Biomaterials 31 (2010) 7332e7339
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Fe3O4 nanoparticles-loaded PEGePLA polymeric vesicles as labels for ultrasensitive immunosensors Qin Wei a, Ting Li b, Gaolei Wang a, He Li a, c, ***, Zhiyong Qian d, **, Minghui Yang a, * a
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China Department of Surgery, The Second Xiang-ya Hospital, Central South University, Changsha 410011, China c School of Medical and Life Sciences, University of Jinan, Jinan 250022, PR China d State Key Lab of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 March 2010 Accepted 4 June 2010 Available online 9 July 2010
A class of immuno-labels based on poly(ethylene glycol)epoly(lactic acid) (PEGePLA) polymeric vesicles was developed in this study. To fabricate these immune-labels, the uniform Fe3O4 nanoparticles (NPs) were loaded into the vesicles followed by conjugating secondary antibody (Ab2) onto the vesicles surface, named Ab2ePEGePLAeFe3O4. The resulting Ab2ePEGePLAeFe3O4 demonstrated high catalytic activity towards H2O2, and the sensitivity of the sandwich-type immunosensor using this label for prostate specific antigen (PSA) detection increased greatly. The immunosensor based on this label exhibited high sensitivity, wide linear range (0.005e10 ng/mL), low detection limit (2 pg/mL), good reproducibility, selectivity and stability. These labels for immunosensors may provide many potential applications for the ultrasensitive detection of different cancer biomarkers. In addition, this technique also has the potential to be extended to the loading of other interesting material for preparing various kinds of labels to meet the different requirements in immunoassays. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Fe3O4 nanoparticles Label Polymeric vesicles Prostate specific antigen Sandwich type
1. Introduction The detection of trace amount of proteins in biological samples is of great importance in clinical diagnosis, biomedical research, food quality control and environmental monitoring [1e4]. Ultrasensitive methods are urgently needed for disease diagnosis to detect cancer biomarkers present at ultralow levels during the early stages of disease [5e8]. Among the currently available analytical methods, immunosensors based on antibodyeantigen interaction are one of the most widely used analytical techniques to achieve high sensitivity and selectivity [9,10]. For sandwich-type immunosensors, signal amplification is typically through coupling different labels to the secondary antibody. As the sensitivity of the immunosensor is mainly determined by what kind of label used, various types of nanomaterials have been investigated as labels for such immunosensors, including metal nanoparticles, quantum dots, carbon nanotubes, and electroactive component-loaded nanoparticles [11e14].
* Corresponding author. Tel.: þ86 531 82765730. ** Corresponding author. *** Corresponding author at: School of Medical and Life Sciences, University of Jinan, Jinan 250022, PR China. E-mail addresses:
[email protected] (H. Li),
[email protected] (Z. Qian),
[email protected] (M. Yang). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.06.014
Recently, hollow spheres for the encapsulation of different sensing materials and used as labels have gained growing interest because high loading level of sensing materials can be achieved. Cai et al. encapsulated organic dye fluorescein diacetate (FDA) into hollow periodic mesoporous organosilicas (PMOs) and with the further attachment of antibody onto sphere surfaces, the functionalized spheres were used as label for immunoassay [15]. However, one drawback of this method is that it is difficult to measure the fluorescence intensity, the encapsulated dyes need to be released from the spheres, which takes about 1 h. Tang et al. loaded enzyme horseradish peroxidase (HRP) into nanogold hollow microsphere and use it as label for immunosensors. But to achieve efficient electron transfer between enzyme and electrode, mediators needed to be immobilized onto electrode surface or added into buffer solution [16]. Polymer vesicles are closed spherical lamellar structures formed from amphiphilic block copolymers in selective solvents. Since the polymer vesicles are robust and have advanced mechanical properties over those vesicles from lipids and surfactants, they are considered to be promising candidates for application in drug delivery system [17e19]. They offer the advantages of high drug loading capacity, sub-cellular size and great biocompatibility. For example, proteins, genes and chemical drugs could be incorporated into the vesicles and delivered to specific cells. Moreover, the surface properties and composition of these vesicles can be easily
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Scheme 1. Schematic representation of the preparation of the Ab2ePEGePLAeFe3O4 polymeric vesicle (A) and immunosensor (B).
