Femtomolar sensing of Alzheimer's tau proteins by water oxidation-coupled photoelectrochemical platform

Journal Pre-proof Femtomolar sensing of Alzheimer's tau proteins by water oxidation-coupled photoelectrochemical platform Kayoung Kim, Chan Beum Park PII:

S0956-5663(20)30072-5

DOI:

https://doi.org/10.1016/j.bios.2020.112075

Reference:

BIOS 112075

To appear in:

Biosensors and Bioelectronics

Received Date: 1 November 2019 Revised Date:

31 January 2020

Accepted Date: 4 February 2020

Please cite this article as: Kim, K., Park, C.B., Femtomolar sensing of Alzheimer's tau proteins by water oxidation-coupled photoelectrochemical platform, Biosensors and Bioelectronics (2020), doi: https:// doi.org/10.1016/j.bios.2020.112075. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

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2nd Revised manuscript

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Femtomolar Sensing of Alzheimer’s Tau Proteins by Water Oxidation-Coupled Photoelectrochemical Platform

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Our answers to the Reviewer #1’s comment are described in a separate file uploaded along with the revised manuscript.

Kayoung Kim and Chan Beum Park* Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), 335 Science Road, Daejeon 305-701, Republic of Korea. *E-mail: [email protected], Tel.: +82-42-350-3340; Fax: +82-42-350-3310

Abstract

15

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder. A key

16

pathogenic event of AD is the formation of intracellular neurofibrillary tangles that are

17

mainly composed of tau proteins. Here, we report on ultrasensitive detection of total tau (t-tau)

18

proteins using an artificial electron donor-free, BiVO4-based photoelectrochemical (PEC)

19

analysis. The platform was constructed by incorporating molybdenum (Mo) dopant and iron

20

oxyhydroxide (FeOOH) ad-layer into the BiVO4 photoelectrode and employing a signal

21

amplifier

22

diaminobenzidine (DAB). Despite the absence of additional electron suppliers, the

23

FeOOH/Mo:BiVO4 conjugated with the Tau5 antibody produced strong current signals at 0 V

24

(vs. Ag/AgCl, 3M NaCl) under the illumination of a white light-emitting diode. The Mo

25

extrinsic dopants increased the charge carrier density of BiVO4-Tau5 by 1.57 times, and the

26

FeOOH co-catalyst promoted the interfacial water oxidation reaction of Mo:BiVO4-Tau5 by

27

suppressing charge recombination. The introduction of HRP-labeled Tau46 capture

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antibodies to the FeOOH/Mo:BiVO4-Tau5 platform produced insoluble precipitation on the

29

transducer by accelerating the oxidation of DAB, which amplified the photocurrent signal of

30

FeOOH/Mo:BiVO4-Tau5 by 2.07-fold. Consequently, the water oxidation-coupled,

31

FeOOH/Mo:BiVO4-based PEC sensing platform accurately and selectively recognized t-tau

32

proteins down to femtomolar concentrations; the limit of detection and limit of quantification

33

were determined to be 1.59 fM and 4.11 fM, respectively.

formed

by

horseradish

peroxidase

(HRP)-triggered

oxidation

of

3,3′-

34 35 36

Keywords: Alzheimer’s disease, tau proteins, Femtomolar sensitivity, BiVO4, water oxidation

37

1

1 2

1. Introduction

3

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder that

4

causes progressive cognitive decline and irreversible memory impairment. It affects more

5

than 10% of people over the age of 65 worldwide, and its incidence is estimated to grow

6

exponentially over the next decades (Chen et al. 2018). Current methods for AD diagnosis

7

(e.g., neuropsychological evaluation, neuroimaging) are primarily based on symptoms,

8

suffering from limitations such as low accuracy and the lack of accessibility (Weimer and

9

Sager 2009). One of the key pathogenic events of AD is the formation of intracellular

10

neurofibrillary tangles that are mainly composed of tau proteins, which exist as six isoforms

11

having 352 to 441 amino acid residues (Verwilst et al. 2018). The tau proteins in the normal

12

adult brain stabilize the microtubule architecture through tubulin-binding motifs and promote

13

axonal outgrowth (Wang and Mandelkow 2015). However, the abnormal phosphorylation of

14

tau proteins in AD patients’ brains causes a reduction in the binding affinity between tau

15

proteins and microtubules, which subsequently leads to the impairment of neuronal function

16

(Polanco et al. 2017). Moreover, the hyperphosphorylated tau proteins self-assemble into

17

toxic insoluble aggregates such as paired helical filaments and tau tangles. Because the

18

tauopathy starts decades before the manifestation of AD symptoms (Blennow et al. 2015;

19

Congdon and Sigurdsson 2018; Nordberg 2015), tracking these neuropathological changes

20

can assist in identifying asymptomatic AD patients. Recent studies have shown that the total-

21

tau proteins—t-tau that includes all tau isoforms irrespective of phosphorylation state—in the

22

blood are the most effective biomarker in discriminating AD patients from normal individuals

23

and predicting the progression of neurodegeneration in the AD brain (Olsson et al. 2016).

24

Clinical studies have revealed that the levels of t-tau proteins in the blood plasma of AD

25

patients are approximately two to three folds higher than those of normal controls (Yang et al.

2

1

2018). However, blood concentrations of pathological t-tau proteins are in the femtomolar

2

levels, beyond the detection range of conventional enzyme-linked immunosorbent assay (Lue

3

et al. 2017; Yang et al. 2018).

