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Magnetic nanoparticles and polydopamine amplified FP aptasensor for the highly sensitive detection of rHuEPO-α
Author’s Accepted Manuscript Magnetic nanoparticles and polydopamine amplified FP aptasensor for the highly sensitive detection of rHuEPO-α Zhu Chen, Hui Li, Yaju Zhao, Meng Xu, Danke Xu www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(18)30552-6 https://doi.org/10.1016/j.talanta.2018.05.061 TAL18702
To appear in: Talanta Received date: 15 March 2018 Revised date: 10 May 2018 Accepted date: 17 May 2018 Cite this article as: Zhu Chen, Hui Li, Yaju Zhao, Meng Xu and Danke Xu, Magnetic nanoparticles and polydopamine amplified FP aptasensor for the highly sensitive detection of rHuEPO-α, Talanta, https://doi.org/10.1016/j.talanta.2018.05.061 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 nanoparticles and polydopamine amplified FP aptasensor for the highly sensitive detection of rHuEPO-α Zhu Chen, Hui Li*, Yaju Zhao, Meng Xu, Danke Xu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210046, China
Corresponding Author Tel/Fax (+) 00862589685835 E-mail: [email protected];
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Abstract In this paper, an amplified fluorescence polarization (FP) aptasensor based on magnetic nanoparticles @ polydopamine (MNP@PDA) was innovatively developed for sensitive detection of recombinant human erythropoietin-alpha(rHuEPO-α). The amplified FP signal was due to the the large mass of protein and MNP@PDA. And this assay can be utilized for target separation or recycling based on the magnetic property of MNP@PDA through magnetic separation. Briefly, rHuEPO-α and MNP@PDA were added successively to react with the labeled aptamer (FAM-P1), which both contributed to the increase of FP signal via the formation of FAM-P1-rHuEPO-α and particularly FAM-P1-MNP@PDA complex. The strong interaction between MNP@PDA and FAM-P1 ensured the high efficiency of mass amplification and magnetic separation. As a result, the detection limit for rHuEPO-α was 0.12 pM, 4 orders of magnitude lower than original assay. Besides, three kinds of rHuEPO-α injections, NuPIAO, Epogen and ESPO were used to evaluate the selectivity of this assay in complex matrix with reasonable standard deviation. In a word, this work provides a simple, rapid, non-modified, highly sensitive and selective sensing platform for the detection of rHuEPO-α.
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Graphical Abstract
Keywords: aptamer; FP; rHuEPO-α; MNP@PDA; mass amplification; magnetic separation 1. Introduction Erythropoietin (EPO) primarily produced in kidney is one kind of glycoprotein hormones which participate in the regulation of red blood cell production[1]. EPO mainly functions by stimulating red blood cells to promote cell multiplication and differentiation with increasing red blood cells in a short time. In clinic, rHuEPO is produced in large scale to serve as the routine pharmaceutical for the treatment of chronic renal failure as well as for anemia with renal disease and cancer[2]. Generally, rHuEPO can be classified into two subtypes: rHuEPO-α and rHuEPO-β[3]. However, the majority of rHuEPO injections on the market are composed of rHuEPO-α, for example: NuPIAO, Epogen, ESPO, Aranesp and so on. Various rHuEPO-α injections are widely employed to treat patients associated with dialysis and radiation treatment caused by diabetes, cancers and so on. So it is very important to identify the activity of rHuEPO-α injections for better effect of injection treatment. The typical method for the detection of rHuEPO-α is based on antibody-assisted immunoassay. As recognition molecules for rHuEPO-α, its batch-to-batch reproducibility and specificity are no doubt needed to be improved [4]. Regarding simple synthesis, flexible modification, good stability and low cost, aptamer is a good alternate for antibody. Xie group[5] firstly screened a few aptamers of rHuEPO-α from a large random sequence pool through systematic evolution exponential enrichment(SELEX) process.
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Subsequently, Xie group[2, 4, 6] developed three fluorescent methods for the detection of rHuEPO-α and the optimized detection limit was 53.6 pM through a fluorescent off-on assay. And it is still urgent to develop simple and rapid methods for rHuEPO-α analysis , which will be of great value for clinical diagnosis and treatment. Regarding rHuEPO-α with larger molecular weight, FP is an effective method due to its high sensitivity to the rotational rate depending on the physical properties and local environment of the fluorophore.[7-9] However, direct FP method without any amplification exhibits limited signal and substantial background due to the relatively small size of the target and its aptamers[10]. To address the above limitations, various nanoparticles are utilized to amplify FP signal due to its easy synthesis, modifiable property and especially large mass[11-13]. Briefly, nanoparticle based FP methods were mainly divided into two categories: labeled and non-labeled assays. Generally, labeled assays require certain aptamer-probe modification on the surface of nanomaterials. For example, we[14] reported a novel type of bivalent aptasensor based on the modification of silver nanoparticles for the detection of lactoferrin in milk. Compared with labeled ones, non-labeled assays construct more simple and convenient methods for target detection without any modification. Graphene oxide(GO)[15-17], carbon nanotube(CNT)[18] and carbon nanoparticles(CNP)[19] were synthesized as quenchers for fluorophores, then were utilized to develop FP methods free of modification based on competitive assays. The design mechanism was due to the competitive binding with the probe between targets and quenchers. Huang group [15] used GO as a amplifier to detect small molecules by fluorescence anisotropy(FA) method and the mass of GO contributed to the increase of signal. Later, Liang group[18] introduced CNT as a mass amplifier into FP system. Besides, PDA with superior quenching ability was also an ideal quencher [20-21] for its outstanding ability to bind single strand DNA (ssDNA). And PDA was used for our following study. In this work, we have developed a sensitive FP aptasensor for rHuEPO-α detection based on its aptamer and MNP@PDA. To fabricate the amplified aptasensor, rHuEPO-α preferentially bound with FAM-P1, resulting in the increase of FP. Then upon adding MNP@PDA, redundant FAM-P1 was absorbed and quenched 97.15% by MNP@PDA in 3 min, causing the extreme increase of FP. It was proved that the mass amplification tremendously improved the sensitivity of this FP aptasensor. As a result, the obtained detection limit for rHuEPO-α was 0.12 pM, 4 orders of magnitude lower than original assay. Finally, three representative kinds of rHuEPO-α injections, NuPIAO, Epogen and ESPO, were used to evaluate the selectivity of this aptasensor and the analyzed results showed the superiority of this method again. 2. Materials and Methods 2.1.Materials and Reagents Dopamine hydrochloride(C8H11NO2·HCl), citric acid(C6H8O7)，ferric chloride, hexahydrate (FeCl3 • 6H2O), polyethylene glycol 20000(PEG 20000), ammonium bicarbonate (NH4HCO3), glycol ((CH2OH)2) were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). All active proteins including rHuEPO-α, immunoglobulin (lgG), hemoglobin (HB), human serum albumin (HSA), bovine serum albumin (BSA) and lysozyme were also obtained from Sangon. 20 mM Tris-HCl buffer (pH 7.4, containing 140 mM NaCl, 5mM MgCl2 and 5 mM KCl) was used in this experiment. The aptamer sequence[6] synthesized by Sangon is denoted as 3
FAM-P1(fluorescein isothiocyanate), 5’-FAM-GATTGAAAGGTCTGTTTTTGGGGTTGGTTTGGGTCAATA-3’. Actual samples of rHuEPO-α injection : NuPIAO produced by 3SBio Inc., Epogen produced by Amgen Co., Ltd., ESPO produced by Kyowa Hakko kirin China pharmaceutical Co., Ltd., were selected for actual application detection . 2.2. Apparatus FP signals and dynamic experiment were measured on BioTek (Synergy H1, U.S.A.). Excitation wavelength was set at 485 ± 20 nm, and emission wavelength was recorded at 528±20 nm. Scanning electron microscope (SEM) (S-4800, Hitachi, Japan) was used for collecting SEM images. A UV-3600 Spectrophotometer (Shimadzu) was used to obtain UV-Visible spectra. Dynamic light scattering was used to measure the size of nanoparticles. X-ray diffraction(XRD) and SEM coupled with energy dispersive spectrometer (EDS) were used to confirm the formation of MNP. Fourier transform infrared spectroscopy (FTIR) experiment was conducted for the information of functionalization between MNP and PDA. 2.3. Preparation of MNP@PDA The preparation of MNP@PDA nanoparticles was clarified as follows. In our experiment, MNP was synthesized via a simple hydrothermal reaction based on the high temperature reduction of Fe3+ salts with citric acid. Briefly, 1.62 g FeCl3•6H2O and 200 mg citric acid were dissolved in 80 mL glycol with vigorous ultrasonic for 20 min. Then, 5.0 g NH4HCO3 and 2.0 g PEG 2000 were subsequently added to the above mixture, accomplished by a lot of gas. A homogeneous solution was formed by stirring constantly for 30 min. Next, the yellowish-brown mixture was transferred into a autoclave to react at 200 ℃ for 8 h. The obtained brownish-black suspension was washed by water and ethanol for three times respectively. Finally, the synthesized MNP was dried at 60 ℃ for the following experiment. The encapsulated process was synthesized according to a reported method with some modifications [22] . Generally, 100 mg prepared MNP was dissolved into the Tris-HCl buffer (10 mM, pH 8.5) with vigorous ultrasonic for 10 min. Then, 100 mg dopamine hydrochloride was added with constant stirring. After stirring for 12 h, MNP@PDA was obtained. The suspension was centrifuged and washed/resuspended with water for three times. The precipitate was dried and stored at 4 ℃ for future use. The MNP@PDA nanoparticles was sonicated to form 10 mg/mL stock solutions for experimental use. 2.4. Competitive assay of amplified FP aptasensor For direct target detection, an aliquot of rHuEPO-α reacted with 100 nM FAM-P1 at 37 ℃ for 15 min under rapid shaking and FP signal was recorded in time. For MNP@PDA enhanced assay, FAM-P1 and the target were pre-incubated for 20 min. Then MNP@PDA was added into the above mixture and reacted without light for 15 min at 37 ℃ under rapid shaking. For target separation experiment, appropriate NaOH was added to destroy the conjugation condition of target with its aptamer. Then the matched magnet plate was used to attract MNP@PDA in solution to the bottom of 96-well plate and the supernatant was transferred into another well for recycling.
