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Article

Boosting the Sensitivity of a Photoelectrochemical Immunoassay by Using SiO@polydopamine CoreShell Nanoparticles as a Highly Efficient Quencher 2

Huaijia Xue, Jinjin Zhao, Qing Zhou, Deng Pan, Yue Zhang, Yuanjian Zhang, and Yanfei Shen ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00050 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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ACS Applied Nano Materials

1

Boosting the Sensitivity of a Photoelectrochemical

2

Immunoassay by Using SiO2@polydopamine Core-Shell

3

Nanoparticles as a Highly Efficient Quencher

4

Huaijia Xue,† Jinjin Zhao,‡ Qing Zhou,† Deng Pan,† Yue Zhang,† Yuanjian Zhang †

5

and Yanfei Shen*, †

6

†

7

University, Nanjing 210009, China.

8

‡

9

Medical University, Weihui 453100, Henan, China

10

Medical School, School of Chemistry and Chemical Engineering, Southeast

Department of Clinical Laboratory, The First Affiliated Hospital of Xinxiang

Email: [email protected]

1

ACS Paragon Plus Environment

ACS Applied Nano Materials 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

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Page 2 of 29

Abstract:

2

By using SiO2@polydopamine (PDA) core-shell nanoparticles (NPs) as a quencher

3

for a CdTe quantum dots (QDs) probe, a “signal-off” photoelectrochemical (PEC)

4

immunosensor with boosted sensitivity was reported. In the proposed SiO2@PDA NPs,

5

PDA that was prepared by self-polymerization of dopamine had many features, such as

6

broad absorption in the ultraviolet-visible (UV-vis) region, high fluorescence quenching

7

efficiency, and surface functional groups that can further conjugate with biomolecules.

8

More importantly, the coupling of SiO2 NPs improved the dispersibility and loading of

9

PDA and increased the charge transfer impedance. All these factors jointly led to

10

enhanced PEC quenching efficiency of the CdTe probe, which was up to 10 times that

11

of PDA alone. Using the detection of CA125 as an example, the PEC immunosensor

12

showed a detectable range of 1 mU·mL-1 to 100 U·mL-1 and an ultralow detection limit of

13

0.3 mU·mL-1 (S/N = 3), the most sensitive sensor for CA125 reported so far. The

14

constructed PEC immunosensor also had high selectivity and good accuracy for detecting

15

tumor markers. This work shows the great potential of SiO2@PDA for boosting sensitivity

16

in “signal-off” PEC clinical diagnosis.

17 18

Keywords: SiO2@PDA, CdTe quantum dots, signal off, photoelectrochemical biosensor,

19

CA125

2


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1

Introduction

2

As a blooming technique, the photoelectrochemical (PEC) immunoassay has attracted

3

intense attention.1, 2 In principle, the photoelectrode current generated by light excitation

4

is used as the output signal. Such a configuration results in the complete separation of

5

the excitation source and detection signal, ensuring high sensitivity and low

6

background in sensing.3-5 Compared to conventional immunoassays, such as the

7

enzyme immunoassay (EIA)6, enzyme-linked immunosorbent assay (ELISA)7, and

8

immunoradiometric assay (IRMA)8, the PEC immunoassay was more sensitive and

9

does not require the participation of enzymes. Thus, the PEC assay combines the

10

advantages of both electrochemical and optical methods and has features of simple

11

instruments, ease of miniaturization and low cost.9

12

The “signal-off” PEC assay has been applied in a wide range of applications,

13

owing to its intrinsic qualities of simple organization and convenient operation, but is

14

often criticized for its relatively low sensitivity compared to the “signal-on”

15

strategy.10-13 Addressing this challenge, in general, requires both a highly efficient

16

cooperative quencher and a photoactive probe.14-17 For this purpose, tremendous

17

research has been undertaken on the development of various highly efficient

18

photoactive probes, such as semiconducting metal oxides and quantum dots (QDs), in

19

the PEC assay.18-21 In contrast, studies of PEC quenchers are still in infancy.22

20

Bioinspired polydopamine (PDA) has attracted increasing interest, as it can

21

spontaneously form a homogeneous ultrathin coating on diverse types of inorganic

22

and organic surfaces23-27 with high biocompatibility.28, 29 Moreover, PDA has strong

23

absorption in the whole ultraviolet-visible (UV-vis) and near-infrared (NIR) regions

24

and has abundant functional groups that can facilitate further loading of interesting

25

bioconjugates.30,

26

biomedical and biological applications.32-37 For instance, thanks to a large surface area,

27

SiO2 can act as a carrier for loading secondary antibodies (Ab2) to form secondary

28

antibody-coated SiO2 conjugates for signal amplification.38-41 In addition, due to poor

29

conductivity, SiO2 can also be used as a label of Ab2 for the “signal-off” PEC

31

In addition, SiO2 nanoparticles (NPs) have been widely used in

3



Page 4 of 29

1

immunosensor.42 In this context, coupling PDA and SiO2 into a nanohybrid is very

2

attractive for application as a highly efficient signal-off quencher in PEC

3

immunosensors to improve sensing sensitivity.