4

Here, we report on ultrasensitive, photoelectrochemical (PEC) detection of t-tau

5

proteins using an artificial electron donor-free, bismuth vanadate (BiVO4)-based sensing

6

platform. In PEC analysis, a photoelectrode generates excited charge carriers by harnessing

7

light energy; the photogenerated majority charges are transferred to the counter electrode

8

through the external circuit, while the minority carriers participate in redox reactions with

9

sacrificial scavengers at the semiconductor/electrolyte interface (Farka et al. 2017). Typically,

10

sacrificial electron suppliers (e.g., ascorbic acid, uric acid, glutathione, and triethanolamine)

11

are required to suppress the electron-hole recombination (Kang et al. 2010). However, the

12

accumulation of oxidized scavengers impedes continuous redox reactions at the interface

13

(Lee et al. 2018a; Son et al. 2018), disturbing the accurate sensing of target analytes. We

14

envisioned that a PEC sensing platform using water as an electron donor would be highly

15

desirable because it would not require an additional electron supplier.

16

We have designed iron oxyhydroxide deposited, molybdenum-doped BiVO4

17

(FeOOH/Mo:BiVO4)-based platform for water oxidation-coupled, PEC sensing of

18

femtomolar t-tau proteins, as depicted in Figure 1. The monoclinic BiVO4 is a ternary oxide-

19

based semiconducting material that has intrinsic advantages such as substantial absorption of

20

visible light (Kim et al. 2015), favorable band-edge position (Cooper et al. 2014), and earth

21

abundance (Lee and Choi 2018). Along with the beneficial intrinsic properties of BiVO4, it

22

has been regarded as a top-performer in water oxidation among numerous metal oxide-based

23

light absorbers (Jang et al. 2017; Kim et al. 2016a; Kim and Lee 2019). The hexavalent Mo

24

ion (Mo6+) was introduced as a donor-type dopant to facilitate electron-hole separation in

25

bulk BiVO4. The FeOOH as an electrocatalyst to accelerate the water oxidation reaction at 3

1

the surface of Mo:BiVO4. We demonstrate that the FeOOH/Mo:BiVO4-based PEC sensing

2

platform can generate strong and stable current signals in the absence of a sacrificial electron

3

donor under the illumination of a white light-emitting diode (LED). To extend the detection

4

limit of the sensing platform down to femtomolar levels, we have adopted a horseradish

5

peroxidase (HRP)-labeled, sandwich-type PEC sensing configuration. HRP accelerates the

6

formation of insoluble precipitates onto the transducer by oxidizing 3,3 ′-diaminobenzidine

7

(DAB) in the presence of H2O2 (Fan et al. 2012; Lathwal and Sikes 2016; Li et al. 2015),

8

resulting in significant changes in the photocurrent of the sensing platform. We verify that the

9

FeOOH/Mo:BiVO4-based PEC sensing platform can accurately and selectively recognize t-

10

tau proteins in blood plasma by amplifying the photocurrent signal via peroxidase-catalyzed

11

oxidation of DAB.

12

2. Experimental section

13

2.1 Chemicals and Materials

14

Human tau441 was purchased from the rPeptide Co., USA. The fluorine-doped SnO2 (FTO)

15

substrates were obtained from Nippon Sheet Glass Co., Ltd. Monoclonal antibody reactive to

16

210-241 residues of tau proteins (clone Tau5) was purchased from EMD Millipore Co., USA.

17

The Tau5 antibody recognizes all phosphorylated and non-phosphorylated isoforms of tau

18

proteins. The HRP-conjugated antibody (clone Tau46) that reacts with the phosphorylation-

19

independent epitope in the c-terminals of the human tau proteins was obtained from Santa

20

Cruz Biotechnology Inc, USA. Bismuth(III) nitrate pentahydrate (98.0%), Potassium iodide

21

(≥ 99.0%), Nitric acid (HNO3, 70%), p-benzoquinone (≥ 98.0%), vanadyl acetylacetonate

22

(98.0%), dimethyl sulfoxide (DMSO, ≥ 99.9%), Sodium hydroxide (NaOH, ≥ 97.0%),

23

Bis(acetylacetonato)dioxomolybdenum(VI), Iron(II) sulfate heptahydrate (FeSO4, ≥99.0%),

24

Tween 20, (3-Aminopropyl) triethoxysilane (99%), N,N-disuccinimidyl carbonate (≥95%),

25

bovine serum albumin (≥98%), 3,3′-Diaminobenzidine tetrahydrochloride hydrate (≥96%), 4

1

sodium phosphate dibasic dehydrate (≥99.0%), and sodium phosphate monobasic

2

monohydrate (≥98%) were purchased from Sigma Aldrich Chemical Co., USA. Hydrogen

3

peroxide (H2O2, 35 % (w/w) in H2O) was obtained from Junsei Chemical Co.Ltd, Japan.

4 5

2.2 Synthesis of pristine BiVO4 photoelectrodes

6

We fabricated nanoporous BiVO4 electrodes according to the literature (Kim and Choi

7

2014b). First, we adjusted the pH of 0.4 M KI solution to 1.7 using HNO3, followed by the

8

addition of 0.04 M Bi(NO3)3 into the solution. After vigorous stirring for a few minutes, we

9

slowly added the absolute ethanol containing 0.23M p-benzoquinone into the solution. We

10

electrodeposited the BiOI on FTO substrates in a typical three-electrode configuration using

11

an Ag/AgCl (3 M NaCl) electrode as a reference electrode and a Pt mesh as a counter

12

electrode. We immersed the FTO substrates into the plating solution and applied bias of -0.1

13

V (vs. Ag/AgCl, 3M NaCl) for 4 min at room temperature. Afterward, we prepared 0.2 M

14

vanadyl acetylacetonate dissolved in dimethyl sulfoxide (DMSO) and applied it to the

15

electrodeposited BiOI electrode. After annealing the sample at 450 °C for 2 h, we soaked the

16

resulting samples in 1M NaOH solution for 30 min with gentle stirring. The as-synthesized

17

BiVO4 electrodes were rinsed with deionized (DI) water.

