Aptasensors for the selective detection of alpha-synuclein oligomer by colorimetry, surface plasmon resonance and electrochemical impedance spectroscopy

Sensors and Actuators B 245 (2017) 87–94

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Aptasensors for the selective detection of alpha-synuclein oligomer by colorimetry, surface plasmon resonance and electrochemical impedance spectroscopy Kai Sun 1 , Ning Xia 1 , Lijuan Zhao, Ke Liu, Wenjing Hou, Lin Liu ∗ Henan Province of Key Laboratory of New Optoelectronic Functional Materials, College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan 455000, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 18 August 2016 Received in revised form 18 January 2017 Accepted 26 January 2017 Available online 27 January 2017 Keywords: Aptasensors Colorimetry Surface plasmon resonance Electrochemical impedance spectroscopy Parkinson’s disease Alpha-synuclein oligomer

a b s t r a c t Soluble alpha-synuclein (␣-syn) oligomer is believed to be a reliable molecular biomarker for diagnosis of Parkinson’s disease (PD). Thus, it is critical to develop a simple method for the selective detection of ␣-syn oligomer with low cost as well as high sensitivity. In this paper, we reported the label-free detection of ␣syn oligomer using a DNA aptamer as the bioreceptor with the techniques of gold nanoparticles (AuNPs)based colorimetric assay, surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS). In the colorimetric assay, the aptamer adsorbed onto the surface of AuNPs to prevent the saltinduced aggregation of the nanoparticles. However, the specific binding of ␣-syn oligomer to aptamer prevented the absorption of aptamer onto the surface of AuNPs. As a result, the aggregation of AuNPs was triggered by high concentration of salt with a color change from red to blue. In the SPR- or EISbased surface analysis, specific binding of ␣-syn oligomer to the aptamer immobilized onto the gold film or electrode surface led to an increase in the SPR dip shift or the electron-transfer resistance. The detection limits of the colorimetry, SPR and EIS were found to be 10 nM, 8 pM and 1 pM, respectively. The amenability of this method to ␣-syn oligomer analysis in a biological matrix was demonstrated by assay of ␣-syn oligomer in serum. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Parkinson’s disease (PD), the second most common neurodegenerative disorder after Alzheimer’s disease, affects approximately seven million people globally. A hallmark of PD is that surviving dopaminergic cells contain cytosolic filamentous inclusions known as the Lewy bodies [1,2]. A major component in Lewy bodies is the alpha-synuclein (␣-syn) aggregates, whose monomeric constituent is a synuclein protein of unknown function primarily found in neural tissue [1,3,4]. ␣-Syn monomer makes up as much as 1% of all proteins in the cytosol of brain cells. However, it can aggregate first into small oligomeric species and then into higher molecular weight fibril [5,6]. Among them, soluble oligomer is viewed as primarily neurotoxic and responsible for neuronal death in preclinical PD [7–11]. Also, the elevated levels of ␣-syn oligomer has been detected in the cerebrospinal fluid

∗ Corresponding author. E-mail address: [email protected] (L. Liu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.snb.2017.01.171 0925-4005/© 2017 Elsevier B.V. All rights reserved.

and plasma of PD patients [12–19]. Thus, ␣-syn oligomer has been considered not only as the therapeutic target but also as the diagnostic marker [20–24]. Recently, a few new methods have been developed for the detection of ␣-syn monomer with improving sensitivity, such as electrochemical immunosensors [25,26], mass spectrometry [27], fluorescent immunoassay [28], and capillary electrophoresis [29,30]. These methods are feasible in laboratory studies, but expensive, labor-intensive and/or less specific for clinical assays. Moreover, assay of ␣-syn monomer only might be unable to discriminate between PD patients and healthy controls or other types of dementia because the levels of ␣-syn monomer may differ by gender and age [31,32]. In view of the high neurotoxicity of ␣syn oligomer in PD, the direct detection of ␣-syn oligomer would be more reliable for PD diagnosis than assay of ␣-syn monomer [29,33]. Currently used method for clinical detection of ␣-syn oligomer is the enzyme-linked immunosorbent assay (ELISA) [12–19]. However, this method requires the relatively expensive and less stable antibody for molecular recognition. Moreover, the reported antibody of ␣-syn oligomer would also recognize ␣-syn monomer and other ␣-syn aggregates and metabolites to some extent

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[21,34]. Additionally, the organic dyes-based fluorescence assays (e.g. thioflavin T or ThT) have been commonly used for monitoring the formation of ␣-syn aggregates in laboratory investigations. However, most of the dyes cannot be used to discriminate ␣syn oligomer from other ␤-sheets of ␣-syn aggregates, which is detrimental to the accurately quantitative assay of ␣-syn oligomer [8,35–38]. Aptamer is an excellent example of functional bioreceptors selected in vitro. After nearly 30 years’ endeavor, DNA and RNA aptamers have been identified as binding tightly to a broad range of targets (e.g., proteins, peptides, amino acids, drugs, metal ions and even whole cells) [39–42]. Because of its advantageous characteristics over antibody (e.g., small sizes, long-term stability, simple preparation procedure and low preparation cost), aptamer has emerged as a good alternative to antibody in the design of novel electrochemical, optical and mass-sensitive biosensor devices that have exhibited high sensitivity and selectivity [43,44]. Recently, Ikebukuro’s group has selected a DNA aptamer that strongly bound to ␣-syn oligomer but not to ␣-syn monomer or fibril [34]. The dissociation constant (Kd ) of the aptamer for ␣-syn oligomer was estimated to be 60–70 nM [34]. This value is close to that between antibody and ␣-syn monomer [26], suggesting that the aptamer shows a high binding affinity to ␣-syn oligomer. In the present work, we attempted to develop a label-free aptasensor for the selective detection of ␣-syn oligomer. Among kinds of aptasensors, metal nanoparticles-based liquid-phase colorimetric assay has received considerable attention as it enables color visualization without a specific instrument [45]. Surface plasmon resonance (SPR) has also been shown as a promising technique for analyte concentration determination and kinetic studies of biomolecular interactions due to its attractive features, such as high sensitivity, label-free and real-time measurements, and relatively simple procedure [46–50]. Furthermore, for assay of non-electroactive protein in a single label-free and quantitative manner, electrochemical impedance spectroscopy (EIS) is particularly powerful because of its inherently favorable attributes of high innate sensitivity, facile miniaturization, low detection cost, and less sample consumption [51,52]. For this consideration, herein, we reported the label-free detection of ␣-syn oligomer using a DNA aptamer as the bioreceptor by gold nanoparticles (AuNPs)-based colorimetric assay, SPR and EIS. The analytical performances of the three aptasensors were compared and their practical applications for assays of ␣-syn oligomer in serum were demonstrated.

