Ultrasensitive Detection of Amyloid-Î² Using ... - ACS Publications

Subscriber access provided by BUFFALO STATE

Article

Ultra-sensitive detection of amyloid-# using PrPC on the highly conductive AuNPs-PEDOT-PTAA composite electrode Jieling Qin, Misuk Cho, and Youngkwan Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02266 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 4, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 17 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

Analytical Chemistry

Ultra-sensitive detection of amyloid-β using PrPC on the highly conductive AuNPs-PEDOT-PTAA composite electrode

Jieling Qin, Misuk Cho*, and Youngkwan Lee* School of Chemical Engineering, Sungkyunkwan University, 16419 Suwon, Korea Fax: +82-31-290-7272 Tel.: +82-31-290-7248 *Correspondence: [email protected] (Misuk Cho) and [email protected] (Youngkwan Lee) *These authors contributed equally. ABSTRACT: A highly sensitive electrochemical impedance sensor for amyloid beta oligomer (AβO) was fabricated using a PrPC bio-receptor linked with poly(thiophene-3-acetic acid)(PTAA) transducer. An additional thin layer of poly(3,4-ethylene dioxythiophene) embedded with gold nanoparticles (AuNPs-PEDOT) was employed to provide high electrical conductivity and a large surface area. The sensing performace was investigated in terms of sensitivity and detection range. The fabricated sensor exhibited extremely low detection limit at a sub-femto molar level with a wide detection range from 10-8 to 104 nM and its utility was established in mice infected with Alzheimer’s disease (AD) mouse. The developed AβO sensor could be utilized for the early diagnosis of AD. Key words: Alzheimer’s disease, amyloid beta oligomer, PEDOT, gold nanoparticles, PTAA

1

ACS Paragon Plus Environment

Analytical Chemistry 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

Alzheimer’ disease (AD) is the most common cause of dementia and is a rapidly growing social and medical problem throughout the world. 1-3 The peptide with amyloid beta oligomer (AβO) is the major component of the senile plaques found in AD. Thus, AβO have been regarded as the reliable molecular biomarkers and therapeutic targets for the diagnosis and prognosis of AD.4-8 Recently, novel techniques have been developed to probe for AβO. A highly selective and sensitive enzyme-linked immunosorbent assay (ELISA) has been utilized for the detection of AβO in body fluids. However, this ELISA has several inherent shortcomings, which are the labor-intensiveness and relatively high cost of the enzyme-linked antibodies for AβO recognition. Alternatively, electrochemical sensors have increased tremendously in popularity in recent years owing to their high sensitivity, speed, and good portability.9-11 To enhance the sensing performance of the electrochemical AβO sensor, several strategies have been reported, such as the use of signal tag or amplification,12-15 the development of active electrode materials with high conductivity or large surface areas,16,17 and the immobilization of some agents on substrates to block foreign substances, etc.11-12,18 Han’s group prepared an electrochemical AβO sensor based on antibodies, in which ferrocene attached to Zn-MOF as an electrochemical signal tag, and gold nanoparticles (AuNPs) for the amplification of signal from ferrocene.16 Wu et al. reported an electrochemical analysis of Aβ using a nanostructured biochip with gold nanoparticles to provide a large surface area and to magnify a detection signal.14 Kaushik et al. developed an electrochemical Aβ immunosensor with specific Aβ antibodies on a gold substrate and utilized bovine serum albumin (BSA) to block the non-binding sites.18 Electrochemical Aβ immunosensors are generally fabricated through the immobilization of a bioreceptor on the electrode for the specific binding of AβO with high affinity, and the extremely small changes in signal is transformed and amplified by the electrical impedance. From this perspective, the improvement of the sensitivity and detection should be directly dependent on the high conductivity and large surface area of the electrode. In order to achieve high conductivity and large surface area, conducting polymers (CPs) and metal nanoparticles, which are characterized by low-cost fabrication and easy 2