18 19

2.3 Synthesis of FeOOH/Mo:BiVO4 photoelectrode

20

We electrodeposited the BiOI film on the FTO substrate using the identical synthetic method

21

described above. To make Mo:BiVO4 photoelectrode, we prepared vanadyl acetylacetonate

22

(0.2M) dissolved in DMSO, followed by the addition of the different amount of the DMSO

23

solution containing 0.2 M molybdenum acetylacetonate. The Mo precursor solutions having

24

the different molar ratios of molybdenum acetylacetonate to vanadyl acetylacetonate (i.e.,

25

0.05, 0.1, 0.3, 1.0, 3.0 mol%) were applied to the as-prepared BiOI films. Subsequently, the

5

1

samples were annealed at 450 °C for 2 h with the heating rate of 2 °C min−1. After cooling in

2

air, we immersed the resulting samples in 1M NaOH solution for 30 min at room temperature.

3

We deposited FeOOH layer on the as-synthesized Mo:BiVO4 photoelectrode by using photo-

4

assisted electrochemical deposition method. We dissolved 0.1 M FeSO4 in DI water purged

5

with N2 for 1h. The Mo: BiVO4 photoelectrode was immersed in the FeSO4 solution with an

6

Ag/AgCl (3M NaCl) reference electrode and a Pt mesh. We applied 0.25 V (vs Ag/AgCl, 3M

7

NaCl) for 20 min with illumination to the backside of Mo: BiVO4 photoelectrode. After that,

8

an anodic bias of 1.2 V (vs Ag/AgCl, 3M NaCl) was additionally applied under the dark

9

condition for 1 min. The as-fabricated FeOOH/Mo:BiVO4 photoelectrodes were washed with

10

DI water several times.

11 12

2.4 Characterization of FeOOH/Mo:BiVO4 photoelectrode

13

We observed the morphology of the as-prepared FeOOH/Mo:BiVO4 photoelectrode using an

14

S-4800 field emission scanning microscope (Hitachi High-technologies Co., Japan) and Talos

15

F200X transmission electron microscope (Thermo Fisher Scientific Inc., USA). The

16

elemental mapping images of FeOOH/Mo:BiVO4 photoelectrode was obtained using energy

17

dispersive X-ray spectroscopic analysis in Talos F200X scanning transmission electron

18

microscope (Thermo Fisher Scientific Inc., USA). We examined the crystal structure and

19

chemical composition of the as-synthesized FeOOH/Mo:BiVO4 photoelectrodes using X-ray

20

diffractometer (Rigaku Co., Japan) and a K-alpha X-ray photoelectron spectroscope (Thermo

21

Scientific, USA), respectively. The dispersive Raman spectroscope (Horiba Jobin-Yvon Ltd.,

22

France) with 633 nm laser as light source was used for identifying the doping sites of Mo

23

ions in the BiVO4 crystal lattice. We measured UV-Vis absorption of FeOOH/Mo:BiVO4

24

photoelectrodes using a V-650 spectrophotometer (Jasco Inc., Japan). The Fourier-transform

25

infrared spectra of FeOOH/Mo:BiVO4 photobioelectrode were obtained using Nicolet iS50

6

1

Spectrometer (Thermo Fisher Scientific Inc., USA) and iS50 ATR accessory. All ATR-FTIR

2

spectra were recorded over the range of 4000 to 400 cm−1 by collecting 32 scans with a

3

resolution of 1.928 cm-1.

4 5

2.5. Preparation of t-tau proteins and blocking/washing buffer

6

We prepared a stock solution of t-tau proteins by dissolving tau proteins (100 µg) in the DI

7

water of 100µL. The t-tau proteins of femtomolar to picomolar concentrations were obtained

8

by serially diluting the stock solution using a phosphate-buffered saline solution (10 mM, pH

9

7.4). For detecting of tau proteins spiked in human plasma, we dilute the blood plasma to

10

1/10. The 0.01 M phosphate buffer solution (pH 7.4) containing 0.1% Tween 20 was used as

11

a washing buffer. The blocking buffer was prepared by dissolving 3% (w/w) bovine serum

12

albumin in PBS. To obtain tau proteins spiked in blood plasma, human plasma (Cat. # P9523,

13

Sigma-Aldrich Chemical Co., USA) was diluted into 1/10, and the prepared t-tau proteins

14

solutions were spiked into the diluted human plasma.

15 16

2.6 Fabrication of FeOOH/Mo:BiVO4-Tau5 photobioelectrode

17

We applied O2 plasma (power: 80 W) to the as-prepared FeOOH/Mo:BiVO4 photoelectrode

18

for 30 min. The hydroxyl-terminated photoelectrode was subsequently reacted with 3% (3-

19

Aminopropyl) triethoxysilane in 95% ethanol and further cured at 110 °C. Afterward, we

20

immersed the samples into sodium bicarbonate buffer (50 mM, pH 8.5) containing 20 mM

21

N,N-disuccinimidyl for 3h at room temperature, followed by washing with DI water and

22

drying with N2 gas. Next, we uniformly placed the 10 mM PBS solution containing Tau5

23

antibody (300

24

photoelectrode and incubated them overnight in a moisture atmosphere at 4 °C.

