Ultrasensitive Electrochemical Immunoassay Based on Cargo

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Ultrasensitive Electrochemical Immunoassay based on Cargo Release from Nanosized PbS Colloidosomes Xiujuan Han, Hongfang Zhang, and Jianbin Zheng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04807 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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Analytical Chemistry

Ultrasensitive Electrochemical Immunoassay based on Cargo Release from Nanosized PbS Colloidosomes Xiujuan Han, Hongfang Zhang*, Jianbin Zheng* Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of Chemistry & Materials Science, Northwest University, Xi’an 710127, China

ABSTRACT: Colloidosome is a novel nanostructure composed of millions of colloid particles. In this work, nanosized PbS colloidosomes were initially prepared and applied as nanoprobes for ultrasensitive immunoassay. The colloidosomes were simply prepared in mild conditions by assembling the elementary ca. 8 nm PbS nanoparticles at the water-in-oil interface of emulsion droplets. To enhance the rigidity and biocompatibility of the colloidosomes, interfacial polymer was introduced by utilizing self-polymerization of dopamine. By treating with dilute nitric acid, a bursting release of lead ions from the colloidosomes occurred and the lead ions can be detected easily by anodic stripping voltammetry. In this way, a colloidosome-based electrochemical immunoassay was developed by using the nanosized PbS colloidosomes as electroactive labels. The proposed method featured a linear calibration range from 10 fg·mL-1 to 100 ng·mL-1 with a low detection limit of 3.4 fg·mL-1 for the detection of human epididymis protein 4. This work introduced a new member for the family of colloidosomes and offered a novel perspective for the rational implementation of various colloidosomes for novel low-abundance cancer biomarkers analysis.

Colloidosomes (CSs), firstly termed by Dinsmore et al. at 2002,1 refers to solid and hollow capsules composed of colloid particles and usually prepared by self-assembly of colloid particles on emulsion droplets. As witnessed by the pioneering studies, the initial interest for CSs was their important internal volume combined with the properties of the nanobuilding blocks in the shell, which facilitated the capsulation of active ingredients such as drugs, flavors, proteins, bacteria or *

Corresponding author. E-mail: [email protected] (Hongfang Zhang)

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even living cells.2-5 Different with the simplex phospholipid bilayer capsules of liposomes, the nanobuilding blocks of CSs are theoretically unlimited. As a hollow structure,6-12 the packing colloidal particles of CSs is a wide variety nanoparticles (NPs) include iron oxide,6 gold,7 silver,8 polymer,9 silica,10 metal-organic frameworks,11 and the combination of these NPs.12 As a matter of course, the specific physical properties of CSs as novel nanostructure were noticed. For instance, hollow core–shell α-Fe2O3 CSs were evaluated as a lithium-ion battery anode13 and carbon CSs were demonstrated as good electrocatalysts for oxygen reduction reaction.14 The identification and detection of disease biomarkers have become important issues for the early disease diagnosis.15,

16

Electrochemical immunoassay is a sensitive technique for the early

disease diagnosis through the accurate determination of specific protein biomarkers at ultralow levels. To quantitatively transform the immuno-recognition event into sensitive electrochemical signals, different tracing nanoprobes were designed.17 Among them, nanoparticles of metal (eg. Cu, Ag) or metal sulfides (eg. CdS, PbS) are frequently applied because of their intrinsic redox properties. Wang’s group18 pioneered this concept by using ZnS, CdS, PbS, and CuS colloidal crystals for the simultaneous analysis of four proteins. These nanocrystal labels exhibited detect limit (DL) of ～ 10 ng·mL-1 for β2-microglobulin, IgG, bovine serum albumin (BSA), and C-reactive protein. Using the same concept, Chen et al.19 obtained DL of 3.4 pg·mL-1 for the detection of IgG1 with CdS quantum dot-doped BSA as the signal tracing nanoprobes. Both the studies applied the magnetic separation and voltammetric measurement procedures. Thus the ca. tenfold increase of the average size of the CdS nanoparticles utilized in the latter study owned much to the dramatic decrease of DL (10 ng·mL-1 vs. 3.4 pg·mL-1) when compared with it obtained by Wang’s group. 18 Zhu’s team20 ingeniously designed a sandwich type electrochemical immunoassay by using the rolling circle amplification products as the specific templates for the subsequent Cu NPs formation. The DL for prostate specific antigen was as low as 0.02 fg·mL-1 because cascade signal amplification was easily achieved by dissolving the CuNPs and detecting the released copper ions. Quite understandably, the immunoassay sensitivity is greatly connected with the metal ions concentration that the metal-containing nanocrystal tracers can supply. Of course, this does not mean that super-large tracers are needed. Large tracers definitely create steric hindrance that


