 and 2.17 for HSA and Trf, respectively.

12


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Figure 3. (A) Binding isotherms of HSA and Trf on MCNTs@D-EMIP and MCNTs@D-ENIP. (B) Adsorption kinetics of HSA and Trf on MCNTs@D-EMIP and MCNTs@D-ENIP. The error bars represent the standard deviation of three parallel experiments.

Thermo-sensitive Property. The thermo-sensitive polymer can undergo a reversible volume transition between the swollen and the shrinkable phases. To investigate the thermo-sensitive property of MCNTs@D-EMIP, we incubated it with HSA and Trf to examine its adsorption amount over the temperature change between 20℃ and 45℃. The results were shown in Figure 4A which displayed that MCNTs@D-EMIP had higher adsorption amount at low temperature than that of high temperature35,36. As for pNIPAM, when the temperature is lower than LCST of pNIPAM, the pNIPAM hydrogel is highly swollen, and when the temperature is above LCST, the hydrogel is at a state of shrinking. Based on the property of pNIPAM, the adsorption amounts of MCNTs@D-EMIP for target proteins underwent a switch by a temperature swing. Due to the properties of the thermo-sensitive polymer layer, the swollen and shrinkable states were present in this process and the imprinting cavities also had a volume change36. The results proved that MCNTs@D-EMIP could realize temperature controlled adsorption and release of the two target proteins.

13



Figure 4. (A) Changes of the adsorption amounts of MCNTs@D-EMIP for HSA and Trf by a temperature swing between 20℃ and 45℃. (B) Binding specificity of MCNTs@D-EMIP and MCNTs@D-ENIP for competitive proteins including BSA, Mb and Cyt C. (C) The selective adsorption amounts of MCNTs@D-EMIP and MCNTs@D-ENIP for HSA, Trf, Cyt C and Mb in the protein mixture (HSA, Trf, Cyt C and Mb). (D) Results of a comparison experiment of difference between metal chelation and hydrogen bond. The error bars represent the standard deviation of three parallel experiments.

Specificity Study. To examine the specificity of MCNTs@D-EMIP towards HSA and Trf, the specific test was performed by recognizing different kinds of proteins as competitors including BSA, Mb and Cyt C at the concentration of 0.5 mg mL-1. The adsorption results were shown in Figure 4B which demonstrated that the adsorption amounts and imprinting factors of MCNTs@D-EMIP for HSA and Trf were much higher than those of competitive proteins. Besides, as shown in Table S1, the selectivity coefficients (K, Equation S5) of MCNTs@D-EMIP for BSA, Mb and Cyt C were 1.63, 1.77 and 2.16, respectively, which indicated that MCNTs@D-EMIP had specific recognition ability 14


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towards HSA and Trf. In addition, in order to further study the recognition ability for HSA and Trf in mixture protein solution, MCNTs@D-EMIP was used to adsorb target proteins from a mixture protein solution containing HSA, Trf, Cyt C and Mb with each concentration of 0.5 mg mL-1. Figure 4C displayed that MCNTs@D-EMIP still exhibited specific recognition ability for HSA and Trf under the influence of Cyt C and Mb. MCNTs@D-EMIP and MCNTs@D-ENIP had high adsorption capacity for Cyt C because Cyt C had relative small molecular weight and size. Comparison of Different Immobilization Methods. In this report, zinc acrylate was selected as main functional monomer, which could form metal chelation and six-membered ring with functional groups of two types of template epitopes. Metal chelation and six-membered ring was stronger than hydrogen bond and it could immobilize more template epitopes to generate more imprinted sites to increase the final recognition efficiency. In order to examine the influence of metal chelation force on the final recognition ability, AA (AA only had hydrogen bond with templates) was used to prepare control imprinting polymers (MCNTs@D-EMIP-AA) to adsorb target proteins. As shown in Figure 4D, MCNTs@D-EMIP had more adsorption amounts and higher imprinting factors for HSA and

Trf

than

those

of

MCNTs@D-EMIP-AA,

which proved

that

MCNTs@D-EMIP contained more imprinted sites than MCNTs@D-EMIP-AA and also indicated that metal chelation and six-membered ring had stronger force to immobilize templates than only hydrogen bond. Therefore, choosing a suitable immobilization method played an important role for final recognition ability. Reusability. Reusability and stability is a significant property for the application of imprinting polymers. In order to investigate the reusability of MCNTs@D-EMIP, here we conducted the experiment of reusability for four circles and the results were shown in Figure S5. The results indicated that the adsorption capacity of MCNTs@D-EMIP and MCNTs@D-ENIP