4

Herein, we report the preparation and utilization of SiO2@PDA core-shell NPs

5

as a new kind of signal-off quencher for the CdTe QDs probe in a PEC immunosensor.

6

Taking the detection of CA125, an ovarian cancer biomarker, as an example, the

7

proposed “signal-off” PEC immunosensor (Scheme 1) not only showed high selectivity,

8

stability and reproducibility but also exhibited much improved sensitivity due to the

9

enhanced quenching by the cooperative SiO2 core and PDA shell nanostructures.

10 11

Scheme 1 (a) Preparation of Ab2-modified SiO2@PDA core-shell NPs and (b) the

12

assembling steps of the CA125 PEC immunosensor using SiO2@PDA NPs as the

13

signal-off quencher and CdTe QDs as the photoactive probe.

14 15 16

Experimental section

4


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1

Reagents

2

CA125 antigen, monoclonal capture antibody (Ab1) and labeled antibody (Ab2)

3

were purchased from Beijing Key-Bio Biotech Co., Ltd. (Beijing, China). Carboxylic

4

group-functionalized CdTe QDs were received from Beida Jubang Science and

5

Technology Co., Ltd. (Beijing, China). Chitosan (CS), glutaraldehyde (GA),

6

dopamine (DA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

7

(EDC), N-hydroxysuccinimide (NHS), Proclin 300, (3-aminopropyl)-triethoxysilane

8

(APTES), hexaamimineruthenium(II) chloride (Ru(NH3)6Cl), and tetraethoxysilane

9

(TEOS) were obtained from Sigma-Aldrich (USA). Phosphate buffer solution (PBS)

10

and bovine serum albumin (BSA) were obtained from Sangon Biotech (Shanghai,

11

China) and Sunshine Biotechnology Co., Ltd. (Nanjing, China), respectively.

12

Potassium ferricyanide (K3[Fe(CN)6]) and potassium ferrocyanide (K4[Fe(CN)6])

13

were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai,

14

China). Indium tin oxide (ITO, 6.2-6.8 Ω/sq) slices were obtained from Zhuhai Kaivo

15

Optoelectronic Technology Corporation (Zhuhai, China). Other chemicals such as

16

ascorbic acid (AA), acetone, ammonia, and ethanol were obtained from Sinopharm

17

Chemical Reagent Co., Ltd. (Shanghai, China). All reagents were of analytical grade

18

and used as received unless otherwise specified. Ultrapure water (18.2 MΩ·cm,

19

Milli-Q) was used in all experiments.

20

Apparatus

21

Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and

22

PEC measurements were performed on a CHI 660e electrochemical workstation (CH

23

Instruments, Shanghai, China) at room temperature with a three-electrode system. A

24

modified ITO electrode with an area of 0.16 cm2, an Ag/AgCl (KCl-saturated)

25

electrode, and a platinum wire were used as the working electrode, the reference

26

electrode, and the counter electrode, respectively. 0.1 M AA in PBS (0.02 M, pH = 6)

27

was utilized as the PEC electrolyte.1, 43 A 150 W xenon lamp was used as the source

28

of visible light (Zolix Instruments CO., Ltd, Beijing, China) for PEC measurements. 5



Page 6 of 29

1

UV-visible (UV−vis) absorption spectra were collected from a Cary100 UV−vis

2

spectrophotometer (Agilent, Singapore). Scanning electron microscopy (SEM)

3

images were obtained using a Zeiss Ultra Plus (Germany). Transmission electron

4

microscopy (TEM) was performed with a JEM-2100 transmission electron

5

microscope (JEOL, Japan) and FEI-TF20 TEM system at an accelerating voltage of

6

200 kV (FEI, USA). Fluorescence spectra were recorded on a FluoroMax-4

7

spectrofluorometer with xenon discharge lamp excitation (Horiba, Japan).

8

Time-resolved fluorescence spectroscopy was recorded on a PluoroLog 3-TCSPC

9

steady/transient fluorescence spectrometer (Horiba, Japan) with excitation at λ = 280

10

nm. A freeze dryer (Ailang Instruments CO., Ltd, Shanghai, China) was applied for

11

drying SiO2 NPs during sample preparation. Fourier transform infrared (FTIR)

12

spectra were recorded with a Nicolet iS10 FTIR spectrometer (Thermo, USA)

13

equipped with an attenuated total reflection (ATR) setup. Particle size distribution

14

was analyzed via the dynamic light scattering (DLS) technique using a NanoBrook

15

Omni instrument (Brookhaven, USA).

