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Magnetic fluorescent molecularly imprinted nanoparticles for detection and separation of transferrin in human serum
Author’s Accepted Manuscript Magnetic fluorescent molecularly imprinted nanoparticles for detection and separation of transferrin in human serum Yi-Da Zhang, Qiang-Wei Huang, Chen Ma, XiaoYan Liu, Hai-Xia Zhang www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(18)30608-8 https://doi.org/10.1016/j.talanta.2018.06.002 TAL18744
To appear in: Talanta Received date: 24 February 2018 Revised date: 26 May 2018 Accepted date: 1 June 2018 Cite this article as: Yi-Da Zhang, Qiang-Wei Huang, Chen Ma, Xiao-Yan Liu and Hai-Xia Zhang, Magnetic fluorescent molecularly imprinted nanoparticles for detection and separation of transferrin in human serum, Talanta, https://doi.org/10.1016/j.talanta.2018.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Magnetic fluorescent molecularly imprinted nanoparticles for detection and separation of transferrin in human serum Yi-Da Zhang, Qiang-Wei Huang, Chen Ma, Xiao-Yan Liu, Hai-Xia Zhang* College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China *Email: [email protected]
Abstract: Transferrin (TrF) is an important glycoprotein and disease biomarker that controls iron ion balance in the human body. Isolation and detection of TrF have important implication for the early detection of disease. Thus, a magnetic fluorescent molecularly imprinted nanoparticles (FMINPs) was prepared for extraction and fluorescence detection of TrF. The FMINPs was prepared with two steps, the first step was the synthesis of magnetic TrF imprinted nanoparticle and the second step was introducing a near-infrared fluorescent compound (CyA) on the imprinted nanoparticles, which has a strong near infrared fluorescence emission at 730 nm while excitation at 690 nm and a large fluorescence signal quenching after adsorption of TrF. The concentration of TrF can be determined by the change of the fluorescence signal. FT-IR, TEM and fluorescence spectrophotometer were used to verify the successful preparation and the fluorescence performance of the FMINPs. Under the optimized experimental conditions, the prepared FMINPs had a great fluorescence performance, offering the lower relative standard deviation (7.7%), good analytical range (0.025-0.175 mg/mL, R2=0.998) and lower detection limit (0.0075 mg/mL) for TrF. This method provides a new solution for the direct detection and separation of TrF in human serum samples.
Graphical Abstract
Keywords: Fluorescence detection; Molecularly imprinted nanoparticles; Transferrin; Fluorescence quenching; Near infrared fluorescence spectroscopy.
1. Introduction TrF as an iron ion transport protein causes much interest in the drug targeting studies due to its non-toxicity, non-immunogenicity, and biodegradability [1]. TrF is of great use in drug carriers as it binds to the TrF receptor on the surface of cancer cells [2]. The effective cell uptake by TrF mediated pathway, generated great potential in delivery of anticancer drugs, proteins, and gene therapy into multiplying malignant cells [3-6]. Furthermore, TrF is a glycoprotein that can be used as a biomarker of neurological diseases in the body [7] and its concentration can reflect the emergence of some diseases such as thalassemia, bacterial infection, diabetes, atransferrinemia even chronic alcohol abuse [8-10]. Therefore, the detection of TrF for the early diagnosis have great significance. However, there is little study on the extraction, separation and detection of TrF. Since the structure of TrF is complex, complicated in composition, and easily degenerated, it is difficult to use a method for rapid separation and detection of TrF. At present, the popular instrumental method used for the detection of proteins is mass spectrometry [11-13]. Unfortunately, the cost of protein mass spectrometry is generally high and it is difficult to directly quantify proteins without pretreatment. In addition, biological samples, especially protein samples,
can be easily hydrolyzed or denatured prior to analysis due to sample preparation or purification [14,15]. Therefore, it is necessary to establish a high selective and fast response method for detection of TrF. Molecular imprinted technique is using template molecule, functional monomer and crosslinker to prepare the molecular imprinted materials (MIM). After removing the template, many cavities are formed that can specifically match the template molecules. Therefore, MIM has great selectivity to the analyte. Currently, preparation of MIM for protein has received a great attention. Liu Y. et al. prepared molecular imprinted polymers (MIP) to detect bovine hemoglobin using a deep eutectic solvent as monomer [16]. Liu L. et al. prepared the MIP for the quantitative detection of serum transferrin receptor [17]. The molecular imprinted method for the detection of protein has a number of advantages, however it has some drawbacks as well. Detaching protein from the cavities makes the detection time-consuming and increases the risk of protein degeneration because of using acidic solution as the eluent. So establishing a method for rapid detection of proteins without inactivating is important. Much attention has given to the detection of glycoproteins based on the boronic acid affinity MIM, due to its unique reversible covalent binding of the cis-diol groupin glycoproteins. The boric acid molecular imprinted approach combining the merits of molecular imprinted technology and boronate affinity, lead to a higher specific recognition for glycoproteins and greatly improves the selectivity of the MIM [18-20]. Magnetic materials have very important role in separation[21,22]. Magnetic MIM share the characteristics of special selective recognition ability and the magnetic separation capability, which avoid the traditional operation steps of centrifuge and filtration, and the solid-liquid separation can be rapidly achieved by the effect of the external magnetic field [23]. Fluorometric analysis as a rapid detection method [24], has been successfully applied to the cell imaging and the proteins detection [25-27]. Wei et al. prepared a fluorescent molecularly imprinted polymer using a fluorescent boric acid monomer, and applied to detect glycoprotein [28]. Tang et al. prepared a MIM using novel CdTe nanocrystals as core based on boronic acid affinity for glycoprotein detection [29]. Lu et al. prepared a MIM based on functional monomer-template-QDs sandwich structure to ratiometric fluorescence sensor to detect bovine
hemoglobin in bovine urine and blood [30]. These works improved the combining of fluorescence and molecular imprinted technique. In this work, a new-type of fluorescent molecularly imprinted nanoparticles (FMINPs) was prepared. First, we modified Fe3O4 nanoparticles using 3-Carboxybenzeneboronic acid based on the interaction of -COOH group in this molecule and Fe ion on the surface of Fe3O4 nanoparticles [31]. Then the imprinted material of TrF was obtained using silica surface imprinted technique. A near-infrared semi-cyanine fluorescent compound CyA was introduced on the surface of the imprinted material to make the material fluorescent. The adsorption of TrF on FMINPs can cause a fluorescence decrease,which offers a rapid and direct method for quantification of TrF. The new method avoided the re-eluting of TrF from FMINPs, which reduced the time-consuming and the risk of de-activity of TrF.