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causes poor antigen–antibody binding interaction and inversely affect the performance of immunoassay. From this point of view, nanosized CSs are excellent candidates for signal tracers of electrochemical immunoassay because of the tunable size and rich metal component distributed at the nanobuilding shells. According to the calculation of Duan et al.,21 one Fe3O4 colloidosome was composed of about 6.1×108 nanoparticles (8.0 nm of average size). This means that if the metal CSs are utilized as the tracing nanoprobes for electrochemical immunoassay, the cargo release of the CSs shell would give rise to a “burst” of metal ions. And the electric signal will be amplified exceptionally when compared with it using the basic metal NPs as tracing nanoprobes. Inspired by this exciting consideration, we try to develop a proof-of-concept colloidosome-based electrochemical immunoassay.

Anti-HE4 GA

C60-Chit

GCE

BSA HE4

Anti-HE4

BSA

PbS CSs -

Current/A

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


-1.0

-0.8

-0.6

Potential/V

-0.4

-0.2

Pb2+ Pb2+ Pb2+ pH 5.0 HAc-NaAc Pb2+ Pb2+ Pb2+ Pb2+ Pb2+ DPV Pb2+

1 M HNO3 Release

Scheme 1 Schematic illustration of the PbS CSs-based electrochemical immunoassay.

Herein, the nanosized, polydopamine coated PbS CSs were initially prepared by a reverse water-in-oil emulsion system. And an ultrasensitive immunoassay based on the PbS CSs was developed. As shown in Scheme 1, the functional fullerene-chitosan (C60-Chit) nanocomposite was immobilized onto a glassy carbon electrode (GCE) to covalently immobilize the capture antibodies (Ab1) and expectedly improve the detection sensitivity.22 Human epididymis protein 4 (HE4), a tumor marker of great importance for its potential to reflect the differentiation stage of the ovarian carcinoma,23 was choose as the model target. After the PbS CSs was introduced onto



the electrode surface by the formation of the sandwich-type of immunocomplex, Pb2+ released from the CSs was detected. As a result, the developed immunoassay exhibited a ultralow detection limit of 3.4 fg·mL-1. The strategy offered a novel perspective for the rational implementation of CSs for HE4 detection and could be integrated with other recognition elements to broaden its applications in bioassay.

EXPERIMENTAL METHODS Reagents and Materials. Oleylamine (OLA, 80-90%), PbCl2 (99.999%) and glutaraldehyde (GA) were purchased from Aladdin. HE4, monoclonal anti-HE4 antibody (Ab1) and polyclonal anti-HE4 (Ab2) were obtained from Linc-Bio Science Co. Ltd. (Shanghai, China). Bovine serum albumin (BSA) was obtained from Beijing Biosynthesis Biotechnology Co. Ltd. (Beijing, China). 3-mercaptopropionic acid (MPA, 99%) was purchased for local reagents companies and used as received. All other chemicals were analytical grade. Phosphate buffered saline (PBS, pH 7.4) was prepared by mixing Na2HPO4 and KH2PO4. PBS containing 0.05% Tween-20 (PBST) was used as the washing solution. Characterization. Scanning electron microscopy (SEM) images were obtained by using a JSM-6390A scanning electron microscopy (JEOL). The transmission electron microscopy (TEM) images were determined using a Tecnai G2 F20 S-TWIN (FEI, USA). All powdered X-ray diffraction (XRD) experiments were recorded on a D8 ADVAHCL* diffractometer (Bruker) using Cu Kα radiation. Inductively coupled plasma optical emission spectrometry (ICP-OES) was carried out on an Optima 2100DV (Perkin Elmer). All voltammetric experiments were carried out with a CHI660E electrochemical workstation (Chenhua Instruments). A conventional three-electrode system with either a bare or modified GCE as the working electrode, a platinum electrode as the counter electrode and a saturated calomel electrode as the reference electrode was applied. Preparation of the PbS CSs. In typical experiment, 10 mg of PbS NPs was dispersed in 2 mL of oil phase, containing 50vol% vegetable oil and 50vol% heptane under ultrasonication for 10 min. Then, 400 L of Tris-HCl buffer (10 mM, pH 8.5) was added to the PbS NPs dispersion (the volume ratio of the oil to water, Ro/w, was 5), and the two-phase system was sonicated for 1 h in


