Cu Nanoclusters-Encapsulated Liposomes: Toward Sensitive

Subscriber access provided by MT ROYAL COLLEGE

Article

Cu Nanoclusters-Encapsulated Liposomes: Toward Sensitive Liposomal Photoelectrochemical Immunoassay Li-Ping Mei, Xin-Yuan Jiang, Xiao-Dong Yu, Wei-Wei Zhao, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04789 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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

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

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

Analytical Chemistry

Cu Nanoclusters-Encapsulated Liposomes: Toward Sensitive Liposomal Photoelectrochemical Immunoassay Li-Ping Mei,† Xin-Yuan Jiang,† Xiao-Dong Yu,† Wei-Wei Zhao,*,†,‡ Jing-Juan Xu† and Hong-Yuan Chen*,† †

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative

Innovation Center of Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China. ‡

Department of Materials Science and Engineering, Stanford University, Stanford,

California 94305, United States

* To whom correspondence should be addressed.

* E-mail: [email protected]; [email protected]

* E-mail: [email protected]

1

ACS Paragon Plus Environment

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

Page 2 of 25

Abstract: Herein we report the strategy of liposome-mediated Cu2+-induced exciton trapping upon CdS quantum dots (QDs) for amplified photoelectrochemical (PEC) bioanalysis application. Specifically, the Cu nanoclusters (NCs)-encapsulated liposomes were firstly fabricated and then processed with antibodies bound to their external surfaces. After the sandwich immunocomplexing, the confined liposomal labels were subjected to sequential lysis treatments for the release of Cu NCs and numerous Cu2+ ions, which were then directed to interact with the CdS QDs electrode. The interaction of Cu2+ ions with CdS QDs could generate CuxS and form the trapping sites to block the photocurrent generation. Since the photocurrent inhibition is closely related with the Cu NCs-loaded liposomal labels, a novel and general “signal-off” PEC immunoassay could thus be tailored with high sensitivity. Meanwhile,

a

complementary

“signal-on”

fluorescent

detection

could

be

accomplished by measuring the fluorescence intensity originated from the Cu NCs. This work features the first use of Cu NCs in PEC bioanalysis and also the first NCs-loaded liposomal PEC bioanalysis. More importantly, by using other specific ions/reagents-semiconductors interactions, this protocol could serve as a common basis for the general development of a new class of liposome-mediated PEC bioanalysis.

Keywords: Photoelectrochemical; Immunoassay; Liposomes; Cu Nanoclusters; CdS QDs

2


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


The developments of chemical and nanotechnological approaches have been opening up attractive possibilities in bioassay and related applications.1-3 Compared with other techniques, photoelectrochemical (PEC) bioanalysis provides a new methodology that affords an elegant route for sensitive biomolecular detection.4-15 Currently, the impetus for advanced PEC bioanalysis has been growing much faster as evidenced by increasing academic articles.16-25 On the other hand, liposomes are unique spherical vesicles composed of a phospholipid bilayer and an aqueous cavity capable of shipping particular functional species, and the liposomes-based detection systems have demonstrated as powerful approaches in the biomolecular detection.26-28 However, the application of liposomes in PEC bioanalysis has been rarely studied. Motivated by the versatility of liposomes, our group then described the first proof-of-concept

for

liposomal

enediol-ligands-encapsulated

liposomes

PEC for

bioanalysis

directed

by

sensitization

using of

TiO2

nanoparticles and the “signal-on” PEC immunoassay was achieved.29 Soon after, Zhuang’ group reported another interesting liposomes-amplified PEC immunoassay by using the alkaline phosphatase (ALP)-encapsulated liposomes label and the graphene/g-C3N4 nanohybrids photoelectrode.30 More recently, Tang’s group ingeniously utilized the dopamine-loaded liposomes to enhance the photocurrent of Mn2+-doped Zn3(OH)2V2O7 nanobelts and thus realized the in-situ amplified PEC immunoassay of aflatoxin B1.31 Despite these desirable progress, achieving advanced protocol for liposomal PEC bioanalysis remains a challenge.