2. Experimental 2.1 Reagents and materials 1,2,3,3-tetramethyl-3H-indolium iodide was obtained from Aladdin (Shanghai, China); Transferrin (TrF), bovine hemoglobin (Bhb), ovalbumin (OVA), bovine serum albumin (BSA), γ-globin (γ-Glo), horseradish peroxidase (HRP) and myoglobin (Myo) were purchased from BioDee Biotechnology (Beijing, China); Ferric chloride, anhydrous sodium acetate, ethylene glycol, sodium lauryl sulfate, glacial acetic acid were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China); Ethylsilicate (TEOS) was purchased from Energy Chemical (Shanghai, China); Inter-diphenol was purchased from Sinopharm Reagent (Shanghai, China); 3-Carboxybenzeneboronic acid was purchased from Sukailu Chemical Technology (Suzhou, China). The human serum was obtained from people's hospital of Gansu province (Lanzhou, China) and stored at -20°C for further use. 2.2 Instruments The materials were characterized by Fourier transform infrared (FT-IR) spectroscopy (VEREEX 70V FT-IR, USA), Elemental Analyzer (Vario EL, Germany) and Transmission Electron Microscope (Tecnai G2 TF20, USA). Fluorescence spectrophotometer (RF-5301pc, Japan) was used to test fluorescence properties under the conditions including excitation
wavelength λex 690 nm and the emission wavelength λem 725 nm, the slit width 15/15 nm; The solution pH was adjusted with pH meter (PHSJ-3F, China). 2.3 Preparation of FMINPs The Fe3O4 nanoparticles was obtained according to the reference [23]. CyA was synthesized according to the reference [25].The synthesis details were shown in supporting materials. Fe3O4 (200 mg) was dissolved in ethanol (30 mL) and ultrasonic dispersion for 30 min, then added 3-Carboxybenzene boronic acid (50 mg, the structure is shown in Fig. 1). The reaction system was kept persistent stir for 2.0 h. The prepared boronic acid modified Fe3O4 (Fe3O4@B(OH)2) was freeze-dried for use. Fe3O4@B(OH)2 (20 mg) was ultrasonically dispersed in phosphate buffer saline (PBS, 10 mL, 50 mM, pH=7.4) for 30 min. Then, TrF (1.0 mg) was added to the solution and kept oscillation for 1.5 h, in which ethanol (160 mL), TEOS (40 mL, 10 mM) and ammonia solution (2.8 mL, 25%) were added sequentially. The reaction system endured a mechanical stirring for 3.5 h to obtain a molecular imprinted material containing TrF. The template was eluted in a oscillation shaker using 10 mL aqueous solution (2% SDS + 2% HAc) for 3×20 min. At last, the molecularly imprinted nanoparticles (MINPs) were washed with ultrapure water and freeze-dried. The corresponding non-imprint material (NINPs) was prepared in the same manner except for no adding template molecules. The prepared MINPs (20 mg) or NINPs (20 mg) was ultrasonically dispersed in a 5.0 mL centrifuge tube with PBS (3.0 mL, 50 mM, pH=5.0). CyA (5 mg, the structure is shown in Fig. 1) dissolved in absolute ethanol (1.0 mL) was added in the above centrifuge tube and oscillated for 2.0 h. Finally, the materials were freeze-dried to get FMINPs and FNINPs. 2.4 Fluorescence performance experiments The FMINPs or FNINPs (2.0 mg) were dispersed in TrF solution (1.0 mL) with different concentrations (0.025-0.175 mg/mL) and oscillated for 1.0 h. After adsorption, the FMINPs and FNINPs were separated from the solution by a magnet. Re-disperse the materials with 1.0 mL of ethanol/water (1:1, v/v) to detect the fluorescence signal. The fluorescence quenching performance can be described by the Stern-Volmer equation [32]:
F0 Ae kCTrF B F
F0 and F are the fluorescence intensity of FMINPs or FNINPs before and after the adsorption of TrF, respectively. CTrF is the concentration of TrF, and A, B and k is the constant. 2.5 Selectivity experiments TrF, Bhb, OVA, BSA, γ-Glo, HRP and Myo solution (each 0.1 mg/mL) were used to detect the selectivity of the FMINPs and FNINPs. FMINPs or FNINPs (each 2.0 mg) was added to 1.0 mL of the relative protein solution and oscillated for 1.0 h. Then the fluorescence quenching performance of FMINPs or FNINPs was calculated using fluorescence ratio F0/F. 2.6. Determination of TrF in serum samples First, the human serum was thawed at room temperature and then diluted 50-fold, 75-fold, 100-fold with PBS solution (50 mM, pH=7.4), respectively. The FMINPs (2.0 mg) were ultrasonically dispersed in 1.0 mL of the prepared serum solution, then the mixture was oscillated for 1.0 h at 25°C. After the absorption, the FMINPs were separated from the solution by a magnet. Re-disperse the FMINPs with 1.0 mL of ethanol/water (1:1, v/v) to detect the fluorescence signal. The amount of TrF in human serum was calculated with the Stern-Volmer equation. 2.7 Reusability and repeatability of FMINPs. FMINPs or FNINPs (each 2.0 mg) were dispersed in TrF (0.10 mg/mL, 1.0 mL) solution and oscillated for 1.0 h. After adsorption, the materials were separated from the solution by a magnet. Then, SDS-HAc (2% w/v-2% w/v) was used as the eluant to remove the adsorbed TrF on the materials. The elution was performed with 20 min ×2. After elution, the materials were separated by a magnet and re-dispersed in 1.0 mL of ethanol/water (1:1, v/v) to detect the fluorescence signal. Four cycles of adsorption and elution were operated and fluorescence intensities was recorded and compared. Three batches of FMINPs were prepared using the same steps. The CyA amount adsorbed on the MINPs was first evaluated. MINPs (20 mg) was dispersed in 4.0 mL of CyA solution (5 mg in pH 5.0 PBS/ absolute ethanol (v/v, 3:1)) with vortex for 30 s. Then the materials were isolated from the solution with magnetic field. The fluorescence signals of CyA solution before/after the adsorption were recorded and compared to calculate the amount of adsorbed CyA. The obtained FMINPs were dispersed in 1.0 mL of ethanol/water (1:1, v/v) to detect the fluorescence signal, which was used to evaluate the reproducibility of the synthesized materials.
