Pt Nanoparticles Enables Label-free

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Collision of Aptamer/Pt Nanoparticles Enables Labelfree Amperometric Detection of Protein in Rat Brain Yue Zhang, Jinpeng Mao, Wenliang Ji, Taotao Feng, Zixuan Fu, Meining Zhang, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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Analytical Chemistry

Collision of Aptamer/Pt Nanoparticles Enables Label-free Amperometric Detection of Protein in Rat Brain Yue Zhang,† Jinpeng Mao,† Wenliang Ji,† Taotao Feng,† Zixuan Fu,† Meining Zhang,†,* Lanqun Mao‡ †

Department of Chemistry, Renmin University of China, Beijing 100872, China.

‡

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical

Chemistry for Living Biosystems, Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100190, China.

*Corresponding

Author. E-mail: [email protected].

ABSTRACT Single particle collision is emerging as a powerful and sensitive technique for analyzing small molecules, however, its application in biomacromolecules detection, e.g. protein, in complex biological environments is still challenging. Here, we present the first demonstration on the single particle collision that can be developed for the detection of platelet derived growth factor (PDGF), an important protein involved in the central nervous system in living rat brain. The system features Pt nanoparticles (PtNPs) conjugated with the PDGF recognition aptamer, suppressing the electrocatalytic collision of PtNPs towards the oxidation of hydrazine. In the presence of PDGF, the stronger binding between targeted protein and the aptamer disrupts the aptamer/PtNPs conjugates, recovering the electrocatalytic performance of PtNPs, and allowing quantitative, selective, and highly sensitive detection of PDGF in cerebrospinal fluid of rat brain.

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INTRODUCTION Proteins are building blocks and central protagonists of cells, contributing to all processes of nerve system at the molecular level, such as signal, learning and memory.1-3 The changes of protein concentration or structure can trigger many brain related diseases (e.g., Alzheimer’s disease (AD)), serving as a biomarker for diseases diagnosis.4-6 Although there are several methods to detect the disease-related proteins, such as colorimetry, fluorescence, electrochemical immunosensors, and surface plasmon resonance (SPR),7-10 most of them require biolabeling, enzyme amplification, or sophisticated operating procedures. Mass spectrometry is one powerful method in identification and quantification of proteome, especially monitor multiple proteins simultaneously with the aid of database of protein sequences, isobaric tagging, chromatography or electrophoresis techniques.11-14 However, a label-free, portable strategy is highly needed for sensitively detecting proteins in the complex brain cerebral fluid.15 Due to the advantages of portability and miniaturization, electrochemical method has been proven as one of the powerful techniques for sensing of a wide variety of neurochemicals.16-20 Since its establishment in 2004, electrochemical collision has been emerging as one of new electrochemical techniques and drawn increasing attention.21-23 This strategy has been used for analytical purposes by measuring the current arising from either bulk electrolysis of the redox-active nanoparticles (NPs), or from redox-inactive NPs blocking the current at microelectrode,24 or from electrocatalytic

amplification

(ECA)

when

NPs

catalyzes

an

inner-sphere-electron-transfer reaction that is kinetically sluggish on the underlying microelectrode. Because it could respond signal at one nanoparticle, electrochemical collision method is very different from traditional ensemble measurements limited to

2


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provide averaged signal of a large number of nanoparticles. So far, electrochemical collision method has been proved to be one powerful strategy and explored to a broad applications ranging from the insightful fundamental studies to single nanoparticle electrocatalysis,25-29

single

detection,30,31

molecular

bioelectrochemistry,32,33

exocytosis,34 and biosensing.35,36 Here, we for the first time demonstrate the collision of electrocatalytic amplification arising from PtNPs can be developed into a strategy for the detection of protein in the complex matrix of cerebral fluid in rat brain. As shown in Scheme 1a, electrocatalytic amplification occurs when a PtNP approaches a noncatalytic electrode surface (i.e. carbon fiber microelectrode (CFME)) in the presence of redox indicator, N2H4.37 Compared to blocking method, this method generates a larger current change and has little edge effect,24 thus process higher sensitivity and more accuracy. To demonstrate the particle collision can be employed for protein detection, we selected PDGF, a disease-related protein in cerebrospinal fluid (CSF) of rat brain, as an example.38-40 Aptamer targeting PDGF was conjugated onto the surface of PtNPs through the coordination interaction between the nitrogen atoms of the unfolded aptamer and the PtNPs,41,42 forming aptamer/PtNPs, which can greatly suppress the electrocatalytic collision current towards the oxidation of N2H4 (Scheme 1b). In the presence of PDGF, the stronger binding between protein and aptamer displaces the aptamer from PtNPs surface, inducing current recovery (Scheme 1c) that provide a quantitative response. This turn-on mode for protein assay does not require sophisticated biolabeling or washing procedure, and can detect target protein sensitively. Furthermore, one collision corresponds to tens of proteins, even to several proteins, at one time which enables this strategy bear high sensitivity. Therefore, this strategy offers a simple and sensitive means to directly detect the protein in rat brain

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without complex labeling process.

