Magnetic nanoparticles and polydopamine amplified FP aptasensor for the highly sensitive detection of rHuEPO-α
Graphical abstract
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. 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], [8], [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], [12], [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], [16], [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.
Section snippets
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, 5 mM
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
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 strategy for the detection of rHuEPO-α
Acknowledgement
This work was supported financially by National Natural Foundation of China (Grant Nos. 21775068, 21475060, 21405077), Natural Science Foundation of Jiangsu Province (BK20140591).
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