16

Synthesis of Ab2-SiO2@PDA core-shell NPs

17

SiO2 NPs were synthesized according to the literature with a slight

18

modification.40 First, SiO2 NPs were prepared by mixing 80 mL ethanol, 3.6 mL

19

ammonia, and 4.85 mL ultrapure water with continuous stirring. Then, 3.1 mL TEOS

20

and 8 mL ethanol were quickly added to the reaction mixture with magnetic stirring at

21

55 °C for 5 h. The SiO2 NPs were obtained after centrifugation, washed with ethanol

22

three times, and freeze-dried. After that, 60 mg DA was added dropwise to a solution

23

containing 35 mg SiO2 NPs, 30 mg Tris, and 25 mL water, and the resulting solution

24

was stirred at room temperature for 72 h to obtain the SiO2@PDA NPs. Then, 60 μL

25

of 100 μg·mL-1 Ab2 was added to 400 μL of 0.1 mg·mL-1 SiO2@PDA solution. Ab2

26

and SiO2@PDA can be stably conjugated because quinone functional groups on the

27

PDA film can be covalently coupled to the amine-terminated antibody via Michael

28

reaction.44 After the mixture was stirred at 4 °C for 12 h, 1% BSA was added to the 6


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1

solution and incubated at 37 °C for 1 h to block the nonspecific sites. Finally, the

2

unbound Ab2 was removed by successive centrifugation, and the as-obtained

3

Ab2-SiO2@PDA was redispersed in 400 μL of 0.02 M PBS (pH 7.4). Scheme 1a

4

shows the process of preparation of Ab2-SiO2@PDA core-shell NPs.

5

Fabrication of the photoelectrochemical immunosensor

6

Scheme 1b shows a diagram of the fabrication of the PEC immunosensor. Prior

7

to surface modification, ITO electrodes were ultrasonically cleaned sequentially in

8

acetone, ethanol and water for 20 min and then blown dry with nitrogen. Then, a

9

mixture of 10 μL of CdTe QDs (5 μM) and chitosan (CS, 0.05 wt% in acetic acid)

10

was dropped onto a piece of an ITO slice (0.16 cm2), kept in a silica gel dryer

11

overnight at room temperature and then dried at 80 °C for 2 h. The CdTe-CS/ITO

12

electrode was obtained after washing with water to remove the excess CdTe-CS. Ab1

13

was immobilized on the CdTe-CS/ITO electrode by a classic cross-linking reaction

14

via GA. In short, 10 μL of 1% GA solution was dropped onto the electrode and kept

15

for 1 h at room temperature, after which the excess GA was washed out with water.

16

Then, 10 μL of 10 μg·mL-1 Ab1 was dropped onto the electrode surface and incubated

17

at 4 °C for 12 h. After rinsing with 0.02 M PBS (pH = 7.4), the electrode was blocked

18

by 1% (w/v) BSA in 0.02 M PBS at 37 °C for 12 h. After rinsing, the electrode was

19

incubated with 10 μL CA125 at different concentrations (0.001-100 U·mL-1) for 2 h at

20

37 °C, followed by washing with PBS. Finally, 10 μL of Ab2-SiO2@PDA solution

21

was dropped onto the modified ITO and incubated for 2 h at 37 °C. The CA125

22

immunosensor was finally obtained after rinsing with PBS. CV experiments were

23

performed by using a glassy carbon electrode (GCE, Ф = 3 mm). Before modification,

24

the GCE was carefully polished with 0.3 μm and 0.05 μm alumina slurry repeatedly,

25

washed with ultrapure water in an ultrasonic bath, and dried in air.

26

Detection of CA125 in serum.

27

Spiked serum collected from healthy people was diluted 10-fold with 0.01 M

28

PBS (pH = 7.4) solution. The spiked serum samples were analyzed by the PEC

29

immunosensor preformed after adding 0.001-100 U·mL-1 CA125 antigen. For each 7



Page 8 of 29

1

sample, four replicates were independently measured. The concentration of each

2

spiked sample was calculated according to the calibration curve.

3

Results and discussion

4

Characterization of SiO2@PDA NPs

5

The morphologies of the as-obtained SiO2 NPs and SiO2@PDA core-shell NPs

6

were first characterized by TEM. Figure 1a shows that the SiO2 NPs were uniform

7

spheres and exhibited a uniform size distribution with a diameter of approximately 69

8

± 6 nm (see DLS result in Figure 1a inset). After the polymerization of the dopamine

9

precursor (Scheme 1a) in the colloidal SiO2 dispersion, a coating with a thickness of

10

~10 nm formed on the surface of SiO2 (Figure 1b), demonstrating successful

11

modification of polydopamine (PDA). It should be noted that the DLS measurements

12

(Figure 1c inset) showed that the PDA in aqueous solution was ca. 21 ± 3 nm.

13

Meanwhile, elemental line scanning analysis of a single SiO2@PDA NP showed that

14

the center of the NP was elemental Si, whereas elemental N was concentrated at the

15

outer edge of the particle (Figure 1d). Since elemental N is a characteristic element of

16

PDA, the result of the elemental line scanning analysis again confirmed the core-shell

17

structure of the as-prepared SiO2@PDA NPs.