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2.5. Application of MNP@PDA assay in rHuEPO-α injections Three representative copies of rHuEPO-α injections, NuPIAO (10000 IU, 2.78 mM), Epogen (3000 IU, 0.83 mM) and ESPO (3000 IU, 0.83 mM), were used to test the performance of this aptasensor under complicated conditions. Primarily, rHuEPO-α injections were dissolved and diluted to a befitting level and processed by the above steps, incubated without light for 15 min at 37 °C under rapid and frequent shaking. Finally, the data of FP was collected and analyzed to evaluate the effectiveness of this assay. 3. Results and discussion 3.1. Preparation and characterization of MNP@PDA Core–shell MNP@PDA nanoparticles were synthesized by growing PDA layers onto the surface of MNP nanoparticles. Scheme 1(A) showed the construction strategy of MNP@PDA. The citric acid induced the reduction of Fe3+ to Fe2+. Then the obtained Fe3O4, also named MNP, was encapsulated by PDA through the self-polymerization of dopamine in the alkaline condition. To confirm the successful synthesis of core-shell structure based MNP@PDA, SEM was used to characterize the surface morphology of nanocomposite. As revealed by Fig 1(A and B), MNP@PDA nanoparticles with about 38 nm magnetic core and 16 nm PDA-based polymeric thin layer were obtained, measured by dynamic light scattering. Furthermore, from Figure 1(B), we knew that MNP@PDA nanoparticles showed larger size and smoother surface. Also, these core nanoparticles were homogenously coated with an amorphous layer, which could be ascribed to the stable formation of a PDA film on the surface of MNP. For the further confirmation of MNP formation, SEM coupled with EDS and XRD were used to characterize MNP. In EDS spectrum, the main elements including Fe, O and C all gave an obvious peaks in the element analysis diagram of MNP in Fig 1(C). The weaker peak of C was attributed to the interference of solvent. So it indicated that MNP was composed of O and Fe elements. Weight percentage（wt%） and atom percentage（at%） all revealed that there were approximately 3 Fe for every 4 O. No other elements were found, implying that the as-prepared MNP were highly pure. Besides, the position and intensity of seven characteristic peaks in XRD spectrum in Fig. 2(A) were almost unanimous with standard diffraction spectrum of Fe3O4 which showed diffraction peaks at 21.55, 30.15, 35.42, 43.12, 53.86, 57.31, and 62.80°[23]. So we can easily conclude that MNP was successfully synthesized via this simple hydrothermal reaction. To further confirm the proper coat of PDA, FTIR, zeta potenial and UV-Vis expeiments were conducted for the information of functionalization between MNP and PDA. FTIR experiment of MNP@PDA, showed in Fig 2(B), contained the characteristic peaks of PDA at the wavenumber of 3420 and 1630cm-1 [20] and Fe– O stretching modes of MNP at 578 cm-1[22]. lt’s no doubt to conclude that PDA was successfully coated on MNP. The zeta potentials of MNP, MNP@PDA were measured to be -19.1 ± 0.9 mV and 23.2 ± 2.2 mV respectively, exhibiting the successful modification of PDA on MNP. Besides, UV-Vis spectrum in Fig 2(C) showed the broad band absence of both MNP and MNP@PDA, which resulted in a spectral overlap with most fluorophores, thus demonstrating the distinct quenching ability. The latter, MNP@PDA, showed broader bond absence and significantly increased absence, about twice as much as MNP. It further served as a proof of the suitable coat of PDA on MNP and indicated the preferable quenching ability of MNP@PDA. Taken together, these facts all supported the successful preparation of core-shell MNP@PDA nanocomposite. The
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core structure, MNP, ensured the large mass and the shell, appropriate coating of PDA, exhibited good quenching property. These two properties of MNP@PDA ensured more promising application in FP aptasensors. 3.2. The sensing scheme of FP aptasensor Our design scheme was based on the noncovalent binding of MNP@PDA with ssDNA, which contributed to the quenching of FAM-P1 mainly through resonance energy transfer(FRET) [21] . The quenching ability of MNP@PDA was evaluated by scanning fluorescence emission spectra and dynamic experiment of FAM-P1 in the presence and absence of MNP@PDA. From Fig 3(A and B)，we can exactly know the outstanding quenching ability of MNP@PDA with up to 97.15% fluorescence quenching efficiency and it was worth noting that the quenching action reached equilibrium within 3 minutes. These two specificity were favorably comparable with PDA and superior than most quenchers[21], further exhibiting the excellent ability of MNP@PDA in binding ssDNA. Besides, compared with PDA, the obtained nanocomposite, MNP@PDA, possessed more promising application potential especially in FP assay due to its large mass. Above all, MNP@PDA is an ideal quencher platform due to the strong interaction with ssDNA. The scheme of the sensing platform was shown in Scheme 1(B). Briefly, FAM-P1exhibited low FP signal in a dependent state, denoted as FP0. Then certain rHuEPO-α was added and specifically bound with FAM-P1. In this system, FP signal was composed of FP1 and FP2, respectively caused by FAM-P1-rHuEPO-α complex and unbinding FAM-P1. FP1 exhibited increased signal due to lower rotation rate of FAM-P1-rHuEPO-α complex. Next, upon adding MNP@PDA, the strong interaction between redundant FAM-P1 and MNP@PDA extremely hindered the rotational rate of dye due to its extraordinary large mass. The formation of FAM-P1-MNP@PDA complex induced sharp increase of FP2, denoted as FP3. FP1 kept unaltered as FAM-P1 preferentially recognized rHuEPO-α and couldn’t be quenched by MNP@PDA any more. Overall, FP signal of these three systems exhibited following relationships: FP0 < FP1+FP2 << FP1+FP3. For MNP@PDA amplified system, increasing rHuEPO-α caused less MNP@PDA-FAM-P1 complex. And rapidly decreased FP signal was observed due to the reduced MNP@PDA-FAM-P1 complex. So FP signal and the concentration of rHuEPO-α showed a negative correlation while ΔFP exhibited a positive correlation with rHuEPO-α. Besides, we designed a target separation process based on the magnetic property of MNP@PDA. In target separation experiment, appropriate NaOH was added to destroy the binding condition of target with its aptamer. Then all probes dissociated from rHuEPO-α and were absorbed on the surface of MNP@PDA. Subsequently, the matched magnet plate was used to attract MNP@PDA in solution to the bottom of 96-well plate and the supernatant was transferred into another well. Finally, the process of target separation and recycle was realized by this way. 3.3.Optimization of the assay conditions. First, in order to quantitatively evaluate the effect of MNP@PDA on FAM-P1, we explored the optimized concentration of MNP@PDA. If the amount of MNP@PDA is too small, the quenching effect will not function fully in amplifying design. However, too much MNP@PDA would form precipitation and result in a poor output signal. With the concentration of FAM-P1 set as 100 nM, different concentrations of MNP@PDA (0, 0.05, 0.10, 0.20, 0.30 or 0.40 mg/mL) were 6