3



Page 4 of 25

Metal nanoclusters (NCs), with discrete band gap, have gathered tremendous attention and elucidated new frontiers in material science.32,33 Due to the enhanced quantum confinement, these ultrasmall metal NCs exhibit interesting and unusual size-dependent optical, electronic and catalytic properties, and also excellent photostability and biocompatibility/low toxicity.34,35 Although with great potential, the research for NCs-based PEC bioanalysis is surprisingly quite limited. Yue et al. has pioneered the use of Au NCs/gold photoelectrode for the PEC enzymatic bioanalysis of glucose.36 Our group then demonstrated that Ag NCs could efficiently quench the photoresponse of CdS quantum dots (QDs) for energy transfer-based PEC bioanalysis.37. These findings have sparked our new interest in exploiting innovative PEC bioanalysis with the use of different NCs. Significantly, Cu NCs, a new class of economic, nontoxic, and excellent phosphors and catalysts, has demonstrated its wide applicability in many fields such as biolabeling and biocatalysis.34 We thus assume that the proper synergy of Cu NCs and liposomes as well as their elegant implementation will certainly present an ideal platform for highly intriguing PEC bioanalysis. In this work, we communicate the development of Cu NCs-loaded liposomal labels (denoted as CNL) and their employment against CdS QDs to realize an exquisite PEC immunoassay of human cardiac troponin T (cTnT) as a model target (see Supporting Information for experimental details). As in Figure S1, Cu NCs and liposomes were prepared by protein-directed synthesis34 and thin lipid film hydrated method38, respectively, and the as-fabricated Cu NCs were then incorporated within the

4


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


liposomes to prepare the CNL probes for the further labeling of antibodies to their external surfaces (the resultant bioconjugates were denoted as Ab2-CNL). As shown in Scheme 1, after the sandwich immunocomplexing, the confined liposomal labels were subjected to sequential lysis treatments for the release of Cu NCs and numerous Cu2+ ions, which were then directed to interact with the CdS QDs electrode. As previously reported,39,40 the interaction of Cu2+ ions with CdS QDs could generate CuxS, forming the trapping sites with new energy levels to block the photocurrent generation. Since the signal inhibition is intimately associated with the released Cu2+ ions, a novel liposome-mediated PEC immunoassay could thus be tailored with high sensitivity, which to our knowledge has not been reported. Meanwhile, a complementary fluorescent detection could be performed to corroborate the PEC result by measuring the fluorescence intensity originated from the Cu NCs. Dual-response analysis with two different transduction mechanisms have been proposed in this work, which are based on the photoelectrochemical photocurrent of the electrode and fluorescence intensity of Cu NCs, respectively. Benefiting from the different mechanisms and relatively independent signal transduction, there was no interference between these two signaling routes in the dual-response analysis. This work features the first use of Cu NCs in PEC bioanalysis and also the first NCs-loaded liposomal PEC bioanalysis. Besides, sensitive detection could be achieved by this protocol due to the release of numerous Cu2+ ions on the basis of CNL.

More

generally,

upon

the

introduction

of

other

specific

5



Page 6 of 25

ions/reagents-semiconductors interactions, this protocol could serve as a common basis for the development of a new class of liposome-mediated PEC bioanalysis. Scheme 1. Illustration of the Cu NCs-Loaded Liposomal PEC Immunoassay Accompanied by A Complementary Fluorescent Detection

EXPERIMENTAL SECTION Chemicals and Apparatus. Monoclonal mouse anti-cardiac Troponin T (Ab1, 4T19-1C11, and Ab2, 4T19-9G6) from human heart tissue were purchased from HyTest (Turku, Finland). Human cTnT antigen (Ag, L4C00201) was obtained from Linc-Bio Science Co. LTD (Linc-Bio). Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol

(DPPG),

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and cholesterol were obtained from Shanghai Advanced Vehicie Technology (A.V.T.) Pharmaceutical Ltd. Chloroform, methanol, and Triton X-100 and all other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd (China). MaxsorpTM modified 96-well plates were provided by Thermo Fisher Scientific Inc. All chemicals were analytical reagent