18 19

8


(c)

Number (%)

(b)

80 40 0

60 70 80 Size (nm)

40 0

80 40 0

60 90 120 Size (nm)

(d)

80

18

21 24 Size (nm)

Intensity (a.u.)

(a)

Number (%)

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


Number (%)

Page 9 of 29

50 nm

Si

N

0

1

30

60

Distance (nm)

90

120

2

Figure 1. TEM images of SiO2 NPs (a), SiO2@PDA NPs with SiO2/DA mass ratio of

3

7:12 (b) and PDA (c). Elemental line scanning over a single SiO2@PDA NP indicated

4

by the dashed line in panel (d). The insets in (a), (b) and (c) show the DLS size

5

distribution histograms of SiO2, SiO2@PDA and PDA, respectively.

6 7

Complementarily, the FTIR spectra also confirmed the successful synthesis of

8

SiO2@PDA (Figure S1). Both PDA and SiO2@PDA showed absorption bands at

9

approximately 1600 cm-1 and 1502 cm-1, corresponding to the stretching vibration of

10

the aromatic ring and bending vibration of N-H and the shearing vibration of N-H,

11

respectively.45 Moreover, a band at 1286 cm-1 and a broad peak at 3200-3660 cm-1

12

could be ascribed to the stretching vibration of phenolic C-O46 and the stretching

13

vibrations of phenolic O-H and N-H, respectively. All these results demonstrated the

14

successful coating of PDA on SiO2 NPs.

15

To investigate the quenching effect of SiO2@PDA on CdTe QDs, UV-vis and 9



Page 10 of 29

1

fluorescence measurements were first performed. As shown in Figure 2a, under

2

excitation at 240 nm, approximately 3 nm-sized (Figure S2) CdTe QDs exhibited

3

strong emission in the range from 470 nm to 650 nm with an emission peak centered

4

at 535 nm. Meanwhile, the UV-vis spectrum of PDA showed wide absorption from

5

400 nm to 650 nm. The spectral overlap between the emission spectrum of CdTe QDs

6

and the absorption spectrum of PDA demonstrated the possibility of energy transfer

7

between them, presumably leading to an interfilter effect.47,

8

observed that SiO2@PDA showed much higher absorption than PDA at the same

9

PDA concentration, and the minor scattering effect of SiO2 NPs in solution was also

10

noticed but was negligible with respect to the much-enhanced UV-vis absorption by

11

SiO2@PDA (Figure 2a). As shown in the inset of Figure 2a, only the PDA in aqueous

12

solution was ready to be aggregated; while the formation of PDA@SiO2 resulted in

13

much better dispersibility, which was a favorable factor for a more effective

14

absorption of UV-vis light. Thus, compared with only PDA, the much enhanced

15

absorption of PDA@SiO2 could be ascribed to its better dispersibility in aqueous

16

solution. As a result, the steady-state fluorescence of CdTe QDs was more quenched

17

by SiO2@PDA (8.5% remained) than PDA (41%) by a factor of nearly 5 times under

18

identical conditions (Figure 2b and 2c). Therefore, rather than only PDA, SiO2@PDA

19

is very promising as a quencher for CdTe QDs for enhancing sensing sensitivity.

48

Moreover, it was

20

Time-resolved fluorescence spectroscopy was used to further understand the

21

mechanism of enhanced quenching. A similar fluorescence decay time was observed

22

for CdTe before (40.61 ns) and after (38.76 ns) the addition of SiO2@PDA, indicating

23

that the addition of SiO2@PDA did not cause a significant change in the electronic

24

structure and emission kinetics of pristine CdTe QDs (Figure 2d).48 Therefore,

25

fluorescence quenching by SiO2@PDA could be caused by the interfilter effect.49

26

10


Page 11 of 29

(a)

(b) SiO2@PDA

SiO2@PDA

Abs

CdTe

PDA

1.2

0.6

2000000

CdTe

PDA

CdTe + PDA

FL Intensity (a.u.)

SiO2

1500000

CdTe + SiO2@PDA

1000000

500000

SiO2 0.0 400

0

500

600

500

550

Wavelength (nm)

Wavelength (nm)

600

(d)

100

50

0

CdTe CdTe + PDA CdTe + SiO2@PDA

Intensity (a.u.)

(c)

Normalized FL intensity (%)

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


CdTe - Fit CdTe + SiO2@PDA - Fit

0

300

1

600

Time (ns)

900

2

Figure 2. (a) UV-vis absorption spectra of PDA and SiO2@PDA and fluorescence

3

spectra of CdTe QDs. The inset shows the photo of SiO2, SiO2@PDA and PDA

4

dispersions after resting for 24 h after preparation. (b) The fluorescence spectra of

5

CdTe QDs (0.016 μM) and that in the presence of PDA or SiO2@PDA. The

6

concentration of PDA was kept the same (52 μg·mL-1) for both PDA and the

7

SiO2@PDA NP dispersion. (c) The normalized fluorescence intensity histogram using

8

the data derived from (b). (d) Fitting curves of time-resolved fluorescence spectra

9

monitored at λ = 533 nm under excitation of λ = 280 nm for CdTe QDs with and

10

without SiO2@PDA. The inset shows the fitted lifetime in nanoseconds.