µg/mL) on

the

N-hydroxysuccinimide-activated

25

7

FeOOH/Mo:BiVO4

1

2.7 Detection of t-tau protein

2

We incubated the FeOOH/Mo:BiVO4-Tau5 photobioelectrodes with 50 µL of blocking

3

solution for 2 h at 4 °C and washed them with the washing buffer thoroughly. Subsequently,

4

70 µL of t-tau proteins solution with different concentrations was dropped onto the resulting

5

FeOOH/Mo:BiVO4-Tau5 photobioelectrodes for an incubation of 60 min at room temperature,

6

followed by washing with washing buffer and PBS. The obtained samples were further

7

incubated with HRP-Tau46 antibody solution (30 µL, 200 µgmL-1) for 60 min and rinsed with

8

washing buffer and PBS. Finally, we applied the solutions containing 5 mM 3,3'-

9

diaminobenzidine tetrahydrochloride to the HRP-Tau46 labeled FeOOH/Mo:BiVO4-Tau5

10

photobioelectrodes, followed by the incubation for 10 min at room temperature.

11 12

2.8 Electrochemical and photoelectrochemical analysis

13

We conducted linear sweep voltammetric and chronoamperometric analyses with

14

potentiostat/galvanostat (WMPG1000, Wonatech Co., Korea). The Mott-Schottky plots and

15

impedance spectra were obtained using an impedance analyzer (ZIVE SP1, Wonatech Co.,

16

Korea) under the following conditions: frequency ranging from 100 kHz to 0.1 Hz, the

17

applied bias of 100 mV, and AC potential amplitude of 10 mV. The numerical fitting of EIS

18

data was conducted using Zman software (WonATech Co., Ltd., Korea). In all measurements,

19

we adopted a three-electrode configuration in which the Ag/AgCl (3M NaCl) and Pt coil were

20

used as a reference electrode and as a counter electrode, respectively, and used 0.1 M PBS as

21

an electrolyte. In the photoelectrochemical analysis, white LED light (Prism, Korea) was

22

irradiated through the FTO contact (i.e., back-side illumination). The photocurrent signal (i.e.,

23

|∆I|/I0) was calculated using the following equation; |I-I0|/I0, where I is the final photocurrent

24

of the FeOOH/Mo:BiVO4-Tau5 after HRP-catalyzed oxidation of DAB, and I0 indicates the

25

photocurrent of the FeOOH/Mo:BiVO4-Tau5 prior to the specific binding of t-tau proteins.

8

1

The limit of detection (LOD) and limit of quantification (LOQ) were estimated using the

2

following equation:

3

σ and S indicate the residual standard deviation of the linear regression and the slope of the

4

regression line, respectively.

×σ/S, where κ is the statistical confidence level (LOD: 3.3, LOQ: 10),

5 6

3. Results and Discussion

7

3.1 Characterization of FeOOH/Mo:BiVO4 photoelectrode

8

We prepared the Mo:BiVO4 photoelectrode by electrochemically depositing bismuth

9

oxyiodide (BiOI) on a fluorine-doped SnO2 (FTO) substrate and then converting it to

10

Mo:BiVO4 via thermal and chemical treatments. We doped BiVO4 using Mo ions because

11

Mo6+ can replace the V5+ sites of BiVO4 due to their suitable size, charge, and coordination

12

preferences (Luo et al. 2013; Luo et al. 2011; Ye et al. 2010). As displayed in Figure S1A, as-

13

prepared BiOI film was composed of extremely thin plate-like nanocrystals that have a

14

perpendicular orientation on the substrate. After drop-casting of vanadium and Mo precursor

15

solutions onto BiOI and the subsequent annealing step, BiOI nanoplates were transformed

16

into columnar particles of Mo:BiVO4. The plan-view scanning electron microscopic images

17

of Mo:BiVO4 in Figure S1B show the formation of a three-dimensional nanoporous network

18

structure. To identify the doping sites in the BiVO4 crystal lattice, we carried out Raman

19

spectroscope and X-ray diffraction (XRD) analyses. As shown in Figure S2, the Raman peak

20

corresponding to the symmetric stretching mode of V-O bonds in Mo:BiVO4 was down-

21

shifted by 5.6 cm-1 compared to that of pristine BiVO4 with the increasing concentration of

22

the Mo precursor. We ascribe a down-shifted Raman band (around 828 cm-1) of Mo:BiVO4 to

23

an increase in V-O bond length resulting from the replacement of V5+ by Mo6+ (Luo et al.

24

2011; Merupo et al. 2015; Rettie et al. 2013). The Mo ions did not affect the monoclinic

25

scheelite structure of BiVO4 according to the XRD spectra of Mo:BiVO4 samples (Figure

9

1

S3). The results indicate that Mo ions substituted the V5+ site in the BiVO4 lattice without

2

forming any impurity phase. X-ray photoelectron spectroscopy (XPS) spectra of Mo:BiVO4

3

further supported the successful doping of Mo ions into BiVO4. As shown in Figure S4, Bi 4f

4

and V2p peaks of Mo: BiVO4 slightly shifted to higher binding energy by ~ 0.4 eV compared

5

to those of pristine BiVO4. The up-shifted binding energies of Bi4f and V2p peaks in

6

Mo:BiVO4 samples are attributed to the relatively higher electronegativity of the dopants

7

(Mo6+: 2.16 > V5+: 1.63) (Parmar et al. 2012; Yao et al. 2008). In addition, the peaks of

8

Mo3d5/2 and Mo3d3/2 newly appeared at 232.2 eV and at 235.3 eV, respectively, and no peak

9

(at 229.5 eV) associated with Mo oxide was observed in the XPS spectra of Mo:BiVO4. These

10

results indicate that the Mo ions were successfully doped into BiVO4’s lattice in a hexavalent

11

oxidation state (Huang et al. 2018; Tang et al. 2016). To optimize Mo-doping levels, we

12

investigated the current density-voltage characteristics of pristine and Mo:BiVO4 samples

13

under dark conditions and the illumination of a white LED (power density: 6 mW/cm2,

14

wavelength : 441~667 nm, Figure S5). The Mo:BiVO4 photoelectrode synthesized using 0.1

15

mol% of the Mo precursor showed the highest photocurrent density over the entire potential

16

range (Figure S6). A further increase of the Mo precursor resulted in much lower

17

photocurrent density. We ascribe the result to excessive doping of Mo, which should decrease

18

the depletion width and forms the impurities at the grain boundaries of BiVO4 (Park et al.