3. Results and discussion 3.1 Design and preparation of FMINPs The synthesis steps of FMINPs are shown in Fig. 1. Briefly, the Fe3O4 provided a support for 3-carboxyphenylboronic acid anchoring, due to the iron ion on Fe3O4 nanoparticle can form a complex with the carboxyl group [31]. Then, the -B(OH)2 group provided a site for specific binding to TrF. The TEOS was cross-linking itself, which helped TrF molecules be confined inside. Followed with removal of TrF from the material and electrostatic adsorption of CyA, the FMINPs were obtained. Fluorescent compound CyA was adsorbed onto the silica layer under pH=5.0 by the electrostatic force. The isoelectric point (IP) of silica is 2.5, which is negatively charged at pH>2.5. The N+ in CyA molecule can attract the negative charge on the surface of silica.
Fig. 1 Synthesis scheme of the FMINPs
3.2 Characterization of the FMINPs Fe3O4, Fe3O4@B(OH)2 and FMINPs were characterized by SEM. As shown in Fig. 2A and Fig. 2B, the size of both Fe3O4 and Fe3O4@B(OH)2 was about 10 nm, and the surface of Fe3O4 can not be observed with significant change after modification of 3-carboxyphenylboronic acid. From
the image of FMINPs (Fig. 2C), it is clear that a 5-8 nm silica layer (the location of the arrow) has formed on the surface of Fe3O4@B(OH)2. The elemental analysis results are shown in Table 1, the percentages of carbon and hydrogen in Fe3O4@B(OH)2 were up to 2.63% and 1.65%, which indicated that the 3-carboxyphenylboronic acid was grafted on the surface of Fe3O4. After the CyA adsorption, the nitrogen element was appeared in magnetic FMINPs, demonstrated that CyA was successfully adsorbed in silica layer and the upload amount was 0.0695 mg/mg by the calculation. The materials was also demonstrated by FT-IR characterization (Shown in supporting information, Fig. S2) to prove that FMINPs was obtained with CyA adsorption.
Table 1. Elemental analysis of Fe3O4, Fe3O4@B(OH)2 and FMINPs N (%)
C (%)
H (%)
Fe3O4
0
0.22
0.30
Fe3O4@B(OH)2
0
2.632
1.652
FMINPs
0.36
3.254
1.985
Fig. 2 The characterization of synthesized materials TEM image of Fe3O4 (A), Fe3O4@B(OH)2 (B) and FMINPs (C); Insets picture is magnetic response of FMINPs to external magnetic field.
3.3 Fluorescence properties of FMINPs and FNINPs The fluorescence signals of FMINPs and FNINPs were investigated after the adsorption of TrF with different concentrations. As shown in Fig. 3A and Fig. 3B, when the excitation wavelength is 690 nm, a strong and symmetrical emission appears at 725 nm for the blank FMINPs and FNINPs, the stable fluorescence of CyA showed the silica layer was fluorescence permeable [33]. When TrF was adsorbed on the materials, the fluorescence signal of FMINPs gradually decreased with higher TrF concentration, the quenching reached the maximum with 0.15 mg/mL of TrF solution, which meant the amount of TrF detected by the FMINPs reached
saturation. For FNINPs, less quenching was obtained. The fluorescence quenching may be from photoelectron transfer (PET) effect. The isoelectric point (IP) of TrF is 5.2, when pH=7.4, the protein surface is of electron-rich. The N+ moiety in CyA serves both as part of the electrostatic binding site for protein and a receptor for electron transfer. When CyA and TrF were contacted, electron-rich TrF would transfer electron to CyA, then the entire electron cloud density of CyA changed, which led to the fluorescence signal quenching. The Stern-Volmer plots of FMINPs and FNINPs are shown in Fig. 4. The Stern-Volmer plot bends upward for TrF from 0.025 to 0.175 mg/mL and the detection limit is 0.0075 mg/mL. The fluorescence quenching is caused by the combination of dynamic quenching and static quenching [32, 34]. The regression equations were y 2.34e R2=0.998 and y 0.226e
0.0010CTrF
0.00087CTrF
1.8774 to FMINPs with
1.0524 to FNINPs with R2=0.948, respectively. The
difference between FMINPs or FNINPs was obvious.