added separately into the system with subsequent records of fluorescence intensity and FP signal. Fig 4(A) showed the signal changes of fluorescence and FP along with the increased concentration of MNP@PDA. When the amount of MNP@PDA increased, both the fluorescence quenching and FP value exhibited a MNP@PDA-dependent manner, in which FP value was positively relevant while the fluorescent intensity was negatively relevant. It was clear that the fluorescent intensity changed sharply with less than 0.2 mg/mL MNP@PDA and remained unchanged after that point. Besides, FP value ensured an increasing trend with MNP@PDA, but changed slower after the point of 0.2 mg/mL MNP@PDA. So in short, 0.2 mg/mL MNP@PDA was chosen for the following experiments. In this amplifying design, a critical consideration that impacts on the rHuEPO-α/MNP@PDA competitive interactions with FAM-P1 is the pre-incubation time of the reaction. In Fig 4(B), FAM-P1 and rHuEPO-α were pre-incubated before adding MNP@PDA. FP signal of this system at different incubation time (0, 5, 10, 15 , 20 , 30, 40, 50, 60 min) were recorded to evaluate the optimal pre-reaction time. It was obvious that FP signal was unstable within 20 min due to the competitive reaction between rHuEPO-α and MNP@PDA with FAM-P1. Then FP signal reached an equilibrium situation after 20 min pre-incubation. Hence, the pre-incubation time for competitive binding was optimized to be 20 min. Besides, we chose 15 min as the reaction time of FAM-P1/protein/MNP@PDA system for fully competition between the protein and MNP@PDA and adequately quenching of FAM-P1. The study of reusability and stability of MNP@PDA on developed aptasensor were conducted to achieve the recycle of this assay. The reusable experiment was based on the magnetic property of MNP@PDA. MNP@PDA was added into the pre-incubated mixture of FAM-P1 and 10 ng/mL rHuEPO-α, then reacted without light for 15 min at 37 ℃ under rapid shaking. Afterwards, the matched magnet plate was used to attract MNP@PDA in solution to the bottom of 96-well plate and removed the supernatant. For second round, 10 ng/mL rHuEPO-α was added into the former well containing recycled MNP@PDA. Data in Fig 5(A) have showed the high recovery rate of MNP@PDA due to the magnetic separation. No doubt the recycled MNP@PDA can be used for 5 rounds without any signal lose. Besides, the stability of MNP@PDA was carried out through the comparison of FP signal between fresh MNP@PDA and MNP@PDA for 6 months, in 1 ng/mL, 10 ng/mL, 100 ng/mL rHuEPO-α system respectively. Fig 5(B) well exhibited the consistency of FP signal between two groups. So we concluded that MNP@PDA functioned well in 6 months at room temperature . The excellent reusability and stability of MNP@PDA further widen the depth and width of this aptasensor application. 3.4. Detection of rHuEPO-α by the sensing platform To further verify the feasibility of our design, 10 ng/mL and 500 ng/mL rHuEPO-α at different detecting systems were measured, analyzed in Fig 6(A). In FAM-P1 system, FP0 exhibited a extremely low signal. Upon adding 500 ng/mL rHuEPO-α, FP1+FP2 increased to a certain degree along with the formation of FAM-P1-rHuEPO-α. Subsequently, with the addition of MNP@PDA, FP1+FP3 was approximately enhanced by 5 times higher than FP0 and ΔFP was greatly enhanced by 10 times due to the mass amplification. While adding 10 ng/mL rHuEPO-α, FP1+FP2 was almost unchanged owing to the weaker detection limit of original assay. FP1+FP3 was enhanced by 11 times in MNP@PDA enhanced system. These phenomenon excellently
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proved the outstanding detection ability of amplified assay. Besides, Fig 6 (B) showed the fluorescent intensity change of this system with 10 ng/mL rHuEPO-α, which was consistent with the trend of FP signal. Taken together, all these phenomenon supported the feasibility of the sensing platform. To evaluate the sensitivity of this MNP@PDA-amplified FP method, various concentrations of rHuEPO-α were activated in probe solutions for a fixed reaction time respectively. Fig 7 (A and B) showed the excellent linear relationship between ΔFP response of original and amplified sensing system with the logarithm of rHuEPO-α concentrations. For original system, increasing proteins caused more FAM-P1-rHuEPO-α complex, and resulted in the increase of FP signal. Hence, ΔFP value and log C showed a positive correlation. The linear range was 156 ng/mL−25 μg/mL with the detection limit of 62.4 ng/mL(1.73 nM), R2 = 0.996. For amplified aptasensor, with increasing rHuEPO-α, more FAM-P1 bound to rHuEPO-α with rapid rotational rate. Then linearly decreased FP signal were observed to monitor the concentration of target and there was a positive correlation between ΔFP and log C in the range of 11 pg/mL− 8 μg/mL, R2 =0.999. On the basis of S/N >3, the detection limit was estimated to be 4 pg/mL (0.12 pM), which was about 4 orders of magnitude lower than the assay without amplification. Table 1 have listed the present detection methods and results for aptasensors of rHuEPO-α. Obviously, our aptasensor, which has the potential for the rapid and real-time monitoring of the target in homogeneous solution, exhibits excellent performance compared with reported methods. The selectivity of this amplified strategy was evaluated by four negative proteins: HB, HSA, lgG and lysozyme. These four negative proteins were tested as parallel controls with the standard procedure and the concentration of the negative proteins was 105 times as many as rHuEPO-α (10 ng/mL). Fig 7(C) showed that ΔFP response of negative proteins was almost unchanged compared with blank system while sharply changed upon the addition of rHuEPO-α. So the results demonstrated that the present MNP@PDA amplified aptasensor was highly selective to rHuEPO-α, which was promising to be applied in some complicated situations. 3.5. Quantitative detection of rHuEPO-α injections Three samples of rHuEPO-α injections: NuPIAO (10000 IU), Epogen (3000 IU) and ESPO (3000 IU) were chosen to verify the feasibility of this analytical method in complicated matrix. Three representative samples were diluted to 10 ng/mL and processed by this system respectively. Final results were substituted into the obtained linear formula to calculate the theoretical concentrations of three samples. It turned out that the theoretical concentrations of selected samples were consistent with their labeled concentrations. Also, relative standard deviations of the results were in a reasonable range under the optimal condition, listed in Table 2, which further validated the feasibility of proposed FP aptasensor in actual samples. 4. Conclusions In this work, we have designed a successful FP aptasensor for the detection of rHuEPO-α based on MNP@PDA. This method was simple, rapid, highly sensitive without any modification process. Due to the large mass of MNP@PDA, the amplified aptasensor was proved to be capable of detecting low concentrations of rHuEPO-α, up to 0.12 pM, which was 4 orders of magnitude lower than original assay. Moreover, selective recognition made the proposed assay an excellent