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an ultrasound bath to generate homogeneous water-in-oil emulsions. The mixtures were left unperturbed for 1 h after sonication. After the formation of a Pickering emulsion, 0.40 mg dopamine powder (DA/Tris-HCl=1 mg·mL-1) was added to the emulsion, followed by ultrasonication for 10 min and left unperturbed for 10 min, giving rise to a dark gray emulsion composed of a large number of PbS CSs. In order to transfer the CSs from the organic to the aqueous phase, the system was centrifuged for 5 min at 3000 rpm. After centrifugation, the heptane phase was carefully removed from the centrifugation tubes, and the aqueous phase containing the CSs was collected. Preparation of PbS CSs based nanoprobe. 100 L of the dispersion of the as-synthesized PbS CSs was mixed with 100 μL of pH 7.4 phosphate buffer. Then, 2 L 1 mg·mL-1 Ab2 was added and was left to stand overnight at 4 °C, followed by adding 20 μL of the 1% BSA solution for 1 h reaction. After centrifugation and washing thrice with a pH 7.4 phosphate buffer, the PbS CSs-Ab2 nanoprobe was finally obtained and redispersed in 0.1 mL of pH 7.4 phosphate buffer for further use. Fabrication of sandwich immunosensor. Prior to each experiment, GCE was sequentially polished with 0.3 m and 0.05 m of alumina powder, respectively. And the polished GCE was successively sonicated with ethanol and distilled water to remove any adsorbed substances on the electrode surface. Firstly, 6 L of 1 mg·mL-1 C60-Chit dispersion was dropped onto the electrode surface and dried at room temperature to allow it to chemisorb onto the surface. The modified electrode was washed with water and incubated with 6 μL of 5% GA for 2.5 h at 4 °C in refrigerator, followed by washing with ultrapure water to remove unreacted reagents. Then, 6 μL of 10 μg·mL-1 Ab1 was spread on the modified electrode at 4 °C overnight, and excess antibodies were removed with PBS and PBST, respectively. Afterward, 6 μL of 1% BSA solution was added to the electrode surface And reacted for 1 h to block the nonspecific sites. After another washing with PBS and PBST, the immunosensor was obtained and stored at 4 °C prior to use. Measurement procedure. To carry out the immunoreaction and electrochemical measurement, the immunosensor was incubated at room temperature for 1 h with different concentrations of HE4. After washing with PBS and PBST, the immunosensor was further incubated with 6 μL of PbS CSs-Ab2 nanoprobe for 45 min at room temperature. After finally being washed with PBS to



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remove nonspecifically bounded nanoprobes, the electrode was immersed into 200 μL of HNO3 solution (1 M) for 10 min to dissolve the probes. The resulting solution was mixed with 4.8 mL of HAc-NaAc buffer (0.1 M, pH 5.0) containing 0.2 mM Hg2+ for the detection of metal ions by differential pulse anodic stripping voltammetry (DPASV). The detection was recorded in the potential range from −1.2 V to 0 V, with deposition potential of −1.0 V, deposition time of 240 s, amplitude of 100 mV, pulse width of 50 ms and quiet time of 2 s.