11 12

The quenching effect of SiO2@PDA for PEC sensing was further verified by

13

modifying Ab2 with different labels such as PDA and SiO2@PDA. As shown in

14

Figure 3a, compared to that with Ab2, the incubation of the immunosensor with 11



Page 12 of 29



 2  1  100% 1 , where

1

Ab2-PDA caused about a quenching efficiency of ca. 7% (

2

η1 and η2 are respectively reduced photocurrent without and with quencher)50, and the

3

Ab2-SiO2 caused 30% quenching due to the hindrance effect and the poor

4

conductivity

of

SiO2.

However,

a

much

enhanced

quenching

efficiency

5

 2  1  100% 1 upon the incubation with Ab2-SiO2@PDA (70%) was observed.

6

A quenching efficiency of 70% for the Ab2-SiO2@PDA was much larger than the sum

7

of Ab2-PDA (7%) and Ab2-SiO2 (30%), demonstrating that PDA and SiO2 have a

8

synergistical effect for quenching CdTe QDs. Ruling out the hindrance effect by Ab2,

9

the relative quenching efficiency of Ab2 for SiO2@PDA was up to 10 times higher

10

than that of PDA when the concentration of CA125 was 10 U·mL-1 (Figure 3a).

11

Moreover, as control, BSA-SiO2@PDA was prepared, and the photocurrent change of

12

the PEC immunosensor upon the incubation with BSA-SiO2@PDA was much smaller

13

than that with Ab2 and Ab2-SiO2@PDA, implying that BSA can effectively block the

14

non-specific absorption of nanohybrid. The electron transfer resistance (Rct) of the

15

modified

16

Ab2-SiO2/Ag/BSA/Ab1/CdTe-CS/ITO

17

Ab2-SiO2@PDA/Ag/BSA/Ab1/CdTe-CS/ITO was evaluated by EIS. As shown in

18

Figure 3b, a semicircle in high frequencies and a line in low frequencies were

19

observed in the Nyquist plots, implying that the photoelectrodes can be described by a

20

modified Randles circuit.51, 52 As shown in Figure S3, the curve (solid line) coincides

21

well with the experimental impedance data (black solid square), indicative of an

22

accurate equivalent circuit. From the fitting results of the equivalent circuit model, Rct

23

of

24

PDA/Ag/BSA/Ab1/CdTe-CS/ITO

25

demonstrating that both PDA and SiO2 contributed to the residence on the electrode

26

surface (Figure S3 and Table S1). Nevertheless, with respect to the profound

27

quenching efficiency, the stronger quenching of SiO2@PDA over that of PDA alone

28

for CdTe QDs should still be more dominated by the interfilter effect of PDA via an



electrode

such

as

Ab2-PDA/Ag/BSA/Ab1/CdTe-CS/ITO, and

SiO2@PDA/Ag/BSA/Ab1/CdTe-CS/ITO and

was

larger

than

those

of

SiO2/Ag/BSA/Ab1/CdTe-CS/ITO,

12


Page 13 of 29

1

effective loading on SiO2 NPs. Therefore, by homogenous coating of PDA on SiO2

2

NPs, a significantly improved quenching was observed up to 10 times that of PDA

3

alone, and this result was highly anticipated to enhance the sensitivity of the

4

“signal-off” PEC immunosensor, an often criticized weak point compared to its

5

“signal-on” counterpart.

6

(a)

(b) 0.6

-Z" (Ω)

70%

30% 7% }

0.2

0.0

7

500

• Ab2-PDA • Ab2-SiO2 • Ab2-SiO2@PDA

400

0.4

ΔI/I0

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


55%

Ab 2 -PDAb -SiO 2 @PDA PDA Ab 2 A 2 -SiO 2 -SiO 2@ Ab 2 BSA

300 200 100 0

0

100

200

300

400

500

Z' (Ω)

8

Figure 3. (a) Photocurrent change of the modified ITO (Ag/BSA/Ab1/CdTe-CS/ITO)

9

upon the incubation with Ab2, Ab2-PDA, Ab2-SiO2, Ab2-SiO2@PDA, and

10

BSA-SiO2@PDA,

respectively.

(b)

Nyquist

plots

of

11

Ab2-PDA/Ag/BSA/Ab1/CdTe-CS/ITO, Ab2-SiO2/Ag/BSA/Ab1/CdTe-CS/ITO, and

12

Ab2-SiO2@PDA/Ag/BSA/Ab1/CdTe-CS/ITO in 20 mM PBS solution containing 5.0

13

mM [Fe(CN)6]3-/4-. The insets show the equivalent circuit for fitting. Rs, the solution

14

resistance; Y0, the modified layer/solution interface capacitance; Rct, the electron

15

transfer resistance; Wd, Warburg diffusion impedance.