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strategy for the detection of rHuEPO-α in some complicated situations. In all, the designed approach substantially present a general sensing platform for biomolecules. Acknowledgement This work was supported financially by National Natural Foundation of China (Grant Nos. 21775068, 21475060, 21405077), Natural Science Foundation of Jiangsu Province (BK20140591). Reference [1] R. Abellan, R. Ventura, S. Pichini, A. F. Remacha, J. A. Pascual, R. Pacifici, R. D. Giovannandrea, P. Zuccaro, J. Segura, Erythropoietin (EPO) as an indirect biomarker of recombinant human EPO misuse in sport. J. Pharm. Biomed. Anal. 35(2004) 1169-1177. [2] R. Shen, L. Guo, Z. Y. Zhang, Q. W. Meng, J. W. Xie, Highly sensitive determination of recombinant human erythropoietin-α in aptamer-based affinity probe capillary electrophoresis with laser-induced fluorescence detection. J. Chromatogr., A. 1217（2011） 5635-5641. [3] W. E. Owen, W. L. Roberts, Pediatric reference intervals for thyroglobulin using the Beckman Coulter Access 2 immunoassay. Clin.Chim.Acta. 340（2014） 213-217. [4] J. F. Sun, A. T. Guo, Z. Y. Zhang, L. Guo, J. W. Xie, A Conjugated Aptamer-Gold Nanoparticle Fluorescent Probe for Highly Sensitive Detection of rHuEPO-α. Sensors. 11(2011) 10490-10501. [5] Z. Y. Zhang, L. Guo, A. T. Guo, H. Xu, J. J.Tang, X. J. Guo, J. W. Xie, In vitro lectin-mediated selection and characterization of rHuEPO-a-binding ssDNA aptamers. Bioorg. Med. Chem. 18(2010), 8016-8025. [6] J. F. Sun, L. Guo, H. Xu, J. J.Tang, J. W. Xie. Self-assembly of quantum dots/denatured BSA-oligonucleotides bioconjugate and its applicationon aptameric gold nanoparticles- based biosensor for the determination of rHuEPO-a. Biosens. Bioelectron. 43(2013), 446-452. [7] Z. Y. Zhang, C. Ravelet, S. Perrier, V. Guieu, E. Fiore, E. Peyrin, Single-Stranded DNA Binding Protein-Assisted Fluorescence Polarization Aptamer Assay for Detection of Small Molecules. Anal. Chem. 84(2012) 7203−7211. [8] G. Gokulrangan, J. R. Unruh, D. F. Holub, B. Ingram, C. K. Johnson, G. S. Wilson, DNA aptamer-based bioanalysis of IgE by fluorescence anisotropy. Anal. Chem. 77（2005） 1963−1970. [9] S. Perrier, C. Ravelet, V. Guieu, J. Fize, B. Roy, C. Perigaud, E. Peyrin, Rationally designed aptamer-based fluorescence polarization sensor dedicated to the small target analysis. 25(2010), 1652−1657. [10] H. K. Huang, J. Qin, K. Hu, X. Q. Liu, S. L. Zhao, Y. Huang, Novel autonomous protein-encoded aptamer nanomachines and isothermal exponential amplification for ultrasensitive fluorescence polarization sensing of small molecules. RSC Adv. 6(2016) 86043–86050. [11] D. Zhang, M. Lu, H. J. Wang, Fluorescence Anisotropy Analysis for Mapping Aptamer– Protein Interaction at the Single Nucleotide Level. Am. Chem. Soc.133(2011) 9188−9191. [12] Z. Y. Zhu, T. Schmidt, M. Mahrous, V. Guieu, S. Perrier, C. Ravelet, E. Peyrin, Optimization of the structure-switching aptamer-based fluorescence polarization assay for the sensitive tyrosinamide sensing. Anal. Chim. Acta 707(2011) 191−196.
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Scheme 1. The synthesis process of MNP@PDA (A). MNP@PDA amplified FP aptasensor for the rapid detection of rHuEPO-α (B).
Figure 1. SEM images of MNP (A) and MNP@PDA (B). (C)SEM coupled with EDS images of MNP.
Figure 2. XRD images of MNP (A). (B)FTIR spectra of MNP@PDA . (C) UV-Visible spectra of MNP@PDA (a) and MNP (b).
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Figure 3. (A) Fluorescence emission spectra of 200 nM FAM-P1 in the absence(a) and presence(b) of 0.4 mg/mL MNP@PDA. (B) Fluorescence quenching curves of 200 nM FAM-P1 in the presence of 0.4 mg/mL MNP@PDA as a function of time.
Figure 4. (A) Normalized FL intensity and FP signal of FAM-P1 upon the addition of different concentrations of [email protected] concentration of FAM-P1 was 100 nM. The concentration of PDANS was 0, 0.05, 0.10, 0.20, 0.30, and 0.40 mg/mL respectively. (B) The influence of the pre-incubation time between FAM-P1 and the target before adding MNP@PDA. The concentration of FAM-P1, rHuEPO-α and MNP@PDA was 100 nM, 10 ng/mL, and 0.20 mg/mL, respectively.
Figure 5. (A) The reusable study of MNP@PDA in this aptasensor. The concentration of FAM-P1, rHuEPO-α and MNP@PDA was 100 nM, 10 ng/mL, and 0.20 mg/mL, respectively. (B) The stability of MNP@PDA in this aptasensor. The concentration of FAM-P1 and MNP@PDA was 100 nM and 0.20 mg/mL, respectively.
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Figure 6. (A) FP signal of different sensing systems with 10 ng/mL or 500 ng/mL rHuEPO-α . (B) The fluorescence intensity of FAM-P1 (a) , FAM-P1/ MNP@PDA (c), FAM-P1/ MNP@PDA / rHuEPO-α(b). The concentration of FAM-P1, rHuEPO-α and MNP@PDA was 100 nM, 10 ng/mL, and 0.20 mg/mL, respectively.
Figure 7. The linear relationship between ΔFP and log C of rHuEPO-α, MNP@PDA amplified (A), original (B). (C)The selectivity of this assay with 10 ng/mL rHuEPO-α, 1mg/mL HB, HAS, lgG and lysozyme. Table 1. The comparison of rHuEPO-α analysis by different aptasensors
Method/technique
Linear range
Detection limit
Ref.
Aptamer affnity probe and capillary electrophoresis based fluorescence assay
0.2-100 nM
0.2 nM
2
Gold nanoparticles and CdTe QDs based fluorescence off-on assay
5-60 pM
53.6 pM
6
Aptamer-gold nanoparticles probe based fluorescence off-on assay
2-500 nM
0.92 nM
4
MNP@PDA based fluorescence Polarization assay
0.3 pM-222 nM
0.12 pM
\
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Table 2. Samples detection of actual rHuEPO-α injections Recombinant human erythropoietin-alpha injection samples
Labeled amount/vial
Detected amount/vial
NuPIAO from 3SBio Inc.
10000 IU
9972 ± 316 IU
Epogen from Amgen Co., Ltd.
3000 IU
3028 ± 74 IU
ESPO from Kyowa Hakko kirin China pharmaceutical Co., Ltd.
3000 IU
3069 ± 42 IU
Highlights: 1. A novel nanocomposite MNP@PDA was synthesized with 97.15% quenching efficiency for FAM in 3 min. 2. The enhanced FP signal was due to the mass amplification of MNP@PDA and rHuEPO-α. 3. Magnetic separation can be utilized for target separation or recycle based on the magnetic property of MNP@PDA. 4. The detection limit of this assay was 0.12 pM, and this assay can be used to detect rHuEPO-α in actual samples.
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PII: DOI: Reference:
S0039-9140(18)30552-6 https://doi.org/10.1016/j.talanta.2018.05.061 TAL18702
To appear in: Talanta Received date: 15 March 2018 Revised date: 10 May 2018 Accepted date: 17 May 2018 Cite this article as: Zhu Chen, Hui Li, Yaju Zhao, Meng Xu and Danke Xu, Magnetic nanoparticles and polydopamine amplified FP aptasensor for the highly sensitive detection of rHuEPO-α, Talanta, https://doi.org/10.1016/j.talanta.2018.05.061 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 nanoparticles and polydopamine amplified FP aptasensor for the highly sensitive detection of rHuEPO-α Zhu Chen, Hui Li*, Yaju Zhao, Meng Xu, Danke Xu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210046, China
Corresponding Author Tel/Fax (+) 00862589685835 E-mail: [email protected];
[email protected]
Abstract In this paper, an amplified fluorescence polarization (FP) aptasensor based on magnetic nanoparticles @ polydopamine (MNP@PDA) was innovatively developed for sensitive detection of recombinant human erythropoietin-alpha(rHuEPO-α). The amplified FP signal was due to the the large mass of protein and MNP@PDA. And this assay can be utilized for target separation or recycling based on the magnetic property of MNP@PDA through magnetic separation. Briefly, rHuEPO-α and MNP@PDA were added successively to react with the labeled aptamer (FAM-P1), which both contributed to the increase of FP signal via the formation of FAM-P1-rHuEPO-α and particularly FAM-P1-MNP@PDA complex. The strong interaction between MNP@PDA and FAM-P1 ensured the high efficiency of mass amplification and magnetic separation. As a result, the detection limit for rHuEPO-α was 0.12 pM, 4 orders of magnitude lower than original assay. Besides, three kinds of rHuEPO-α injections, NuPIAO, Epogen and ESPO were used to evaluate the selectivity of this assay in complex matrix with reasonable standard deviation. In a word, this work provides a simple, rapid, non-modified, highly sensitive and selective sensing platform for the detection of rHuEPO-α.