2. Experimental 2.1. Chemicals and reagents ␣-Syn monomer, bovine serum albumin (BSA), immunoglobin G (IgG), lysozyme, thrombin, 6-mercapto-1hexanol (MCH), tris(carboxy-ethyl)phosphine (TCEP), serum, trisodium citrate, KH2 PO4 and K2 HPO4 were purchased from Sigma-Aldrich. The ␣-syn oligomer-specific aptamers (5 -TTTTTGGTGGCTGGAGGGGGCGCGAACG-3 and 5 - HS-(CH2 )6 TTTTTGGTGGCTGGAGGGGGCGCGAACG-3 ) were synthesized and purified by Sangon Biotech. Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade and obtained from Beijing Chemical Reagent Co., Ltd. (Beijing, China). All solutions were prepared with ultrapure water from a Millipore system. The citrate-stabilized AuNPs with a size of 13 nm were prepared using a trisodium citrate reduction method and diluted with 2 mM phosphate-buffered saline solution (PBS buffer, pH 7.2). The particle concentration of the AuNPs suspension was determined based on a molar absorptivity of 2.7 × 108 M−1 cm−1 at 520 nm. The preparation of ␣-syn oligomer follows the reported procedure [5].

In brief, ␣-syn monomer was dissolved in the 2 mM PBS, filtered with 0.2 ␮m filter membrane and then incubated at room temperature for a given time. The concentration of ␣-syn monomer was determined by absorption measurement with a NanoDrop spectrophotometer (ND-1000, Thermo Scientific) using a theoretical extinction coefficient of 5960 M−1 cm−1 . 2.2. Colorimetric assay 50 ␮L of aptamer solution was first mixed with 50 ␮L of ␣-syn oligomer at a given concentration to react for 10 min. Then, 170 ␮L of AuNPs was added to the mixture. After 5-min incubation, 30 ␮L of PBS containing 0.8 M NaCl was added to the suspension. After reaction for 10 min again, the color change of the mixed solution was recorded by a digital camera. The absorbance spectra were collected with a Cary-60 UV–vis spectrophotometer. 2.3. SPR detection The immobilization of aptamer on the SPR sensing chips was carried out based on the Au-S interaction. Briefly, the cleaned Au films provided by Biosensing Instrument Inc. (Tempe, AZ) were immersed in the mixed solution of 10 ␮M thiolated aptamer (5 - HS-(CH2 )6 -TTTTTGGTGGCTGGAGGGGGCGCGAACG-3 ), 50 ␮M TCEP and a given concentration of MCH overnight to form the aptamer/MCH self-assembled monolayers (SAMs). Upon the completion of the surface modifications, the resultant chips were rinsed with ethanol and deionized water, dried with nitrogen and then stored at 4 ◦ C for use. For the ␣-syn oligomer detection, the prepared sensing chip was assembled onto the SPR instrument (BI-SPR 3000, Biosensing Instrument Inc., Tempe, AZ) for measurements. After a stable baseline was obtained, the ␣-syn sample was delivered onto the SPR flow cell using a syringe pump. 2.4. Impedance analysis The cleaned gold electrode with a diameter of 2 mm was incubated with a PBS solution containing 10 ␮M thiolated aptamer, 50 ␮M TCEP and 1.2 mM MCH overnight. After thoroughly washing the electrode with ethanol/water, 10 ␮L of ␣-syn sample at a given concentration was cast onto the sensor surface for 30 min. After having been rinsed with water again, the electrode was placed in a mixed solution of 5 mM [Fe(CN)6 ]3−/4− (1:1) and 0.1 M KCl for impedance measurement on a CHI 660E (CH Instruments, Shanghai, China) electrochemical workstation. Electrochemical impedance spectroscopy was collected at the potential of 0.245 V in the frequency range of 0.01–500 kHz. The auxiliary electrode and the reference electrode were platinum wire and Ag/AgCl, respectively. For the signal-amplified detection, the aptamer-covered electrode was incubated with the ␣-syn sample, washed with water, and then exposed to 50 ␮L of AuNPs suspension. After having been washed thoroughly with water, the electrode was placed in the solution of [Fe(CN)6 ]3−/4− for impedance measurement. 3. Results and discussion 3.1. Colorimetric assay Since the report of the most classical colorimetric sensor proposed by Mirkin’s group [53], AuNPs have shown tremendous potential in colorimetric aptasensors because of their high extinction coefficients and distance-dependent optical properties. Usually, AuNPs-based aptasensors can be divided into two categories: DNA-functionalized AuNPs aptasensors and unfunctionalized AuNPs aptasensors. In the latter, aptamer could bind to the unmodified AuNPs and stabilize them against salt-induced

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Fig. 1. (A) Illustration of the aptamer/AuNPs-based colorimetric detection of ␣-syn oligomer. (B) UV–vis absorption spectra of AuNPs in various systems: curve a, PBS; curve b, NaCl; curve c, aptamer + NaCl; curve d, aptamer + ␣-syn monomer + NaCl; curve e, aptamer + ␣-syn oligomer + NaCl; curve f, aptamer + ␣-syn fibril + NaCl. The inset shows the photos of AuNPs in the system. The final concentrations of AuNPs, atpamer, NaCl, and ␣-syn sample (equivalent monomer) were 4.2 nM, 200 nM, 100 mM and 10 ␮M, respectively. (C) AFM images of the ␣-syn sample in the format of monomer, oligomer and fibril.

aggregation through the interactions between gold and nitrogencontaining bases (Fig. 1A) [54]. Upon binding to its target, the aptamer loses its ability to protect AuNPs from being salt-induced aggregated. Thus, the color change of unmodified AuNPs could be observed by the naked eyes. The label-free method has allowed for the detection of metal ions, small molecules and proteins [54–56]. In this work, we also found that the aptamer of ␣-syn oligomer enhanced the physical stability of AuNPs and effectively resisted the NaCl-induced aggregation; thus, the color of the solution remained red and only one absorption peak at 520 nm was observed (Fig. 1B).