tailored during the preparation process, which makes it easy for the conjugation of antibodies and receptors onto vesicle surface to achieve target delivery [20,21]. The Fe3O4 nanoparticles (NPs)embedded polymeric vesicles has been extensively studied for MRI application [22,23]. However, to our best knowledge, there is still no report of using this kind of vesicles as label for the fabrication of immunosensors. In this paper, poly(ethylene glycol)epoly(lactic acid) (PEGePLA) polymeric vesicles were prepared and used to simultaneously encapsulate Fe3O4 NPs and immobilize secondary antibody (Ab2) for the fabrication of electrochemical immunosensors. Using prostate specific antigen (PSA) as model analyte, and with the immobilization of primary antibody (Ab1) onto graphene sheet (GS) surface, an ultrasensitive immunosensor was prepared. This simple, economic and sensitive immunosensor could find wide potential application in clinical analysis for the detection of different cancer biomarkers.
other chemicals were of analytical reagents grade and used without further purification. Phosphate buffered saline (PBS, 0.1 M, pH 7.4) was used as electrolyte for all electrochemistry measurement. Double distilled water was used throughout the experiments. All electrochemical measurements were performed on a CHI 760D electrochemical workstation (Shanghai CH Instruments Co., China). 1H NMR spectrum of the samples was performed on a Varian 400 instrument (Varian, USA) using CDCl3 as the solvents at 25 C. Gel permeation chromatography (GPC) analysis was carried out at ambient temperature using the 270maxÔ Viscotek GPC system equipped with triple detectors, including a refractive index detector, a viscometer detector, and a light-scattering detector. THF was used as an eluent with a flow rate of 1 ml/min and polystyrene (PS) standards were used for column calibration. The amphiphilic block copolymer solutions in THF (5 mg/ml) were prepared. The size and size distribution of the polymeric vesicles were determined by dynamic light scattering (DLS) (Beckman Coulter PCS submicron particle size analyzer) with an angle detection at 90 . Transmission electron microscope (TEM) images were obtained from a JEOL JEM-2010 microscope (Japan). A conventional three-electrode system was used for all electrochemical measurements: a glassy carbon electrode (GC, 3 mm in diameter) as the working electrode, an Ag/AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode.
2. Materials and methods 2.1. Apparatus and reagents
2.2. Preparation of carboxyl functionalized poly(ethylene glycol)epoly(D,L-lactide) copolymer (HOOCePEGePLA)
D,L-lactide was purchased from SigmaeAldrich (USA) and recrystallized twice from ethyl acetate. Stannous (II) octoate (Sn(Oct)2), 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were obtained from Sinopharm Chemical Reagent Co., Ltd (China) and used as received. Hydrophobic Fe3O4 NPs were prepared by following the procedures reported by Sun et al. [24]. a-Hydroxy-u-carboxy poly(ethylene glycol) (HOePEGeCOOH) with a Mw of 3500 was purchased from Jenkem Technology Co., Ltd (China). Graphite was purchased from Shanghai Carbon Co., Ltd (China). Prostate specific antigen (PSA) and anti-PSA antibody were purchased from Dingguo Biochemical Reagents (Beijing, China). All
The HOOCePEGePLA diblock copolymer were synthesized by ring-opening polymerization in dry toluene under argon atmosphere according to the method adapted by Oliver et al. with slight modification [25]. Typically, 0.2 g of HOOCePEGeOH (Mw: 3000), 1.4 g of recrystallized D,L-lactide and Sn(Oct)2 (0.1% of D,L-lactide in molar amount) were added into a flame dried flask equipped with distillation unit under argon atmosphere. The polymerization was carried out under reflux at 130 C for 5 h. After cooling to room temperature, the copolymer was recovered by precipitation into the cold diethyl ether. HOOCePEGePLA was purified by re-dissolving in THF and precipitating in hexane for three times then dried under