6


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


and used without further purification. Ag, Ab1, and Ab2 were diluted to the desired concentrations with the 10 mM phosphate buffer solution (PBS, pH 7.4). All of the aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore). Transmission electron microscopy (TEM) was obtained on a JEOL model 2000 instrument operating at 200 kV accelerating voltage. Size, and surface charge of the Cu NCs-loaded liposomes were measured with a Model 90 Plus particle size analyzer (Brookhaven Instruments) with Zeta PALS particle sizing software version 2.32. The fluorescence emission spectra were obtained on a Shimadzu fluorescence S-3 spectrophotometer (RF-5301PC, Shimadzu Co., Japan). Photoelectrochemical (PEC) measurements were conducted with a home-made PEC system equipped with a 500 W Xe lamp and a monochromator. Photocurrents were performed with a CHI 660A electrochemical workstation (China) in a three-electrode system: a modified CdS QDs electrode with a geometrical circular area (5.0 mm in diameter) as the working electrode, a Pt wire as the counter electrode and a saturated Ag/AgCl electrode as the reference electrode. Fabrication of TGA stabilized CdS QDs modified ITO Electrodes. The TGA stabilized CdS QDs was prepared and applied according to the previous literature.41 Preparation of Cu NCs-loaded liposomes. Firstly, BSA stabilized Cu nanoclusters was prepared through a modified protein-directed synthesis method.34 Subsequently, Cu NCs loaded liposome were prepared through a thin lipid film hydration method.38 DPPC, cholesterol, DPPG, and DPPE (in a molar ratio of 10:10:1:0.4, 42.8 µmol and 37.78 mg in total) were firstly dissolved in 16 mL of the mixture of chloroform and

7



Page 8 of 25

methanol (4:1, v:v), followed by 10 min sonication at 45 °C under nitrogen. Then, the organic solvent was removed by rotary evaporation under reduced pressure at 45 °C to form a thin lipid film. 10 mL BSA-stabilized Cu NCs was added to hydrate the lipid mixture film. The treatment of lipid mixture and the conjugation of Ab2 to CNL were conducted on the basis of our previous work.29 Finally, the resulting CNL and Ab2 conjugated CNL were both stored in 10 mM PBS (pH 7.4) at 4 °C for further use. Immunoassay Development. Ab1 were introduced into 96-well plates by dropping 40 µL of 0.25 mg mL−1 Ab1 and allowed to incubate at 4 °C for at least 12 h. Finally, it was rinsed with a washing buffer solution (10 mM pH 7.4 phosphate buffer containing 0.05 % Tween 20) and then incubated with a blocking buffer solution (10 mM pH 7.4 phosphate buffer containing containing 3.0 % (w/v) BSA) for 2 h to block nonspecific binding sites at 4 °C. After being rinsed with the washing buffer solution, the 96-well plates were used as a PEC immunosensor and incubated with 40 µL of various concentration human cTnT antigens (Ag) solution at 37 °C for 30 min. The CNL (40 µL) was added and incubated for 1 h, and then the well was washed using washing buffer solution

three times. Subsequently, 20 µL of Triton X-100 (1.0 %)

was added and incubated for 30 min to lyse the CNL. The released Cu NCs were converted to Cu2+ with 40 µL HNO3 (0.1 mM). Finally, 20 µL of the lysis solution was dropped on the CdS QDs modified electrode for PEC detection.

RESULTS AND DISCUSSION

8


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


Figure 1. (a) TEM image and (b) the fluorescent spectra of the resultant BSA–Cu NCs. Insets in a and b show the corresponding size distribution histogram and photographs of BSA–Cu NC aqueous solution under visible and UV light, respectively.

Materials Characterization. The water-soluble, stable and fluorescent copper nanoclusters (Cu NCs) were synthesized through protein-directed method,29 using BSA and hydrazine hydrate as the stabilizing agent and reducing agent, respectively. As shown in Figure 1a, transmission electron microscopy (TEM) showed the resultant Cu nanocrystals to have a high monodispersity with a mean diameter of 2.79 ± 1.2 nm, as demonstrated by the size distribution histogram in Figure 1a inset. The oxidation state of copper in the Cu NCs was further characterized through X-ray Photoelectron Spectroscopy (XPS) analysis. As shown in Figure S2, the binding energy of Cu 2p3/2 was revealed to be 932.3 eV, which was assigned to the binding energy of Cu(0).34,42 As was reported, the valence state of the prepared Cu NCs is most likely between 0 9



Page 10 of 25

and +1 owing to the indiscernible binding energies (∼0.1 eV difference) of Cu(0) and Cu(1). 42 There was no peak around 942.0 eV, indicating no presence of Cu2+ in this system.42 Figure 1b depicts the normalized excitation (Ex) and emission (Em) spectra of the Cu NCs in aqueous solution. As shown, optimal Ex wavelength emerged at λex = 550 nm and the corresponding emission maximum appeared in the visible region centered at λem = 638 nm, the results of which were similar with previous literature.34 Incidentally, the Figure 1b inset shows the corresponding photograph and fluorescence images of the Cu NCs, demonstrating that the resulting Cu NCs exhibited a light-yellow color and emitted a red fluorescence under a 365 nm UV lamp.