19

2014; Zhang et al. 2016).

20

We deposited a FeOOH layer on the Mo:BiVO4 photoelectrode using a photo-assisted

21

electrochemical method. FeOOH is an effective electrocatalyst that interfaces well with

22

BiVO4, capable of oxidizing water at moderate potentials by suppressing charge

23

recombination and minimizing the kinetic barrier for water oxidation (McDonald and Choi

24

2012; Seabold and Choi 2012; Zhang et al. 2018). As shown in Figure 2A, as-synthesized

25

FeOOH/Mo:BiVO4 showed an identical surface morphology to Mo:BiVO4 film’s nanoporous

10

1

network structure. According to our analyses using transmission electron microscopy (TEM)

2

and energy-dispersive X-ray spectroscopic elemental mapping (Figures 2B, 2C), an

3

amorphous FeOOH layer of approximately 5 nm thickness evenly covered the surface of the

4

crystallized Mo:BiVO4 photoelectrode. To unveil the chemical states of the FeOOH layer, we

5

performed the XPS analysis. The Fe 2p peaks in Figure 2D suggest that the oxidation of Fe2+

6

to Fe3+ occurred at the surface of Mo:BiVO4 during the deposition of FeOOH (Lee et al.

7

2018b; Seabold and Choi 2012). The deconvolution of the O1s peak indicates the presence of

8

two chemically distinct species: an Fe-O-Fe bond at 529.0 eV and an Fe-O-H bond at 530.5

9

eV (Liu et al. 2016; Seabold and Choi 2012). The as-synthesized FeOOH/Mo:BiVO4 film

10

strongly absorbed visible light below approximately 510 nm, and its bandgap determined

11

based on a Tauc plot was 2.5 eV (Figure 2E). Due to the small grain composition,

12

nanoporous FeOOH/Mo:BiVO4 exhibited a slightly larger optical bandgap than BiVO4

13

samples (≈2.4 eV) prepared by other synthetic methods, such as the vacuum deposition

14

process (Berglund et al. 2011), metal-organic decomposition (Pilli et al. 2011), or

15

electrodeposition (Seabold and Choi 2012). The absorption edge of FeOOH/Mo:BiVO4 film

16

was consistent with those of Mo:BiVO4 and pristine BiVO4 (Figure S7), which was

17

attributed to the uniform deposition and thin thickness of the FeOOH layer (Zhang et al.

18

2018).

19 20

3.2 Tau5 antibody-conjugated FeOOH/Mo:BiVO4 for photocurrent generation

21

We employed the FeOOH/Mo:BiVO4 photoelectrode as a transducer for sensing t-tau

22

proteins. Through surface silanization and N-hydroxysuccinimide-assisted covalent bonding,

23

we conjugated the monoclonal Tau5 antibody to the surface of the FeOOH/Mo:BiVO4

24

photoelectrode (Figures S8, S9; see the experimental section for details). Note that the Tau5

25

antibody recognizes 210-241 residues of all the phosphorylated and non-phosphorylated tau

11

1

isoforms (Chauhan et al. 2015; LoPresti et al. 1995). We investigated the capability of the

2

Tau5-conjugated FeOOH/Mo:BiVO4 photoelectrode (FeOOH/Mo:BiVO4-Tau5) to generate a

3

photocurrent signal in the absence of an organic electron donor. According to the linear sweep

4

voltammetric analysis under LED illumination (Figure 3A), FeOOH/Mo:BiVO4-Tau5

5

produced a substantial amount of anodic photocurrent over the potential of -0.15 V (vs.

6

Ag/AgCl, 3M NaCl) in a sacrificial electron donor-free phosphate buffer (pH 7.4). In contrast,

7

Mo:BiVO4-Tau5 and pristine BiVO4-Tau5 required additional anodic potentials of

8

approximately 200 mV for water oxidation than FeOOH/Mo:BiVO4-Tau5. Concomitantly,

9

FeOOH/Mo:BiVO4-Tau5 generated a 4.10 and 6.23 times higher photocurrent signal than

10

Mo:BiVO4-Tau5 and pristine BiVO4-Tau5, respectively, at 0 V (vs. Ag/AgCl, 3M NaCl)

11

(Figure 3B).

12

To explore the origin of strong photocurrents generated by FeOOH/Mo:BiVO4-Tau5,

13

we carried out impedimetric and voltammetric analyses. According to the results of Mott-

14

Schottky analyses (Figure 3C, Table S1), the charge carrier densities of FeOOH/Mo:BiVO4-

15

Tau5 and Mo:BiVO4-Tau5 were 1.54 and 1.57 times higher, respectively, than that of pristine

16

BiVO4-Tau5. These results imply that the Mo ions doped into BiVO4 increased the carrier

17

density of the transducer. The increase in the number of charge carriers facilitates electron-

18

hole separation and improves charge transport properties in bulk BiVO4 (Park et al. 2013).