RESULTS AND DISCUSSION Characterization of PbS CSs. To prepare the PbS CSs, mono-dispersed PbS NPs with average diameter of 8 nm were prepared and characterized (Supporting Information, Figure S1). The CSs were synthesized through controlled agglomeration of the PbS NPs at the droplet interfaces in the water-in-oil emulsion, as illustrated in Figure 1A. To acquire cross-linkable and stable PbS colloidosomes, the colloidosomes was designed to be coated with polydopamine. Therefore, the aqueous phase was chosen to be the pH 8.5 tris-HCl buffer which was a proper medium for the self-polymerization of dopamine.24 The organic phase was the mixture of vegetable oil and heptane (V/V=1/1) which is an excellent medium for the dispersion of hydrophobic PbS NPs.

A

Oil phase

Dopamine addition

Ultrasonation

Centrifugation

Water phase

Water phase: Tris-HCl buffer

B

Oil phase: vegetable oil/heptane (1/1)

C

PbS QDs

Polydopamine

D

Figure 1 Preparation and characterization of PbS CSs. (A) Schematic illustration of the preparation of PbS CSs. (B-D) SEM images of PbS CSs. Scale bar is 1 m for B, 400 nm for C and 100 nm for D.

The morphology of the coarse products can be observed clearly from the scanning electron


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microscopy (SEM) image (Figure 1B) which exhibits spherical particles with size of ca. 100 to 500 nm. Magnifying one big particles (Figure 1C), the three-dimensional spherical structures assembled by single nanoparticles can be revealed. The hexagonally close packed surface, depicts more closely in the further magnification (Figure 1D), emphasizes the high order of the building block particles of the CSs. EDS spectrum (Supporting Information, Figure S2) displays distinct signal of Pb, S, C and N, confirming the elemental composition of the CSs. Therefore, nanosized PbS CSs were prepared by inverse w/o Pickering emulsions containing oil-soluble PbS NPs. Since the coarse PbS CSs are not as homogeneous as expected, the material was centrifuged, washed with ethanol and re-dispersed in water. After that, the large CSs and the surplus NPs were separated

and

PbS

CSs

with

relatively

narrow

size

distribution

were

obtained.

Furthermore, effect of concentration of dopamine (CDA), loading amount of PbS NPs (mPbS), oil-to-water volume ratio (Ro/w) and component of oil phase on the formation of CSs was investigated respectively. Average size of CSs doesn’t change much after addition of low concentration of DA (Figure 2A and 2B). However, when CDA increases from 0.5 to 2 mg·mL-1, a significant decrease in size of CSs is observed (Figure 2C). The possible reason is that the presence of high concentration of DA in the aqueous phase influences the size of emulsion droplets. Moreover, when CDA is held constant, an increase of mPbS from 5 to 20 mg lead to a gradual increase in CSs size (Figure 2 D to F), demonstrating that more and more colloid particles are involved into the self-assembly of PbS NPs onto the emulsion droplets. Since CSs were usually prepared by self-assembly of colloid particles on emulsion droplets, effect of Ro/w on the formation of the CSs was investigated by changing volume of water. As shown in Figure 2G to I, the average diameter of PbS CSs rarely changes with increasing Ro/w values from 2 to 10, indicating that the size of emulsion droplets is not influenced greatly by the volume of water. However, the reduction of water volume decreases the number of emulsion droplets which is depicted by the density reduction of CSs on the SEM images. The size is also tuned by changing the composition of the oil phase. The reported oil phase for CSs preparation includes toluene,6 heptane,25 and vegetable oil.1, 26 Considering the homogeneous dispersion of PbS NPs, three types of organic phase were studied. As shown in Figure 2J-L, comparing with toluene and vegetable oil/toluene (1/1), vegetable oil/heptane (1/1) is an ideal organic phase for the preparation of



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Effect of CDA: (A) 0, (B) 0.5, (C) 2 mg·mL-1

A

B

C

Effect of mPbS: (D) 5, (E) 15, (F) 20 mg

D

E

F

Effect of Ro/w, (G) 2, (H) 8, (I) 10

G

H

I

Effect of oil phase, (J) toluene, (K) vegetable oil/toluene (1/1), (L) vegetable oil/heptane (1/1)