Fig. 3 Fluorescence curves of FMINPs (A) and FNINPs (B) with adsorption of TrF. (2.0 mg of FMINPs was dispersed in 1.0 mL of TrF solution with different concentration. After adsorption, the FMINPs was re-dispersed in 1.0 mL of ethanol/water (1:1, v/v))
Fig. 4 Stern-Volmer plots for FMINPs and FNINPs under different TrF concentration.
3.4 Selectivity of FMINPs Bhb (MW:65.4 KDa, IP:6.7), BSA (MW:66.4 KDa, IP:4.7), OVA (MW:44.5 KDa, IP:4.7), γ-Glo (MW:150 KDa, IP:5.5), HRP (MW:40.0 KDa, IP:3-9) and Myo (MW:16.7 KDa, IP:7.07) were used to examine the selectivity of the FMINPs. These proteins have different IP and molecular weights (MW) for TrF (MW:77 KDa, IP:5.2) [35]. TrF is the template molecule for synthesis of imprinted materials, in which the special cavities were formed corresponding to its shape, size, and spatial arrangement. As shown in Fig. 5, for FMINPs, TrF has the ability to preferentially occupy these cavities, and the fluorescence of the FMINPs has undergone a drastic decrease after absorption of TrF. For FNINPs, fluorescence quenching to all the studied proteins including TrF was quite similar, suggesting that the FNINPs can not effectively distinguish TrF and other proteins. As a typical glycoprotein, HRP may bind some of the boric acid molecules in the imprinted cavities to cause adsorption, so that the fluorescence intensity has a certain degree of annihilation. Myo contains heme which has a porphyrin structure, and the emission energy of CyA is partly absorbed by the heme and causes a certain annihilation [36]. This is the reason that HRP and Myo have higher adsorption than other proteins on FMINPs. Thanks to the tiny amount of HRP and Myo in serum, they can not affect the quantification of TrF with the new method.
Fig. 5 Selective adsorption of different proteins by FMINPs and FNINPs at pH=7.4 (PBS, 50 mM). 3.5 Application to real samples analysis
The FMINPs was used for selective and direct detection of TrF in serum samples. The results were shown in table 2. When serum was diluted 50-fold, 75-fold and 100-fold respectively, the TrF concentrations were determined as 0.0700, 0.0461 and 0.0340 mg/mL with the Stern-Volmer plot, which can be calculated as 3.503, 3.462 and 3.403 mg/mL TrF in blank serum, respectively. The average concentration is 3.456 mg/mL with less than 8% error, which is within the normal scope (the content of TrF in human serum is 2.000-3.500 mg/mL [37, 38]). Therefore, it indicated that the proposed method is capable for the TrF analysis in complex samples.
Table 2. Analysis of TrF in serum samples. n=3 Diluted Human Serum
Founda
Concentration of TrF in
RSD
with PBS Buffer
(mg/mL)
Human Serum (mg/mL)
(%)
50-fold
0.0700
3.503 ± 0.228
6.5
75-fold
0.0461
3.462 ± 0.266
7.7
100-fold
0.0340
3.403 ± 0.235
6.9
3.6 Evaluation of the synthesized materials The fluorescence intensities of FMINPs and FNINPs were detected after each cycle of adsorption and elution. As shown in Fig. 6A (The numbers 1-4 in Fig. 6A means the elution times), FMINPs can be used for three replicate experiments with the fluorescence intensities decreased less than 10%. FNINPs can not be used repeatedly.
Fig. 6 Reusability of FMINPs and FNINPs with 4 times elution and the reproducibility of the FMINPs with different batches.
The FMINPs prepared at different batches was compared with the fluorescence signal of each other. In Fig. 6B, three different batches (1, 2, 3 means different batch) of FMINPs showed nearly same fluorescence intensities after the adsorption of CyA, which can prove that the FMINPs can be prepared with good reproducibility. The proposed FMINPs were compared with the similar imprinted materials previously reported with fluorescence detection for proteins, with respect to analytical range and detection limit. As can be seen in Table 3, the FMINPs exhibited the competitive analytical range and the detection limit.
Table 3. Comparison of the FMINPs with other protein imprinted materials reported Analytical range
Detection limit
(μM)
(μM)
0.05-1
0.02
29
UCNPs -MIP
1-24
0.73
39
CdTe QDs-MIP
0.97-24
0.41
40
CdTe QDs-Bhb-MIP
0.02-2.0
0.0063
41
0.325-2.27
0.098
(0.025-0.175 mg/mL)
(0.0075 mg/mL)
Method PGMA/EDMA-MIP a
FMINPs a
Ref
This work
Upconversion nanoparticles
4. Conclusion In conclusion, a new material FMINPs was prepared combining fluorescent detection and boric-acid based surface molecular imprinted technology. FMINPs with good fluorescence performance and selectivity, can be directly used to detect TrF dispensing with the protein elution. The method demonstrated a new way for glycoprotein analysis in complex samples. However, the fluorescence quenching signals were used to quantify the protein with non-linear curve is the shortage. It is necessary to improve the material with fluorescence increasing signal for the quantification of protein.
Acknowledgements: This research was financially supported by the National Natural Science Foundation of China (No. 21575055) and the Research Funds for the Central Universities (lzujbky-2017-k09).
Compliance with ethical standards The study using serum as sample has been approved by the People's hospital of Gansu province Ethics Committee and the Lanzhou University Ethics Committee, and has been performed in accordance with the ethical standards. Informed consent was obtained from all individual participants included in the study.
Conflict of interest The authors declare that they have no conflict of interest.