Scheme 1. Schematic illustration of the strategy for PDGF detection. (a) Schematic of one collision event of a single PtNPs produces one transient current step, which is (b) suppressed upon the formation of aptamer/PtNPs, and (c) then recovered in the presence of PDGF.

EXPERIMENTAL SECTION Reagents and Solutions. Chloroplatinic acid (H2PtCl6·6H2O) and BSA was purchased from Chemical Reagent Co. Ltd. (Beijing, China). Sodium citrate, ascorbic acid (AA), dopamine (DA), citric acid, hydrazine hydrate (N2H4·H2O) were purchased from Sigma-Aldrich. Other chemicals were of analytical grade at least and were used as received. The 10 mM phosphate buffer solution contains NaH2PO4 and Na2HPO4 was adjusted to pH 7.4 with sodium hydroxide. Artificial cerebrospinal fluid (aCSF) was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 4


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mM) into deionized water and then adjusting the pH to 7.4 as our previous work.43 All the aqueous solutions were prepared with Milli-Q water. Unless stated otherwise, all experiments were carried out at room temperature. The DNA (5’-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3’) was the aptamer of PDGF following a previously reported.40 The FAM-aptamer was the aptamer modified the fluorescent carboxyfluorescein (FAM) dye at the 5’ end of the sequence. All the oligonucleotides and proteins were synthesized or purified by Sangon Biotechnology Co. Ltd. (Shanghai, China). Fabrication of Carbon Fiber Microelectrode. Carbon fiber microelectrode (CFME) were fabricated as described previously.44 The exposed CF was cut to ca. 500 μm with a surgery scalpel under a microscopy. Prior to use in electrochemistry, the CFME were first sonicated in acetone, 3 M HNO3, 1.0 M KOH, and deionized water sequentially, each for 3-5 min. Then, the electrodes were subject to electrochemical activation, first with potential-controlled amperometry at +2.0 V for 30 s and at -1.0 V for 10 s, and then with cyclic voltammetry in 0.5 M H2SO4 within a potential range from 0 to +1.0 V at a scan rate of 0.1 V·s−1 until a stable cyclic voltammogram was obtained. Synthesis and Characterization of PtNPs. 25 nm PtNPs was synthesized and characterized following a previously reported seed-mediated method.45 Briefly, 7.76 mL of 0.2% H2PtCl6·6H2O was added to 100 mL of boiling deionized water and reacted for 1.0 min. Then 2.37 mL of 1% sodium citrate and 0.05% citric acid solution was added to above solution and boiled for 30 s. Then 1.18 mL of a solution containing 0.08% NaBH4, 1% sodium citrate, and 0.05% citric acid solution were added to above solution and boiled for 10 min. After cooling to room temperature (25 °C), ca. 5 nm PtNPs seed solution was obtained.

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To synthesize 25 nm PtNPs, 1.0 mL of the PtNPs seed solution was added to 29.0 mL of deionized water at 25 °C. Then 0.023 mL of 0.40 M H2PtCl6·6H2O solution, 0.5 mL of 1% sodium citrate, and 1.25% ascorbic acid solution were added to above solution while stirring condition. Then the mixed solution was heated to boiling at the rate of 10 °C·min−1, and reacted for 30 min. After cooling to room temperature (25 °C), the solution was filtrated to remove large particle using 0.22 µm membrane before use. The concentration of prepared 25 nm PtNPs solution was determined and calculated as following.45 Briefly, the UV-vis absorbance spectra of series concentration of H2PtCl6 were measured to establish a calibration curve. Then, 600 μL 25 nm PtNPs solution was added to 3 mL aqua regia, heated to fully evaporate the solvent, which is then dissolved in 5 mL of 0.1 M HCl for absorbance spectra measurement. Pt ion concentration was calculated from absorbance and molar absorptivity from calibration plot. The density of Pt atoms per cubic centimeter was calculated from the density of Pt (21.45 g/cm3), the atomic mass of Pt (195 g/mol), and Avogadro’s number (6.02 × 1023 atoms/mol). The amount of Pt atoms per NPs was calculated to be 523310 by multiplying the density of Pt atoms (6.62 × 1022 atoms/cm3) by the average volume (cm3/NP). The size of the PtNPs was ca. 25 nm statisticed from TEM image (Figure S1). The PtNPs concentration of was calculated to be 53 pM through dividing the Pt ion concentration by the amount of Pt atoms per NPs. Preparation and Characterization of Aptamer/PtNPs. The aptamer/PtNPs were prepared through mixing 140 μL of 25 nm PtNPs solution with 10 μL aptamer with different concentration and then incubated for 30 min in centrifuge tube at room temperature. The amount of aptamers on per PtNPs was determined according to the