19

We analyzed the interfacial charge transport property of each Tau5-conjugated photoelectrode

20

using Nyquist plots that were fitted to the Randles circuit model (Figures 3D, S10). Note that

21

the Randles model is the most commonly used equivalent circuits describing the

22

electrochemical reactions across the interface of the semiconductor (Suni 2008). We observed

23

that the charge transfer resistance of FeOOH/Mo:BiVO4-Tau5 was 4.27 and 24.4 times lower

24

than those of Mo:BiVO4-Tau5 and pristine BiVO4-Tau5, respectively. These results were

25

further supported by the surface charge transport efficiency (ηsurface) of each sample; ηsurface

12

1

can be estimated according to the following equation:

2

ηsurface= JH2O/JNa2SO3

3

where JH2O and JNa2SO3 are the photocurrents for water oxidation and sodium sulfite oxidation,

4

respectively. Because sulfite oxidation is a thermodynamically and kinetically facile process,

5

JNa2SO3 is representatively used to calculate a photoelectrode’s ηsurface value (Kim and Choi

6

2014a). As shown in Figure S11, the ηsurface values for FeOOH/Mo:BiVO4-Tau5 were more

7

than 1.95 times higher than those for pristine BiVO4-Tau5 and Mo:BiVO4-Tau5 over the

8

entire potential range. These results verify the FeOOH layer’s capability to efficiently capture

9

the photogenerated holes and accelerate water oxidation reactions at the transducer’s surface

10

(Figure S12). Nonetheless, approximately over 15% of surface-reaching holes at

11

FeOOH/Mo:BiVO4-Tau5 were not engaged in the water oxidation reaction, instead being

12

lost to surface recombination, which was ascribed to the intrinsic limitations of FeOOH’s

13

catalytic activity toward water oxidation reaction (Kim and Choi 2014a; Louie and Bell

14

2013). Further studies to suppress the electron-hole recombination at water oxidation-coupled

15

PEC platform are needed for accurate and reliable sensing of targeted biomarkers.

16 17

3.3. Detection of t-tau proteins by sandwich-type PEC sensor

18

We investigated the changes in the photocurrent of FeOOH/Mo:BiVO4-Tau5 at 0 V (vs.

19

Ag/AgCl, 3M NaCl) upon exposure to the targeted t-tau proteins. As shown in Figure S13A,

20

the specific bindings between t-tau proteins and Tau5 antibodies reduced the photocurrent

21

from FeOOH/Mo:BiVO4-Tau5 by 15.1%. To amplify the degree of the photocurrent’s change,

22

we additionally captured the targeted t-tau proteins by using an HRP-labeled Tau46 antibody

23

that reacts with the phosphorylation-independent epitope in the C-terminal of the t-tau protein

24

(Asai et al. 2015). Upon exposure to DAB and H2O2, the photocurrent signal (i.e., |∆I|/I0) of

25

the sensing platform was enhanced by approximately 107% compared to that in the absence

13

1

of signal amplifying agents. In contrast, negligible amplification of the photocurrent was

2

observed in the sole presence of either DAB or H2O2 (Figure S13B). According to the

3

literature (Lathwal and Sikes 2016), HRP forms insoluble and insulating precipitates on the

4

transducer surface by accelerating the oxidation of DAB by H2O2. As shown in Figure S14,

5

new FTIR peaks at 1508 cm-1 and 1636 cm-1 were observed after HRP-catalyzed oxidation of

6

DAB, indicating the formation of insoluble precipitates having benzenoid and quinoid imine

7

units on the transducer (Kellenberger et al. 2011). Through electrochemical impedance

8

spectroscopic analysis, we confirmed that HRP-triggered oxidation of DAB increased the

9

charge transfer resistance of FeOOH/Mo:BiVO4-Tau5 by up to 589 ohm, which was 1.43

10

times higher than the Rct caused by t-tau proteins (Figure S15). In contrast, no significant

11

changes in the Rct of FeOOH/Mo:BiVO4-Tau5 were observed in the sole presence of H2O2 or

12

DAB. These results suggest that the insoluble deposits generated by the oxidation of DAB

13

impeded the redox rea1ctions between the photogenerated holes and water at the surface of

14

FeOOH/Mo:BiVO4, leading to the reduction of photocurrents by 21.2%. Figure S16 shows

15

stepwise transient photocurrent responses of FeOOH/Mo:BiVO4-based sensing platform; the

16

sequential exposure of blocking solution, targeted t-tau proteins, and HRP-labeled Tau46

17

antibody gradually decreased the photocurrent of FeOOH/Mo:BiVO4 decorated with Tau5

18

antibody. Upon the additional introduction of DAB and H2O2, the photocurrent of the sensing

19

platform was further depressed. These results verify the successful construction of

20

FeOOH/Mo:BiVO4-based PEC sensing platform. Overall, our results indicate that the

21

FeOOH/Mo:BiVO4-based platform can detect t-tau proteins by amplifying the photocurrent

22

signal via an HRP-catalyzed DAB oxidation reaction.

23 24

3.4. Femtomolar sensing performance of FeOOH/Mo:BiVO4-Tau5 PEC sensor

14

1

To evaluate the sensitivity and precision of the FeOOH/Mo:BiVO4-Tau5 PEC platform, we

2

investigated the correlation between the variation in photocurrents at 0 V (vs. Ag/AgCl, 3M

3

NaCl) and the concentrations of t-tau proteins. As shown in Figure 4A, the changes in the

4

sensor’s photocurrent exhibited a linear dependency on the logarithmic concentration of t-tau

5

proteins with the coefficient of determination (R2) of 0.999. We estimated the limit of

6

detection (LOD) and the limit of quantification (LOQ) based on the results of the linear

7

regression analysis with confidence levels of 3.3 and 10, respectively. The values of LOD and

8

LOQ were 1.59 fM and 4.11 fM, respectively, approximately 100 times lower than the levels

9

of t-tau proteins in AD patients’ blood (i.e., 170~890 fM, Table S2) . In addition to the

10

femtomolar sensitivity, the PEC platform showed high reliability in sensing t-tau proteins,

11

evidenced by the values of coefficient-of-variation below 7.81% (Figure S17). To

12

demonstrate the specificity of the sensing platform toward t-tau proteins, we exposed non-