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Highlights 1. The FMINPs were fluorescent by introducing near-infrared fluorescent CyA. 2. The FMINPs exhibits great fluorescence performance and selectivity for transferrin (TrF). 3. TrF can be rapidly detected in serum samples with the proposed method . 4. The FMINPs can be prepared with good reproducibility.
PII: DOI: Reference:
S0039-9140(18)30608-8 https://doi.org/10.1016/j.talanta.2018.06.002 TAL18744
To appear in: Talanta Received date: 24 February 2018 Revised date: 26 May 2018 Accepted date: 1 June 2018 Cite this article as: Yi-Da Zhang, Qiang-Wei Huang, Chen Ma, Xiao-Yan Liu and Hai-Xia Zhang, Magnetic fluorescent molecularly imprinted nanoparticles for detection and separation of transferrin in human serum, Talanta, https://doi.org/10.1016/j.talanta.2018.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Magnetic fluorescent molecularly imprinted nanoparticles for detection and separation of transferrin in human serum Yi-Da Zhang, Qiang-Wei Huang, Chen Ma, Xiao-Yan Liu, Hai-Xia Zhang* College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China *Email: [email protected]
Abstract: Transferrin (TrF) is an important glycoprotein and disease biomarker that controls iron ion balance in the human body. Isolation and detection of TrF have important implication for the early detection of disease. Thus, a magnetic fluorescent molecularly imprinted nanoparticles (FMINPs) was prepared for extraction and fluorescence detection of TrF. The FMINPs was prepared with two steps, the first step was the synthesis of magnetic TrF imprinted nanoparticle and the second step was introducing a near-infrared fluorescent compound (CyA) on the imprinted nanoparticles, which has a strong near infrared fluorescence emission at 730 nm while excitation at 690 nm and a large fluorescence signal quenching after adsorption of TrF. The concentration of TrF can be determined by the change of the fluorescence signal. FT-IR, TEM and fluorescence spectrophotometer were used to verify the successful preparation and the fluorescence performance of the FMINPs. Under the optimized experimental conditions, the prepared FMINPs had a great fluorescence performance, offering the lower relative standard deviation (7.7%), good analytical range (0.025-0.175 mg/mL, R2=0.998) and lower detection limit (0.0075 mg/mL) for TrF. This method provides a new solution for the direct detection and separation of TrF in human serum samples.
Graphical Abstract
Keywords: Fluorescence detection; Molecularly imprinted nanoparticles; Transferrin; Fluorescence quenching; Near infrared fluorescence spectroscopy.
1. Introduction TrF as an iron ion transport protein causes much interest in the drug targeting studies due to its non-toxicity, non-immunogenicity, and biodegradability [1]. TrF is of great use in drug carriers as it binds to the TrF receptor on the surface of cancer cells [2]. The effective cell uptake by TrF mediated pathway, generated great potential in delivery of anticancer drugs, proteins, and gene therapy into multiplying malignant cells [3-6]. Furthermore, TrF is a glycoprotein that can be used as a biomarker of neurological diseases in the body [7] and its concentration can reflect the emergence of some diseases such as thalassemia, bacterial infection, diabetes, atransferrinemia even chronic alcohol abuse [8-10]. Therefore, the detection of TrF for the early diagnosis have great significance. However, there is little study on the extraction, separation and detection of TrF. Since the structure of TrF is complex, complicated in composition, and easily degenerated, it is difficult to use a method for rapid separation and detection of TrF. At present, the popular instrumental method used for the detection of proteins is mass spectrometry [11-13]. Unfortunately, the cost of protein mass spectrometry is generally high and it is difficult to directly quantify proteins without pretreatment. In addition, biological samples, especially protein samples,
can be easily hydrolyzed or denatured prior to analysis due to sample preparation or purification [14,15]. Therefore, it is necessary to establish a high selective and fast response method for detection of TrF. Molecular imprinted technique is using template molecule, functional monomer and crosslinker to prepare the molecular imprinted materials (MIM). After removing the template, many cavities are formed that can specifically match the template molecules. Therefore, MIM has great selectivity to the analyte. Currently, preparation of MIM for protein has received a great attention. Liu Y. et al. prepared molecular imprinted polymers (MIP) to detect bovine hemoglobin using a deep eutectic solvent as monomer [16]. Liu L. et al. prepared the MIP for the quantitative detection of serum transferrin receptor [17]. The molecular imprinted method for the detection of protein has a number of advantages, however it has some drawbacks as well. Detaching protein from the cavities makes the detection time-consuming and increases the risk of protein degeneration because of using acidic solution as the eluent. So establishing a method for rapid detection of proteins without inactivating is important. Much attention has given to the detection of glycoproteins based on the boronic acid affinity MIM, due to its unique reversible covalent binding of the cis-diol groupin glycoproteins. The boric acid molecular imprinted approach combining the merits of molecular imprinted technology and boronate affinity, lead to a higher specific recognition for glycoproteins and greatly improves the selectivity of the MIM [18-20]. Magnetic materials have very important role in separation[21,22]. Magnetic MIM share the characteristics of special selective recognition ability and the magnetic separation capability, which avoid the traditional operation steps of centrifuge and filtration, and the solid-liquid separation can be rapidly achieved by the effect of the external magnetic field [23]. Fluorometric analysis as a rapid detection method [24], has been successfully applied to the cell imaging and the proteins detection [25-27]. Wei et al. prepared a fluorescent molecularly imprinted polymer using a fluorescent boric acid monomer, and applied to detect glycoprotein [28]. Tang et al. prepared a MIM using novel CdTe nanocrystals as core based on boronic acid affinity for glycoprotein detection [29]. Lu et al. prepared a MIM based on functional monomer-template-QDs sandwich structure to ratiometric fluorescence sensor to detect bovine
hemoglobin in bovine urine and blood [30]. These works improved the combining of fluorescence and molecular imprinted technique. In this work, a new-type of fluorescent molecularly imprinted nanoparticles (FMINPs) was prepared. First, we modified Fe3O4 nanoparticles using 3-Carboxybenzeneboronic acid based on the interaction of -COOH group in this molecule and Fe ion on the surface of Fe3O4 nanoparticles [31]. Then the imprinted material of TrF was obtained using silica surface imprinted technique. A near-infrared semi-cyanine fluorescent compound CyA was introduced on the surface of the imprinted material to make the material fluorescent. The adsorption of TrF on FMINPs can cause a fluorescence decrease,which offers a rapid and direct method for quantification of TrF. The new method avoided the re-eluting of TrF from FMINPs, which reduced the time-consuming and the risk of de-activity of TrF.