Figure 2. (a) TEM image and (b) DLS of the hollow liposomes. (c) SEM image and (d) DLS of Cu NCs-loaded liposome. Insets in b and d provide the images of liposome and CNL, respectively.

As shown in Figure 2a of the TEM image, the as-fabricated liposomes, with average size of ca. 40 ± 10 nm, appeared as quasi-spherical shape unilamellar 10


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


vesicles with their thin shell well-demarcated, and no rupture of the capsule wall was observed. Such morphology obviously indicated that a typical vesicle structure had been established. As displayed in Figure 2b, the dynamic light scattering (DLS) analysis was also performed and the result proofed that the liposomes had a narrow size distribution, with a diameter of approximately 99.5 nm. Given that the drop of liposomes on the TEM grids for the vacuum imaging would render their shrinkage to some degree with a certain deformation, the DLS value was consistent with that of TEM characterization. After encapsulating Cu NCs through hydrating Cu NCs and thin lipid film, Figure 2c and d record the scanning electron microscope (SEM) and DLS value (with radius (R) determined as 109 nm) of the as-fabricated CNL, revealing their similar morphology to the hollow liposomes and that the encapsulation of Cu NCs would not affect the liposomal structure. And insets in b and d displayed the color difference between liposome and CNL. Assuming the liposomes were ideal spheres and the bilayer thickness (T) was 4 nm,43 the number of lipids per liposome (Ntot) and liposome per L (Nlip) were calculated as 1.27×105 and 2.01×1014, respectively, according to the following equation 1 and equation 2.38,44 As a result, according to equation 3,44 the number of encapsulated Cu NCs per liposome (NEnc) was calculated as ∼1.48×107, which was expected to amplify the detection signals (see Table S1 and associated description for detailed calculation process). 4π×R2 +R-T2 1 A Mlipid ×NA = 2 Ntot

Ntot = Nlip

11



NEnc =

MEnc ×NA N(lip)

Page 12 of 25

(3)

where Mlipid and MEnc are the molar concentration of lipid and encapsulated material per mL of the Cu NCs, respectively. As described above, liposomes can serve as good signal reporters due to their large surface area and relatively large encapsulation volume. Therefore, they can load numerous hydrophilic and hydrophobic signaling molecules across a wide spectrum of sensing modalities within their cores or bilayers. By introducing liposomes, one-to-one biological binding event could be converted into one-to-many signal output occurrence, and signal intensity could be amplified dramatically. Due to the self-quenching fluorescence of Cu NCs in the liposomes, the leakage of Cu NCs from liposomes could be monitored by the increase of the fluorescence intensity during storage. In this work, no significant changes in fluorescence intensity were observed over one month, indicating the high stability of the as-fabricated CNL. In fact, we had planned to use Cu2+-encapsulated liposomes. However, as demonstrated in Figure S3, the experiment was failed due to the severe leakage of Cu2+ ions. To minimize the leakage of signal molecule from liposomes, Cu NCs was then chosen, tested, and used in subsequent experiments.

12


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


Figure 3. (a) Photocurrent response of CdS QDs/ITO before (curve a) and after (curve b) incubation with the lysis solution corresponding to 50 ng mL−1 antigen, inset is the enlarge view of curve b. (b) The corresponding XPS results. ∆I is the photocurrent decrement and the PEC tests were performed in 10 mM PBS solution (pH 7.4) containing 0.1 M AA with 0.0 V applied voltage and 410 nm excitation light.

Immunoassay Development. To evaluate the feasibility of the proposed strategy, Figure 3a gives the photocurrent response of CdS QDs/ITO electrode before (curve a) and after (curve b) incubation of lysis solution (Cu2+) corresponding to 50 ng mL−1 antigen. Significantly, compared with that of CdS QDs/ITO, the photocurrent exhibited greatly quenching after reaction with the lysis solution. Obviously, such a strong inhibition effect would be very useful for the signal amplification in the