13

target analytes to the FeOOH/Mo:BiVO4-Tau5 platform. As shown in Figure 4B, non-target

14

AD biomarkers (e.g., β-amyloid42, β-amyloid40) and non-target proteins (e.g., human serum

15

albumin, human immunoglobulin G) showed no obvious responses despite their high

16

nanomolar concentrations. These results indicate that the FeOOH/Mo:BiVO4-Tau5-based

17

PEC sensor possesses high selectivity toward target t-tau proteins without noticeable

18

interference by non-target proteins. We further examined the assay’s accuracy by detecting t-

19

tau proteins spiked in the pooled blood plasma (Figures 4C, 4D). We observed a linear

20

dependence (R2 =0.993) between the sensor’s photocurrent change and the targeted t-tau

21

proteins’ concentration. The degree of recovery for spiked t-tau proteins was over 92.6%

22

within

23

FeOOH/Mo:BiVO4-Tau5 platform can accurately and reliably detect targeted t-tau proteins

24

down to femtomolar levels without being disturbed by the interfering agents in blood plasma.

the clinically relevant

concentration

15

range.

The results

show that

the

1

The water oxidation-coupled, FeOOH/Mo:BiVO4-based PEC sensing platform exhibits

2

superior sensitivity compared to other analytical techniques. The PEC detection utilizes two

3

different forms of energy (i.e., light and electricity) as an excitation source and a detection

4

signal, respectively (Cohen and Walt 2019); thus, it can minimize background noise signals

5

and facilitate higher sensitivity than common electrochemical methods. Figure 5 compares

6

the detection limit of the AD biomarker-targeting electrochemical biosensors reported so far.

7

The previously reported electrochemical bioassay detected AD biomarkers down to

8

picomolar concentrations (Carneiro et al. 2017; Esteves-Villanueva et al. 2014; Li et al. 2011;

9

Li et al. 2016; Liu et al. 2015; Prabhulkar et al. 2012; Rushworth et al. 2014; Wang et al.

10

2017; Yoo et al. 2017; Yu et al. 2014; Zhou et al. 2016); in contrast, the FeOOH/Mo:BiVO4-

11

based PEC platform showed a much lower detection limit of femtomolar levels. In the case of

12

optical sensors based on surface plasmon resonance, colorimetry, and surface-enhanced

13

Raman scattering for detecting AD biomarkers (e.g., β-amyloid-related species,

14

apolipoprotein, and β-secretase enzyme) (Demeritte et al. 2015; Georganopoulou et al. 2005;

15

Haes et al. 2005; Hu et al. 2017; Kim et al. 2018; Kim et al. 2017; Kim et al. 2016c; Lisi et al.

16

2017; Neely et al. 2009; Ren et al. 2017; Vilela et al. 2017; Xia et al. 2010; Zhou et al. 2015;

17

Zhu et al. 2018), their LOD values were 10 to 1011 folds higher than those achieved by the

18

PEC bioassay. Furthermore, the sensitivity of the FeOOH/Mo:BiVO4-Tau5 detection system

19

has surpassed that of the preceding PEC sensors by over 10-fold (Wang et al. 2018; Xu et al.

20

2018; Zhang et al. 2019), even in the absence of sacrificial electron suppliers. Compared to

21

the other PEC platform targeting Aβ42, the FeOOH/Mo:BiVO4-based analytic system has

22

employed t-tau proteins as a target biomarker because of the femtomolar concentration of t-

23

tau proteins in human plasma; the levels of t-tau proteins in AD patients’ blood plasma are

24

approximately 10-to 102 fold lower than those of Aβ42 (Table S2) (Liu et al. 2018; Mielke et

25

al. 2018; Zetterberg et al. 2013). The extremely low concentrations of t-tau proteins in blood

16

1

plasma call for highly sensitive analytical techniques. Along with the advantageous features

2

of PEC sensing, such as simplicity, cost-effectiveness, and ease of miniaturization, the

3

unprecedented sensitivity shows great promise of the FeOOH/Mo:BiVO4-based PEC

4

platform for AD diagnosis.

5 6

4. Conclusion

7

The incorporation of extrinsic Mo dopants and the FeOOH ad-layer into the BiVO4

8

photoelectrode and the amplification of photocurrent signals via HRP-catalyzed oxidation of

9

DAB allowed the BiVO4-based PEC sensor to detect t-tau proteins down to femtomolar

10

levels. Despite the absence of artificial electron suppliers, FeOOH/Mo:BiVO4 conjugated

11

with the Tau5 antibody generated strong photocurrent signals at 0 V (vs. Ag/AgCl, 3M NaCl).

12

According to our analyses, the doped Mo ions increased the charge carrier density of the

13

BiVO4-Tau5 photoelectrode by 1.57 times, and the FeOOH co-catalyst facilitated interface

14

charge transfer at the surface of Mo:BiVO4-Tau5 by effectively collecting photogenerated

15

holes. To sensitively and accurately detect t-tau proteins, we adopted a signal-amplifying

16

strategy based on HRP-controlled oxidation of DAB. The peroxidase-catalyzed production of

17

insoluble deposits amplified the photocurrent signal (i.e., |∆I|/I0) of FeOOH/Mo:BiVO4-Tau5

18

by 2.07-fold, which enabled the FeOOH/Mo:BiVO4-based PEC sensing platform to identify

19

the t-tau proteins down to femtomolar concentrations with LOD and LOQ values of 1.59 fM

20

and 4.11 fM, respectively. Further improvement of the water oxidation-coupled PEC sensing

21

platform would be achieved by designing more efficient photoelectrodes that minimize the

22

interfacial electron-hole recombination.

23 24

Acknowledgement

17

1

This study was supported by the National Research Foundation (NRF) via the Creative

2

Research Initiative Center (NRF-2015R1A3A2066191), Republic of Korea.