16

13



(a)

(b)

(c)

(d)

(e)

(f) Intensity (a.u.)

0.4

ΔI / I0

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

0.3

Page 14 of 29

CdTe CdTe + SiO2@PDA(7:2) CdTe + SiO2@PDA(7:6) CdTe + SiO2@PDA(7:12) CdTe + SiO2@PDA(7:16)

0.2 0

1

20

40

60

PDA shell thickness (nm)

500

550

Wavelength (nm)

600

2

Figure 4. TEM images of SiO2@PDA NPs prepared with SiO2 to DA mass ratios of

3

7:2 (a), 7:6 (b), 7:12 (c) and 7:16 (d). The calibration curve of photocurrent response

4

(ΔI/I0) and PDA shell thickness in SiO2@PDA at different mass ratios of SiO2 and

5

DA (e). The fluorescence of CdTe QDs (0.016 μM) in the presence of SiO2@PDA

6

with the same amount of SiO2 and different mass ratios of SiO2/DA in the dispersion

7

(f). The color scheme in (a-c) highlights the PDA shell. The concentration of SiO2 in

8

all SiO2@PDA was 10.4 μg·mL-1. 14


Page 15 of 29 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


1

The influence of the mass ratio of SiO2 and DA on the quenching efficiency for

2

CdTe QDs was further studied. The PDA shell thickness successively increased with

3

increasing mass ratios of DA to SiO2 (see TEM images in Figure 4a-d and particle

4

size distribution in Figure S4). Nevertheless, it should be noted that due to the thick

5

PDA shell, SiO2@PDA with a mass ratio of 7:16 was heavily cross-linked among

6

multiple NPs, resulting in poor dispersibility under the current synthesis conditions

7

(Figure 4d). As a result, the elevated quenching level by using ΔI/I0 increased with

8

increasing PDA thickness in SiO2@PDA and reached a plateau (Figure 4e). For

9

instance, ΔI/I0 for SiO2@PDA increased from 0.25 to 0.43 when the PDA shell

10

thickness increased from 4 nm to 10 nm. Notably, the measured ΔI/I0 also coincided

11

with the fluorescence intensities of SiO2@PDA NPs prepared at different ratios of DA

12

to SiO2 with different PDA shell thicknesses (Figure 4f). The concentrations of the

13

solutions in Figure 4f and Figure 2b were different. Therefore, a SiO2/DA mass ratio

14

of 7:12, i.e., a PDA shell thickness of 10 nm, was adopted as the optimized condition

15

for the following biosensing studies.

16 17

Fabrication of the PEC immunosensor

18

The stepwise assembly process of the PEC immunosensor was shown in Scheme

19

1 and optimized according to the Ab1 concentration, electrolyte pH and CdTe

20

concentration (see Figures S5 to S7 and more discussion in the Supporting

21

Information). Successful stepwise assembly was confirmed by CV measurements in

22

20 mM PBS (pH 7.4) using 2.0 mM [Ru(NH3)6]3+/2+ as the electrochemical probe. As

23

shown in Figure S8a, a pair of well-defined redox peaks of [Ru(NH3)6]3+/2+ can be

24

observed on the bare GCE (black curve). When CdTe-CS was modified on the

25

electrode surface, the redox peak currents increased significantly (magenta curve),

26

suggesting that CdTe-CS promoted the electron transfer of [Ru(NH3)6]3+/2+ on the

27

electrode surface. When the electrode was further modified with Ab1, an obvious

28

decrease in the redox current was observed (blue curve). Subsequently, the redox peak 15



Page 16 of 29

1

current was further reduced after the electrode was incubated with BSA and Ag

2

(green curve) because the BSA and Ag protein layers on the electrode blocked the

3

electron transfer. Finally, the redox peak current slightly increased when the electrode

4

was further incubated with Ab2-SiO2@PDA (red curve), and this result was due to the

5

electrostatic repulsion between Ab2-SiO2@PDA and [Ru(NH3)6]3+/2+ being less than

6

that between Ag and [Ru(NH3)6]3+/2+.

7

To further verify the feasibility of the designed immunosensor, the photocurrent

8

generation during the immunosensor fabrication was measured under chopped light

9

irradiation within a standard PEC cell configuration. As shown in Figure S8b, the

10

photocurrents of all electrodes were prompt, steady and reproducible during repeated

11

on/off light irradiation cycles. The CdTe-CS/ITO electrode exhibited an evident

12

photocurrent response (magenta curve, photocurrent = 7.06 μA·cm-2), indicating that

13

CdTe QDs are excellent photoelectric materials for the construction of the PEC

14

immunosensor. The photocurrent significantly decreased after Ab1 was covalently

15

conjugated with CdTe-CS on ITO (blue curve, photocurrent = 4.88 μA·cm-2), and this

16

result could be attributed to the steric hindrance of both the protein blocking layer and

17

the glutaraldehyde cross-linking agent. The photocurrent further decreased gradually