J

K

L

Figure 2 SEM images of PbS CSs prepared with different CDA (A-C), mPbS (D-F), RO/W (G-I), and oil phase (J-L). Scale bar is 500 nm for A-F, and 1 m for G-L.

high-density and homogenous PbS CSs. In the subsequent experiments, PbS CSs prepared with vegetable oil/heptane (1/1) as organic phase and CDA=1 mg·mL-1, Ro/w=5, mPbS=10 mg were applied. And the average diameter of CSs prepared at the selected conditions, assessed via the


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analysis of dynamic lighting scatter, is 112 nm (Supporting Information, Figure S3). Cargo release of Pb2+. DPASV is an excellent technique for the quantitative measurement of lead ions.22,

27

By applying a negative potential for pre-deposition and accumulation, a

well-defined peak owing to the electrochemical oxidation of Pb can be clearly observed at −0.62 V on the voltammogram (Figure 3A) recorded in pH 4.5 HAc-NaAc buffer containing 0.2 mM Hg2+. Owing to the excellent conductivity and deposition capability of the C60-Chit nanocomposite,22 the Pb oxidation peak can be dramatically enhanced by using the C60-Chit modified GCE (C60-Chit/GCE) (Figure 3A). Furthermore, modification of the C60-Chit nanocomposite supplied an excellent for the covalent bonding of the capture antibodies.28

220

A Peak current/A

40 30

Current/A

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


C60-Chit/GCE

20 10

Bare GCE

0 -0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

B

200 180 160 140

0

Potential/V

5

10

15

20

25

30

Time/min

Figure 3 Pb2+ release and detection. (A) DPASV responses toward 1.0 M Pb2+. (B) Variation of Pb2+ peak current obtained from DPASV as a function of release time. Error bars represents the standard deviation of three independent experiments.

With the aim of using the PbS CSs as the electroactive labels for immunoassay, the acidity-triggered Pb2+ release from the nano-assemblies was performed. Briefly, 200 L of 1 M HNO3 solution was added into 6 L of the suspension of the as-prepared PbS CSs to trigger the release of Pb2+. And the solution was diluted to 5.0 mL with HAc-NaAc buffer to obtain a proper detection acidity of pH 4.5. Then, Pb2+ was detected by DPASV using C60-Chit/GCE as working electrode. Figure 3B shows the variation of the stripping peak current with the addition time of HNO3, which ranged from 1 to 30 min. The peak current increases with the increase of the release time till 10 min. For release time longer than 10 min, the peak current increases slowly, showing the approximation to complete release of lead ions within 10 min. The concentration of the released Pb2+ from 6 L of dispersion of PbS CSs, evaluated using the quantitative relationship



between peak current on voltammogram and concentration (Supporting Information, Figure S4A) is 7.9 μM, which is in accordance with the result obtained from ICP-OES (Supporting Information, Figure S4B), demonstrating the “burst” of lead ions. PbS CSs-based electrochemical immunoassay. In this work, anti-HE4 antibodies (Ab1) were covalently crosslinked onto the surface of C60-Chit/GCE to prepare the sensor interface, and the secondary anti-HE4 antibodies (Ab2) were immobilized onto the PbS CSs to introduce electroactive labels for the electrochemical determination of HE4 based on the formation of sandwich type immunocomplex. By sequentially incubating with HE4 and the bioconjugates Ab2-PbS CSs, DPASV response of the immunosensor was recorded. As shown in Figure 4A, the response for blank solution (curve a’) displays a small peak while for 100 pg·mL-1 HE4, a brilliant oxidation peak with peak current of 46.13 μA (curve b’) corresponding to the oxidation of lead was observed, demonstrating the feasibility of the strategy. For comparison, DPASV response of the same immuonsensing interface for 100 pg·mL-1 HE4 by using PbS NPs as electroactive nanoprobes was also shown (curve a and b). The peak current for 100 pg·mL-1 HE4 is only 0.64 μA which is ca. one-hundredth of the peak current for PbS CSs labeling, indicating the dramatic sensitivity difference of the two tracers for immunoassay.