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Fig. 1. NMR characterization of the HOOCePEGePLA copolymer. vacuum. The purified copolymer was characterized by gel permeation chromatography (GPC) and the 1H NMR spectroscopy. 2.3. Preparation of Ab2ePEGePLAeFe3O4 polymeric vesicles The Fe3O4-loaded polymeric vesicles were prepared using a double-emulsion method. Typically, 0.2 ml water solution was added dropwisely into 2 ml chloroform solution containing 10 mg HOOCePEGePLA and 10 mg Fe3O4 NPs under sonication on ice (30 s). This primary emulsion was emulsified by sonication on ice in 4 ml 0.1% PVA aqueous solution. Subsequently, the resulting w/o/w emulsion was diluted by mixing with 40 ml of 0.5% PVA aqueous solution under vigorous stirring. After 2 min, the chloroform solvent was evaporated using a rotary evaporator. The Fe3O4-loaded vesicles formed were then centrifuged; after discarding the supernatant, the vesicles were re-suspended in water solution. Then the pre-determined amount of EDC and NHS was added to the above solution. After activated for 30 min, the antibody solution was added and the mixture was allowed to react for 3 h at room temperature. After the reaction, the solution was dialyzed against water to remove the impurities. Finally, the vesicle solution was filtered with a 0.45 mm filter membrane to remove the large aggregates. Scheme 1A shows the procedure for the preparation of the Ab2ePEGePLAeFe3O4 polymeric vesicles. 2.4. Determination of the Fe3O4 loading contents The loading density of Fe3O4 NPs inside the polymeric vesicles was determined using an atomic absorption spectrophotometer (AAS). Briefly, the freeze-dried vesicles were weighed and then added to 1 M HCl solution to allow the disaggregation of vesicles and complete dissolution of the Fe3O4 NPs. Iron concentration was determined at the specific Fe absorption wavelength (248.3 nm) based on a previously established calibration curve. The Fe3O4 loading density was calculated as the ratio of iron oxide to the total weight of the vesicles.
for 12 h. Then, while immersing the reaction vessel in an ice bath, 30 g of KMnO4 was added slowly. After the addition of KMnO4, the solution was stirred at 100 C for another 12 h to fully oxidize graphite GO. The obtained GO was then thoroughly washed and dried. Thermal exfoliation of GO was achieved by placing GO (100 mg) into a quartz tube under argon atmosphere. The quartz tube was flushed with argon for 10 min, and then quickly inserted into a furnace preheated to 1000 C and held in the furnace for about 1 min. 2.6. Fabrication of the immunosensor Primary anti-PSA antibody (Ab1) was immobilized onto the surfaces of GS through an amidation reaction between the carboxylic acid groups attached to GS and the available amine groups of Ab1. Typically, into 1 mL of GS solution (2 mg/mL), EDC and NHS (100 mM) were added. The mixture was stirred for 4 h and after that, 1 mL of Ab1 solution (100 mg/mL) was added into the mixture. After another 12 h of reaction, the GS solution was centrifuged and washed. The resulting GS-Ab1 conjugates were stored at 4 C in phosphate buffer solution before use. Scheme 1B shows the fabrication procedure of the immunosensor. A glassy carbon electrode was polished repeatedly using alumina powder and then thoroughly cleaned before use. Onto the electrode, 5 mL of GS-Ab1 buffer solution was added. After the GS-Ab1 coated electrode was dried and washed with buffer, it was incubated in 1 wt.% BSA solution for 1 h to eliminate nonspecific binding between the antigen and the electrode surface. Subsequently, PSA buffer solution with a varying concentration was added onto the electrode surface and incubated for 1 h at room temperature, and then the electrode was washed extensively to remove unbounded PSA molecules. Finally, the prepared Ab2ePEGePLAeFe3O4 buffer solution was dropped onto the electrode surface and incubated for another 1 h. After washing, the electrode was ready for measurement. 2.7. Detection of PSA
2.5. Preparation of graphene sheet (GS) GS was prepared from graphite oxide (GO) through a thermal exfoliation method according to previous report [26]. At first, GO powders were produced from graphite by a modification of Hummer’s method [27]. In a typical experiment, 5 g of graphite was oxidized by reacting with 100 mL of concentrated H2SO4 under stirring
The pH 7.4 PBS buffer was used for all the electrochemical measurements. Cyclic voltammetry was recorded in PBS at 100 mV/s. For amperometric measurement of the immunosensor, a detection potential of 0.4 V was selected. After the background current was stabilized, 1 mM H2O2 was added into the buffer and the current change was recorded.