Page 2 of 17

Page 3 of 17 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


preparation, can be considered in the modification of the electrodes.19,20 Specifically, the presence of residual functional groups in CPs can support the immobilization of a bioreceptor, which reduces the complexity associated with electrically insulating organic binders or linking agents. Previously, we reported an electrochemical AβO sensor using poly (pyrrole-2-carboxylic acid) (PPy-2-COOH) transducer and a cellular prion protein (PrPC) receptor.21 The residual -COOH groups in PPy-2-COOH were utilized for linking with the amine-terminated (-NH2) PrPC bioreceptor. The developed PrPC/PPy-2-COOH biosensor showed a relatively low detection limit of 10-7 nM and a narrow detection range from 10-7 to 10 nM. However, for an ultra-small trace of AβO in blood or cerebrospinal fluid,6,22 an ultra-sensitive detection tool is urgently needed. In this work, we attempted to improve the AβO sensing performance of a PrPC modified electrode for the early diagnosis of AD. In the preparation of electrode, the additional thin layer of PEDOT embedded with gold nanoparticles (AuNPs-PEDOT) was electrochemically deposited for the high conductivity and the large surface area of the substrate, followed by the electrochemical deposition of poly(thiophene-3-acetic acid) (PTAA) for the immobilization of NH2-terminated PrPC bioreceptor. PEDOT acted as a conductive pathway between AuNPs and a stabilizer for the AuNPs.23,24 PDOT is a well-known outstanding CP equipped with high conductivity, high transparency, and excellent stability.25-27 Furthermore, the AuNPs can provide a large surface area as well as higher conductivity. The conducting polymer PTAA also has some advantages over PPy-2-COOH because of its more regular conjugated structure resulting in higher conductivity than that of pyrrole-2-carboxylic acid in which the 2-position is blocked by the -COOH group. The fabrication procedures for the modified electrodes are presented in Scheme 1 and each fabrication step was optimized to enhance sensing performance. The designed AuNPs-PEDOT-PTAA/PrPC sensor without any label detected AβO directly with a wide detection range from 10-8 to 104 nM. The AβO sensor can open the possibilities for the easy and early diagnosis of AD.

3



Scheme 1. The stepwise preparation of AuNPs-PEDOT-PTAA/PrPC sensor and its AβO detection EXPERIMENTAL SECTION Materials. 3-Thiophene acetic acid (3-TAA), 3,4-ethylenedioxythiophene (EDOT), anhydrous acetonitrile (ACN), K3Fe(CN)6, K4Fe(CN)6, NaBH4, N-Hydroxysuccinimide (NHS), tetrabutylammonium perchlorate, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and 2-(N-Morpholino)ethanesulfonic acid (MES) from Sigma-Aldrich (St. Louis, MO, USA); Gold (III) chloride from Strem Chemicals, Inc (Newburyport, MA, USA); and Aβ1-42 from AnaSpec, Inc. (Fremont, CA, USA) were purchased. The tissue protein extraction reagent (T-PER, cat no: 78510) and the BCA assay kit (cat no: 23225) were purchased from Thermo Fisher Scientific Korea Ltd. NH2-terminated PrPC(95-110) with the sequence of THSQWNKPSKPKTNMK from Peptron Ltd. (Daejeon, Korea) and Aβ1-42 monomer from AnaSpec, Inc (Fremont, CA, USA) were purchased. The protease inhibitor cocktail (cat no: K272-1) and phosphatase inhibitor cocktail (cat no: K276-1) were purchased from Biovision, Inc. (Milpitas, CA, USA). The bio-samples were stored at -20℃ before use. All chemicals were used without further purification. Phosphate-buffered saline (0.01 M PBS, pH=7.4) containing NaCl, KCl, Na2HPO4, and KH2PO4 was freshly prepared as the solvent. Modification of gold electrode and immobilization of PrPC. A 2 mm diameter gold disc was polished according to previous reports.

28,29

And all the electrolytes were degassed with

nitrogen for 15 min to prevent oxidation prior to polymerization.30 The modified electrode was prepared on the gold electrode as shown in Figure S1. The AuNPs-PEDOT was electrodeposited on the gold electrode followed by the electrodeposition of PTAA as a linking agent with PrPC (Scheme 1). The detailed process and the mechanism of AuNPs linked EDOT were discussed in Supporting information. In the immobilization of PrPC, 4


Page 4 of 17

Page 5 of 17 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


PTAA coated electrode was activated in the MES buffer (100 mM) with the same amount of EDC and NHS (20 mM) for 15 min, and then immersed in PBS (0.01 M, pH 7.4) solution containing NH2-PrPC for 1 h. PTAA and PEDOT-PTAA electrodes were also prepared for the comparison of sensing performance. PTAA was electrodeposited on the gold electrode by cyclic voltammetry (CV) from 0.55-1.9 V at a scan rate of 50 mVs-1 in the ACN containing 1 mg/mL 3-TAA and 50 mM tetrabutylammonium perchlorate for 10 cycles.31 In the case of PEDOT-PTAA electrode, PEDOT was deposited on the gold electrode by CV and PTAA was subsequently deposited under the same condition described above. Treatment of Aβ solution and real sample analysis. The preparation of Aβ monomers, oligomers, and fibrils was performed according to previous studies.32,33 All animals used in the real sample analysis experiments were purchased from the Jackson Laboratory (Bar Harbor, ME, USA), and maintained in the Department of Pharmacy at the Sungkyunkwan University. The experimental samples were prepared as shown in our previous work