60

90

B

A

4 -0.8

a' -0.7

-0.6

-0.5

Potential/V

0

-0.4

a

a' -1.2

-0.8

-0.4

60

30

Current/ A

b' b

Current/A

a

5

20

-1

100 ngmL

90

6

Current/ A

40

Current/A

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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60

-1

1 fgmL

30

0

-4

-2

0

2

-1

logCHE4/pg mL

4

6

0 0.0

-1.2

Potential/V

-0.8

-0.4

0.0

Potential/V

Figure 4 (A) DPASV responses toward blank (a, a’) and 100 pg·mL-1 HE4 (b, b’) by using PbS NPs (a, b) and PbS CSs (a’, b’) as labels. Inset is the partial magnification for curve a and a’. (B) DPASV responses of the electrochemical immunoassay toward different concentration of HE4 and the corresponding calibration curve (inset). Error bars represents the standard deviation of three independent experiments.

DPASV response of the immunosensor toward different concentrations of HE4 was recorded. As shown in Figure 4B, the peak current increases with the increasing HE4 antigen concentration.


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A good linear dependence between the current and the logarithm of HE4 level can be achieved within the dynamic working range from 10 fg·mL-1 and to 100 ng·mL-1 (Inset of Figure 4B). The linear regression equation could be fitted to be I (A) =10.51×logC (pg·mL-1)+26.65 (R2=0.9970). The DL is calculated to be 3.4 fg·mL-1, which is about two to four orders of magnitude lower than the level of the other reported methods (Table 1). Table 1 Comparison of analytical characteristics for HE4 assay Method

Linear range

photoelectrochemical sensor Electronic sensor

Detection limit*

Ref.

0.025-4 ng·mL-1

15.4 pg·mL-1

[29]

ng·mL-1

pg·mL-1

[30]

1.5-25

100

LSPR biosensor

10-10000 pM

4 pM

[31]

Chemiluminescent immunoassay

20-1500 pM

0.18 pM

[32]

ELISA

15-900 pM

15 pM

[33]

ELISA

4-400 pM

6.8 fM

[34]

Electrochemical immunoassay

3-300 pM

0.06 pM

[35]


20-40000 pM

12 pM

[23]


0.00001-100 ng·mL-1

3.4 fg·mL-1

* Molecular weight of HE4 is about 25

this work

kDa. [34]

To demonstrate the sensitivity enhancement effect of PbS CSs, we compared the stripping peak current of Pb2+ released from the other lead-containing tracers for electrochemical immunoassay (Table 2). Although the target for these assays is different, we still can convince the relationship between the stripping current and DL. Therefore, it can be concluded that the large stripping peak current of Pb2+ was a decisive factor of the high sensitivity of the ultimate electrochemical immunoassay. In this work, even for the low concentration of 10 fg·mL-1 HE4, the related Pb2+, determined by ICP-OES, was 3.7 M. Therefore, the mechanism for the ultrahigh sensitivity of the PbS CSs-based immunoassay was attributed to the following three aspects. Firstly, the cascade Pb2+ releasing capability of the PbS CSs exceptionally amplified the antibody-antigen interaction. Secondly, the unique ability to preconcentrate target metal ions and the inherent detection sensitivity of DPASV ensured the intensity of the electrochemical signal of the released Pb2+. Thirdly, the signal of the released Pb2+ was further amplified a hundredfold by the application of C60-Chit nanocomposite.



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Table 2 Comparison of electrochemical immunoassay using Pb2+-based labels Labels*

Target**

Peak current of Pb2+ ***

Detection limit

Reference

(pg·mL-1) PbS CSs

HE4

45.76 μA for 0.1 ng·mL-1 HE4

0.0034

This work

PbS@PDA

IgM

0.1 μA for 0.1 ng·mL-1 of IgM

1.3×107

[36]

AuNPs-Ab2-Pb2+

AFP

9.1 μA for 0.1 ng·mL-1 AFP

3.1

[37]

rApo-Pb

CEA

11.5 μA for 0.1 ng·mL-1 CEA

0.35

[38]

CEA

ng·mL-1 CEA

0.08

[39]



[40]

0.01

[18]

Au/BSA-Pb2+ PbS QDs PbS QDs

Lysozyme BSA

14.2 μA for 0.1 0.7 μA for 1

ng·mL-1

1.5 μA for 50

Lysozyme

ng·mL-1

BSA

*PbS@PDA: polydopamine particles loaded with PbS quantum dots. rApo-Pb: recombinant apoferritin-encoded Pb nanoparticles. QDs: quantum dots. **IgM: Immunoglobulins M. AFP: alpha-fetoprotein. CEA: carcinoembryonic antigen. ***peak currents for all references are calculated from the according relationship between peak current and concentration of target.