However, in the presence of ␣-syn oligomer, the aptamer lost its ability to protect AuNPs against the NaCl-induced aggregation, which was confirmed by transmission electron microscope (TEM) observations: significant aggregation of AuNPs in the presence of ␣-syn oligomer and monodisperse of AuNPs in the absence of ␣syn oligomer (Fig. S1). Meanwhile, the solution color changed from red to blue and the red-shifted band corresponding to the aggregated AuNPs was intensified, which was accompanied by a decrease in the absorbance at 520 nm. After addition of ␣-syn sample in the format of monomer, oligomer or fibril (Fig. 1C) to the aptamer

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Fig. 2. (A) The photographic images of AuNPs in the presence of aptamer, NaCl and different concentrations of ␣-syn oligomer. (B) Dependence of A650 /A520 on concentrations of ␣-syn oligomer (equivalent monomer). The final concentrations of AuNPs, atpamer and NaCl were 4.2 nM, 150 nM and 80 mM, respectively.

solution, we found that only ␣-syn oligomer allowed for the aggregation of AuNPs in the presence of high concentration of salt and the sequent color change from red to blue, which can be contributed to the specific interaction between ␣-syn oligomer and the aptamer. The result also confirmed that the secondary structure of ␣-syn is indispensable for the interaction with the aptamer. For the colorimetric detection of ␣-syn oligomer, a series of experimental conditions were first optimized, including the concentrations of NaCl as well as aptamer and the incubation time for the formation of ␣-syn oligomer. The A650 /A520 ratio (wherein A650 and A520 represent the absorption intensity of the solution at 650 nm and 520 nm, respectively) was used to evaluate the performances. A lower A650 /A520 indicates that AuNPs disperse well in the solution, while a higher A650 /A520 is related with the aggregated AuNPs. When the concentration of NaCl was 80 mM, the A650 /A520 reached to the maximum value (Fig. S2), indicating that AuNPs aggregated almost completely. Thus, 80 mM of NaCl was used to in the detection assay. With the increasing concentration of aptamer in the range from 0 to 500 nM, the A650 /A520 decreased and began to level off when the concentration of aptamer is over 150 nM (Fig. S3), suggesting the saturated surface coverage of aptamer on the AuNPs. Thus, 150 nM aptamer was used in the following experiments. The formation of ␣-syn oligomer depends on the incubation time of ␣-syn monomer. We also found that the A650 /A520 reached to the maximum for 84 h incubation of ␣-syn monomer, indicating that the optimal incubation time for the formation of ␣-syn oligomer was around 84 h (Fig. S4). Thus, in the following assays, 84 h was set as the standard condition for the preparation of ␣-syn oligomer. Under the optimized experimental conditions, we investigated the detection sensitivity of the colorimetric assay. As shown in the inset of Fig. 2A, the solution color changed from red to blue gradually in the presence of increasing concentration of ␣-syn oligomer. The visible color change could be easily distinguished by naked eyes from the blank test (without ␣-syn oligomer) when the target concentration was over 0.2 ␮M. The UV–vis spectra investigations revealed that the A650 /A520 increased with the increasing concentration of ␣-syn oligomer and began to level off beyond

5 ␮M (Fig. 2B). According to the linear regression equation of A650 /A520 = 0.334 [␣-syn] + 0.112 (␮M) (R = 0.996) fitting from the linear relationship between A650 /A520 and the concentration of ␣-syn oligomer (equivalent monomer) in the range of 20 nM to 3 ␮M, a detection limit of 10 nM for the equivalent monomer was achieved. To explore the specificity of the colorimetric assay, three interfering proteins (BSA, IgG and thrombin) were chosen for the selectivity investigations. In contrast to the control, these three proteins did not cause significant enhance in A650 /A520 (Fig. S5). The results confirmed that the aptamer is highly specific to ␣syn oligomer. Unfortunately, for the detection of ␣-syn oligomer in serum, we found the serum itself even at the concentration of 2% showed many UV–vis absorption peaks in the range of 400 ∼ 600 nm (Fig. S6). More importantly, the serum itself also prevented the NaCl-induced aggregation of AuNPs (Fig. S7). The presence of some matrix components in serum solution may be responsible for this result [57]. Therefore, the colorimetric assay is inadvisable for quantitative detection of ␣-syn oligomer in serum. However, this method requires very simple sample handling procedure and minimum instrumental investment, which may offer a useful means for quantitative detection of ␣-syn oligomer in other laboratory investigations, such as understanding the dynamics and mechanism of ␣-syn aggregation/fibrillation processes under different experimental conditions and screening of effective agents that can prevent the formation of neurotoxic ␣-syn oligomer. 3.2. SPR detection SPR sensors are mass-sensitive techniques capable of registering mass change by the associated change in refractive index at the surface [47]. Because SPR can determine the binding of aptamer and the target, this technology has been used in systematic evolution of ligands by exponential enrichment (SELEX) process and aptamerbased sensing applications [40,58,59]. In this detection format, a selective sensing surface is formed by immobilizing the aptamer on a gold chip surface. When the target is injected at a constant flow rate, the change in the resonance angle occurring at the surface can be measured, thus allowing for the label-free detection in a single-site binding configuration. Herein, the interaction between the aptamer and different forms of ␣-syn was investigated using SPR. As shown in Fig. 3A, in sharp contrast to the ␣-syn oligomer (curve b), no significant binding was detected for the monomer (curve a) and fibril (curve c). To test whether the striking differences between the oligomer and fibrillar aggregates are not just due to the difference in the size of these assemblies, long fibrils were fragmented by sonication. Also, in this case, no significant binding to the aptamer was detected by SPR (curve d). These results are clearly indicative of configuration-dependent binding of ␣-syn oligomer to the immobilized aptamer. For the affinity biosensors, it is critical that the probe molecules attached onto the sensor surface maintain their specificity and activity without non-specific binding. Self-assembly of thiolated DNA probes on gold surface is the most common type of attachment chemistries for design of affinity biosensors. However, because DNA probes can also be absorbed onto the gold surface through the coordinating interactions between the electron-rich nitrogen atoms and the electron-deficient gold surface, linear thiol molecules (e.g. MCH) are usually employed to block the unreacted gold surface, displace non-specifically adsorbed DNA probes and orient the pre-immobilized DNA molecules for more efficient probetarget interaction. Thus, the distribution and density of aptamers immobilized on the gold surface play a crucial role in the sensing performances [60]. Herein, we also investigated the effect of the aptamer/MCH ratio on the biosensor performance (Fig. 3B). It can be observed that the gold surface modified with aptamer/MCH