13



Page 14 of 25

proposed PEC bioanalytical system. As shown in Figure 3b, XPS was further applied to study the changes of characteristic peaks before (curve a) and after (curve b) reaction with the lysis solution, using the C 1s binding energy of 284.6 eV as the internal marked standard. As shown, curve a verified the presence of Cd, S, O, C, and N

peaks,

and

N

peak

was

introduced

by

the

use

of

polyelectrolyte

poly(diallyldimethylammonium chloride) (PDDA) during the preparation of CdS QDs/ITO electrode. After incubation with Cu2+, both the Cd and N peak intensities exhibited clear reduction, whereas a new characteristic peak of Cu 2p3/2 emerged at around 931.4 eV,45,46 suggesting the existence of a copper element on the CdS QDs/ITO. These results revealed that the ion exchange between Cd2+ and Cu2+ was occurred on the surface of CdS QDs,47 as illustrated in Figure 3b inset. The photophysics of CdS QDs before and after reaction with Cu2+ was depicted in Figure S4. The newly formed recombination centers (CuxS) on the CdS QDs surface would open a new pathway for the electron–hole recombination and thus inhibit the photocurrent intensity of CdS QDs. As shown in Figure 4a, the Cu 2p3/2 peak was further deconvoluted into three parts located at 932.4, 931.4, and 929.2 eV for the high-resolution Cu 2p XPS spectrum. As reported in the previous work,45 the main peak at 931.4 eV of Cu+ specie was produced from the interaction between Cu2+ and thiol groups on the surface of QDs, indicating the presence of CuxS (x = 1, 2) on the surface of original CdS QDs. Besides, the one weak peak at 932.4 eV was attributed to CuS,48 while the other at 929.2 eV was assigned to the tiny amount of CuO.49 Concurrently, due to the fluorescence property of the Cu NCs, an auxiliary fluorescent

14


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


assay may also be conducted by measuring the fluorescence intensity originated from the Cu NCs. As shown in Figure 4b, the fluorescence spectra of Cu NCs released from surfactant-lysed liposomes corresponding to 0.1 pg mL−1 antigen (curve a) exhibited strong increase to that corresponding to 50 ng mL−1 sample (curve b), demonstrating the feasibility of such a complementary “signal-on” fluorescent assay to support the PEC results in the proposed system.

Figure 4. (a) High-resolution Cu 2p XPS. (b) Fluorescence intensity of the lysis solutions corresponding to 0.1 (curve a) and 50 (curve b) ng mL−1 antigen, respectively.

15



Page 16 of 25

Figure 5. (a) Derived calibration curve of the photocurrent decrement. (b) Selectivity of the proposed immunoassay to cTnT by comparing to the interfering proteins at 10 pg mL−1 level: IgG, CRP, p53, CEA, and IgA.

Since the signal decrease extent associated closely with the target concentration, a novel liposomal PEC immunoassay can be achieved by recording the photocurrent variation. Figure 5a exhibited the decrement of photocurrent after reaction with the lysis solution, and Figure 5a inset displayed the derived calibration curve. As shown, the photocurrent signal decreased linearly with the increasing antigen concentrations from 0.1 to 2 pg mL−1, and the lowest detection limit (LOD) was estimated to be 0.03 pg mL−1 (S/N = 3). The reduction of the signal decrease over 2 pg mL−1 was due to the near saturation of the chemical displacement process on the CdS QDs surface. 16


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


Table 1 compared some other reported works addressing cTnT antigen.50-54 As presented, this PEC biosensor possessed comparable analytical performance among these cTnT biosensors. More significantly, it simultaneously provided a fluorescent analysis to corroborate the PEC result. Table 1 Comparisons of some cTnT assays.

Method

Linear range

Detection limit

Reference

DPV

0.01–0.1 ng mL−1

0.006 ng mL−1

50

Electrochemical

0.2–1.0 ng mL−1

0.1 ng mL−1

51

＜ 50 mg mL−1

100 ng mL−1

52

Piezoelectric

0.1-0.5 ng mL−1

0.0015 ng mL−1

53

Capacitance

-

0.2 ng mL−1

54

PEC

0.1-2 pg mL−1

0.033 pg mL−1

FL

0.5 to 5 pg mL−1

0.167 pg mL−1

Surface Plasmon resonance

This work

Furthermore, the reproductivity of this liposomal PEC immunoassay was estimated by an interassay of analyzing 50 ng mL−1 sample with five electrodes. The relative standard deviation was of 6.3%, indicating a favorable reproductivity. As shown in Figure 5b, the selectivity of this work was investigated by using the IgG, C-reactive protein (CRP), p53, carcino embryonie antigen (CEA), IgA as interfering agents, and the results demonstrated that these species slightly cause the signal decrease and thus the satisfactory selectivity. Figure 6a illustrated the photocurrent response of modified electrode corresponding to 10 ng mL−1 antigen upon irradiation repeated every 10 s. The irradiation process was repeated 20 times over 400 s, and the photocurrent shows no obvious variation, indicating the stable response of the assay. Simultaneously, as 17