21

and

described in the supporting information. Characterization. Transmission electron microscopy (TEM, JEM ARM 200F) was performed to confirm that the AuNPs were well-dispersed. The VSP Potentiostat (Princeton applied research, USA) with VSP EC-Lab software was utilized for all the electrochemical experiments. A conventional three-electrode system was employed with a prepared electrode on a gold disc, an Ag/AgCl, and a platinum plate as the working, reference, and counter electrodes, respectively. The applied potential from 0 to 0.5 V in the PBS (pH 7.4) containing 20 mM Fe(CN)63−/4− at a scan rate of 10 mVs-1 was applied for the CV measurements. A sine wave with 10 mV amplitude with a frequency range of 0.1 Hz -100 kHz in 20 mM Fe(CN)63−/4− /PBS (pH 7.4) was used to the as-prepared electrodes for the EIS measurements. The EIS data were subsequently analyzed from ZSimpWin EIS DATA analysis software (Perkin-Elmer, Version 2.00) with an appropriate equivalent circuit.

RESULTS AND DISCUSSION Preparation and characterization of PTAA, PEDOT-PTAA, and AuNPs-PEDOTPTAA electrodes. Figure 1 shows the CV curves recorded during the electropolymerization 5



of PTAA on various substrates: 1A on gold, 1B on PEDOT, 1C on AuNPs-PEDOT. The time for polymerization was fixed at 10 cycles. The behavior of CV was in good agreement with that obtained by others

29,34.

The current density was increased with successive cycling,

indicating the successful formation of PTAA. The preparation and electrochemical properties of the substrate are described in Supporting Information (Figure S2 and Table S1). In the deposition of PTAA on bare gold surface, only a minute increase in the current at 1.4 V was observed. However, the relatively large increase of the current density was observed for the deposition on the AuNPs-PEDOT surface. So the effect of the additional conductive layer of AuNPs-PEDOT can be explained for the facile formation of PTAA layer. Figure 1D shows the typical TEM image of the PTAA-AuNPs-PEDOT electrode in which 5-10 nm AuNPs were well dispersed in the PEDOT and PTAA.

Figure 1. Electrodeposition of PTAA (inset, amplification of PTAA) (A), PTAA on PEDOT (B), PTAA on AuNPs-PEDOT(C) by CV with applied potential 0.55-1.9 V at 50 mVs-1 scan rate for 10 cycles; the TEM image of AuNPs-PEDOT-PTAA (D). The electrochemical properties of the electrodes were evaluated by EIS (in Figure 2). In order to select the suitable model for the experimental results, four representative models were 6


Page 6 of 17

Page 7 of 17 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


applied and tested to compared with the experiment data in Figure S3.35 In the inset, the solution resistance (Rs), the double layer capacitance (C), the electron-transfer resistance (Ret), and the Warburg diffusion resistance (Zw) are represented.36,37 The Ret values for the modified electrodes were summarized in a table below. The semicircle domains representing Ret were clearly presented depending on the electrode types, implying a relative resistance value to the ferrocyanide redox-probe dissolved in the solution. The highly conductive AuNPs-PEDOT-PTAA electrode exhibited the lowest Ret value, which would be suitable for detecting insignificant changes in Ret, resulted from sensing extremely small amount of analyte. This function is extremely important for early diagnosis of AD. The electrochemically active specific surface (SA) was estimated from the specific capacitance and loading mass.38-40 As shown in the table, the AuNPs-PEDOT-PTAA electrode exhibited the highest SA of 175 cm2g-1 due to the incorporation of Au nanoparticles, which would allow the electrically active sites for a wide sensing range. The detailed data were represented in Table S1.

Figure 2. Nyquist plots of AuNPs-PEDOT-PTAA (a), PEDOT-PTAA (b), PTAA (c) electrode in 20 mM Fe(CN)63-/4/PBS. Sensing performance of PrPC immobilized three different electrodes. The process for immobilization of PrPC on the AuNPs-PEDOT-PTAA electrode and its binding with AβO were monitored by EIS analysis. The gradual increase of the Ret values after each steps confirmed the successful immobilization as shown in Figure 3A.21 The AuNPs-PEDOT corresponded to an almost straight line demonstrating a diffusion controlled process in the whole range. After the electrodeposition of AuNPs-PEDOT-PTAA, an increased Ret value in 7



the EIS was monitored indicating that the successful formation of AuNPs-PEDOT-PTAA hindered the electron transfer slightly to the electrochemical probe. When the PrPC was immobilized on the surface of AuNPs-PEDOT-PTAA, the bioreceptor may further prevent the Fe(CN)6