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layer (between 0.1 nM and 0.5 ␮M), the SPR response was found to be linearly dependent on the concentration (see the inset) with an equation of  = 131.2 [␣-syn] + 3.4. The high fraction of coverage by ␣-syn oligomer benefits from the optimal density of aptamer on the sensor surface. The detection limit of this method was calculated to be 8 pM from 3 of the signal of the lowest concentration of sample. 3.3. Impedance analysis

Fig. 3. (A) SPR sensorgrams after injections of ␣-syn monomer (curve a), ␣-syn oligomer (curve b) and ␣-syn fibril before (curve c) and after sonication (curve d). The concentration of ␣-syn sample (equivalent monomer) was 5 ␮M. (B) Dependence of SPR dip shift on the aptamer/MCH concentration ratio. (C) Dependence of SPR dip shift on the concentration of ␣-syn oligomer (equivalent monomer).

at a ratio of 1:120 exhibited the highest response. For lower ratio of aptamer/MCH (1:500), the result is acceptable since increasing the amount of MCH decreased the absorption number of aptamer molecules on sensor surface, thus reducing the binding amount of ␣-syn oligomer. However, high density of aptamers also lowered the efficiency (1:10). This should be attributed to the coordinating interaction between aptamer and gold surface and the steric hindrance effect [60]. Furthermore, we investigated the dependence of the SPR dip shift on the concentration of ␣-syn oligomer (equivalent monomer). As shown in Fig. 3C, the SPR dip shift increased sharply within the concentration range of 0.1 nM to 0.5 ␮M but began to level off beyond 0.5 ␮M. The relative standard deviations (RSDs), shown as the error bars, are all less than 14%. The different size of ␣-syn oligomer should be responsible for such high error bars. When the surface coverage is less than 89% of a full mono-

EIS is simple and sensitive to study the change in various surface processes and properties. Aptasensors based on EIS have been proven to be the most effective and sensitive for label-free detection of proteins. AuNPs possess distinct physical and chemical attributes that make them excellent materials for the fabrication of signal-amplified electrochemical aptasensors. The strategies introducing AuNPs into the DNA-based electrochemical biosensors can be sub-categorized into four main types [61,62]: (1) as the electrode materials with the large surface area and excellent electronic conductivity, (2) as the carriers to load large amounts of electrochemically active species, (3) as the tracers to directly produce a electrochemical signal, and (4) as the nanocatalysts to obtain the amplified electrochemical signal. In our design, the impedimetric aptasensor for the amplified detection of ␣-syn oligomer was based on the excellent electron transfer ability of AuNPs and their unique interaction with the DNA aptamer. It employs the AuNPs as the signal amplifiers to reduce the background resistance and utilizes the electron-transfer resistance change (Ret = R’et −Ret , where R’et and Ret represent the electron-transfer resistance of the sensing electrode with and without attachment of ␣-syn oligomer before the incubation of AuNPs, respectively) as the response signal (Fig. 4A) [63,64]. As shown in Fig. 4B, there is a significant increase in the electron transfer resistance after the attachment of ␣-syn oligomer on the aptamer-covered electrode surface (cf. curve a and curve b). This demonstrated that ␣-syn oligomer was captured by the anchored aptamer on electrode surface. Predictably, capture of AuNPs by the aptamer-covered electrode resulted in a great decrease in the Ret (curve c), indicating that AuNPs absorbed onto the electrode surface indeed facilitated the electron-transfer of ferricyanide and thus reducing the background resistance (cf. curve a and curve c). Interestingly, when ␣-syn oligomer was anchored onto the electrode surface followed by the capture of AuNPs, a larger semicircle portion at high frequency relating to the electron transfer limited process was observed (curve d). The electrontransfer resistance was closed to that obtained by incubating the sensing electrode with ␣-syn oligomer only (curve b), indicating that the formation of aptamer-oligomer complex on the electrode surface prevented the capture of AuNPs. The Ret between curve d and curve c is higher than that between curve b and curve a, confirming that the signal was amplified by AuNPs. The quantitative assay of ␣-syn oligomer was conducted by measuring the difference of Ret . As shown in Fig. 5, Ret increased with increasing concentration of ␣-syn oligomer with or without the signal amplification of AuNPs. The regression equations in the linear ranges were found to be Ret = 9896 [␣-syn] (␮M) + 67 (without signal amplification) and Ret = 24308 [␣-syn] (␮M) + 112 (with signal amplification), and the detection limits were calculated to be 3 and 1 pM, respectively. Electrochemical aptasensors have shown great potential to determine target analytes in clinical diagnostics during the past decade. To explore the specificity and amenability of this impedimetric aptasensor for quantifying ␣-syn oligomer in a biological sample, ␣-syn monomer and fibril, four interfering proteins (BSA, lysozyme, IgG and thrombin) and serum were tested. As shown in Fig. 6, compared to the control, the six interferences and the serum caused negligible Ret , indicating that the established electro-