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Graphical Abstract
Keywords: aptamer; FP; rHuEPO-α; MNP@PDA; mass amplification; magnetic separation 1. Introduction Erythropoietin (EPO) primarily produced in kidney is one kind of glycoprotein hormones which participate in the regulation of red blood cell production[1]. EPO mainly functions by stimulating red blood cells to promote cell multiplication and differentiation with increasing red blood cells in a short time. In clinic, rHuEPO is produced in large scale to serve as the routine pharmaceutical for the treatment of chronic renal failure as well as for anemia with renal disease and cancer[2]. Generally, rHuEPO can be classified into two subtypes: rHuEPO-α and rHuEPO-β[3]. However, the majority of rHuEPO injections on the market are composed of rHuEPO-α, for example: NuPIAO, Epogen, ESPO, Aranesp and so on. Various rHuEPO-α injections are widely employed to treat patients associated with dialysis and radiation treatment caused by diabetes, cancers and so on. So it is very important to identify the activity of rHuEPO-α injections for better effect of injection treatment. The typical method for the detection of rHuEPO-α is based on antibody-assisted immunoassay. As recognition molecules for rHuEPO-α, its batch-to-batch reproducibility and specificity are no doubt needed to be improved [4]. Regarding simple synthesis, flexible modification, good stability and low cost, aptamer is a good alternate for antibody. Xie group[5] firstly screened a few aptamers of rHuEPO-α from a large random sequence pool through systematic evolution exponential enrichment(SELEX) process.
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Subsequently, Xie group[2, 4, 6] developed three fluorescent methods for the detection of rHuEPO-α and the optimized detection limit was 53.6 pM through a fluorescent off-on assay. And it is still urgent to develop simple and rapid methods for rHuEPO-α analysis , which will be of great value for clinical diagnosis and treatment. Regarding rHuEPO-α with larger molecular weight, FP is an effective method due to its high sensitivity to the rotational rate depending on the physical properties and local environment of the fluorophore.[7-9] However, direct FP method without any amplification exhibits limited signal and substantial background due to the relatively small size of the target and its aptamers[10]. To address the above limitations, various nanoparticles are utilized to amplify FP signal due to its easy synthesis, modifiable property and especially large mass[11-13]. Briefly, nanoparticle based FP methods were mainly divided into two categories: labeled and non-labeled assays. Generally, labeled assays require certain aptamer-probe modification on the surface of nanomaterials. For example, we[14] reported a novel type of bivalent aptasensor based on the modification of silver nanoparticles for the detection of lactoferrin in milk. Compared with labeled ones, non-labeled assays construct more simple and convenient methods for target detection without any modification. Graphene oxide(GO)[15-17], carbon nanotube(CNT)[18] and carbon nanoparticles(CNP)[19] were synthesized as quenchers for fluorophores, then were utilized to develop FP methods free of modification based on competitive assays. The design mechanism was due to the competitive binding with the probe between targets and quenchers. Huang group [15] used GO as a amplifier to detect small molecules by fluorescence anisotropy(FA) method and the mass of GO contributed to the increase of signal. Later, Liang group[18] introduced CNT as a mass amplifier into FP system. Besides, PDA with superior quenching ability was also an ideal quencher [20-21] for its outstanding ability to bind single strand DNA (ssDNA). And PDA was used for our following study. In this work, we have developed a sensitive FP aptasensor for rHuEPO-α detection based on its aptamer and MNP@PDA. To fabricate the amplified aptasensor, rHuEPO-α preferentially bound with FAM-P1, resulting in the increase of FP. Then upon adding MNP@PDA, redundant FAM-P1 was absorbed and quenched 97.15% by MNP@PDA in 3 min, causing the extreme increase of FP. It was proved that the mass amplification tremendously improved the sensitivity of this FP aptasensor. As a result, the obtained detection limit for rHuEPO-α was 0.12 pM, 4 orders of magnitude lower than original assay. Finally, three representative kinds of rHuEPO-α injections, NuPIAO, Epogen and ESPO, were used to evaluate the selectivity of this aptasensor and the analyzed results showed the superiority of this method again. 2. Materials and Methods 2.1.Materials and Reagents Dopamine hydrochloride(C8H11NO2·HCl), citric acid(C6H8O7)，ferric chloride, hexahydrate (FeCl3 • 6H2O), polyethylene glycol 20000(PEG 20000), ammonium bicarbonate (NH4HCO3), glycol ((CH2OH)2) were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). All active proteins including rHuEPO-α, immunoglobulin (lgG), hemoglobin (HB), human serum albumin (HSA), bovine serum albumin (BSA) and lysozyme were also obtained from Sangon. 20 mM Tris-HCl buffer (pH 7.4, containing 140 mM NaCl, 5mM MgCl2 and 5 mM KCl) was used in this experiment. The aptamer sequence[6] synthesized by Sangon is denoted as 3
FAM-P1(fluorescein isothiocyanate), 5’-FAM-GATTGAAAGGTCTGTTTTTGGGGTTGGTTTGGGTCAATA-3’. Actual samples of rHuEPO-α injection : NuPIAO produced by 3SBio Inc., Epogen produced by Amgen Co., Ltd., ESPO produced by Kyowa Hakko kirin China pharmaceutical Co., Ltd., were selected for actual application detection . 2.2. Apparatus FP signals and dynamic experiment were measured on BioTek (Synergy H1, U.S.A.). Excitation wavelength was set at 485 ± 20 nm, and emission wavelength was recorded at 528±20 nm. Scanning electron microscope (SEM) (S-4800, Hitachi, Japan) was used for collecting SEM images. A UV-3600 Spectrophotometer (Shimadzu) was used to obtain UV-Visible spectra. Dynamic light scattering was used to measure the size of nanoparticles. X-ray diffraction(XRD) and SEM coupled with energy dispersive spectrometer (EDS) were used to confirm the formation of MNP. Fourier transform infrared spectroscopy (FTIR) experiment was conducted for the information of functionalization between MNP and PDA. 2.3. Preparation of MNP@PDA The preparation of MNP@PDA nanoparticles was clarified as follows. In our experiment, MNP was synthesized via a simple hydrothermal reaction based on the high temperature reduction of Fe3+ salts with citric acid. Briefly, 1.62 g FeCl3•6H2O and 200 mg citric acid were dissolved in 80 mL glycol with vigorous ultrasonic for 20 min. Then, 5.0 g NH4HCO3 and 2.0 g PEG 2000 were subsequently added to the above mixture, accomplished by a lot of gas. A homogeneous solution was formed by stirring constantly for 30 min. Next, the yellowish-brown mixture was transferred into a autoclave to react at 200 ℃ for 8 h. The obtained brownish-black suspension was washed by water and ethanol for three times respectively. Finally, the synthesized MNP was dried at 60 ℃ for the following experiment. The encapsulated process was synthesized according to a reported method with some modifications [22] . Generally, 100 mg prepared MNP was dissolved into the Tris-HCl buffer (10 mM, pH 8.5) with vigorous ultrasonic for 10 min. Then, 100 mg dopamine hydrochloride was added with constant stirring. After stirring for 12 h, MNP@PDA was obtained. The suspension was centrifuged and washed/resuspended with water for three times. The precipitate was dried and stored at 4 ℃ for future use. The MNP@PDA nanoparticles was sonicated to form 10 mg/mL stock solutions for experimental use. 2.4. Competitive assay of amplified FP aptasensor For direct target detection, an aliquot of rHuEPO-α reacted with 100 nM FAM-P1 at 37 ℃ for 15 min under rapid shaking and FP signal was recorded in time. For MNP@PDA enhanced assay, FAM-P1 and the target were pre-incubated for 20 min. Then MNP@PDA was added into the above mixture and reacted without light for 15 min at 37 ℃ under rapid shaking. For target separation experiment, appropriate NaOH was added to destroy the conjugation condition of target with its aptamer. Then the matched magnet plate was used to attract MNP@PDA in solution to the bottom of 96-well plate and the supernatant was transferred into another well for recycling.