Page 18 of 25

shown in Figure 6b and inset, a complementary “signal-on” fluorescent assay was also performed by measuring the fluorescence intensity of the Cu NCs. The fluorescent intensity decrement was proportional to the antigen concentrations in a linear range from 0.5 to 5 pg mL−1 with the LOD of 0.17 pg mL−1. Table S2 compared this work with other reported liposome-amplified PEC immunoassays.29-31 The comparison revealed that the protocol in this work possessed a relatively high sensitivity, which was due to the outstanding signal amplification ability of the CNL system. To further assess its suitability for practical application, this protocol was applied to detect cTnT antigen in normal human serum samples, with the results presented in Table S3. The concentration of cTnT antigen was calculated to be 9.28 pg mL−1 in normal human serum samples by standard addition, and the results revealed that the developed assay was compatible with actual samples. As a proof of principle, the good performance of this protocol manifested the potential of the proposed liposomal PEC bioanalysis, future research will optimize e.g. the acidolysis condition of Cu NCs for enhanced property.

18


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


Figure 6. (a) Stability of the modified ITO electrode corresponding to 10 ng mL−1. (b) Fluorescent intensity vs. cTnT antigen concentration. Data were recorded in 10 mM PBS solution containing 0.1 M AA with applied potential of 0.0 V and excitation light of 410 nm. (∆I is the photocurrent decrement).

CONCLUSIONS In conclusion, we have presented a new protocol for sensitive liposomal PEC immunoassay using CNL and simple CdS QDs electrode. The release of numerous Cu2+ ions from the liposome complexes and the subsequent in situ formation of trapping sites upon the QDs are crucial to the operation of the system, based on which a novel and sensitive PEC immunoassay could thus be realized without any

19



Page 20 of 25

complicated enzymatic amplification. Meanwhile, a complementary “signal-on” fluorescent detection could also be accomplished by measuring the fluorescence intensity originated from the Cu NCs. This work features the first use of Cu NCs in PEC bioanalysis and also the first NCs-loaded liposomal PEC bioanalysis. Based on this simple protocol, general PEC bioanalysis can be envisioned by encapsulating specific metal NCs or other reagents (eg. Ag NCs or alloyed nanoparticles55-57) within the liposomes, corresponding to different targets of interest, and monitoring these specific species-semiconductors interactions using the corresponding electrodes. Such attractive potentials and prospects make these functional liposomes a useful addition to the armory of advanced PEC bioanalysis.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.xxxxxxx. Characteristics of Cu NCs and liposome (PDF), the stability of Cu2+ loaded liposome, the photophysics of CdS QDs before and after reaction with Cu2+, and Determination of cTnT antigen in normal human serum samples. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected] *E-mail: [email protected]

20


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


Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Financial support from the National Natural Science Foundation of China (Grant nos. 21327902 and 21675080), the Natural Science Foundation of Jiangsu Province (Grant BK20170073), and the Scientific Research Foundation of Graduate School of Nanjing University (2016CL04) is appreciated. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

REFERENCES

(1) Liu, B.; Liu, J. Anal. Methods 2017, 9, 2633-2643. (2) Taniguchi, M. Bull. Chem. Soc. Jpn. 2017, 90, 1189-1210. (3) Komiyama, M.; Yoshimoto, K.; Sisido, M.; Ariga, K. Bull. Chem. Soc. Jpn. 2017,

90, 967-1004. (4) Zhang, N.; Zhang, L.; Ruan, Y.-F.; Zhao, W.-W.; Xu, J.-J.; Chen, H.-Y. Biosens.

Bioelectron. 2017, 94, 207-218. (5) Willner, I.; Patolsky, F.; Wasserman, J. Angew. Chem. Int. Ed. 2001, 40, 1861-1864. (6) Zhao, W.-W.; Xu, J.-J.; Chen, H.-Y. Chem. Soc. Rev. 2015, 44, 729-741. (7) Zhao, W.-W.; Xu, J.-J.; Chen, H.-Y. Chem. Rev. 2014, 114, 7421-7441. (8) Zhao, W.-W.; Xu, J.-J.; Chen, H.-Y. TrAC, Trends Anal. Chem. 2016, 82, 307-315. (9) Haddour, N.; Chauvin, J.; Gondran, C.; Cosnier, S. J. Am. Chem. Soc. 2006, 128,