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Fig. 4. (A) Schematic illustration of the impedance analysis with and without the signal amplification of AuNPs. (B) EIS of the aptamer-covered electrodes after incubation with different solutions: (curve a, PBS control; curve b, ␣-syn oligomer; curve c, AuNPs; curve d, ␣-syn oligomer and AuNPs). The concentrations of ␣-syn oligomer (equivalent monomer) and AuNPs were 2 ␮M and 1.4 nM, respectively.

the quantitative assay. Note that 2% serum showed serious interference in the unmodified AuNPs-based colorimetric assay (Fig. S6 and Fig. S7); thus, the proposed electrochemical method would be more suitable for determining ␣-syn oligomer in clinical investigations. Actually, we also found that the undiluted serum sample showed no significant interference in the electrochemical assay. This result is understandable since the DNA/MCH-covered sensing electrodes can improve the specificity of electrochemical assays and thus allow for the direct detection of DNA and proteins in serum [65,66].

4. Conclusion

Fig. 5. Dependence of Ret on the concentration of ␣-syn oligomer (equivalent monomer) with (red dot curve) and without (black dot curve) the signal amplification of AuNPs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Selectivity of the sensing protocol (from bar 1 to bar 9: ␣-syn monomer, ␣syn fibril, BSA, lysozyme, IgG, thrombin, serum, ␣-syn oligomer in PBS and ␣-syn oligomer in serum). The concentration of ␣-syn sample was 0.2 ␮M and that of the interfering proteins was 20 ␮g/mL.

In summary, we reported the colorimetry, SPR and EIS detection of ␣-syn oligomer with a DNA aptamer as the bioreceptor. Among the three aptasensors, AuNPs-based colorimetric assay exhibits simple detection principle and easy manipulation procedure. Although the colorimetric method shows low sensitivity and poor anti-interference ability for assay of ␣-syn oligomer in a serum sample, it may offer a useful means for assessing the mechanism of ␣-syn aggregation/fibrillation processes and screening of effective drugs that can prevent the formation of neurotoxic ␣-syn oligomer. SPR exhibits a low detection limit (8 pM) that is comparable to that achieved by impedimetric assay (1 pM) based on the signal amplification of AuNPs with a simple procedure. The two surface analysis techniques require less sample consumption and simple procedure for preparation of sensor surface and obviate the utilization of complex instruments. Furthermore, the selectivity and interference studies (e.g. ␣-syn monomer and fibril, interfering proteins and serum) indicated that the impedimetric aptasensor shows extraordinary selectivity towards ␣-syn oligomer. Because soluble ␣-syn oligomer is the most important toxic species in the brain of PD patient and the direct assay of ␣-syn oligomer may be more reliable for the early diagnosis of PD than assay of ␣-syn monomer or fibril, the proposed SPR or EIS biosensor could potentially serve as a viable alternative for facile clinical diagnosis of PD.

Acknowledgments chemical method showed extraordinary selectivity towards ␣-syn oligomer and no detectable ␣-syn oligomer was found in the serum sample. The high selectivity could be principally attributed to the strong and specific interaction between ␣-syn oligomer and the aptamer. We also found that the Ret for assay of ␣-syn oligomer added in a 2% serum sample was close to that obtained in the buffer solution, demonstrating that serum did not interfere with

Partial support of this work by the National Natural Science Foundation of China (Nos. 21205003, 21305004), the Joint Fund for Fostering Talents of National Natural Science Foundation of China and Henan Province (U1304205) and the Program for Science and Technology Innovation Talents at the University of Henan Province (15HASTIT001) is gratefully acknowledged.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.01.171.