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2.5. Application of MNP@PDA assay in rHuEPO-α injections Three representative copies of rHuEPO-α injections, NuPIAO (10000 IU, 2.78 mM), Epogen (3000 IU, 0.83 mM) and ESPO (3000 IU, 0.83 mM), were used to test the performance of this aptasensor under complicated conditions. Primarily, rHuEPO-α injections were dissolved and diluted to a befitting level and processed by the above steps, incubated without light for 15 min at 37 °C under rapid and frequent shaking. Finally, the data of FP was collected and analyzed to evaluate the effectiveness of this assay. 3. Results and discussion 3.1. Preparation and characterization of MNP@PDA Core–shell MNP@PDA nanoparticles were synthesized by growing PDA layers onto the surface of MNP nanoparticles. Scheme 1(A) showed the construction strategy of MNP@PDA. The citric acid induced the reduction of Fe3+ to Fe2+. Then the obtained Fe3O4, also named MNP, was encapsulated by PDA through the self-polymerization of dopamine in the alkaline condition. To confirm the successful synthesis of core-shell structure based MNP@PDA, SEM was used to characterize the surface morphology of nanocomposite. As revealed by Fig 1(A and B), MNP@PDA nanoparticles with about 38 nm magnetic core and 16 nm PDA-based polymeric thin layer were obtained, measured by dynamic light scattering. Furthermore, from Figure 1(B), we knew that MNP@PDA nanoparticles showed larger size and smoother surface. Also, these core nanoparticles were homogenously coated with an amorphous layer, which could be ascribed to the stable formation of a PDA film on the surface of MNP. For the further confirmation of MNP formation, SEM coupled with EDS and XRD were used to characterize MNP. In EDS spectrum, the main elements including Fe, O and C all gave an obvious peaks in the element analysis diagram of MNP in Fig 1(C). The weaker peak of C was attributed to the interference of solvent. So it indicated that MNP was composed of O and Fe elements. Weight percentage（wt%） and atom percentage（at%） all revealed that there were approximately 3 Fe for every 4 O. No other elements were found, implying that the as-prepared MNP were highly pure. Besides, the position and intensity of seven characteristic peaks in XRD spectrum in Fig. 2(A) were almost unanimous with standard diffraction spectrum of Fe3O4 which showed diffraction peaks at 21.55, 30.15, 35.42, 43.12, 53.86, 57.31, and 62.80°[23]. So we can easily conclude that MNP was successfully synthesized via this simple hydrothermal reaction. To further confirm the proper coat of PDA, FTIR, zeta potenial and UV-Vis expeiments were conducted for the information of functionalization between MNP and PDA. FTIR experiment of MNP@PDA, showed in Fig 2(B), contained the characteristic peaks of PDA at the wavenumber of 3420 and 1630cm-1 [20] and Fe– O stretching modes of MNP at 578 cm-1[22]. lt’s no doubt to conclude that PDA was successfully coated on MNP. The zeta potentials of MNP, MNP@PDA were measured to be -19.1 ± 0.9 mV and 23.2 ± 2.2 mV respectively, exhibiting the successful modification of PDA on MNP. Besides, UV-Vis spectrum in Fig 2(C) showed the broad band absence of both MNP and MNP@PDA, which resulted in a spectral overlap with most fluorophores, thus demonstrating the distinct quenching ability. The latter, MNP@PDA, showed broader bond absence and significantly increased absence, about twice as much as MNP. It further served as a proof of the suitable coat of PDA on MNP and indicated the preferable quenching ability of MNP@PDA. Taken together, these facts all supported the successful preparation of core-shell MNP@PDA nanocomposite. The
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core structure, MNP, ensured the large mass and the shell, appropriate coating of PDA, exhibited good quenching property. These two properties of MNP@PDA ensured more promising application in FP aptasensors. 3.2. The sensing scheme of FP aptasensor Our design scheme was based on the noncovalent binding of MNP@PDA with ssDNA, which contributed to the quenching of FAM-P1 mainly through resonance energy transfer(FRET) [21] . The quenching ability of MNP@PDA was evaluated by scanning fluorescence emission spectra and dynamic experiment of FAM-P1 in the presence and absence of MNP@PDA. From Fig 3(A and B)，we can exactly know the outstanding quenching ability of MNP@PDA with up to 97.15% fluorescence quenching efficiency and it was worth noting that the quenching action reached equilibrium within 3 minutes. These two specificity were favorably comparable with PDA and superior than most quenchers[21], further exhibiting the excellent ability of MNP@PDA in binding ssDNA. Besides, compared with PDA, the obtained nanocomposite, MNP@PDA, possessed more promising application potential especially in FP assay due to its large mass. Above all, MNP@PDA is an ideal quencher platform due to the strong interaction with ssDNA. The scheme of the sensing platform was shown in Scheme 1(B). Briefly, FAM-P1exhibited low FP signal in a dependent state, denoted as FP0. Then certain rHuEPO-α was added and specifically bound with FAM-P1. In this system, FP signal was composed of FP1 and FP2, respectively caused by FAM-P1-rHuEPO-α complex and unbinding FAM-P1. FP1 exhibited increased signal due to lower rotation rate of FAM-P1-rHuEPO-α complex. Next, upon adding MNP@PDA, the strong interaction between redundant FAM-P1 and MNP@PDA extremely hindered the rotational rate of dye due to its extraordinary large mass. The formation of FAM-P1-MNP@PDA complex induced sharp increase of FP2, denoted as FP3. FP1 kept unaltered as FAM-P1 preferentially recognized rHuEPO-α and couldn’t be quenched by MNP@PDA any more. Overall, FP signal of these three systems exhibited following relationships: FP0 < FP1+FP2 << FP1+FP3. For MNP@PDA amplified system, increasing rHuEPO-α caused less MNP@PDA-FAM-P1 complex. And rapidly decreased FP signal was observed due to the reduced MNP@PDA-FAM-P1 complex. So FP signal and the concentration of rHuEPO-α showed a negative correlation while ΔFP exhibited a positive correlation with rHuEPO-α. Besides, we designed a target separation process based on the magnetic property of MNP@PDA. In target separation experiment, appropriate NaOH was added to destroy the binding condition of target with its aptamer. Then all probes dissociated from rHuEPO-α and were absorbed on the surface of MNP@PDA. Subsequently, the matched magnet plate was used to attract MNP@PDA in solution to the bottom of 96-well plate and the supernatant was transferred into another well. Finally, the process of target separation and recycle was realized by this way. 3.3.Optimization of the assay conditions. First, in order to quantitatively evaluate the effect of MNP@PDA on FAM-P1, we explored the optimized concentration of MNP@PDA. If the amount of MNP@PDA is too small, the quenching effect will not function fully in amplifying design. However, too much MNP@PDA would form precipitation and result in a poor output signal. With the concentration of FAM-P1 set as 100 nM, different concentrations of MNP@PDA (0, 0.05, 0.10, 0.20, 0.30 or 0.40 mg/mL) were 6
added separately into the system with subsequent records of fluorescence intensity and FP signal. Fig 4(A) showed the signal changes of fluorescence and FP along with the increased concentration of MNP@PDA. When the amount of MNP@PDA increased, both the fluorescence quenching and FP value exhibited a MNP@PDA-dependent manner, in which FP value was positively relevant while the fluorescent intensity was negatively relevant. It was clear that the fluorescent intensity changed sharply with less than 0.2 mg/mL MNP@PDA and remained unchanged after that point. Besides, FP value ensured an increasing trend with MNP@PDA, but changed slower after the point of 0.2 mg/mL MNP@PDA. So in short, 0.2 mg/mL MNP@PDA was chosen for the following experiments. In this amplifying design, a critical consideration that impacts on the rHuEPO-α/MNP@PDA competitive interactions with FAM-P1 is the pre-incubation time of the reaction. In Fig 4(B), FAM-P1 and rHuEPO-α were pre-incubated before adding MNP@PDA. FP signal of this system at different incubation time (0, 5, 10, 15 , 20 , 30, 40, 50, 60 min) were recorded to evaluate the optimal pre-reaction time. It was obvious that FP signal was unstable within 20 min due to the competitive reaction between rHuEPO-α and MNP@PDA with FAM-P1. Then FP signal reached an equilibrium situation after 20 min pre-incubation. Hence, the pre-incubation time for competitive binding was optimized to be 20 min. Besides, we chose 15 min as the reaction time of FAM-P1/protein/MNP@PDA system for fully competition between the protein and MNP@PDA and adequately quenching of FAM-P1. The study of reusability and stability of MNP@PDA on developed aptasensor were conducted to achieve the recycle of this assay. The reusable experiment was based on the magnetic property of MNP@PDA. MNP@PDA was added into the pre-incubated mixture of FAM-P1 and 10 ng/mL rHuEPO-α, then reacted without light for 15 min at 37 ℃ under rapid shaking. Afterwards, the matched magnet plate was used to attract MNP@PDA in solution to the bottom of 96-well plate and removed the supernatant. For second round, 10 ng/mL rHuEPO-α was added into the former well containing recycled MNP@PDA. Data in Fig 5(A) have showed the high recovery rate of MNP@PDA due to the magnetic separation. No doubt the recycled MNP@PDA can be used for 5 rounds without any signal lose. Besides, the stability of MNP@PDA was carried out through the comparison of FP signal between fresh MNP@PDA and MNP@PDA for 6 months, in 1 ng/mL, 10 ng/mL, 100 ng/mL rHuEPO-α system respectively. Fig 5(B) well exhibited the consistency of FP signal between two groups. So we concluded that MNP@PDA functioned well in 6 months at room temperature . The excellent reusability and stability of MNP@PDA further widen the depth and width of this aptasensor application. 