3-/4-ions

from reaching the electrode for the redox reaction.21 Furthermore, the

subsequent binding of AβO with PrPC made the diameter of semicircle increased dramatically. The attached AβO may act as a barrier to block the charge and mass transfer, hindering the access of Fe(CN)63-/4- toward the electrochemical probe and finally increasing the Ret value.41 In the case of progressive amount of AβO bound with PrPC receptor, the Ret value of the EIS increased dramatically from 205.1 Ω cm-2 (in 10-8 nM AβO) to 953.9 Ω cm-2 (in 104 nM AβO), as shown in Figure 3B. The EIS results of PTAA and PEDOT-PTAA electrodes are presented in Figure S4. The relationship between the Ret variation (∆Ret) and logarithmic concentration of AβO (Log Aβ) is shown in Figure 3C, which can be expressed in a calibration curve as ∆Ret = a Log Aβ + b. The value in the bracket is the slope of the curve represented the sensitivity of prepared sensor. The sensitivity and detection range of the prepared sensors are summarized in a table. The overall performance of the AuNPs-PEDOT-PTAA/PrPC sensor was superior to that of the PTAA/PrPC and PEDOT-PTAA/PrPC sensors in terms of sensitivity and detection range. The initial Ret of the synthesized sensors was quite discrepant: 229 Ω for PTAA/PrPC, 184 Ω for PEDOT-PTAA/PrPC, and 99 Ω for AuNPs-PEDOT-PTAA/PrPC. The low Ret value (i.e. high conductivity) of AuNPs-PEDOT-PTAA/PrPC sensor can detect a small change in electrochemical signal by the low concentration of AβO and allow the limit of detection (LOD) up to10-2 fM of AβO. In the impedance sensors, the Ret value was increased with increasing concentrations of target materials. An initial high Ret value may limit the low detection of AβO. Furthermore, the high surface area of the AuNPs-PEDOT-PTAA/PrPC sensor can embrace large amounts and huge molecules of AβO and detect 104 nM of AβO. In the electrochemical impedance sensor, it can’t be overemphasized that high conductivity (initial low resistance) and high surface area are prerequisite for high sensitivity with LOD and wide detection range. The immobilization of PrPC and the binding with AβO on the electrodes were also 8


Page 8 of 17

Page 9 of 17 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


characterized by CV and are shown in Figure S5. The results were consistent with the results of the EIS experiments. Based on these results, we concluded that the sensing performance including the sensitivity and the detection range was deeply dependent on the electrochemical properties of the designed electrode.

Electrode

Initial

PrPC (1 mg/mL)

∆Ret

Ret (Ω)

Ret (Ω)

Detection range

Sensitivity

(nM)

(Ω/ log nM)

429.9

-5

PTAA

229

317

88.3

10 -10

18.4

90.8

450.7

-6

PEDOT-PTAA

184

275.2

AuNPs-PEDOT-PTAA

99

186.2

10 -10

24.2

87.3

773.2

10-8-104

62.3

(Ω)

AβO (10 nM) Ret (Ω)

Figure 3. Nyquist plots of the step immobilization (A) and sensing performance (B) for AuNPs-PEDOT-PTAA based electrode in 20 mM Fe(CN)63−/4− /PBS at constant concentration of PrPC(1 mg/mL); in the table represented the Ret values before/after the immobilization and linear relationship between ∆Ret value and logarithmic AβO concentration with different kinds of electrodes (C) In order to optimize the PTAA content on AuNPs-PEDOT, several sensors were prepared with different TAA concentrations. The EIS responses with the calibration curves of the prepared sensors are shown in Figure S6. Briefly, the sensitivities of the sensors gradually 9



Page 10 of 17

increased as the TAA content increased. The more amount of TAA can be occupied with more affinity materials (PrPC), thus, sensitivity toward the target materials (AβO) was increased

13,42.

In the case of high loading with PTAA, the thickness of the PTAA film was

increased and the initial Ret value was also increased resulting in limited sensing performance. Similar results were reported in the literature.21 Hence, the optimal concentration of TAA was chosen to be 1 mg/mL, in consideration of the sensitivity and detection range. In addition, the sensing performance of the AuNPs-PEDOT-PTAA/PrPC sensor was also compared with other published AβO sensors in Table 1. The AuNPs-PEDOT-PTAA/PrPC sensor exhibited extra low detection at the 10-2 fM level and a wide detection range from 10-8 to 104 nM, which was sensitive enough for the early diagnosis of AD.4,5

Table1. Summary of sensitivity performance of electrochemical amyloid-β sensors Electrode Peptide/MUA/Au a PrPC/POPA/Au b Antibody/AuNP/AAO

Linear range (nM)

Analysis

LOD (nM)