2. Experimental 2.1 Reagents and materials 1,2,3,3-tetramethyl-3H-indolium iodide was obtained from Aladdin (Shanghai, China); Transferrin (TrF), bovine hemoglobin (Bhb), ovalbumin (OVA), bovine serum albumin (BSA), γ-globin (γ-Glo), horseradish peroxidase (HRP) and myoglobin (Myo) were purchased from BioDee Biotechnology (Beijing, China); Ferric chloride, anhydrous sodium acetate, ethylene glycol, sodium lauryl sulfate, glacial acetic acid were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China); Ethylsilicate (TEOS) was purchased from Energy Chemical (Shanghai, China); Inter-diphenol was purchased from Sinopharm Reagent (Shanghai, China); 3-Carboxybenzeneboronic acid was purchased from Sukailu Chemical Technology (Suzhou, China). The human serum was obtained from people's hospital of Gansu province (Lanzhou, China) and stored at -20°C for further use. 2.2 Instruments The materials were characterized by Fourier transform infrared (FT-IR) spectroscopy (VEREEX 70V FT-IR, USA), Elemental Analyzer (Vario EL, Germany) and Transmission Electron Microscope (Tecnai G2 TF20, USA). Fluorescence spectrophotometer (RF-5301pc, Japan) was used to test fluorescence properties under the conditions including excitation
wavelength λex 690 nm and the emission wavelength λem 725 nm, the slit width 15/15 nm; The solution pH was adjusted with pH meter (PHSJ-3F, China). 2.3 Preparation of FMINPs The Fe3O4 nanoparticles was obtained according to the reference [23]. CyA was synthesized according to the reference [25].The synthesis details were shown in supporting materials. Fe3O4 (200 mg) was dissolved in ethanol (30 mL) and ultrasonic dispersion for 30 min, then added 3-Carboxybenzene boronic acid (50 mg, the structure is shown in Fig. 1). The reaction system was kept persistent stir for 2.0 h. The prepared boronic acid modified Fe3O4 (Fe3O4@B(OH)2) was freeze-dried for use. Fe3O4@B(OH)2 (20 mg) was ultrasonically dispersed in phosphate buffer saline (PBS, 10 mL, 50 mM, pH=7.4) for 30 min. Then, TrF (1.0 mg) was added to the solution and kept oscillation for 1.5 h, in which ethanol (160 mL), TEOS (40 mL, 10 mM) and ammonia solution (2.8 mL, 25%) were added sequentially. The reaction system endured a mechanical stirring for 3.5 h to obtain a molecular imprinted material containing TrF. The template was eluted in a oscillation shaker using 10 mL aqueous solution (2% SDS + 2% HAc) for 3×20 min. At last, the molecularly imprinted nanoparticles (MINPs) were washed with ultrapure water and freeze-dried. The corresponding non-imprint material (NINPs) was prepared in the same manner except for no adding template molecules. The prepared MINPs (20 mg) or NINPs (20 mg) was ultrasonically dispersed in a 5.0 mL centrifuge tube with PBS (3.0 mL, 50 mM, pH=5.0). CyA (5 mg, the structure is shown in Fig. 1) dissolved in absolute ethanol (1.0 mL) was added in the above centrifuge tube and oscillated for 2.0 h. Finally, the materials were freeze-dried to get FMINPs and FNINPs. 2.4 Fluorescence performance experiments The FMINPs or FNINPs (2.0 mg) were dispersed in TrF solution (1.0 mL) with different concentrations (0.025-0.175 mg/mL) and oscillated for 1.0 h. After adsorption, the FMINPs and FNINPs were separated from the solution by a magnet. Re-disperse the materials with 1.0 mL of ethanol/water (1:1, v/v) to detect the fluorescence signal. The fluorescence quenching performance can be described by the Stern-Volmer equation [32]:
F0 Ae kCTrF B F
F0 and F are the fluorescence intensity of FMINPs or FNINPs before and after the adsorption of TrF, respectively. CTrF is the concentration of TrF, and A, B and k is the constant. 2.5 Selectivity experiments TrF, Bhb, OVA, BSA, γ-Glo, HRP and Myo solution (each 0.1 mg/mL) were used to detect the selectivity of the FMINPs and FNINPs. FMINPs or FNINPs (each 2.0 mg) was added to 1.0 mL of the relative protein solution and oscillated for 1.0 h. Then the fluorescence quenching performance of FMINPs or FNINPs was calculated using fluorescence ratio F0/F. 2.6. Determination of TrF in serum samples First, the human serum was thawed at room temperature and then diluted 50-fold, 75-fold, 100-fold with PBS solution (50 mM, pH=7.4), respectively. The FMINPs (2.0 mg) were ultrasonically dispersed in 1.0 mL of the prepared serum solution, then the mixture was oscillated for 1.0 h at 25°C. After the absorption, the FMINPs were separated from the solution by a magnet. Re-disperse the FMINPs with 1.0 mL of ethanol/water (1:1, v/v) to detect the fluorescence signal. The amount of TrF in human serum was calculated with the Stern-Volmer equation. 2.7 Reusability and repeatability of FMINPs. FMINPs or FNINPs (each 2.0 mg) were dispersed in TrF (0.10 mg/mL, 1.0 mL) solution and oscillated for 1.0 h. After adsorption, the materials were separated from the solution by a magnet. Then, SDS-HAc (2% w/v-2% w/v) was used as the eluant to remove the adsorbed TrF on the materials. The elution was performed with 20 min ×2. After elution, the materials were separated by a magnet and re-dispersed in 1.0 mL of ethanol/water (1:1, v/v) to detect the fluorescence signal. Four cycles of adsorption and elution were operated and fluorescence intensities was recorded and compared. Three batches of FMINPs were prepared using the same steps. The CyA amount adsorbed on the MINPs was first evaluated. MINPs (20 mg) was dispersed in 4.0 mL of CyA solution (5 mg in pH 5.0 PBS/ absolute ethanol (v/v, 3:1)) with vortex for 30 s. Then the materials were isolated from the solution with magnetic field. The fluorescence signals of CyA solution before/after the adsorption were recorded and compared to calculate the amount of adsorbed CyA. The obtained FMINPs were dispersed in 1.0 mL of ethanol/water (1:1, v/v) to detect the fluorescence signal, which was used to evaluate the reproducibility of the synthesized materials.