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previous literature.46 First, the fluorescence intensity of series concentration of FAM-aptamer (from 0 to 6 nM) was detected to establish a calibration curve (Figure S2). Then 53 pM PtNPs solution was mixed with 5 nM FAM-aptamer to form FAM-aptamer/PtNPs conjugation. Then the mixture was centrifuged for 10 min at the speed of 12000 r·min-1. The fluorescence spectra of supernatant were measured to calculate the amount of free FAM-aptamer. The number of FAM-aptamer strands per PtNPs was calculated to be 68 by subtracting the amount of free FAM-aptamer in supernatant from the total amount of FAM-aptamer and then divided by the concentration of PtNPs. Apparatus and Measurements. The electrochemical measurement was carried out in faraday cage with three electrode systems at room temperature using the computer-controlled CHI 660D electrochemical analyzer (Shanghai Chenhua Instrument Corporation, China). CFME or platinum disc electrode (1.5 mm diameter) was used as working electrode, platinum wire as counter electrode and Ag/AgCl electrode as reference electrode, respectively. The method of electrochemical collision measurements was as follows. 50 pM aptamer/PtNPs was firstly incubated with 10 μL protein solution with different concentration for 30 min in centrifuge tube. Then 50 μL product were added to 5 mL 10 mM phosphate buffer solution containing 10 mM N2H4 for electrochemical collision measurement with amperometric method. The amperometric responses were all recorded using the same sampling rate at 0.02 s-1. The step current (i.e. the minimum current to the maximum current of each current step) was used as signal for quantification. The morphology of the PtNPs was characterized by transmission electron microscope (TEM; JEM-2100F, Hitachi, Ltd., Japan). The size distribution of PtNPs was statisticed using Nano Measurer 1.2.0 software. UV-Vis Spectroscopy (UV-3600, 7



Shimadzu, Japan) was used to detect the concentration of PtNPs. Dynamic light scattering (Zetasizer NanoZS 90, Malvern Instruments Ltd, Malvern) was used to detect the zeta potential and hydrodynamic diameter of the nanoparticles’ dispersion solution. Scanning electron microscopy (SEM; SU8010 SEM, Hitachi, Ltd., Japan) was used for characterization the aggregation of NPs. Fluorescence experiments were measured using fluorescence spectrophotometer (F-4600; Hitachi Ltd., Japan) equipped with a Xenon lamp excitation source. The fluorescence spectrophotometer was set in the synchronous mode with the slit widths of 10 nm and 10 nm for excitation and emission, respectively. The PMT voltage was set at 400 V. Detection the Concentration of PDGF in Cerebrospinal Fluid of Rat Brain. Adult male Sprague-Dawley rats (300-350 g) were purchased from Health Science Center, Peking University. The animals were housed on a 12:12 h light-dark schedule with food and water ad libitum. All animal procedures were approved by the Animal Care and Use Committee at National Center for Nanoscience and Technology of China and performed according to their guidelines. Cerebrospinal fluid (CSF) was obtained as our previous work.47 Briefly, the rat was anaesthetized with chloral hydrate and were placed in a stereotaxic frame. A hollow steel tube (id. 0.5 mm) connected with a microdialysis tube was implanted into the lateral ventricle (AP: -3.7 mm, L: 4.8 mm from the bregma, V: 6.4 mm from the surface of the skull). The cerebrospinal fluid was collected at a flow rate of 1 μL·min-1 in microdialysis injector driven by a microinjection pump (CMA/100; CMA Microdialysis AB, Stockholm, Sweden). When we measured the concentration of PDGF in CSF, we firstly diluted CSF sample 100 times by aCSF, then 10 µL of diluted CSF was added to centrifuge tube containing 150 μL 50 pM aptamer/PtNPs for 30 min incubation. Then 50 μL product were added to 5 mL 10 mM phosphate buffer solution containing 10 mM N2H4 for 8


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electrochemical collision measurement.