18

after incubation of the modified ITO with BSA (orange curve, photocurrent = 3.94

19

μA·cm-2) and Ag (green curve, photocurrent = 3.25 μA·cm-2) due to the increased

20

steric hindrance of these protein molecules. Notably, upon the capture of

21

Ab2-SiO2@PDA (red curve, photocurrent = 1.75 μA·cm-2), the photocurrent shows a

22

much larger depression, which could be ascribed to three aspects: (i) the quenching

23

effect of PDA on CdTe QDs (see Figure S9 and more discussion in the Supporting

24

Information); (ii) the formation of SiO2@PDA increased the dispersibility and loading

25

of the PDA, which further enhanced the quenching of CdTe QDs; and (iii) the poor

26

electron conductivity and steric hindrance of SiO2 effectively suppressed the electron

27

transfer.53 This phenomenon also could be explained by the electron transfer

28

mechanism of the PEC immunosensor (see Figure S9 and more discussion in the

29

Supporting Information). Therefore, both the CV and photocurrent measurements

30

during the stepwise assembly of the immunosensor demonstrated the successful 16


Page 17 of 29

1

fabrication of the “signal-off” PEC immunosensor.

2

Performance of the PEC immunosensor

3

It was supposed that after the sandwich immunoreaction, the Ab2-SiO2@PDA

4

probe would be quantitatively captured via the formation of an immunocomplex, and

5

the signal decrease of CdTe would be correlated to the amount of captured CA125

6

based on the quenching of CdTe by SiO2@PDA on the electrode. As shown in Figure

7

5a, the photocurrent decreased gradually with increasing CA125 concentration under

8

the optimized conditions. The calibration curve showed a good linear relationship

9

between the photocurrent decrease and logarithmic value of target CA125

10

concentrations in the range from 1 mU·mL-1 to 100 U·mL-1 with a correlation

11

coefficient R2 of 0.992, indicating that CA125 can be quantitatively detected by the

12

proposed method (Figure 5b). The linear regression equation is ΔI/I = 0.0704 lg C

13

(U·mL-1) + 0.408, and the detection limit (LOD) of 0.3 mU·mL-1 was estimated in

14

terms of a signal-to-noise ratio of 3. In addition, compared with previous reports for

15

detecting CA125, the prepared PEC immunosensor showed superior performance, as

16

summarized in Table 1. Specially, the LOD of PEC immunosensor was much lower

17

than commercial ELISA kit (LOD = 2 U·mL-1)54. Therefore, the highly efficient

18

fluorescence quenching of SiO2@PDA core-shell NPs is very promising for the

19

“signal-off” PEC immunoassay.

20

(b)

-2

0

0.5 -1

2

-2

1 -1

-3

0

y = 0.0704 x + 0.408 2

R = 0.992

-2 0.2

-4 -1

22

0.4

0.3

-3

21

0.6

ΔI/I0

(a)

Photocurrent (A·cm )

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


lg CCA125 (U·mL

-3

)

-2

-1

0

-1

lg CCA125 (U·mL

1

2

)

Figure 5. (a) Photocurrent responses and (b) calibration curve of the PEC 17



Page 18 of 29

1

immunoassay for the detection of different concentrations of target CA125 from 1

2

mU·mL-1 to 100 U·mL-1. The error bars show the standard deviation of four parallel

3

measurements.

4 5

6

Table 1 Comparison of various methods for CA125 detection Method

Linear range (U·mL-1)

LOD (U·mL-1)

Reference

Commercial ELISA kit

1.56-100

2

54

Electrochemistry

0.01-50

0.005

55

Microarray system

0-1280

17

56

Electrochemiluminescence

0.001-1

0.1

57

Chemiluminescence resonance energy transfer

0.1-600

0.05

58

Electrochemistry

2-14

1.29

59

Surface plasmon resonance

1.0-80

0.4

60

Fluorescence

5-20

5

61

Electrochemistry and colorimetry

0.0064-256

0.005

62

PEC

0.005-80

0.0013

63

PEC

0.001-100

0.0003

This work

Selectivity, stability and reproducibility

7

To investigate the selectivity of the “signal-off” PEC immunosensor, the

8

modified electrodes (Ag/BSA/Ab1/CdTe-CS/ITO) were incubated with several

9

biomarkers/proteins and potential interferents, such as alpha-fetoprotein (AFP),

10

carcinoembryonic antigen (CEA), tumor necrosis factor-α (TNF), albumin (AL),

11

triglyceride (TR), cholesterol (CH) contained in serum and a mixture. As shown in

12

Figure 6a, the photocurrent responses of the immunosensor for AFP, CEA, and TNF

13

were much lower than that for CA125 and the mixture, indicating that these

14

interfering proteins did not cause obvious signal variation. In addition, the

15

photocurrent response of AL, TR and CH in serum were significantly lower than that

16

of CA125 and the mixture, demonstrating that potential interferents and possible 18


Page 19 of 29

1

matrix effects in the serum did not interfere with signal changes in the PEC sensor.