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Fig. 2. TEM images of (a) Fe3O4 NPs; (b) blank PEGePLA vesicles; (c) Ab2ePEGePLAeFe3O4 vesicles and (d) GS.
3. Results and discussion 3.1. Synthesis and characterization of HOOCePEGePLA diblock copolymer The copolymer was synthesized through ring-opening polymerization of D,L-lactide using HOOCePEGeOH as macroinitiator. The feed ratio was controlled to achieve the final copolymer composition (HOOCePEG3kePLA20.4k). The structure of copolymer was confirmed by 1H NMR (Fig. 1) and the length of the PLA block was calculated by comparing the integrals of the characteristic peaks of PEG (e.g., the singlet of eCH2eOe at 3.65 ppm) with that of the characteristic peaks of PLA (e.g., the multiplet of eCH (CH3)eOe at 5.2 ppm) in the 1H NMR spectroscopy. GPC measurements demonstrated the successful synthesis of the diblock copolymer by revealing a unimodal molecular weight distribution in the GPC chromatogram (data not shown). The absolute Mw and the polydispersity index (PDI, Mw/Mn) of the copolymer (HOOCePEG3kePLA20.4k) were 21,200 and 1.215 respectively, which were consistent with that determined by 1H NMR. 3.2. Characterization of the Ab2ePEGePLAeFe3O4 nanoparticles The Fe3O4-loaded polymeric vesicles coded with antibody, denoting Ab2ePEGePLAeFe3O4, were prepared by doubleemulsion method. Compared to conventional surfactant-based liposomes, polymer-based vesicles have much thicker membrane
walls, are more durable/robust, and offer the intriguing possibility of tunable physicochemical and biological properties by simply varying the block copolymer structure [28,29]. The thick membrane wall and aqueous core of polymeric vesicles make them very attractive for material loading purposes, which have been widely demonstrated for drug delivery. In this work, because of the high sensitivity of Fe3O4 NPs toward H2O2 detection, Fe3O4 NPs were selected and encapsulated into the PEGePLA polymeric vesicles [30e33]. With a large amount of Fe3O4 NPs (37 wt.%) loaded into the vesicles, the sensitivity of the resulting vesicles toward H2O2 could increased greatly. After the conjugation of Ab2 onto the vesicle surface, Ab2ePEGePLAeFe3O4 can be used as ideal label for the fabrication of electrochemical immunosensors. Fig. 2a shows the TEM image of Fe3O4 NPs with a uniform size distribution (6 nm). Fig. 2b and c show the morphology of blank and Fe3O4loaded vesicles respectively. Fig. 2b clearly demonstrates that the preparation procedure yielded spherical hollow vesicles with a hydrophobic membrane evidenced by the contrast. The mean diameter of the polymeric vesicles was about 220 nm Fig. 2c revealed that a large number of individual Fe3O4 NPs were incorporated uniformly into the hydrophobic membrane of the vesicles and the diameter of the Fe3O4-loaded vesicles was about 230 nm. These Fe3O4 NPs formed a cluster in the polymeric vesicle. Fig. 2d is the TEM image of the GS, which shows a paper-like structure with irregular size. The size and size distribution of the polymeric vesicles were further determined by DLS. The mean hydrodynamic diameter of blank vesicles was 231 3 nm (Fig. 3a), the polydispersity was 0.107 0.003; while the mean hydrodynamic
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diameter was 240 2 nm (Fig. 3b) and the polydispersity was 0.115 0.002 for Fe3O4-loaded polymeric vesicles. The slight increase in diameter for the Fe3O4-loaded vesicles compared with that of the blank polymeric vesicles may be attributed to the incorporation of a large amount of Fe3O4 NPs resulting from a favorable interaction between Fe3O4 NPs and the hydrophobic membrane of the vesicles.