RESULTS AND DISCUSSION The electrochemical oxidation of N2H4 at CFME is a sluggish process, which cannot reach steady-state at +0.5 V (Figure S3). However, this oxidation process occurs readily at -0.2 V at Pt substrate. The oxidation peak current gradually decreases with increasing the amount of aptamer modified onto the surface of platinum electrode because of the decreased active area and increased tunneling electron distance towards the oxidation of N2H4 (Figure S4). This is quite similar to the sluggish kinetics obtained at PtNPs capped with citrate ions or modified with SH-DNA.48 This kinetics of the oxidation of N2H4 largely depends on the surface chemistry of Pt, which forms the basis of the detection of PDGF using particle collision demonstrated here. To get high sensitivity and suitable potential, we chose N2H4 as indicator for electrochemical collision measurement. Other inner-sphere redox reaction such as oxygen reduction isn’t considered because the collision current is limited by its poor solubility.

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Figure 1. (A) Typical amperometric response recorded with CFME in 10 mM phosphate buffer containing 10 mM N2H4 and 0.5 pM PtNPs. (B) Histogram of step current resulting from PtNPs. Typical amperometric response of aptamer/PtNPs before (C) and after (D) incubated with 0.5 nM PDGF for 30 min. (E) Histogram of step current resulting from aptamer/PtNPs after incubated with 0.5 nM PDGF. (F) Typical amperometric response recorded with CFME in 10 mM phosphate buffer 10


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containing 10 mM N2H4 (black curve) in the presence of 0.5 nM aptamer (red curve) or 0.5 nM PDGF (blue curve). Applied potential, +0.10 V vs. Ag/AgCl.

Figure 1A shows a typical amperometric response recorded with CFME in 10 mM phosphate buffer containing 10 mM N2H4 and 0.5 pM PtNPs when the CFME is polarized at +0.10 V. As shown, we can clearly observe discrete transient step current and the current slowly decay. The decay of step current probably result from the inactivation of PtNPs.49 Although this decay might be alleviated in higher concentration buffer because of the better buffer capacity in the diffusion layer,50 the current of N2H4 oxidation became unstable at macrosized Pt electrode (id. 1.5 mm) in 50 mM phosphate buffer comparing with 10 mM phosphate buffer, which might be ascribed to the hydrogen production (Figure S5). Therefore, we used 10 mM phosphate buffer in our collision measurement. The main distribution of the step current was 0.13 ± 0.05 nA (Figure 1B), which was about four times of that induced by ca. 5 nm PtNPs (i.e., 30 ± 10 pA, Figure S6). This enhanced current of 25 nm PtNPs might be ascribed to the large surface area. Moreover, when a negative potential was applied, the transient current decreased, and even disappeared (Figure S7), suggesting the transient current step was attributed to the collision of PtNPs at the CFME. However, we did not observe the discrete current with the addition of aptamer/PtNPs into phosphate buffer containing N2H4 when the CFME was applied at the same potential for electrocatalytic oxidation of N2H4 (i.e., +0.10 V) (Figure 1C). Whereas, after incubation of the aptamer/PtNPs in phosphate buffer containing 0.5 nM PDGF, we observed the transient current again (i.e., 0.13 ± 0.03 nA) (Figure 1D, 1E), which is similar with that of bare PtNPs. In contrast, we could not observe the transient current in phosphate buffer containing N2H4 with the only addition of 0.5 11



nM aptamer, or 0.5 nM PDGF in Figure 1F. In addition, we observed that the collision frequency of bare PtNPs (i.e. 119 steps/200s) and aptamer/PtNPs incubated with PDGF (i.e. 103 steps/200s) is similar (Figure 1A and 1D), which indicate that the aptamer/PtNPs after incubated with protein was well dispersed. The scanning electron microscopy image of aptamer/PtNPs and aptamer/PtNPs after incubating with 0.5 nM PDGF also show no aggregation of particle (Figure S8). These results indicate that the anchor of aptamer to the surface of PtNPs deactivates the electrocatalysis of PtNPs for the oxidation of N2H4 and the formation of aptamer/PDGF dissociates the aptamer from the surface of PtNPs and further enables the recovery the current of collision event.