2

Hence, the proposed PEC immunosensor exhibited good selectivity.

3

As shown in Figure 6b, the proposed immunosensor shows a stable photocurrent

4

response under several continuous on/off irradiation cycles for 300 s, indicating that

5

the immunosensor has excellent stability. In addition, to further validate the stability

6

of the immunosensor, the prepared immunosensor for 0.1 U·mL-1 CA125 in 20 mM

7

PBS (pH 7.4) was stored at 4 °C under darkness, and the constructed immunosensor

8

still retained 98.8%, 89.3%, and 88.6% of the initial response after storage for 2, 7

9

and 14 days (Figure 6c), respectively, suggesting that the constructed immunosensor

10

had satisfactory stability. In addition, the reproducibility of the PEC immunosensor

11

was assessed by five independent immunosensors64, 65 in the presence of 0.1 U·mL-1

12

target CA125 (Figure S10). As a result, the proposed immunosensors showed good

13

reproducibility with a relative standard deviation (RSD) of 3.1%. These results

14

demonstrated the satisfactory precision and reproducibility of this immunosensor.

15

(a)

(b)

(c)

on

0.25

0.00

16

-2

Photocurrent (A·cm

ΔI/I0

0.50

Relative activity (%)

)

0

CA CA 1 12 A 25 5 F P CA + A 12 CFP 5 E CA + C A 12 T E A 5 N + F CA TN 12 A F 5 L CA + A 12 T L 5 R CA + T 12 C R 5 H + CH M ix

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


-1

-2 0

off 100

200

Time (s)

300

80

40

0

1

2

7

14

Time (d)

17

Figure 6. (a) Selectivity of the proposed PEC immunosensor to 10 U·mL-1 CA125 in

18

comparison to the interfering biomarkers/proteins at the 0.1 μg·mL-1 level and the

19

mixed sample. (b) Stability of the proposed biosensor incubated with 10 U·mL-1

20

CA125 under several on/off irradiation cycles for 300 s. (c) Stability of the

21

immunosensor for CA125 (0.1 U·mL-1) detection after 1, 2, 7, and 14 days.

22

Preliminary analysis of real samples

23

To further validate the reliability of this immunosensor, the proposed

24

immunosensor is applied for the detection of CA125 in human serum samples. As 19



Page 20 of 29

1

shown in Table 2, the proposed method can detect CA125 in serum samples in a

2

wide concentration range of 0.001-100 U·mL-1, with good recoveries varying in the

3

range of 95.67%-104.72%, indicating that this method has good sensitivity and

4

reliability for the detection of CA125 in real serum samples.

5 6

Table 2 Recovery tests of CA125 antigen in serum samples (n = 4)a sample

an

Added

Found

RSD (%)

Recovery (%)

(U·mL-1)

(U·mL-1)

1

1.00*10-3

9.567*10-4

1.09

95.67

2

1.00*10-2

1.010*10-2

2.84

101.0

3

1.00*10-1

9.859*10-2

4.46

98.59

4

1.00

0.9831

1.42

98.31

5

10.0

10.08

2.36

100.8

6

100

104.7

1.14

104.7

is the number of repeated measurements

7 8

Conclusions

9

In this work, SiO2@PDA core-shell NPs were explored as a highly effective

10

quencher in PEC immunosensing. It was revealed that as a carrier, the uniform SiO2

11

NPs significantly improved the dispersibility and loading of PDA, thus maximizing

12

the advantage of PDA in effectively quenching of PEC probes via inner filter effect in

13

the whole UV-vis region by a factor up to 10 times. Moreover, the poor electron

14

conductivity and steric hindrance of SiO2 NPs was also favor of quenching PEC

15

probes by suppressing the electron transfer. As a result, by detecting CA125 (an

16

ovarian cancer biomarker) as an example, the SiO2@PDA-based PEC immunosensor

17

demonstrated a linear range from 1 mU·mL-1 to 100 U·mL-1 and an ultralow detection

18

limit of 0.3 mU·mL-1 (S/N = 3), the most sensitive sensor for CA125 ever reported so

19

far. By following the similar principle, the proposed SiO2@PDA could be developed

20

as a simple and economic “signal-off” PEC platform for immunoassay of other 20


Page 21 of 29 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


1

biomarkers with boosted sensitivity for early clinical diagnosis.

2

Acknowledgments

3

This work is supported by the National Natural Science Foundation of China

4

(21675022 and 21775018), the Natural Science Foundation of Jiangsu Province

5

(BK20170084 and BK20160028) and the Fundamental Research Funds for the

6

Central Universities.

7 8

Supporting Information Available

9

FTIR spectra of DA, SiO2, PDA and SiO2@PDA; HRTEM image of CdTe;

10

simulated EIS result and the fitting results of photoelectrodes; size distribution

11

histogram of SiO2@PDA NPs with the different SiO2:DA mass ratio; optimization of

12

experimental conditions; feasibility verification, the electron-transfer mechanism and

13

reproducibility of the PEC immunosensor.

14

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