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Fig. 4. Cyclic voltammograms of (A) PEGePLA and (B) PEGePLAeFe3O4 modified electrode in N2-saturated PBS without (a, black) and with (b, red) 4 mM H2O2.
through antibodyeantigen binding. Fig. 4A shows the Ab2ePEGePLA vesicles modified electrode in N2 saturated PBS in the absence and presence of H2O2. It can be seen that after the addition of 4 mM H2O2, only a very small current change was observed, which may be due to the response of the GC electrode. On the contrary, a dramatic increase of the reduction current was observed for Ab2ePEGePLAeFe3O4 modified electrode (Fig. 4B). The high sensitivity observed may be attributed to 1) a large amount of Fe3O4 NPs loaded into the vesicles; 2) the high catalytic activity of Fe3O4 NPs itself towards H2O2.
3.3. Characterization of the Ab2ePEGePLAeFe3O4 modified electrode
3.4. Performance of the immunosensor
Before using Ab2ePEGePLAeFe3O4 as labels for the preparation of immunosensors, we investigated the performance of Ab2ePEGePLAeFe3O4 for the detection of H2O2. For sandwich-type immunosensors, the sensitivity is mainly determined by the sensitivity of the label. In this work, the electrochemical signal of the immunosensor was mainly from the encapsulated Fe3O4 toward H2O2 reduction. Since a large number of Fe3O4 NPs was loaded into the vesicles as evidenced from Fig. 2c and AAS, and due to the intrinsic good catalytic activity of Fe3O4 NPs towards H2O2, we hypothesis the sensitivity of the immunosensor could be greatly enhanced when the vesicle was captured onto electrode surface
Since the high sensitivity of Ab2ePEGePLAeFe3O4 toward H2O2 detection has been demonstrated, immunosensors using Ab2ePEGePLAeFe3O4 as labels were built and characterized. Aside from the high amount of Fe3O4 NPs loaded into the PEGePLA vesicles, a relatively high amount of Ab2 was also conjugated onto the vesicles surface based on the carboxylic groups on the vesicle surface. Thus, when PSA was present on the electrode, the Ab2ePEGePLAeFe3O4 labels could be easily captured onto the electrode surface via the specific antibodyeantigen interaction and the amount of labels captured is in accordance with the PSA concentration.
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Fig. 5. Amperometric response of the immunosensor for detecting of different concentration of PSA at 0.4 V vs. Ag/AgCl toward addition of 1 mM H2O2 in N2saturated PBS. (a) 10, (b) 5, (c) 1, (d) 0.5, (e) 0.1 ng/mL.
To build the immunosensor, in this work, the Ab1 was immobilized onto GS surface through the carboxylic group on GS surface, which was subsequently coated onto the electrode surface. The large surface area of GS could increase the Ab1 loading and its good electrical conductivity could facilitate the transfer of electrons [34]. Fig. 5 shows the amperometric response of the immunosensor after reacted with different concentrations of PSA towards 1 mM H2O2. A detection potential of 0.4 V was selected and in N2 saturated PBS, it can be seen that the current change increased according with increase of PSA concentration, which suggested that different PSA concentration could be determined. The standard calibration curve for the PSA detection is shown in Fig. 6. The current response increased with the increase of PSA concentration in the range from 0.005 to 10 ng/mL, with a detection limit of 2 pg/mL based on S/N ¼ 3. The serum PSA concentration ranges from 1 to 4 ng/mL and 4 to 10 ng/mL for normal person and cancer patient, respectively, which fall in the linear range of this immunosensor [35]. The low detection limit may be attributed to three aspects: 1) the high loading level of Fe3O4 NPs into the
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vesicles increased the sensitivity of the vesicles to H2O2; 2) the large amount of Ab2 conjugated onto vesicles surface enhanced the access chance of the antibodyeantigen interaction, especially when the PSA concentration is very low; 3) the large surface area of GS increased the Ab1 onto electrode surface. The detection limit of this immunosensor is better than those of previously reported PSA immunosensors using labels based on HRP (1 ng/mL) [36], carbon nanotube-HRP (4 pg/mL) [37], or alkaline phosphatase encapsulated liposome (7 pg/mL) [38]. To further characterize the specificity of the immunosensor, we mixed the 1 ng/mL of PSA with 100 ng/mL human IgG, BSA, glucose and vitamin C, respectively and then detected the immunosensor response. In comparison with the amperometric response obtained from pure PSA, the current variation due to interference substances is less than 9%, indicating the good selectivity of the immunosensor and thus this immunosensor can be used for the detection of PSA in serum samples (Fig. 7). Since reproducibility is a very important feature for immunosensors, it was necessary to check it for the developed
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Fig. 7. Amperometric response of the immunosensor to 1 ng/mL PSA (1), 1 ng/mL PSA þ 100 ng/mL human IgG (2), 1 ng/mL PSA þ 100 ng/mL BSA (3), 1 ng/mL IgG þ 100 ng/mL glucose (4) and 1 ng/mL PSA þ 100 ng/mL vitamin C (5). Error bar ¼ RSD (n ¼ 5).