Specificity, reproducibility and stability of the immunoassay. The response of the immunoassay for several biomolecules that might coexist with HE4 in the real serum samples were detected. Compared with the current response obtained for 100 pg·mL-1 HE4, the variation in current caused by the mixture of HE4 and equal concentration of carcinoembryonic antigen (CEA), immunoglobulin G (IgG), human serum albumin (HSA) or excessive level of glucose (Glu) was less than 5% (Supporting Information, Figure S5A) , indicating the satisfying specificity of the developed method toward HE4. To investigate the reproducibility of the developed assay, a batch of five parallel experiments was conducted under the same condition, where 1 pg·mL-1 of HE4 was detected (Supporting Information, Figure S5B). As a result, a relative standard deviation of 5.9% was achieved, indicating good reproducibility of the developed method. The stability of the immunosensor was also evaluated by storing the immunosensor and the probes at 4 °C for two weeks. The electrochemical response only decreased 3.3% for HE4 compared with the freshly prepared, which indicated the proposed immunosensor has good stability Application in Analysis of Serum Samples. To corroborate the analytical accuracy of the new developed electrochemical immunoassay by using PbS CSs-Ab2 as electrochemical labels to detect HE4 in real samples, serum samples diluted with PBS buffer (1:10) were spiked with different concentrations of HE4. The recoveries of the spiked HE4 ranged from 95.5% to 111.8% (Supporting Information, Table S1), indicating great accuracy and reliability of the proposed great accuracy and reliability of the proposed immunosensor the quantification of HE4 in biological


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samples.

CONCLUSIONS In summary, we have presented a novel method to prepare nanometer-sized (112 nm) PbS CSs from inverse w/o Pickering emulsion containing PbS NPs (8 nm) and devised a colloidosome-based electrochemical immunosensing platform for the ultrasensitive detection of the ovarian cancer biomarker HE4. Introduction of C60-Chit in this system was conducive to amplify the detection sensitivity to Pb2+ during the measurement, whereas utilization of PbS CSs was dramatic to enhance the released amount of Pb2+ signal tags. The developed immunoassay exhibited an ultralow detection limit of 3.4 fg·mL-1 and potential application in patient serum sample analysis. In addition, this system also provides a new technological platform for the study of other biomarkers. Moreover, the voltammetric signal can be changed by various CSs (e.g., CuS and CdS) and even to offer a highly sensitive and selective simultaneous bioelectronic detection of several protein targets to meet the requirements of specific applications.

Supporting Information Supporting Information Available: Synthesis of oleylamine-capped colloidal PbS NPs; Preparation of MPA-capped water-soluble PbS NPs. Figure S1, SEM, TEM images and XRD pattern of the as-synthesized PbS NPs; Figure S2, EDS spectrum of PbS CSs; Figure S3, DLS intensity distribution of PbS CSs; Figure S4 Quantitative determination of Pb2+; Figure S5 Electrochemical responses of the developed immunoassay; Table S1 Detection of HE4 in spiked serum samples.

ACKNOWLEDGEMENTS This work is financially supported by National Natural Science Foundation of China (No. 21775120), Natural Science Foundation of Shaanxi Province of China (No. 2018JM2017) and Scientific Research Foundation of Shaanxi Provincial Key Laboratory (No. 16JS101).

CONFLICT OF INTERESTS The authors declare no competing financial interest.



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FOR TOC ONLY:

PbS nanoparticles Oil phase

Inverse w/o Pickering emulsion Self-assembly PbS Colloidosomes Water phase

Current/A

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Potential/V


Ultrasensitive Electrochemical Immunoassay Based on Cargo

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