Reference

0.48-12

SWV

2.4 x10-1

11

10-3-103

EIS

5 x10-4

10

2.2x10-4-2.2#

c

PrPC/Cysteine/Au d

5

Ad-PrPC/MCH/AgNPs-β-CD/Au e

2x10-2-102

An βA Abs/DTSP/Au

x10-3-2

Antibody-aptamer/graphene/Au

g

10-7 -10

PrPC/AuNPs-PEDOT-PTAA/Au

10-8 -104

a

Peptide: I10 peptide;

b

14

3

x10-3

43

8

x10-3

12

1

x10-2

18

1

x10-1

13

EIS

10-7

21

EIS

10-8

Our work

LSV EIS

0.5-30

PrPC/PPy-2-COOH/Au h

EIS CV

10-2-102

f

x10-4#

DPV

2.2

PrPC/POPA: the poly (tyramine and 3-(4-hydroxyphenyl) propionic acid) copolymer with the

attachment of cellular prion protein; c Antibody/AuNP/AAO: Aβ antibody/gold nanoparticle decorated anodic aluminum oxide;

d

PrPC/Cysteine: Cysteine-containing PrPC /alkaline phosphatase-conjugated PrPC; e Ad-PrPC: adamantine labeled

PrPC; f An βA Abs/DTSP: self-assembled monolayer of dithiobis (succinimidyl propionate) with the immobilization anti-Aβ antibodies;

g

Antibody-aptamer/graphene/Au: the antibodies of Aβ oligomers and a nanocomposite of gold nanoparticles

with aptamer and thionine (aptamer-Au-Th) were used as the recognition element;

h

PPy-2-COOH: Poly

#

(pyrrole-2-carboxylic acid); Value was expressed in ng/mL (pg/mL) and converted to nM, the Mw of Aβ monomer was 4514.1 g/mol.

Real sample testing. The procedure (Figure S7) for sampling and analysis of mice tissue was carried out according to the literature

21

and more details are given in the Supporting

Information. The analysis of AD mice using the AuNPs-PEDOT-PTAA/PrPC sensor is shown in Figure 4, and the data is summarized in the table below. In the 0.05 mg/mL wild type mice 10


Page 11 of 17 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


(WT), 9.25 x 10-9 nM AβO was detected using the AuNPs-PEDOT-PTAA/PrPC sensor with 2.2% RSD. In the 0.05 mg/mL AD mice, 1.94 x 10-6 nM AβO was detected with 1.4% RSD. The recoveries of different concentrations of spiked AβO in AD mice were from 99.1 to 102 %, and the RSDs were less than 5% for three independent measurements. These results indicated that the AuNPs-PEDOT-PTAA/PrPC based sensor was promising for AβO detection with satisfactory in actual use.

Sample

Spiked

After spiking

RSD

Recovery

(× 10-3 nM)

(× 10-3 nM)

(%)

(%)

a. AuNPs-PEDOT-PTAA/PrPC b. In 0.05 mg/mL WT, 9.25 x10-9 nM (RSD 2.2 %) AβO c. In 0.005 mg/mL AD, 1.94 x10-6 nM (RSD 1.4 %) AβO

1

0.99(c')

1.5

99.1

d. In 0.05 mg/mL AD, 1.81 x10 nM (RSD 2.5 %) AβO

1

1.40(d')

3.8

102

e. In 0.5 mg/mL AD, 1.53 x10 nM (RSD 2.3 %) AβO

1

1.15(e')

1.2

99.8

-5

-4

WT: wild type, normal mice, AD: Alzheimer’s disease mice, RSD: relative standard deviation for three independent measurements, Recovery: (after spiking–before spiking)/(spiked)x100%.

Figure 4. Nyquist plots of the AβO detection using AuNPs-PEDOT-PTAA/PrPC sensor during the ex vivo test: the initial AuNPs-PEDOT-PTAA/PrPC sensor (a), in 0.05 mg/mL WT (b), in 0.005 mg/mL AD (c), in 0.05 mg/mL AD (d), in 0.5 mg/mL AD (e), and with the spiked 0.001 nM AβO [in 0.005 mg/mL AD (c'), 0.05 mg/mL AD (d'), 0.5 mg/mL AD (e')]. The detection results were summarized in a table. Stability

and

selectivity

of

AuNPs-PEDOT-PTAA

based

sensor.

The

AuNPs-PEDOT-PTAA/PrPC sensor was stored at 4℃ to readout the current response every 7 days for a month. No significant change was observed during the stability test, indicating the sufficient stability of the sensor for determination of the AβO. 11



For the selectivity, the AuNPs-PEDOT-PTAA/PrPC sensor was also tested in Aβ monomers and fibrils. The increase in ∆Ret by the logarithmic concentration of various Aβ states is shown in Figure S8. The sensor showed high sensitivity for the Aβ oligomer.