Figure 2. (A) The step current resulting from the amperometric response recorded with CFME in 10 mM phosphate buffer containing 10 mM N2H4 and PtNPs treated with different ratio of aptamer. Applied potential, +0.10 V vs. Ag/AgCl. (B) Fluorescent spectra of 5 nM FAM-aptamer toward successive addition of PtNPs (each addition, 10 pM).

To verify the origin of signal changes in the magnitude of PtNPs collision, we turned the ratio of aptamer to PtNPs from 0:1 to 200:1, and the result was shown in 12


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Figure 2A. With increasing the ratio of aptamer to PtNPs from 0:1 to 100:1, the transient current steps were reduced and no collision current was observed at the highest ratio (100:1). To avoid the interference of free aptamer in detection, we chose the 100:1 as the optimum ratio to prepare aptamer/PtNPs for the subsequent detection of PDGF. To study and confirm the efficient absorption of aptamer on PtNPs, we used a sequence concentration of PtNPs to titrate 5 nM fluorescently FAM labeled aptamer (FAM-aptamer) solution. The efficient fluorescence quenching between FAM and PtNPs provides direct evidence for the strong absorption of FAM-aptamer on PtNPs (Figure 2B). Zeta potential measurements also showed an increased negative potential with aptamer/PtNPs (-9.93 ± 1.2 mV) compared to PtNPs alone (-1.74 ± 0.5 mV) (Figure S9). The hydrodynamic size of PtNPs and aptamer/PtNP was 32 ± 8 nm and 40 ± 5 nm (Figure S10), respectively, further indicating that the aptamer anchored to the surface of PtNPs and the adsorption and desorption of the aptamer onto PtNPs are the main reasons for the suppression and recovery of transient current.

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Figure 3. (A) Relationship between the step current of aptamer/PtNPs and PDGF concentration. Inset, plot of step current against the logarithmic function of the concentration of PDGF. (B) The image of PtNPs and aptamer/PtNPs solution in different media. (C) The step current recorded with CFME in 10 mM phosphate buffer containing 10 mM N2H4 aptamer/PtNPs incubated with different protein (i.e. 0.5 nM bovine serum albumin (BSA), human serum albumin (HSA), β-galactosidase (EA), glucose oxidase (GOD), hemoglobin (Hem), horseradish peroxidase (HRP), myoglobin (MyO), Trypsin (Try) and PDGF). Applied potential, +0.10 V vs. Ag/AgCl. We studied the collision event towards the different concentrations of PDGF in 10 mM phosphate buffer containing 10 mM N2H4 and aptamer/PtNPs (the ratio is 100:1). As shown in Figure 3A, the transient current is linearly correlated with the concentration of PDGF in the range of 0.1 pM to 0.1 nM (ΔI (nA) = 0.21

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lg(CPDGF/nM) + 0.048). The detection limit was down to 0.1 pM, which is lower than the reported literatures.7,51 It is worth noting that treatment with nM protein resulted in essentially fully restoration of PtNPs catalytic current, suggesting a nearly complete displacement of aptamer from the surface of PtNPs. As the higher ionic strength of the CSF could cause the aggregation of PtNPs, and produce false signal in particle collision mediated detection.52,53 We next studied the stability of the PtNPs and aptamer/PtNPs under the high salt conditions of aCSF. As shown in Figure 3B, PtNPs alone has an obvious color change, suggesting the accumulation of PtNPs in aCSF. In contrast, aptamer/PtNPs showed minimal color change both in aCSF and in water. Moreover, as described above of SEM results (Figure S8) there is no apparent aggregation when the aptamer/PtNPs incubated with 0.5 nM PDGF. The excellent stability of aptamer/PtNPs in high salt biological relevant samples probably attributed to the strong protection of aptamer at the surface of PtNPs. We also investigated the selectivity of aptamer/PtNPs to the detection of PDGF. The response of the aptamer/PtNPs conjugates is very selective toward PDGF (Figure 3C). The addition of a variety of biologically relevant proteins, including BSA, HSA, generated no perceptible transient current in 10 mM phosphate buffer containing 10 mM N2H4 and aptamer/PtNPs. The high selectivity along with the turn-on model of our developed strategy encouraged us to explore the utility of the system for detection of PDGF in rat brain.

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Figure 4. (A) Typical amperometric response recorded with CFME in 10 mM phosphate buffer containing 10 mM N2H4 and aptamer/PtNPs incubated with CSF of rat brain for 30 min. Applied potential, +0.10 V vs. Ag/AgCl. (B) Histogram of the step current of aptamer/PtNPs incubated with CSF of rat brain for 30 min.