References [1] M.G. Spillantini, R.A. Crowther, R. Jakes, M. Hasegawa, M. Goedert, a-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 6469. [2] C.C. Bi, A.P. Wang, Y.C. Chu, S. Liu, H.J. Mu, W.H. Liu, Z.M. Wu, K.X. Sun, Y.X. Li, Intranasal delivery of rotigotine to the brain with lactoferrin-modified PEG-PLGA nanoparticles for Parkinson’s disease treatment, Int. J. Nanomed. 11 (2016) 6547. [3] H.A. Lashuel, D. Hartley, B.M. Petre, T. Walz, P.T. Lansbury Jr., Neurodegenerative disease – amyloid pores from pathogenic mutations, Nature 418 (2002) 291. [4] K. Vekrellis, M. Xilouri, E. Emmanouilidou, H.J. Rideout, L. Stefanis, Pathological roles of ␣-synuclein in neurological disorders, Lancet Neurol. 10 (2011) 1015. [5] N. Lorenzen, S.B. Nielsen, A.K. Buell, J.D. Kaspersen, P. Arosio, B.S. Vad, W. Paslawski, G. Christiansen, Z. Valnickova-Hansen, M. Andreasen, J.J. Enghild, J.S. Pedersen, C.M. Dobson, T.P.J. Knowles, D.E. Otzen, The role of stable ␣-synuclein oligomers in the molecular events underlying amyloid formation, J. Am. Chem. Soc 136 (2014) 3859. [6] P.J. Salveson, R.K. Spencer, J.S. Nowick, X-ray crystallographic structure of oligomers formed by a toxic ␤-hairpin derived from ␣-synuclein: trimers and higher-order oligomers, J. Am. Chem. Soc 138 (2016) 4458. [7] B. Winner, R. Jappelli, S.K. Maji, P.A. Desplats, L. Boyer, S. Aigner, C. Hetzer, T. Loher, M. Vilar, S. Campioni, C. Tzitzilonis, A. Soragni, S. Jessberger, H. Mira, A. Consiglio, E. Pham, E. Masliah, F.H. Gage, R. Riek, In vivo demonstration that ␣-synuclein oligomers are toxic, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 4194. [8] L. Giehm, S. D. D.E, B. Otzen, Vestergaard, Low-resolution structure of a vesicle disrupting ␣-synuclein oligomer that accumulates during fibrillation, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 3246. [9] K.A. Conway, S.-J. Lee, J.-C. Rochet, T.T. Ding, R.E. Williamson, P.T. Lansbury Jr., Acceleration of oligomerization, not fibrillization, is a shared property of both a-synuclein mutations linked to early-onset Parkinson’s disease: implications for pathogenesis and therapy, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 571. [10] M.S. Celej, R. Sarroukh, E. Goormaghtigh, G.D. Fidelio, J.M. Ruysschaert, V. Raussens, Toxic prefibrillar ␣-synuclein amyloid oligomers adopt a distinctive antiparallel ␤-sheet structure, Biochem. J. 443 (2012) 719. [11] N. Cremades, S.I.A. Cohen, E. Deas, A.Y. Abramov, A.Y. Chen, A. Orte, M. Sandal, R.W. Clarke, P. Dunne, F.A. Aprile, C.W. Bertoncini, N.W. Wood, T.P.J. Knowles, C.M. Dobson, D. Klenerman, Direct observation of the interconversion of normal and toxic forms of a-synuclein, Cell 149 (2012) 1048. [12] R.F. Roberts, R. Wade-Martins, J. Alegre-Abarrategui, Direct visualization of alpha-synuclein oligomers reveals previously undetected pathology in Parkinson’s disease brain, Brain 138 (2015) 1642. [13] E.B.M. Ladinska, Parkinson’s disease: diagnostic relevance of elevated levels of soluble a-synuclein oligomers in cerebrospinal fluid, Future Neurol. 6 (2011) 159. [14] O. Hansson, S. Hall, A. Öhrfelt, H. Zetterberg, K. Blennow, L. Minthon, K. Nägga, E. Londos, S. Varghese, N.K. Majbour, A. Al-Hayani, O.M. El-Agnaf, Levels of cerebrospinal fluid ␣-synuclein oligomers are increased in Parkinson’s disease with dementia and dementia with Lewy bodies compared to Alzheimer’s disease, Alzheimers Res. Ther. 6 (2014) 25. [15] M.J. Park, S.-M. Cheon, H.-R. Bae, S.-H. Kim, K.J. W, Elevated levels of ␣-synuclein oligomer in the cerebrospinal fluid of drug-naïve patients with Parkinson’s disease, J. Clin. Neurol. 7 (2011) 215. [16] X. Wang, S. Yu, F. Li, T. Fengm, Detection of a-synuclein oligomers in red blood cells as a potential biomarker of Parkinson’s disease, Neurosci. Lett. 599 (2015) 115. [17] O.M.A. El-Agnaf, S.A. Salem, K.E. Paleologou, M.D. Curran, M.J. Gibson, J.A. Court, M.G. Schlossmacher, D. Allsop, Detection of oligomeric forms of a-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease, FASEB J. 20 (2006) 419. [18] K.E. Paleologou, C.L. Kragh, D.M.A. Mann, S.A. Salem, R. Al-Shami, D. Allsop, A.H. Hassan, P.H. Jensen, O.M.A. El-Agnaf, Detection of elevated levels of soluble a-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies, Brain 132 (2009) 1093. [19] G. Vivacqua, A. Latorre, A. Suppa, M. Nardi, S. Pietracupa, R. Mancinelli, G. Fabbrini, C. Colosimo, E. Gaudio, A. Berardelli, Abnormal salivary total and oligomeric alpha-synuclein in Parkinson’s disease, PLoS One 11 (2016) e0151156. [20] H.V.S. Ganesh, A.M. Chow, K. Kerman, Recent advances in biosensors for neurodegenerative disease detection, TRAC-Trend. Anal. Chem. 79 (2016) 363. [21] M.R. Sierks, G. Chatterjee, C. McGraw, S. Kasturirangan, P. Schulza, S. Prasad, CSF levels of oligomeric alpha-synuclein and beta-amyloid as biomarkers for neurodegenerative disease, Integr. Biol. 3 (2011) 1188. [22] S.M. Williams, P. Schulz, M.R. Sierks, Oligomeric a-synuclein and b-amyloid variants as potential biomarkers for Parkinson’s and Alzheimer’s diseases, Eur. J. Neurosci. 43 (2016) 3.