3.4. Detection of rHuEPO-α by the sensing platform To further verify the feasibility of our design, 10 ng/mL and 500 ng/mL rHuEPO-α at different detecting systems were measured, analyzed in Fig 6(A). In FAM-P1 system, FP0 exhibited a extremely low signal. Upon adding 500 ng/mL rHuEPO-α, FP1+FP2 increased to a certain degree along with the formation of FAM-P1-rHuEPO-α. Subsequently, with the addition of MNP@PDA, FP1+FP3 was approximately enhanced by 5 times higher than FP0 and ΔFP was greatly enhanced by 10 times due to the mass amplification. While adding 10 ng/mL rHuEPO-α, FP1+FP2 was almost unchanged owing to the weaker detection limit of original assay. FP1+FP3 was enhanced by 11 times in MNP@PDA enhanced system. These phenomenon excellently
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proved the outstanding detection ability of amplified assay. Besides, Fig 6 (B) showed the fluorescent intensity change of this system with 10 ng/mL rHuEPO-α, which was consistent with the trend of FP signal. Taken together, all these phenomenon supported the feasibility of the sensing platform. To evaluate the sensitivity of this MNP@PDA-amplified FP method, various concentrations of rHuEPO-α were activated in probe solutions for a fixed reaction time respectively. Fig 7 (A and B) showed the excellent linear relationship between ΔFP response of original and amplified sensing system with the logarithm of rHuEPO-α concentrations. For original system, increasing proteins caused more FAM-P1-rHuEPO-α complex, and resulted in the increase of FP signal. Hence, ΔFP value and log C showed a positive correlation. The linear range was 156 ng/mL−25 μg/mL with the detection limit of 62.4 ng/mL(1.73 nM), R2 = 0.996. For amplified aptasensor, with increasing rHuEPO-α, more FAM-P1 bound to rHuEPO-α with rapid rotational rate. Then linearly decreased FP signal were observed to monitor the concentration of target and there was a positive correlation between ΔFP and log C in the range of 11 pg/mL− 8 μg/mL, R2 =0.999. On the basis of S/N >3, the detection limit was estimated to be 4 pg/mL (0.12 pM), which was about 4 orders of magnitude lower than the assay without amplification. Table 1 have listed the present detection methods and results for aptasensors of rHuEPO-α. Obviously, our aptasensor, which has the potential for the rapid and real-time monitoring of the target in homogeneous solution, exhibits excellent performance compared with reported methods. The selectivity of this amplified strategy was evaluated by four negative proteins: HB, HSA, lgG and lysozyme. These four negative proteins were tested as parallel controls with the standard procedure and the concentration of the negative proteins was 105 times as many as rHuEPO-α (10 ng/mL). Fig 7(C) showed that ΔFP response of negative proteins was almost unchanged compared with blank system while sharply changed upon the addition of rHuEPO-α. So the results demonstrated that the present MNP@PDA amplified aptasensor was highly selective to rHuEPO-α, which was promising to be applied in some complicated situations. 3.5. Quantitative detection of rHuEPO-α injections Three samples of rHuEPO-α injections: NuPIAO (10000 IU), Epogen (3000 IU) and ESPO (3000 IU) were chosen to verify the feasibility of this analytical method in complicated matrix. Three representative samples were diluted to 10 ng/mL and processed by this system respectively. Final results were substituted into the obtained linear formula to calculate the theoretical concentrations of three samples. It turned out that the theoretical concentrations of selected samples were consistent with their labeled concentrations. Also, relative standard deviations of the results were in a reasonable range under the optimal condition, listed in Table 2, which further validated the feasibility of proposed FP aptasensor in actual samples. 4. Conclusions In this work, we have designed a successful FP aptasensor for the detection of rHuEPO-α based on MNP@PDA. This method was simple, rapid, highly sensitive without any modification process. Due to the large mass of MNP@PDA, the amplified aptasensor was proved to be capable of detecting low concentrations of rHuEPO-α, up to 0.12 pM, which was 4 orders of magnitude lower than original assay. Moreover, selective recognition made the proposed assay an excellent
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strategy for the detection of rHuEPO-α in some complicated situations. In all, the designed approach substantially present a general sensing platform for biomolecules. Acknowledgement This work was supported financially by National Natural Foundation of China (Grant Nos. 21775068, 21475060, 21405077), Natural Science Foundation of Jiangsu Province (BK20140591). Reference [1] R. Abellan, R. Ventura, S. Pichini, A. F. Remacha, J. A. Pascual, R. Pacifici, R. D. Giovannandrea, P. Zuccaro, J. Segura, Erythropoietin (EPO) as an indirect biomarker of recombinant human EPO misuse in sport. J. Pharm. Biomed. Anal. 35(2004) 1169-1177. [2] R. Shen, L. Guo, Z. Y. Zhang, Q. W. Meng, J. W. Xie, Highly sensitive determination of recombinant human erythropoietin-α in aptamer-based affinity probe capillary electrophoresis with laser-induced fluorescence detection. J. Chromatogr., A. 1217（2011） 5635-5641. [3] W. E. Owen, W. L. Roberts, Pediatric reference intervals for thyroglobulin using the Beckman Coulter Access 2 immunoassay. Clin.Chim.Acta. 340（2014） 213-217. [4] J. F. Sun, A. T. Guo, Z. Y. Zhang, L. Guo, J. W. Xie, A Conjugated Aptamer-Gold Nanoparticle Fluorescent Probe for Highly Sensitive Detection of rHuEPO-α. Sensors. 11(2011) 10490-10501. [5] Z. Y. Zhang, L. Guo, A. T. Guo, H. Xu, J. J.Tang, X. J. Guo, J. W. Xie, In vitro lectin-mediated selection and characterization of rHuEPO-a-binding ssDNA aptamers. Bioorg. Med. Chem. 18(2010), 8016-8025. [6] J. F. Sun, L. Guo, H. Xu, J. J.Tang, J. W. Xie. Self-assembly of quantum dots/denatured BSA-oligonucleotides bioconjugate and its applicationon aptameric gold nanoparticles- based biosensor for the determination of rHuEPO-a. Biosens. Bioelectron. 43(2013), 446-452. [7] Z. Y. Zhang, C. Ravelet, S. Perrier, V. Guieu, E. Fiore, E. Peyrin, Single-Stranded DNA Binding Protein-Assisted Fluorescence Polarization Aptamer Assay for Detection of Small Molecules. Anal. Chem. 84(2012) 7203−7211. [8] G. Gokulrangan, J. R. Unruh, D. F. Holub, B. Ingram, C. K. Johnson, G. S. Wilson, DNA aptamer-based bioanalysis of IgE by fluorescence anisotropy. Anal. Chem. 77（2005） 1963−1970. [9] S. Perrier, C. Ravelet, V. Guieu, J. Fize, B. Roy, C. Perigaud, E. Peyrin, Rationally designed aptamer-based fluorescence polarization sensor dedicated to the small target analysis. 25(2010), 1652−1657. [10] H. K. Huang, J. Qin, K. Hu, X. Q. Liu, S. L. Zhao, Y. Huang, Novel autonomous protein-encoded aptamer nanomachines and isothermal exponential amplification for ultrasensitive fluorescence polarization sensing of small molecules. RSC Adv. 6(2016) 86043–86050. [11] D. Zhang, M. Lu, H. J. Wang, Fluorescence Anisotropy Analysis for Mapping Aptamer– Protein Interaction at the Single Nucleotide Level. Am. Chem. Soc.133(2011) 9188−9191. [12] Z. Y. Zhu, T. Schmidt, M. Mahrous, V. Guieu, S. Perrier, C. Ravelet, E. Peyrin, Optimization of the structure-switching aptamer-based fluorescence polarization assay for the sensitive tyrosinamide sensing. Anal. Chim. Acta 707(2011) 191−196.