CONCLUSION Ultra-sensitive at sub-femto molar level AO sensor was demonstrated using PrPC receptor on polythiophene transducer electrode. An additional thin layer of PEDOT with gold nanoparticles was employed for high conductivity as well as high surface area. Poly (thiophene-3-acetic acid) (PTAA) was used for binding NH2-PrPC receptor. The effect of the conductivity (initial resistance, Ret) and surface area of the prepared electrodes on the sensing performances were carefully investigated as a function of sensitivity, limit of detection, and detection range. The impedance response of the AuNPs-PEDOT-PTAA/PrPC sensor showed an extremely low detection of AβO at a sub-femtomolar level with a wide range from 10-8 to 104 nM. Herein, the high sensitivity of the AβO sensor can be used in practical applications for the early diagnosis of AD.

ASSOCIATED CONTENT Supporting information This material is available free of charge via the Internet at http://pubs.acs.org. Additional characterization data and electrochemical performances (PDF).

AUTHOR INFORMATION Corresponding Authors *Give contact information for the author(s) to whom correspondence should be addressed. [*] Prof. Youngkwan Lee Email: [email protected]; Tel: +82 31 290 7248 [*] Dr. Misuk Cho Email: [email protected]: +82 31 290 7326

12


Page 12 of 17

Page 13 of 17 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


ACKNOWLEDGEMENTS This research was sponsored by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT and Future Planning of Korea (NRF-2019R1A2C1003551 and 2019R1A2C1003594).

13



REFENCES (1) Nakamura, A.; Kaneko, N.; Villemagne, V. L.; Kato, T.; Doecke, J.; Doré, 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. High performance plasma amyloid-β biomarkers for Alzheimer’s disease. Nature 2018, 554, 249. (2) He, Y.; Wei, M.; Wu, Y.; Qin, H.; Li, W.; Ma, X.; Cheng, J.; Ren, J.; Shen, Y.; Chen, Z.; Sun, B.; Huang, F.-D.; Shen, Y.; Zhou, Y.-D. Amyloid β oligomers suppress excitatory transmitter release via presynaptic depletion of phosphatidylinositol-4,5-bisphosphate. Nature Communications 2019, 10, 1193. (3) Limbocker, R.; Chia, S.; Ruggeri, F. S.; Perni, M.; Cascella, R.; Heller, G. T.; Meisl, G.; Mannini, B.; Habchi, J.; Michaels, T. C. Trodusquemine enhances Aβ 42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes. Nature communications 2019, 10, 225. (4) Hölttä, M.; Hansson, O.; Andreasson, U.; Hertze, J.; Minthon, L.; Nägga, K.; Andreasen, N.; Zetterberg, H.; Blennow, K. Evaluating Amyloid-β Oligomers in Cerebrospinal Fluid as a Biomarker for Alzheimer’s Disease. PLOS ONE 2013, 8, e66381. (5) Mroczko, B.; Groblewska, M.; Litman-Zawadzka, A.; Kornhuber, J.; Lewczuk, P. Amyloid β oligomers (AβOs) in Alzheimer’s disease. J. Neural Transm. 2018, 125, 177-191. (6) Georganopoulou, D. G.; Chang, L.; Nam, J.-M.; Thaxton, C. S.; Mufson, E. J.; Klein, W. L.; Mirkin, C. A. Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer's disease. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 2273-2276. (7) Roher, A. E.; Esh, C. L.; Kokjohn, T. A.; Castaño, E. M.; Van Vickle, G. D.; Kalback, W. M.; Patton, R. L.; Luehrs, D. C.; Daugs, I. D.; Kuo, Y.-M.; Emmerling, M. R.; Soares, H.; Quinn, J. F.; Kaye, J.; Connor, D. J.; Silverberg, N. B.; Adler, C. H.; Seward, J. D.; Beach, T. G.; Sabbagh, M. N. Amyloid beta peptides in human plasma and tissues and their significance for Alzheimer's disease. Alzheimer's & Dementia 2009, 5, 18-29. (8) Negahdary, M.; Heli, H. An ultrasensitive electrochemical aptasensor for early diagnosis of Alzheimer’s disease, using a fern leaves-like gold nanostructure. Talanta 2019, 198, 510-517. (9) Wen, W.; Yan, X.; Zhu, C.; Du, D.; Lin, Y. Recent Advances in Electrochemical Immunosensors. Anal. Chem. 2017, 89, 138-156. (10) Rushworth, J. V.; Ahmed, A.; Griffiths, H. H.; Pollock, N. M.; Hooper, N. M.; Millner, P. A. A label-free electrical impedimetric biosensor for the specific detection of Alzheimer's amyloid-beta oligomers. Biosens. Bioelectron. 2014, 56, 83-90. (11) Li, H.; Cao, Y.; Wu, X.; Ye, Z.; Li, G. Peptide-based electrochemical biosensor for amyloid β 1–42 soluble oligomer assay. Talanta 2012, 93, 358-363. (12) Xia, N.; Wang, X.; Zhou, B.; Wu, Y.; Mao, W.; Liu, L. Electrochemical Detection of Amyloid-β Oligomers Based on the Signal Amplification of a Network of Silver Nanoparticles. ACS Appl. Mat. Interfaces 2016, 8, 19303-19311. (13) Zhou, Y.; Zhang, H.; Liu, L.; Li, C.; Chang, Z.; Zhu, X.; Ye, B.; Xu, M. Fabrication of an antibody-aptamer sandwich assay for electrochemical evaluation of levels of β-amyloid oligomers. Sci. Rep. 2016, 6, 35186. (14) Wu, C.-C.; Ku, B.-C.; Ko, C.-H.; Chiu, C.-C.; Wang, G.-J.; Yang, Y.-H.; Wu, S.-J. 14