93

[23] N.K. Majbour, N.N. Vaikath, K.D. van Dijk, M.T. Ardah, S. Varghese, L.B. Vesterager, L.P. Montezinho, S. Poole, B. Safieh-Garabedian, T. Tokuda, C.E. Teunissen, H.W. Berendse, W.D.J. van de Berg, O.M.A. El-Agnaf, Oligomeric and phosphorylated alpha-synuclein as potential CSF biomarkers for Parkinson’s disease, Mol. Neurodegener. 11 (2016) 1. [24] Q. Xu, H. Cheng, J. Lehr, A.V. Patil, J.J. Davis, Graphene oxide interfaces in serum based autoantibody quantification, Anal. Chem. 87 (2015) 346. [25] Y. An, X. Jiang, W. Bi, H. Chen, L. Jin, S. Zhang, C. Wang, W. Zhang, Sensitive electrochemical immunosensor for a-synuclein based on dual signal amplification using PAMAM dendrimer-encapsulated Au and enhanced gold nanoparticle labels, Biosens. Bioelectron. 32 (2012) 224. [26] T. Bryan, X. Luo, L. Forsgren, L.A. Morozova-Roched, J.J. Davis, The robust electrochemical detection of a Parkinson’s disease marker in whole blood sera, Chem. Sci. 3 (2012) 3468. [27] S. Slamnoiu, C. Vlad, M. Stumbaum, A. Moise, K. Lindner, N. Engel, M. Vilanova, M. Diaz, C. Karreman, M. Leist, T. Ciossek, B. Hengerer, M. Vilaseca, M. Przybylski, Identification and affinity-quantification of ß-amyloid and ␣-synuclein polypeptides using on-line SAW-biosensor-mass spectrometry, J. Am. Soc. Mass Spectrom 25 (2014) 1472. ´ H. Yang, M. Björkqvist, S. Kim, O.C. Jeong, O. Hansson, T. [28] S. Lee, E. Silajdˇzic, Laurell, A porous silicon immunoassay platform for fluorometric determination of ␣-synuclein in human cerebrospinal fluid, Microchim. Acta 181 (2014) 1143. [29] B.A. Killinger, A. Moszczynska, Characterization of ␣-synuclein multimer stoichiometry in complex biological samples by electrophoresis, Anal. Chem 88 (2016) 4071. [30] M.F. Iwabuchi, M.M. Hetu, W.G. Tong, Sensitive analysis of a -synuclein by nonlinear laser wave mixing coupled with capillary electrophoresis, Anal. Biochem. 500 (2016) 51. [31] B. Mollenhauer, J.J. Locascio, W. Schulz-Schaeffer, F. Sixel-Döring, C. Trenkwalder, M.G. Schlossmacher, ␣-Synuclein and tau concentrations in cerebrospinal fl uid of patients presenting with parkinsonism: a cohort study, Lancet Neurol. 10 (2011) 230. [32] A. Atik, T. Stewart, J. Zhang, Alpha-synuclein as a biomarker for Parkinson’s disease, Brain Pathol. 26 (2016) 410. [33] M.H. Horrocks, L. Tosatto, A.J. Dear, G.A. Garcia, M. Iljina, N. Cremades, M.D. Serra, T.P.J. Knowles, C.M. Dobson, D. Klenerman, Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of ␣-synuclein oligomers, Anal. Chem 87 (2015) 8818. [34] K. Tsukakoshi, K. Abe, K. Sode, K. Ikebukuro, Selection of DNA aptamers that recognize ␣-synuclein oligomers using a competitive screening method, Anal. Chem 84 (2012) 5542. [35] C.W.T. Leung, F. Guo, Y. Hong, E. Zhao, R.T.K. Kwok, N.L.C. Leung, S. Chen, N.N. Vaikath, O.M. El-Agnaf, Y. Tang, W.-P. Gai, B.Z. Tang, Detection of oligomers and fibrils of a-synuclein by AIEgen with strong fluorescence, Chem. Commun. 51 (2015) 1866. [36] N.P. Cook, K. Kilpatrick, L. Segatori, A.A. Martí, Detection of ␣-synuclein amyloidogenic aggregates in vitro and in cells using light-switching dipyridophenazine ruthenium(II) complexes, J. Am. Chem. Soc 134 (2012) 20776. [37] D.A. Yushchenko, J.A. Fauerbach, S. Thirunavukkuarasu, E.A. Jares-Erijman, T.M. Jovin, Fluorescent ratiometric MFC probe sensitive to early stages of a-synuclein aggregation, J. Am. Chem. Soc. 132 (2010) 7860. [38] J.S. Ahn, J.H. Lee, J.H. Kim, S.R. Paik, Novel method for quantitative determination of amyloid fibrils of alpha-synuclein and amyloid beta/A4 protein by using resveratrol, Anal. Biochem 367 (2007) 259. [39] R.Y. Robati, A. Arab, M. Ramezani, F.A. Langroodi, K. Abnous, S.M. Taghdisi, Aptasensors for quantitative detection of kanamycin, Biosens. Bioelectron. 82 (2016) 162. [40] S. Song, L. Wang, J. Li, J. Zhao, C. Fan, Aptamer-based biosensors, TRAC-Trend. Anal. Chem. 27 (2008) 108. [41] K. Tsukakoshi, R. Harada, K. Sode, K. Ikebukuro, Screening of DNA aptamer which binds to a-synuclein, Biotechnol. Lett. 32 (2010) 643. [42] C.H. van den Kieboom, S.L. van der Beek, T. Meszaros, R.E. Gyurcsányi, G. Ferwerda, M.I. de Jonge, Aptasensors for viral diagnostics, TRAC-Trend. Anal. Chem. 74 (2015) 58. [43] I. Willner, M. Zayats, Electronic aptamer-based sensors, Angew. Chem. Int. Ed 46 (2007) 6408. [44] D. Wu, X. Xin, X. Pang, M. Pietraszkiewicz, R. Holyst, X. Sun, Q. Wei, Application of europium multiwalled carbon nanotubes as novel luminophores in an electrochemiluminescent aptasensor for thrombin using multiple amplification strategies, ACS Appl. Mater. Interfaces 7 (2015) 12663. [45] Y.S. Kim, J.H. Kim, I.A. Kim, S.J. Lee, M.B. Gu, The affinity ratio-Its pivotal role in gold nanoparticle-based competitive colorimetric aptasensor, Biosens. Bioelectron. 26 (2011) 4058. [46] H.X. Chen, F.J. Qi, H. Zhou, S.S. Jia, Y.M. Gao, K. Koh, Y.M. Yin, Fe3O4@Au nanoparticles as a means of signal enhancement in surface plasmon resonance spectroscopy for thrombin detection, Sensor. Actuat. B: Chem. 212 (2015) 505. [47] J. Homola, Surface Plasmon Resonance Based Sensors, Springer, Berlin, 2006. [48] T. Kang, S. Hong, H.J. Kim, J. Moon, S. Oh, S.R. Paik, J. Yi, Characterization of surface-confined alpha-synuclein by surface plasmon resonance measurements, Langmuir 22 (2006) 13. [49] Y.H. Kim, J.P. Kim, S.J. Han, S.J. Sim, Aptamer biosensor for lable-free detection of human immunoglobulin E based on surface plasmon resonance, Sensor. Actuat. B: Chem. 139 (2009) 471.