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[13] Y. Huang, X. Q. Liu, H. K. Huang, J. Qin, L. Zhang, S. Zhao, Z.-F. Chen, H. Liang, Attomolar Detection of Proteins via Cascade Strand-Displacement Amplification and Polystyrene Nanoparticle Enhancement in Fluorescence Polarization Aptasensors. Anal. Chem. 87(2015) 8107−8114. [14] Z. Chen, H. Li, W. C. Jia, X. H. Liu, Z. M. Li, F. Wen, N. Zheng, J. D. Jiang, D. K. Xu, Bivalent Aptasensor Based on Silver-Enhanced Fluorescence Polarization for Rapid Detection of Lactoferrin in Milk. Anal. Chem. 89 (2017) 5900-5908. [15] Y. Huang, X. Q. Liu, L. L. Zhang, K. Hu, S. Zhao, B. Fang, Z.-F. Chen,H. Liang, Nicking enzyme and graphene oxide-based dual signal amplification for ultrasensitive aptamer-based fluorescence polarization assays. Biosens. Bioelectron. 63(2015), 178−184. [16] Y. Yu,, Y. Liu,, S. J. Zhen, C. Z. Huang, A graphene oxide enhanced fluorescence anisotropy strategy for DNAzyme-based assay of metal ions. Chem. Commun. 49(2013) 1942-1944. [17] J. H. Liu, C. Y. Wang, Y. Jiang, Y. P. Hu, J. S. Li, S. Yang, Y. H. Li, R. H. Yang, W. H. Tan, C. Z. Huang, Graphene Signal Amplification for Sensitive and Real-Time Fluorescence Anisotropy Detection of Small Molecules. Anal. Chem. 85(2013) 1424-1430. [18] Y. Huang, M. Shi,, L. M. Zhao, S. L. Zhao, K. Hu, Z. F. Chen, Carbon nanotube signal amplification forultrasensitive fluorescence polarization detection of DNA methyltransferase activity and inhibition. Biosens. Bioelectron. 54(2014), 285-291. [19] J. H. Liu, J. Yu, J. R. Chen, R. H. Yang, K. M. Shih, Signal-amplification and real-time fluorescence anisotropy detection of apyrase by carbon nanoparticle. Materials Science and Engineering. 38(2014) 201-211. [20] W. B. Qiang, W. Li, X. Q. Li, X. Chen, D. K. Xu, Bioinspired polydopamine nanospheres: a superquencher for fluorescence sensing of Biomolecules. Chem. Sci. 5(2014) 3018-3024. [21] W. B. Qiang, X. Wang, W. Li,X. Chen, H. Li, D. K. Xu, A fluorescent biosensing platform based on the polydopamine nano-Spheres intergrating with Exonuclease III-assisted target recycling Amplification. Biosens. Bioelectron. 71(2015) 143-149. [22] Y. Zhao, Y. W.Yeh, R. Liu, J. M. You. F. L. Qu, Facile deposition of gold nanoparticles on core-shell Fe3O4@polydopamine as recyclable nanocatalyst. Solid. State. Sci. 45(2015) 9-14. [23] L.Y. Wang·G.Q. Gai·X. F.Xiao·S. Z. Gao, Fabrication of magnetic-luminescent bifunctional composite nanofibers via facile electrospinning. J Mater Sci: Mater Electron. 25(2014) 3147– 3153.
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Scheme 1. The synthesis process of MNP@PDA (A). MNP@PDA amplified FP aptasensor for the rapid detection of rHuEPO-α (B).
Figure 1. SEM images of MNP (A) and MNP@PDA (B). (C)SEM coupled with EDS images of MNP.
Figure 2. XRD images of MNP (A). (B)FTIR spectra of MNP@PDA . (C) UV-Visible spectra of MNP@PDA (a) and MNP (b).
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Figure 3. (A) Fluorescence emission spectra of 200 nM FAM-P1 in the absence(a) and presence(b) of 0.4 mg/mL MNP@PDA. (B) Fluorescence quenching curves of 200 nM FAM-P1 in the presence of 0.4 mg/mL MNP@PDA as a function of time.
Figure 4. (A) Normalized FL intensity and FP signal of FAM-P1 upon the addition of different concentrations of [email protected] concentration of FAM-P1 was 100 nM. The concentration of PDANS was 0, 0.05, 0.10, 0.20, 0.30, and 0.40 mg/mL respectively. (B) The influence of the pre-incubation time between FAM-P1 and the target before adding MNP@PDA. The concentration of FAM-P1, rHuEPO-α and MNP@PDA was 100 nM, 10 ng/mL, and 0.20 mg/mL, respectively.
Figure 5. (A) The reusable study of MNP@PDA in this aptasensor. The concentration of FAM-P1, rHuEPO-α and MNP@PDA was 100 nM, 10 ng/mL, and 0.20 mg/mL, respectively. (B) The stability of MNP@PDA in this aptasensor. The concentration of FAM-P1 and MNP@PDA was 100 nM and 0.20 mg/mL, respectively.
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Figure 6. (A) FP signal of different sensing systems with 10 ng/mL or 500 ng/mL rHuEPO-α . (B) The fluorescence intensity of FAM-P1 (a) , FAM-P1/ MNP@PDA (c), FAM-P1/ MNP@PDA / rHuEPO-α(b). The concentration of FAM-P1, rHuEPO-α and MNP@PDA was 100 nM, 10 ng/mL, and 0.20 mg/mL, respectively.
Figure 7. The linear relationship between ΔFP and log C of rHuEPO-α, MNP@PDA amplified (A), original (B). (C)The selectivity of this assay with 10 ng/mL rHuEPO-α, 1mg/mL HB, HAS, lgG and lysozyme. Table 1. The comparison of rHuEPO-α analysis by different aptasensors
Method/technique
Linear range
Detection limit
Ref.
Aptamer affnity probe and capillary electrophoresis based fluorescence assay
0.2-100 nM
0.2 nM
2
Gold nanoparticles and CdTe QDs based fluorescence off-on assay
5-60 pM
53.6 pM
6
Aptamer-gold nanoparticles probe based fluorescence off-on assay
2-500 nM
0.92 nM
4
MNP@PDA based fluorescence Polarization assay
0.3 pM-222 nM
0.12 pM
\
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Table 2. Samples detection of actual rHuEPO-α injections Recombinant human erythropoietin-alpha injection samples
Labeled amount/vial
Detected amount/vial
NuPIAO from 3SBio Inc.
10000 IU
9972 ± 316 IU
Epogen from Amgen Co., Ltd.
3000 IU
3028 ± 74 IU
ESPO from Kyowa Hakko kirin China pharmaceutical Co., Ltd.
3000 IU
3069 ± 42 IU
Highlights: 1. A novel nanocomposite MNP@PDA was synthesized with 97.15% quenching efficiency for FAM in 3 min. 2. The enhanced FP signal was due to the mass amplification of MNP@PDA and rHuEPO-α. 3. Magnetic separation can be utilized for target separation or recycle based on the magnetic property of MNP@PDA. 4. The detection limit of this assay was 0.12 pM, and this assay can be used to detect rHuEPO-α in actual samples.
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