3. Results and discussion 3.1 Design and preparation of FMINPs The synthesis steps of FMINPs are shown in Fig. 1. Briefly, the Fe3O4 provided a support for 3-carboxyphenylboronic acid anchoring, due to the iron ion on Fe3O4 nanoparticle can form a complex with the carboxyl group [31]. Then, the -B(OH)2 group provided a site for specific binding to TrF. The TEOS was cross-linking itself, which helped TrF molecules be confined inside. Followed with removal of TrF from the material and electrostatic adsorption of CyA, the FMINPs were obtained. Fluorescent compound CyA was adsorbed onto the silica layer under pH=5.0 by the electrostatic force. The isoelectric point (IP) of silica is 2.5, which is negatively charged at pH>2.5. The N+ in CyA molecule can attract the negative charge on the surface of silica.
Fig. 1 Synthesis scheme of the FMINPs
3.2 Characterization of the FMINPs Fe3O4, Fe3O4@B(OH)2 and FMINPs were characterized by SEM. As shown in Fig. 2A and Fig. 2B, the size of both Fe3O4 and Fe3O4@B(OH)2 was about 10 nm, and the surface of Fe3O4 can not be observed with significant change after modification of 3-carboxyphenylboronic acid. From
the image of FMINPs (Fig. 2C), it is clear that a 5-8 nm silica layer (the location of the arrow) has formed on the surface of Fe3O4@B(OH)2. The elemental analysis results are shown in Table 1, the percentages of carbon and hydrogen in Fe3O4@B(OH)2 were up to 2.63% and 1.65%, which indicated that the 3-carboxyphenylboronic acid was grafted on the surface of Fe3O4. After the CyA adsorption, the nitrogen element was appeared in magnetic FMINPs, demonstrated that CyA was successfully adsorbed in silica layer and the upload amount was 0.0695 mg/mg by the calculation. The materials was also demonstrated by FT-IR characterization (Shown in supporting information, Fig. S2) to prove that FMINPs was obtained with CyA adsorption.
Table 1. Elemental analysis of Fe3O4, Fe3O4@B(OH)2 and FMINPs N (%)
C (%)
H (%)
Fe3O4
0
0.22
0.30
Fe3O4@B(OH)2
0
2.632
1.652
FMINPs
0.36
3.254
1.985
Fig. 2 The characterization of synthesized materials TEM image of Fe3O4 (A), Fe3O4@B(OH)2 (B) and FMINPs (C); Insets picture is magnetic response of FMINPs to external magnetic field.
3.3 Fluorescence properties of FMINPs and FNINPs The fluorescence signals of FMINPs and FNINPs were investigated after the adsorption of TrF with different concentrations. As shown in Fig. 3A and Fig. 3B, when the excitation wavelength is 690 nm, a strong and symmetrical emission appears at 725 nm for the blank FMINPs and FNINPs, the stable fluorescence of CyA showed the silica layer was fluorescence permeable [33]. When TrF was adsorbed on the materials, the fluorescence signal of FMINPs gradually decreased with higher TrF concentration, the quenching reached the maximum with 0.15 mg/mL of TrF solution, which meant the amount of TrF detected by the FMINPs reached
saturation. For FNINPs, less quenching was obtained. The fluorescence quenching may be from photoelectron transfer (PET) effect. The isoelectric point (IP) of TrF is 5.2, when pH=7.4, the protein surface is of electron-rich. The N+ moiety in CyA serves both as part of the electrostatic binding site for protein and a receptor for electron transfer. When CyA and TrF were contacted, electron-rich TrF would transfer electron to CyA, then the entire electron cloud density of CyA changed, which led to the fluorescence signal quenching. The Stern-Volmer plots of FMINPs and FNINPs are shown in Fig. 4. The Stern-Volmer plot bends upward for TrF from 0.025 to 0.175 mg/mL and the detection limit is 0.0075 mg/mL. The fluorescence quenching is caused by the combination of dynamic quenching and static quenching [32, 34]. The regression equations were y 2.34e R2=0.998 and y 0.226e
0.0010CTrF
0.00087CTrF
1.8774 to FMINPs with
1.0524 to FNINPs with R2=0.948, respectively. The
difference between FMINPs or FNINPs was obvious.