94

K. Sun et al. / Sensors and Actuators B 245 (2017) 87–94

[50] L. Mihai, A. Vezeanu, C. Polonschii, C. Albu, G.L. Radu, A. Vasilescu, Label-free detection of lysozyme in wines using an aptamer based biosensor and SPR detection, Sensor. Actuat. B: Chem. 206 (2015) 198. ˜ [51] N. de-los-Santos-Álvarez, M. Jesús Lobo-Castanón, A.J. Miranda-Ordieres, P. ˜ Modified-RNA aptamer-based sensor for competitive Tunón-Blanco, impedimetric assay of neomycin B, J. Am. Chem. Soc. 129 (2007) 3808. [52] P. Jolly, N. Formisano, J. Tkac, P. Kasak, C.G. Frost, P. Estrela, Label-free impedimetric aptasensor with antifouling surface chemistry: a prostate specific antigen case study, Sensor. Actuators B: Chem. 209 (2015) 306. [53] R. Elghanian, J.J. Storhoff, R.C. Mucic, R.L. Letsinger, C.A. Mirkin, Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles, Science 277 (1997) 1078. [54] J. Wang, L. Wang, X. Liu, Z. Liang, S. Song, W. Li, G. Li, C. Fan, A gold nanoparticle-based aptamer target binding readout for ATP assay, Adv. Mater. 19 (2007) 3943. [55] Y. Lu, Y. Liu, S. Zhang, S. Wang, S. Zhang, X. Zhang, Aptamer-based plasmonic sensor array for discrimination of proteins and cells with the naked eye, Anal. Chem. 85 (2013) 6571. [56] H. Wei, B. Li, J. Li, E. Wang, S. Dong, Simple and sensitive aptamer-based colorimetric sensing of protein using unmodified gold nanoparticle probes, Chem. Commun. (2007) 3735. [57] C.-C. Chang, C.-P. Chen, C.-H. Lee, C.-Y. Chen, C.-W. Lin, Colorimetric detection of human chorionic gonadotropin using catalytic gold nanoparticles and a peptide aptamer, Chem. Commun. 50 (2014) 14443. [58] E. Luzi, M. Minunni, S. Tombelli, M. Mascini, New trends in affinity sensing: aptamers for ligand binding, TRAC-Trend. Anal. Chem. 22 (2003) 810. [59] G. Lautner, Z. Balogh, V. Bardoczy, T. Meszaros, R.E. Gyurcsanyi, Aptamer-based biochips for label-free detection of plant virus coat proteins by SPR imaging, Analyst 135 (2010) 918. [60] N. Formisano, P. Jolly, N. Bhalla, M. Cromhout, S.P. Flanagan, R. Fogel, J.L. Limson, P. Estrela, Optimisation of an electrochemical impedance spectroscopy aptasensor by exploiting quartz crystal microbalance with dissipation signals, Sensor. Actuators B: Chem. 220 (2015) 369. [61] L. Wu, Y. Yao, Z. Li, X. Zhang, J. Chen, A new amplified impedimetric aptasensor based on the electron transfer ability of Au nanoparticles and their affinity with aptamer, J. Electroanal. Chem. 757 (2015) 243. [62] N. Xia, X. Wang, J. Yu, Y. Wu, S. Cheng, Y. Xing, L. Liu, Design of electrochemical biosensors with peptide probes as the receptors of targets and the inducers of gold nanoparticles assembly on electrode surface, Sens. Actuators B: Chem. 239 (2017) 834.

[63] Y. Yang, C. Li, L. Yin, M. Liu, Z. Wang, Y. Shu, G. Li, Enhanced charge Transfer by gold nanoparticle at DNA modified electrode and its application to label-free DNA detection, ACS Appl. Mater. Interfaces 6 (2014) 7579. [64] N. Xia, X. Wang, X. Wang, B. Zhou, Gold nanoparticle-based colorimetric and electrochemical methods for dipeptidyl peptidase-IV activity assay and inhibitor screening, Materials 9 (2016) 857. [65] J. Hu, Y. Yu, J.C. Brooks, L.A. Godwin, S. Somasundaram, F. Torabinejad, J. Kim, C. Shannon, C.J. Easley, A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices, J. Am. Chem. Soc. 136 (2014) 8467. [66] S.S. Mahshid, S. Camire´ı, F. Ricci, A. Vallée-Be´ılisle, A highly selective electrochemical DNA-based sensor that employs steric hindrance effects to detect proteins directly in whole blood, J. Am. Chem. Soc. 137 (2015) 15596.

Biographies Kai Sun received a PhD degree in Applied Chemistry from Northeast Normal University in 2015. Now he is a lecturer at Anyang Normal University. His research interest is the early diagnosis and drug discovery of neurodegenerative diseases. Ning Xia received her M.S. degree in Applied Chemistry from Fujian Normal University in 2008 and Ph.D. in Analytical Chemistry from Central South University in 2012. Now, she is an associate professor at Anyang Normal University. Her fields of interest are the biomedical applications of surface plasmon resonance and electrochemical biosensors. Lijuan Zhao is a master student in Analytical Chemistry at Anyang Normal University. Her research interests include the synthesis of metal nanomaterials and their applications in electrochemical sensors. Ke Liu will earn her M.S. degree at Anyang Normal University in 2017. Her interest is the biomedical application of surface plasmon resonance. Wenjing Hou will earn his bachelor degree at Anyang Normal University in 2017. Her field of interest is the applications of electrochemical biosensors. Lin Liu is an associate professor at Anyang Normal University. He received his Ph.D. in Analytical Chemistry from Central South University in 2011. His research interests include the early diagnosis and drug delivery of neurodegenerative disease and the development of electrochemical biosensors based on signal amplification of nanomaterials and/or enzyme plus redox cycling.