We further evaluated the developed strategy in detecting PDGF in CSF of rat brain. Figure 4A are the typical amperometric response of aptamer/PtNPs incubated with CSF of rat brain. According to the histogram of step current from the amperometric response in Figure 4B, the step current is 0.03 ± 0.004 nA. The concentration of PDGF in CSF of rat brain is calculated to be 0.28 ± 0.08 nM, which is in the similar level with the results reported in the literature (0-0.5 nM).39 When added 1.10 nM PDGF in CSF, we detected 1.35 nM with a recovery of 98 %.

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Figure 5. (A) The step current resulting from the amperometric response recorded with CFME in 10 mM phosphate buffer containing 10 mM N2H4 and PtNPs with and without incubated with 10 µL CSF. (B) The step current resulting from the amperometric response recorded with CFME in 10 mM phosphate buffer containing 10 mM N2H4 and aptamer/PtNPs incubated with 10 µL aCSF containing different concentration of PDGF without (black column) and with 3 mg·L-1 BSA (red column). Applied potential, +0.10 V vs. Ag/AgCl.

Noting that the bare PtNPs could adsorb protein, which will also suppress the signal. CSF contained a number of proteins (200-400 mg/L) and some small molecule species, such as glucose, ascorbic acid (AA), dopamine (DA).15 As shown in Figure 5A, the collision current decreased ca. 56% when bare PtNPs incubated with CSF. To investigate what extent the protein affect the aptamer/PtNPs in detecting PDGF, we incubated the aptamer/PtNPs with different concentrations of PDGF containing 3 mg·L-1 BSA protein (equal protein concentration in sample),54 the results were shown in Figure 5B. We can observe that protein has no evident affect to the PDGF detection when the concentration of PDGF was lower than 0.1 nM and has great affect when the concentration of PDGF was 1 nM. This result might due that the remained aptamer at surface of PtNPs can repel other protein in the sample when the target protein

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concentration is low.55 On the other hand, because the CSF was diluted 100 times when sample incubated with aptamer/PtNPs and the applied potential was +0.1 V, some small electroactive species, such as ascorbic acid and dopamine could not affect the electrochemical collision current of PtNPs (Figure S11). These results along with good recovery indicate our method is reliable for detecting the protein in CSF.

CONCLUSION In conclusion, we have presented a facile electrochemical collision method for sensitive and selective detecting of PDGF in complexed CSF of rat brain. The method was developed by integration of the collision activity of PtNPs, and the selective recognition of aptamer to proteins. The aptamer/PtNPs conjugates are highly stable under a physiological environment, with collision current selectively generated by PDGF, providing a quantitative response. The high selective and sensitive strategy described here can be generalized and applied to many biological and physiological challenges.

ACKNOWLEDGEMENTS We gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant Nos. 21874152) and Renmin University of China.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Characterization of PtNPs and aptamer/PtNP; N2H4 oxidation at CFME, Pt and 18


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aptamer/Pt electrode; The step current of AA and DA.

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REFERENCE (1) Sharma, K.; Schmitt, S.; Bergner, C. G.; Tyanova, S.; Kannaiyan, N.; Manrique-Hoyos, N.; Kongi, K.; Cantuti, L.; Hanisch, U. K.; Philips, M. A.; Rossner, M. J.; Mann, M.; Simons, M. Nat. Neurosci. 2015, 18, 1819-1831. (2) Korte, M.; Schmitz, D. Physiol. Rev. 2016, 96, 647-693. (3) Yousefzadeh, M. J.; Schafer, M. J.; Noren Hooten, N.; Atkinson, E. J.; Evans, M. K.; Baker, D. J.; Quarles, E. K.; Robbins, P. D.; Ladiges, W. C.; LeBrasseur, N. K.; Niedernhofer, L. J. Aging Cell 2018, 17, e12706. (4) Nakamura, A.; Kaneko, N.; Villemagne, V. L.; Kato, T.; Doecke, J.; Dore, V.; Fowler, C.; Li, Q.-X.; Martins, R.; Rowe, C.; Tomita, T.; Matsuzaki, K.; Ishii, K.; Ishii, K.; Arahata, Y.; Iwamoto, S.; Ito, K.; Tanaka, K.; Masters, C. L.; Yanagisawa, K. Nature 2018, 554, 249-254. (5) Johnson, E. B.; Byrne, L. M.; Gregory, S.; Rodrigues, F. B.; Blennow, K.; Durr, A.; Leavitt, B. R.; Roos, R. A.; Zetterberg, H.; Tabrizi, S. J.; Scahill, R. I.; Wild, E. J.; Group, T.-H. S. Neurology 2018, 90, e717-e723. (6) Papagiannakis, N.; Koros, C.; Stamelou, M.; Simitsi, A. M.; Maniati, M.; Antonelou, R.; Papadimitriou, D.; Dermentzaki, G.; Moraitou, M.; Michelakakis, H.; Stefanis, L. Neurosci. Lett. 2018, 672, 145-149. (7) Lin, B.; Sun, Q. Q.; Liu, K.; Lu, D. Q.; Fu, Y.; Xu, Z. A.; Zhang, W. Langmuir 2014, 30, 2144-2151. (8) Ranallo, S.; Rossetti, M.; Plaxco, K. W.; Vallee-Belisle, A.; Ricci, F. Angew. Chem. Int. Ed. 2015, 54, 13214-13218.