3 4 5 6

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Zhu, X., Zhang, N., Zhang, Y., Liu, B., Chang, Z., Zhou, Y., Hao, Y., Ye, B., Xu, M., 2018.

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Anal. Methods 10(6), 641-645.

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Figure 1. Schematic illustration of water oxidation-coupled, FeOOH/Mo:BiVO4-based photoelectrochemical sensing platform for detecting Alzheimer’s tau proteins of femtomolar levels. Under the illumination of a white light-emitting diode, the FeOOH/Mo:BiVO4 photobioelectrode decorated with the Tau5 antibody generated highly stable and strong current signals at 0 V (vs. Ag/AgCl, 3M NaCl) in the absence of sacrificial charge supplier. We have successfully detected t-tau proteins down to femtomolar concentrations by amplifying photocurrent signals via HRP-catalyzed oxidation of DAB, which formed insoluble deposits on the transducer. Please insert Figure 1 at the top of the second page.

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Figure 2. (A) Top-view SEM and (B) High-resolution TEM images of FeOOH/Mo:BiVO4. (C) Elemental mapping results FeOOH/Mo:BiVO4, analyzed by STEM-EDS. (D) XPS spectra for Fe2p and O1s of FeOOH/Mo:BiVO4. The formation of FeOOH layer was confirmed by the appearance of Fe2p peaks and an O1s peak at 530.5 eV. (E) UV–Vis spectrum and Tauc plot (inset) of FeOOH/Mo:BiVO4 film. The optical bandgap was estimated to be 2.5 eV. Please insert Figure 3 on the page describing Section 3.2.

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Figure 3. (A) Linear sweep voltammetric spectra of pristine BiVO4-Tau5, Mo:BiVO4-Tau5 and FeOOH/Mo:BiVO4-Tau5 photobioelectrodes under dark and light conditions. We used white LED (power density: 6 mW/cm2) as a light source. (B) Transient photoresponse of pristine BiVO4-Tau5, Mo:BiVO4-Tau5 and FeOOH/Mo:BiVO4-Tau5 under LED illumination at 0 V (vs. Ag/AgCl, 3M NaCl). (C) Mott–Schottky plots of FeOOH/Mo:BiVO4-Tau5, Mo:BiVO4-Tau5 and pristine BiVO4-Tau5 obtained in a 0.1 M phosphate buffer saline (pH 7.4). (D) Impedance spectra of pristine, Mo: BiVO4 and FeOOH/Mo:BiVO4 photoelectrodes, which were decorated with Tau5 antibody. The experimental data of each Nyquist curve were indicated as hollow squares, circles, and triangles and the fitted data were represented as solid lines. Please insert Figure 4 on the page describing Section 3.3.

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Figure 4. (A) Changes in photocurrent of the FeOOH/Mo:BiVO4-based PEC platform upon exposure to t-tau proteins of different concentrations. The error bars represent the standard deviation of the mean (n=3). The average values of coefficient-of-variation (CV) is 5.2 ± 2.4%. (B) Photocurrent responses of the FeOOH/Mo:BiVO4-based PEC detection system for t-tau proteins and non-target proteins. The concentrations of negative controls (i.e., HIgG, HSA, Aβ42, and Aβ40) were 1 nM. The error bars indicate means ± standard deviation. The measurements were performed at least three times. The CV value of the positive group is 2.85%.(C) Changes in photocurrents and (D) the degree of recovery of the FeOOH/Mo:BiVO4-Tau5 bioassay upon exposure to the t-tau proteins spiked in human plasma. The data reproducibility was confirmed by two additional experiments. All values represent the mean ± standard deviation. The average CV values for |∆I|/I0 and the degree of recovery are 9.7±6.1 and 6.7±4.0, respectively. Please insert Figure 4 on the page describing Section 3.3.

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Figure 5. Detection limits of AD biomarker-targeting sensing platforms reported so far. The DPV, SWV, and CV represent differential pulse voltammetry, square wave voltammetry, and cyclic voltammetry, respectively. ADDLs and ApoE indicate Aβ-derived diffusible ligands and apolipoprotein E, respectively. References: P1(Zhang et al. 2019), P2 (Xu et al. 2018), P3 (Wang et al. 2018), I1 (Wang et al. 2017), I2 (Esteves-Villanueva et al. 2014), I3 (Yoo et al. 2017), I4 (Rushworth et al. 2014), A1 (Liu et al. 2014), A2 (Liu et al. 2015), D1 (Li et al. 2016), D2 (Yu et al. 2014), D3 (Zhou et al. 2016), R1(Chae et al. 2017), R2 (Oh et al. 2013), R3 (Kim et al. 2016b), S1(Carneiro et al. 2017), and S2(Prabhulkar et al. 2012). Please insert Figure 5 on the page describing Section 3.4.

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Highlights Water oxidation-coupled PEC sensing of t-tau proteins was demonstrated using FeOOH/Mo:BiVO4. Without artificial electron donors, FeOOH/Mo:BiVO4-Tau5 exhibited strong photocurrent signals. The signals of FeOOH/Mo:BiVO4-Tau5 was amplified by peroxidase-catalyzed precipitation. FeOOH/Mo:BiVO4-based PEC platform recognized the t-tau proteins with the detection limit of 1.59 fM. Accurate quantification of the t-tau proteins in human plasma was achieved by FeOOH/Mo:BiVO4-Tau5 assay platform.

CRediT authorship contribution statement Kayoung Kim: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Writing - original draft. Chan Beum Park: Conceptualization, Funding acquisition, Supervision, Writing - review & editing.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Confirmed by the authors (Kayoung Kim, Chan Beum Park) on January 31, 2020 Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), 335 Science Road, Daejeon 305-701, Republic of Korea.