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Fig. 8. Compare of the PSA concentrations in serum samples determined with the proposed immunosensor and the ELISA method. The inset is a plot of the PSA concentration obtained by the two methods.
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immunosensors to confirm the reliability of this method. A series of five electrodes were prepared for detecting 1 ng/mL PSA. The relative standard deviation (RSD) of the five immunosensor was 6.8%, suggesting the precision and reproducibility of the immunosensor was quite good. The stability of the Ab2ePEGePLAeFe3O4 was studied. When not in use, the vesicles were stored in PBS at 4 C. After three weeks, the catalytic current of the immunosensor using these Ab2ePEGePLAeFe3O4 nanoparticles as labels retained about 91% of its initial value. The slow decrease in the current response may be due to the degradation of the PEGePLA vesicles. 3.5. Analysis of PSA in serum samples To investigate the possibility of this immunosensor for real sample analysis, the assay results of clinical serum samples using the proposed method were compared with standard ELISA methods. When the levels of tumor markers were over the calibration range, serum samples were diluted with phosphate buffer. A total of 10 serum samples were analyzed and the results are shown in Fig. 8. PSA contents determined by the two methods agreed well and the plot of PSA contents obtained by the two methods give a straight line with a correlation coefficient of 0.991 (Fig. 8, insert), indicating the reliability of the developed immunosensor. 4. Conclusions In summary, we have demonstrated a general and effective method based on encapsulation of Fe3O4 NPs into PEGePLA polymeric vesicles for the preparation of immuno-labels. Since the vesicles are robust, have advanced mechanical properties and hollow structure, a high Fe3O4 NPs loading (37 wt.%) was achieved which resulted the high sensitivity of the PEGePLAeFe3O4 toward H2O2. With the conjugation of a great amount of Ab2 onto the PEGePLAeFe3O4 vesicles surface, the Ab2ePEGePLAeFe3O4 was successfully used as label which has been demonstrated through the sensitive detection of PSA. In addition, because of the loading capacity of PEGePLA vesicles for various materials, this technique also has the potential to be extended to the loading of other interesting materials, such as quantum dot and fluorescence dyes for the preparation of various kinds of label systems to meet the different requirements in biochemical assays. Acknowledgements We are grateful for the support of the Natural Science Foundation of China (No. 20704027), the Natural Science Foundation of Shandong Province (Y2008B44), the Key Subject Research Foundation of Shandong Province (XTD0705), National High-Tech Project of China (863-Project, 2007AA021902) and the Science and Technology Research Project of Shandong Provincial Education Department (Grant No. J08LC54). Appendix Figures with essential color discrimination. Figs. 4, 8 and Scheme 1 in this article are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10. 1016/j.biomaterials.2010.06.014. References [1] Liu JH, Ju HX. Electrochemical and chemiluminescent immunosensors for tumor markers. Biosens Bioelectron 2005;20:1461e70.
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