21



Page 22 of 25

9693-9698. (10) Stoll, C.; Kudera, S.; Parak, W. J.; Lisdat, F. Small 2006, 2, 741-743. (11) Zhao, W.-W.; Yu, X.-D.; Xu, J.-J.; Chen, H.-Y. Nanoscale 2016, 8, 17407-17414. (12) Chen, K.; Liu, M.; Zhao, G.; Shi, H.; Fan, L.; Zhao, S. Environ. Sci. Technol. 2012, 46, 11955-11961. (13) Ma, W.-G.; Wang, L.-N.; Zhang, N.; Han, D.-X.; Dong, X.-D.; Niu, L. Anal.

Chem. 2015, 87, 4844-4850. (14) Long, Y.-T.; Kong, C.; Li, D.-W.; Li, Y.; Chowdhury, S.; Tian, H. Small 2011, 7, 1624-1628. (15) Zhao, W.-W.; Xu, J.-J.; Chen, H.-Y. Anal. Chem. 2018, 90, 615-627. (16) Li, H.-N.; Mu, Y.-W.; Yan, J.-R.; Cui, D.-M.; Ou, W.-J.; Wan, Y.-K.; Liu, S.-Q.

Anal. Chem. 2015, 87, 2007-2015. (17) Li, C.; Wang, H.; Shen, J.; Tang, B. Anal. Chem. 2015, 87, 4283-4291. (18) Li, X.; Zhu, L.-S.; Zhou, Y.-L.; Yin, H.-S.; Ai, S.-Y. Anal. Chem. 2017, 89, 2369-2376. (19) Kang, Z.; Yan, X.; Wang, Y.; Zhao, Y.; Bai, Z.; Liu, Y.; Zhao, K.; Cao, S.; Zhang, Y. Nano Res. 2016, 9, 344-352. (20) Devadoss, A.; Kuragano, A.; Terashima, C.; Sudhagar, P.; Nakata, K.; Kondo, T.; Yuasa, M.; Fujishima, A. J. Mater. Chem. B 2016, 4, 220-228. (21) Li, J.; Zhang, Y.; Kuang, X.; Wang, Z.; Wei, Q. Biosens. Bioelectron. 2016, 85, 764-770. (22) Hao, N.; Zhang, Y.; Zhong, H.; Zhou, Z.; Hua, R.; Qian, J.; Liu, Q.; Li, H.; Wang,

22


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


K. Anal. Chem. 2017, 89, 10133-10136. (23) Zheng, Y. N.; Liang, W. B.; Xiong, C. Y.; Yuan, Y. L.; Chai, Y. Q.; Yuan, R. Anal.

Chem. 2016, 88, 8698-8705. (24) Yan, Z.; Wang, Z.; Miao, Z.; Liu, Y. Anal. Chem. 2016, 88, 922-929. (25) Zhang, G.-Y.; Zhuang, Y.-H.; Shan, D.; Su, G.-F.; Cosnier, S.; Zhang, X.-J. Anal.

Chem. 2016, 88, 11207-11212. (26) Al-Jamal, W. T.; Kostarelos, K. Acc. Chem. Res. 2011, 44, 1094-1104. (27) Marta, B.; Janos, V. Nanomedicine 2009, 4, 447-467. (28) Qian, R.-C.; Cao, Y.; Long, Y.-T. Angew. Chem. 2016, 128, 729-733. (29) Mei, L.-P.; Liu, F.; Pan, J.-B.; Zhao, W.-W.; Xu, J.-J.; Chen, H.-Y. Anal. Chem. 2017, 89, 6300-6304. (30) Zhuang, J.; Han, B.; Liu, W.; Zhou, J.; Liu, K.; Yang, D.; Tang, D. Biosens.

Bioelectron. 2018, 99, 230-236. (31) Lin, Y.; Zhou, Q.; Tang, D. Anal. Chem. 2017, 89, 11803-11810. (32) Zhang, L.; Wang, E. Nano Today 2014, 9, 132-157. (33) Wilcoxon, J. P.; Abrams, B. L. Chem. Soc. Rev. 2006, 35, 1162-1194. (34) Wang, C.; Wang, C.; Xu, L.; Cheng, H.; Lin, Q.; Zhang, C. Nanoscale 2014, 6, 1775-1781. (35) Shang, L.; Dong, S.; Nienhaus, G. U. Nano Today 2011, 6, 401-418. (36) Zhang, J.; Tu, L.; Zhao, S.; Liu, G.; Wang, Y.; Wang, Y.; Yue, Z. Biosens.