Fig. 3 Fluorescence curves of FMINPs (A) and FNINPs (B) with adsorption of TrF. (2.0 mg of FMINPs was dispersed in 1.0 mL of TrF solution with different concentration. After adsorption, the FMINPs was re-dispersed in 1.0 mL of ethanol/water (1:1, v/v))
Fig. 4 Stern-Volmer plots for FMINPs and FNINPs under different TrF concentration.
3.4 Selectivity of FMINPs Bhb (MW:65.4 KDa, IP:6.7), BSA (MW:66.4 KDa, IP:4.7), OVA (MW:44.5 KDa, IP:4.7), γ-Glo (MW:150 KDa, IP:5.5), HRP (MW:40.0 KDa, IP:3-9) and Myo (MW:16.7 KDa, IP:7.07) were used to examine the selectivity of the FMINPs. These proteins have different IP and molecular weights (MW) for TrF (MW:77 KDa, IP:5.2) [35]. TrF is the template molecule for synthesis of imprinted materials, in which the special cavities were formed corresponding to its shape, size, and spatial arrangement. As shown in Fig. 5, for FMINPs, TrF has the ability to preferentially occupy these cavities, and the fluorescence of the FMINPs has undergone a drastic decrease after absorption of TrF. For FNINPs, fluorescence quenching to all the studied proteins including TrF was quite similar, suggesting that the FNINPs can not effectively distinguish TrF and other proteins. As a typical glycoprotein, HRP may bind some of the boric acid molecules in the imprinted cavities to cause adsorption, so that the fluorescence intensity has a certain degree of annihilation. Myo contains heme which has a porphyrin structure, and the emission energy of CyA is partly absorbed by the heme and causes a certain annihilation [36]. This is the reason that HRP and Myo have higher adsorption than other proteins on FMINPs. Thanks to the tiny amount of HRP and Myo in serum, they can not affect the quantification of TrF with the new method.
Fig. 5 Selective adsorption of different proteins by FMINPs and FNINPs at pH=7.4 (PBS, 50 mM). 3.5 Application to real samples analysis
The FMINPs was used for selective and direct detection of TrF in serum samples. The results were shown in table 2. When serum was diluted 50-fold, 75-fold and 100-fold respectively, the TrF concentrations were determined as 0.0700, 0.0461 and 0.0340 mg/mL with the Stern-Volmer plot, which can be calculated as 3.503, 3.462 and 3.403 mg/mL TrF in blank serum, respectively. The average concentration is 3.456 mg/mL with less than 8% error, which is within the normal scope (the content of TrF in human serum is 2.000-3.500 mg/mL [37, 38]). Therefore, it indicated that the proposed method is capable for the TrF analysis in complex samples.
Table 2. Analysis of TrF in serum samples. n=3 Diluted Human Serum
Founda
Concentration of TrF in
RSD
with PBS Buffer
(mg/mL)
Human Serum (mg/mL)
(%)
50-fold
0.0700
3.503 ± 0.228
6.5
75-fold
0.0461
3.462 ± 0.266
7.7
100-fold
0.0340
3.403 ± 0.235
6.9
3.6 Evaluation of the synthesized materials The fluorescence intensities of FMINPs and FNINPs were detected after each cycle of adsorption and elution. As shown in Fig. 6A (The numbers 1-4 in Fig. 6A means the elution times), FMINPs can be used for three replicate experiments with the fluorescence intensities decreased less than 10%. FNINPs can not be used repeatedly.
Fig. 6 Reusability of FMINPs and FNINPs with 4 times elution and the reproducibility of the FMINPs with different batches.
The FMINPs prepared at different batches was compared with the fluorescence signal of each other. In Fig. 6B, three different batches (1, 2, 3 means different batch) of FMINPs showed nearly same fluorescence intensities after the adsorption of CyA, which can prove that the FMINPs can be prepared with good reproducibility. The proposed FMINPs were compared with the similar imprinted materials previously reported with fluorescence detection for proteins, with respect to analytical range and detection limit. As can be seen in Table 3, the FMINPs exhibited the competitive analytical range and the detection limit.
Table 3. Comparison of the FMINPs with other protein imprinted materials reported Analytical range
Detection limit
(μM)
(μM)
0.05-1
0.02
29
UCNPs -MIP
1-24
0.73
39
CdTe QDs-MIP
0.97-24
0.41
40
CdTe QDs-Bhb-MIP
0.02-2.0
0.0063
41
0.325-2.27
0.098
(0.025-0.175 mg/mL)
(0.0075 mg/mL)
Method PGMA/EDMA-MIP a
FMINPs a
Ref
This work
Upconversion nanoparticles
4. Conclusion In conclusion, a new material FMINPs was prepared combining fluorescent detection and boric-acid based surface molecular imprinted technology. FMINPs with good fluorescence performance and selectivity, can be directly used to detect TrF dispensing with the protein elution. The method demonstrated a new way for glycoprotein analysis in complex samples. However, the fluorescence quenching signals were used to quantify the protein with non-linear curve is the shortage. It is necessary to improve the material with fluorescence increasing signal for the quantification of protein.
Acknowledgements: This research was financially supported by the National Natural Science Foundation of China (No. 21575055) and the Research Funds for the Central Universities (lzujbky-2017-k09).
Compliance with ethical standards The study using serum as sample has been approved by the People's hospital of Gansu province Ethics Committee and the Lanzhou University Ethics Committee, and has been performed in accordance with the ethical standards. Informed consent was obtained from all individual participants included in the study.
Conflict of interest The authors declare that they have no conflict of interest.
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Highlights 1. The FMINPs were fluorescent by introducing near-infrared fluorescent CyA. 2. The FMINPs exhibits great fluorescence performance and selectivity for transferrin (TrF). 3. TrF can be rapidly detected in serum samples with the proposed method . 4. The FMINPs can be prepared with good reproducibility.