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(9) Feng, T.; Qiao, X.; Wang, H.; Sun, Z.; Qi, Y.; Hong, C. J. Mater. Chem. B 2016, 4, 990-996. (10) Shen, M.; Joshi, A. A.; Vannam, R.; Dixit, C. K.; Hamilton, R. G.; Kumar, C. V.; Rusling, J. F.; Peczuh, M. W. Chembiochem 2018, 19, 199-202. (11) Tyanova, S.; Temu, T.; Cox, J. Nat. Protoc. 2016, 11, 2301. (12) Lubeckyj, R. A.; McCool, E. N.; Shen, X.; Kou, Q.; Liu, X.; Sun, L. Anal. Chem. 2017, 89, 12059-12067. (13) Dayon, L.; Turck, N.; García-Berrocoso, T.; Walter, N.; Burkhard, P. R.; Vilalta, A.; Sahuquillo, J.; Montaner J.; Sanchez, J. C. J. Proteome. Res. 2010, 10, 1043-1051. (14) Dahlin, A. P.; Purins, K.; Clausen, F.; Chu, J.; Sedigh, A.; Lorant, T.; Enblad, P.; Lewén, A.; Hillered, L. Anal. Chem. 2014, 86, 8671-8679. (15) Eric. R. Kandel. Principles of Neural Science (The Fourth Edition), McGraw-Hill Companies, 2000. (16) Xiao, T.; Wu, F.; Hao, J.; Zhang, M.; Yu, P.; Mao, L. Anal. Chem. 2017, 89, 300-313. (17) Wu, F.; Yu, P.; Mao, L. Chem. Soc. Rev. 2017, 46, 2692-2704. (18) Dunevall, J.; Fathali, H.; Najafinobar, N.; Lovric, J.; Wigstrom, J.; Cans, A.-S.; Ewing, A. G. J. Am. Chem. Soc. 2015, 137, 4344-4346. (19) Ganesana, M.; Lee, S. T.; Wang, Y.; Venton, B. J. Anal. Chem. 2017, 89, 314-341. (20) Zhang, L.; Tian, Y. Acc. Chem. Res. 2018, 51, 688-696. (21) Quinn, B. M.; van 't Hof, P.; Lemay, S. G. J. Am. Chem. Soc. 2004, 126, 21



8360-8361. (22) Xiao, X.; Bard, A. J. J. Am. Chem. Soc. 2007, 129, 9610-9612. (23) Peng, Y.-Y.; Qian, R.-C.; Hafez, M. E.; Long, Y.-T. ChemElectroChem 2017, 4, 977-985. (24) Dick, J. E.; Renault, C.; Bard, A. J. J. Am. Chem. Soc. 2015, 137, 8376-8379. (25) Kwon, S. J.; Fan, F.-R. F.; Bard, A. J. J. Am. Chem. Soc. 2010, 132, 13165-13167. (26) Chen, L.; Tanner, E. E. L.; Lin, C.; Compton, R. G. Chem. Sci. 2018, 9, 152-159. (27) Robinson, D. A.; Liu, Y.; Edwards, M. A.; Vitti, N. J.; Oja, S. M.; Zhang, B.; White, H. S. J. Am. Chem. Soc. 2017, 139, 16923-16931. (28) Xiang, Z. P.; Deng, H. Q.; Peljo, P.; Fu, Z. Y.; Wang, S. L.; Mandler, D.; Sun, G. Q.; Liang, Z. X. Angew. Chem. Int. Ed. 2018, 57, 3464-3468. (29) Bentley, C. L.; Kang, M.; Unwin, P. R. J. Am. Chem. Soc. 2016, 138, 12755-12758. (30) Zhou, M.; Dick, J. E.; Hu, K.; Mirkin, M. V.; Bard, A. J. Anal. Chem. 2018, 90, 1142-1146. (31) Boika, A.; Bard, A. J. Anal. Chem. 2015, 87, 4341-4346. (32) Lin, C.; Katelhon, E.; Sepunaru, L.; Compton, R. G. Chem. Sci. 2017, 8, 6423-6432. (33) Dick, J. E.; Hilterbrand, A. T.; Strawsine, L. M.; Upton, J. W.; Bard, A. J. Proc. Natl. Acad. Sci. 2016, 113, 6403-6408. (34) Li, X.; Ren, L.; Dunevall, J.; Ye, D.; White, H. S.; Edwards, M. A.; Ewing, A. G. 22