Bioelectron. 2015, 67, 296-302. (37) Zhang, L.; Sun, Y.; Liang, Y.-Y.; He, J.-P.; Zhao, W.-W.; Xu, J.-J.; Chen, H.-Y.

23



Page 24 of 25

Biosens. Bioelectron. 2016, 85, 930-934. (38) Lin, B.-Q.; Liu, D.; Yan, J.-M.; Qiao, Z.; Zhong, Y.-X.; Yan, J.-W.; Zhu, Z.; Ji, T.-H.; Yang, C.-Y. J. ACS Appl. Mat. Interfaces 2016, 8, 6890-6897. (39) Wang, G.-L.; Xu, J.-J.; Chen, H.-Y. Nanoscale 2010, 2, 1112-1114. (40) Tang, J.; Li, J.; Zhang, Y.; Kong, B.; Yiliguma; Wang, Y.; Quan, Y.; Cheng, H.; Al-Enizi, A. M.; Gong, X.; Zheng, G. Anal. Chem. 2015, 87, 6703-6708. (41) Zhang, N.; Ma, Z.-Y.; Ruan, Y.-F.; Zhao, W.-W.; Xu, J.-J.; Chen, H.-Y. Anal.

Chem. 2016, 88, 1990-1994. (42) Jia, X.; Li, J.; Wang, E. Small 2013, 9, 3873-3879. (43) Singh, A. K.; Kilpatrick, P. K.; Carbonell, R. G. Biotechnol. Progr. 1996, 12, 272-280. (44) Zavari-Nematabad, A.; Alizadeh-Ghodsi, M.; Hamishehkar, H.; Alipour, E.; Pilehvar-Soltanahmadi, Y.; Zarghami, N. Anal. Bioanal.Chem. 2017, 409, 1301-1310. (45) Wang, P.; Lei, J.; Su, M.; Liu, Y.; Hao, Q.; Ju, H. Anal. Chem. 2013, 85, 8735-8740. (46) Wang, P.; Ma, X.; Su, M.; Hao, Q.; Lei, J.; Ju, H. Chem. Commun. 2012, 48, 10216-10218. (47) Shen, Q.; Zhao, X.; Zhou, S.; Hou, W.; Zhu, J.-J. J. Phys. Chem. C 2011, 115, 17958-17964. (48) Vegelius, J. R.; Kvashnina, K. O.; Hollmark, H.; Klintenberg, M.; Kvashnin, Y. O.; Soroka, I. L.; Werme, L.; Butorin, S. M. J. Phys. Chem. C 2012, 116, 22293-22300

24


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


(49) Brown Bourzutschky, J. A.; Homs, N.; Bell, A. T. J. Catal. 1990, 124, 52-72. (50) Silva, B. V.; Rodriguez, B. A.; Sales, G. F.; Sotomayor Mdel, P.; Dutra, R. F. Biosens. Bioelectron. 2016, 77, 978-985. (51) Brondani, D.; Piovesan, J. V.; Westphal, E.; Gallardo, H.; Fireman Dutra, R. A.; Spinelli, A.; Vieira, I. C. Analyst 2014, 139, 5200-5208. (52) Liu, J. T.; Chen, C. J.; Ikoma, T.; Yoshioka, T.; Cross, J. S.; Chang, S.-J.; Tsai, J.-Z.; Tanaka, J. Anal. Chim. Acta 2011, 703, 80-86. (53) Fonseca, R. A. S.; Ramos-Jesus, J.; Kubota, L. T.; Dutra, R. F. Sensors 2011, 11, 110785-110797. (54) Bhalla, V.; Carrara, S.; Sharma, P.; Nangia, Y.; Raman Suri, C. Sens. Actuators, B 2012, 161, 761-768. (55) Wang, N.; Han, Y.; Xu, Y.; Gao, C.; Cao, X. Anal. Chem. 2015, 87, 457-463. (56) Wang, N.; Gao, C.; Han, Y.; Huang, X.; Xu, Y.; Cao, X. J. Mater. Chem. B 2015, 3, 3254-3259. (57) Cao, X.; Wang, N.; Jia, S.; Shao, Y. Anal. Chem. 2013, 85, 5040-5046. for TOC only

25


Cu Nanoclusters-Encapsulated Liposomes: Toward Sensitive

Recommend Documents