Page 14 of 17

Page 15 of 17 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


Electrochemical impedance spectroscopy analysis of A-beta (1-42) peptide using a nanostructured biochip. Electrochim. Acta 2014, 134, 249-257. (15) Zhou, Y.; Li, C.; Li, X.; Zhu, X.; Ye, B.; Xu, M. A sensitive aptasensor for the detection of β-amyloid oligomers based on metal–organic frameworks as electrochemical signal probes. Anal. Methods 2018, 10, 4430-4437. (16) Han, J.; Zhang, M.; Chen, G.; Zhang, Y.; Wei, Q.; Zhuo, Y.; Xie, G.; Yuan, R.; Chen, S. Ferrocene covalently confined in porous MOF as signal tag for highly sensitive electrochemical immunoassay of amyloid-β. J. Mater. Chem. B 2017, 5, 8330-8336. (17) Wang, Z.; Liu, T.; Asif, M.; Yu, Y.; Wang, W.; Wang, H.; Xiao, F.; Liu, H. Rimelike Structure-Inspired Approach toward in Situ-Oriented Self-Assembly of Hierarchical Porous MOF Films as a Sweat Biosensor. ACS Appl. Mat. Interfaces 2018, 10, 27936-27946. (18) Kaushik, A.; Shah, P.; Vabbina, P. K.; Jayant, R. D.; Tiwari, S.; Vashist, A.; Yndart, A.; Nair, M. A label-free electrochemical immunosensor for beta-amyloid detection. Anal. Methods 2016, 8, 6115-6120. (19) Yang, J.; Cho, M.; Pang, C.; Lee, Y. Highly sensitive non-enzymatic glucose sensor based on over-oxidized polypyrrole nanowires modified with Ni(OH)(2) nanoflakes. Sensors and Actuators B-Chemical 2015, 211, 93-101. (20) Lin, M.; Cho, M.; Choe, W.-S.; Yoo, J.-B.; Lee, Y. Polypyrrole nanowire modified with Gly-Gly-His tripeptide for electrochemical detection of copper ion. Biosens. Bioelectron. 2010, 26, 940-945. (21) Qin, J.; Jo, D. G.; Cho, M.; Lee, Y. Monitoring of early diagnosis of Alzheimer's disease using the cellular prion protein and poly(pyrrole-2-carboxylic acid) modified electrode. Biosens. Bioelectron. 2018, 113, 82-87. (22) Song, F.; Poljak, A.; Valenzuela, M.; Mayeux, R.; Smythe, G. A.; Sachdev, P. S. Meta-analysis of plasma amyloid-β levels in Alzheimer's disease. Journal of Alzheimer's disease : JAD 2011, 26, 365-375. (23) Sih, B. C.; Teichert, A.; Wolf, M. O. Electrodeposition of Oligothiophene-Linked Gold Nanoparticle Films. Chem. Mater. 2004, 16, 2712-2718. (24) Bourgoin, J.-P.; Kergueris, C.; Lefèvre, E.; Palacin, S. Langmuir–Blodgett films of thiol-capped gold nanoclusters: fabrication and electrical properties. Thin Solid Films 1998, 327-329, 515-519. (25) Kumar, S. S.; Mathiyarasu, J.; Phani, K. L. N.; Yegnaraman, V. Simultaneous determination of dopamine and ascorbic acid on poly (3,4-ethylenedioxythiophene) modified glassy carbon electrode. J. Solid State Electrochem. 2006, 10, 905-913. (26) Aldalbahi, A.; Rahaman, M.; Almoiqli, M. A Strategy to Enhance the Electrode Performance of Novel Three-Dimensional PEDOT/RVC Composites by Electrochemical Deposition Method. Polymers 2017, 9, 157. (27) Lin, Y.-F.; Li, C.-T.; Ho, K.-C. A template-free synthesis of the hierarchical hydroxymethyl PEDOT tube-coral array and its application in dye-sensitized solar cells. J. Mater. Chem. A 2016, 4, 384-394. (28) Yang, J.; Kim, S. E.; Cho, M.; Yoo, I. K.; Choe, W. S.; Lee, Y. Highly sensitive and selective determination of bisphenol-A using peptide-modified gold electrode. Biosens. Bioelectron. 2014, 61, 38-44. 15