Page 22 of 25

Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACS nano 2018, 12, 3010-3019. (35) Ho, T. L. T.; Hoang, N. T. T.; Lee, J.; Park, J. H.; Kim, B. K. Biosens. Bioelectron. 2018, 110, 155-159. (36) Castaneda, A. D.; Brenes, N. J.; Kondajji, A.; Crooks, R. M. J. Am. Chem. Soc. 2017, 139, 7657-7664. (37) Xiao, X.; Fan, F.-R. F.; Zhou, J.; Bard, A. J. J. Am. Chem. Soc. 2008, 130, 16669-16677. (38) Olson, L.; Humpel, C. Exp. Gerontol. 2010, 45, 41-46. (39) Nister, M.; Enblad, P.; Backstrom, G.; Soderman, T.; Persson, L.; Heldin, C. H.; Westermark, B. Br. J. Cancer 1994, 69, 952-956. (40) Lai, R. Y.; Plaxco, K. W.; Heeger, A. J. Anal. Chem. 2007, 79, 229-233. (41) Li, H. X.; Rothberg, L. Proc. Natl. Acad. Sci. 2004, 101, 14036-14039. (42) Wei, H.; Li, B.; Li, J.; Wang, E.; Dong, S. Chem. Commun. 2007, 36, 3735-3737. (43) Liu, X.; Xiao, T.; Wu, F.; Shen, M.-Y.; Zhang, M.; Yu, H.-h.; Mao, L. Angew. Chem. Int. Ed. 2017, 56, 11802-11806. (44) Liu, X.; Zhang, M.; Xiao, T.; Hao, J.; Li, R.; Mao, L. Anal. Chem. 2016, 88, 7238-7244. (45) Dasari, R.; Robinson, D. A.; Stevenson, K. J. J. Am. Chem. Soc. 2013, 135, 570-573. (46) Wu, P.; Hwang, K.; Lan, T.; Lu, Y. J. Am. Chem. Soc. 2013, 135, 5254-5257. (47) Mao, J.; Xu, M.; Ji, W.; Zhang, M. Chem. Commun. 2018, 54, 1193-1196. (48) Castaneda, A. D.; Robinson, D. A.; Stevenson, K. J.; Crooks, R. M. Chem. Sci. 23



2016, 7, 6450-6457. (49) Guo, Z.; Percival, S. J.; Zhang, B. J. Am. Chem. Soc. 2014, 136, 8879-8882. (50) Arulrajan, A. C.; Renault, C.; Lai, S. C. S. Phys. Chem. Chem. Phys. 2018, 20, 11787-11793. (51) Wang, P.; Song, Y. H.; Zhao, Y. J.; Fan, A. P. Talanta 2013, 103, 392-397. (52) Xu, J.; Shi, M.; Chen, W.; Huang, Y.; Fang, L.; Yao, L.; Zhao, S.; Chen, Z. F.; Liang, H. Chem. Commun. 2018, 54, 2719-2722. (53) Jiang, Y.; Shi, M.; Liu, Y.; Wan, S.; Cui, C.; Zhang, L.; Tan, W. Angew. Chem. Int. Ed. 2017, 56, 11916-11920. (54) Felgenhauer, K.; Schliep, G.; Rapic, N. J. Neurol. Sci. 1976, 30, 113-128. (55) McQuistan, A.; Zaitouna, A. J.; Echeverria, E.; Lai, R. Y. Chem. Commun. 2014, 50, 4690-4692.

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for TOC only

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Pt Nanoparticles Enables Label-free

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