for

HSA

only

had

a

slight

decrease

over

several

adsorption-desorption cycles, which proved that MCNTs@D-EMIP had excellent 15



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stability and reusability. Comparison of Different Polymerization Methods in Epitope Imprinting In this report, the thermo-sensitive MCNTs@D-EMIP was prepared by the distillation-precipitation polymerization in only 30 min. Distillation-precipitation polymerization method has the merit of fast reaction rate. In the process of distillation-precipitation polymerization, the solvent will be evaporated with the temperature increases, which would result in that the concentration of the reactants increase gradually. Therefore, the rate of polymerization becomes faster and faster so that the reaction could be finished in a very short time. Here we compared the reaction time of distillation-precipitation polymerization with those of other polymerization methods used in epitope imprinting including free radical polymerization, self-assembly, photo-initiated polymerization, sol-gel polymerization and so on, which were listed in Table 1. From the data we could get the conclusion that distillation-precipitation polymerization method had shorter reaction time than those of other polymerization methods. Table 1. Comparison of Different Polymerization Methods in Epitope Imprinting Carrier

Imprinting method

Polymerization method

Reaction time/h

Refer

Carbon Dots Magnetic particle QCM Magnetic particle Magnetic particles QCM

Epitope imprinting

Free radical polymerization

24

19

Epitope imprinting

Self-assembly

24

27

Epitope imprinting

Photo-initiated polymerization

6

43

Epitope imprinting

Self-polymerization

5

20

Epitope imprinting

Sol-gel polymerization

3

21

Epitope imprinting

Reversed-phase microemulsion Distillation-precipitation polymerization

2

44 This work

MCNTs

Epitope imprinting

0.5

HPLC Analysis of Real Samples. With the aim of investigating the practical application ability in real sample, MCNTs@D-EMIP was used to recognize HSA and Trf in human serum albumin sample and the results of HPLC analysis were provided in Figure 5. As shown in 16


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Figure 5A-a, the peaks of HSA and Trf appeared with strong signals in real sample before adsorption. From Figure 5A-b we could find out that after adsorbed by MCNTs@D-EMIP, the peak of HSA disappeared and the peak of Trf had a sharp decline which indicated that all of HSA and the great mass of Trf were captured by MCNTs@D-EMIP. Because the adsorption amount of MCNTs@D-EMIP for HSA was much more than that of Trf, Trf was not adsorbed completely which lead to a small peak of Trf retained. Figure 5A-c displayed that the peaks of HSA and Trf reappeared in the eluent, which further indicated that HSA and Trf in real sample were adsorbed selectively by MCNTs@D-EMIP. In addition, after adsorbed by MCNTs@D-ENIP, the peaks of HSA and Trf were almost not changed (Figure 5B-b) and there was no targets found in eluent (Figure 5B-c), which proved that MCNTs@D-ENIP did not have the ability to recognize target because of the absence of imprinted sites. These results demonstrated that the MCNTs@D-EMIP showed selective affinity toward HSA and Trf and could be applied in bio-samples.

Figure 5. (A) HPLC analysis of the adsorption of HSA and Trf by MCNTs@D-EMIP from real sample. (B) HPLC analysis of the adsorption of HSA and Trf by MCNTs@D-ENIP from real sample. (a) Mixture of human blood and Trf before adsorption; (b) Supernatant after adsorbed by MCNTs@D-EMIP or MCNTs@D-ENIP; and (c) Eluent eluted by SDS (10%; m/v) and acetic acid (10%; v/v) .

CONCLUSION In conclusion, the thermo-sensitive MCNTs@D-EMIP was prepared successfully within 30 min via the methods of dual-template epitope imprinting, metal chelation 17



imprinting and DPP, and it combined the merits of recognition towards two types of target protein, high imprinting efficiency and short reaction time. The prepared MCNTs@D-EMIP exhibited specific recognition ability towards HSA and Trf simultaneously. Besides, MCNTs@D-EMIP showed thermo-sensitive property towards HSA and Trf. In addition, the results of HPLC analysis indicated that MCNTs@D-EMIP could be applied to recognize the two types of target proteins in bio-sample. In summary, this work provided a rapid strategy to prepare epitope imprinting polymer to recognize two types of targets in bio-samples.

ASSOCIATED CONTENT Supporting Information All the reagents, instruments and HPLC conditions. Size distributions of samples. FT-IR spectra of MCNTs, MCNTs@SiO2, MCNTs@MPS, MCNTs@D-EMIP. Hysteresis loops of MCNTs, MCNTs@SiO2 and MCNTs@D-EMIP. HPLC results of templates in original solution and eluent. Reusability of MCNTs@EMIP and MCNTs@ENIP. Table showing adsorption capacities, imprinting factors and selectivity coefficients of MCNTs@D-EMIP for the target proteins and other proteins.

AUTHOR INFORMATION Corresponding Author *W.-Y. Li. E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 21275078, 21475069 and 21775077) and the Tianjin Natural Science Foundation (No. 16JCZDJC37200).

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Thermosensitive Metal Chelation Dual-Template Epitope Imprinting

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