(29) Su, W. Q.; Cho, M.; Nam, J. D.; Choe, W. S.; Lee, Y. Highly sensitive electrochemical lead ion sensor harnessing peptide probe molecules on porous gold electrodes. Biosens. Bioelectron. 2013, 48, 263-269. (30) Lin, M.; Cho, M. S.; Choe, W. S.; Lee, Y. Electrochemical analysis of copper ion using a Gly-Gly-His tripeptide modified poly(3-thiopheneacetic acid) biosensor. Biosens. Bioelectron. 2009, 25, 28-33. (31) Peng, H.; Zhang, L.; Spires, J.; Soeller, C.; Travas-Sejdic, J. Synthesis of a functionalized polythiophene as an active substrate for a label-free electrochemical genosensor. Polymer 2007, 48, 3413-3419. (32) Qin, J.; Cho, M.; Lee, Y. Ferrocene-Encapsulated Zn Zeolitic Imidazole Framework (ZIF-8) for Optical and Electrochemical Sensing of Amyloid-β Oligomers and for the Early Diagnosis of Alzheimer’s Disease. ACS Appl. Mat. Interfaces 2019, 11, 11743-11748. (33) Stine, W. B.; Dahlgren, K. N.; Krafft, G. A.; LaDu, M. J. In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J. Biol. Chem. 2003, 278, 11612-11622. (34) Malitesta, C.; Guascito, M. R.; Mazzotta, E.; Picca, R. A. X-Ray Photoelectron Spectroscopy characterization of electrosynthesized poly(3-thiophene acetic acid) and its application in Molecularly Imprinted Polymers for atrazine. Thin Solid Films 2010, 518, 3705-3709. (35) Qin, J.; Park, J. S.; Jo, D. G.; Cho, M.; Lee, Y. Curcumin-based electrochemical sensor of amyloid-β oligomer for the early detection of Alzheimer’s disease. Sensor Actuat BChem. 2018, 273, 1593-1599. (36) Kim, S.; Lee, S. H.; Cho, M.; Lee, Y. Solvent-assisted morphology confinement of a nickel sulfide nanostructure and its application for non-enzymatic glucose sensor. Biosens. Bioelectron. 2016, 85, 587-595. (37) Wang, Z.; Liu, T.; Yu, Y.; Asif, M.; Xu, N.; Xiao, F.; Liu, H. Coffee Ring–Inspired Approach toward Oriented Self-Assembly of Biomimetic Murray MOFs as Sweat Biosensor. Small 2018, 14, 1802670. (38) Jung, H.; Lee, S. H.; Yang, J.; Cho, M.; Lee, Y. Ni(OH)2@Cu dendrite structure for highly sensitive glucose determination. RSC Advances 2014, 4, 47714-47720. (39) Lee, S. H.; Yang, J.; Han, Y. J.; Cho, M.; Lee, Y. Rapid and highly sensitive MnOx nanorods array platform for a glucose analysis. Sensor Actuat B- Chem. 2015, 218, 137-144. (40) Chen, W.; Fan, Z.; Gu, L.; Bao, X.; Wang, C. Enhanced capacitance of manganese oxide via confinement inside carbon nanotubes. Chemical Communications 2010, 46, 3905-3907. (41) Su, W.; Lin, M.; Lee, H.; Cho, M.; Choe, W. S.; Lee, Y. Determination of endotoxin through an aptamer-based impedance biosensor. Biosens. Bioelectron. 2012, 32, 32-36. (42) Limbut, W.; Kanatharana, P.; Mattiasson, B.; Asawatreratanakul, P.; Thavarungkul, P. A comparative study of capacitive immunosensors based on self-assembled monolayers formed from thiourea, thioctic acid, and 3-mercaptopropionic acid. Biosens. Bioelectron. 2006, 22, 233-240. (43) Liu, L.; Xia, N.; Jiang, M.; Huang, N.; Guo, S.; Li, S.; Zhang, S. Electrochemical detection of amyloid-β oligomer with the signal amplification of alkaline phosphatase plus electrochemical–chemical–chemical redox cycling. J. Electroanal. Chem. 2015, 754, 40-45. 16


Page 16 of 17

Page 17 of 17 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


Table of Contents Graphic

17


Ultrasensitive Detection of Amyloid-Î² Using ... - ACS Publications

Recommend Documents