A Novel Carbon Quantum Dots Signal Amplification Strategy Coupled

Subscriber access provided by AUT Library

Article

A Novel Carbon Quantum Dots Signal Amplification Strategy Coupled with Sandwich Electrochemiluminescence Immunosensor for the Detection of CA15-3 in Human Serum Dongmiao Qin, Xiaohua Jiang, Guichun Mo, Jinsu Feng, Chunhe Yu, and Biyang Deng ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01607 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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

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

Page 1 of 26 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

ACS Sensors

A Novel Carbon Quantum Dots Signal Amplification Strategy Coupled with Sandwich Electrochemiluminescence Immunosensor for the Detection of CA15-3 in Human Serum Dongmiao Qin, Xiaohua Jiang, Guichun Mo, Jinsu Feng, Chunhe Yu, Biyang Deng* Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004 China

*Corresponding Author: Telephone: +86-773-5845726; Fax: +86-773-2120958. Email: [email protected]

1

ACS Paragon Plus Environment

ACS Sensors 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 26

ABSTRACT: A sensitive sandwich electrochemiluminescence immunosensor was established by employing graphene oxide-PEI-carbon quantum dots (CQDs)-Au nanohybrid as probe to measure carbohydrate antigen 15-3 (CA15-3), a breast cancer biomarker. In this work, the nanocomposites of Ag nanoparticles and polydopamine (AgNPs-PDA) were synthesized by redox reaction between dopamine and Ag+. The nanocomposite with high surface area can provide an efficient substrate for immobilizing initial antibody (Ab1). Carbon quantum dots (CQDs) are fixed on polyethylenimine-functionalized graphene oxide (PEI-GO) by amide bonds. Au nanoparticles are modified on CQDs-decorated PEI-GO substrates. The secondary antibody (Ab2) was immobilized by AuNPs/CQDs-PEI-GO composite. CQDs can be assembled onto the surface of electrode by an incorporation of CA15-3 with Ab1 and Ab2. Under the synergistic action of AgNPs, polydopamine, AuNPs, and PEI-GO, the ECL signal of CQDs is greatly amplified as an excellent conductive material to facilitate electrons transfer rate and further increase electrochemical detection capability. Under optimal conditions, the fabricated immunosensor showed a linear concentration range from0.005 to500 U mL-1, with a detection limit of 0.0017 U mL-1 (signal-to-noise ratio of 3) for CA15-3. The designed ECL immunosensor displayed receivable accuracy, excellent stability, and high specificity. The results of the detection of human serum samples are satisfactory, revealing that the method offers a potential application for the clinical diagnosis of tumor markers. KEYWORDS: Electrochemiluminescence immunosensor, Carbohydrate antigen 15-3, CQDs, Graphene oxide, Serum

2


Page 3 of 26 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

ACS Sensors

Cancer protein biomarkers are particularly associated with oncologic diseases, and their levels in body tissues or physiological fluids can offer information worthy for clinical screening programs, and as well as early cancer detection, with distinct superiorities in terms of costs for societies and healthcare system.1,2 Susceptive and credible technology for the determination of disease biomarkers plays a significant role in early diagnosis, clinical cancer screening, cancer grading, new drug development and molecularly targeted therapy expansion.3-7 Breast cancer is the most widespread life-threatening cancer among women worldwide.7-9 It is an intricate and diversified disease that may be composed of several subtypes with different morphological and clinical implications.10,11 One of the most important biomarkers of breast cancer is the carbohydrate antigen 15-3 (CA15-3).12,13 The concentration of CA15-3 in blood serums could afford information from monitoring of therapy and it can also predict breast cancer recurrence after curative surgery.7,12 Therefore , it is important to develop a dependable analytical method for accurate and selective detection of low concentration levels of CA15-3. So far, various analytical methods for monitoring CA15-3 have been developed, including chemiluminescence immunoassay (ECL),14 electrochemistry (EC),15 enzyme-linked immunosorbent assay (ELISA),16 enzyme immunoassay (EIA),13 radioimmunoassay (RIA),17 optical immunoassay.18 However, all of these conventional immunoassays demand complex operations, costly instruments and long analysis times.7 Therefore, from a health perspective, it is essential to develop an application for testing CA15-3. Electrochemiluminescence method has the characteristics of speediness, sensitivity, easy operation and low cost because electrochemical instrumentation is currently succinct and impressible.19,20 ECL immunosensor is the combination of the ECL and the specific antibody–antigen recognition technology.21 The determination theory is based on antigen-antibody immune recognition of ECL response changes.21,22 So far, great concern has been devoted to ECL research due to distinct advantages of high sensitivity, excellent specificity, rapid response and wide application.22-24 The development of selective and high sensitive methods has turned into one of the most fascinating fields in electrochemistry.25,26 Nanomaterials can be used as biomolecule immobilization matrices and signal markers in ECL immunosensors. Lately, carbon quantum dots have turned into a popular topic in carbon nanomaterials study because of their peculiar tunable optical properties, good light stability, large surface area and high conductivity, as well as good biocompatibility and low cytotoxicity.27,28 In addition, there are a large number of carboxyl groups on its surface, which can be readily marked for electrochemical detection.29,30 Graphene oxide (GO), a kind of two dimensional single-atom-thick nanomaterial rooted in the oxidation 3


ACS Sensors 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 4 of 26

of graphene has received tremendous attention these years.31,32 Due to its outstanding characteristics such as good mechanical properties, good electrical conductivity, large surface area, readily to be functionalized, favorable biocompatibility and fluorescence quenching ability, GO has appealed to comprehensive interest in the preparation of biosensor.33,34 Polyethylenimine (PEI)-GO was synthesized in this paper. The presence of PEI on GO not only provides good solubility, but also offers amino group, which is advantageous for the immobilization of CQDs and AuNPs.30 AuNPs have many advantages including good biocompatibility, quantum size effect and easy coupling to antibodies or antigens, hence, it was chosen as the linkage between GO-PEI-CQDs and antibodies.35 Nanocomplexes of Ag and polydopamine nanoparticles (AgNPs-PDA) were used as immobilized antibody (Ab1), and secondary antibody (Ab2) were immobilized with PEI-GO complexes modified with AuNPs and CQDs. The ECL of CQDs can be obtained by combining CA15-3 with Ab1 and Ab2. Under the synergistic action of AgNPs, polydopamine, AuNPs and PEI-GO, the ECL signal of CQDs was greatly enhanced, and a susceptive sandwich ECL immunosensor was established for the determination of CA15-3 (Scheme 1). Scheme 1

EXPERIMENTAL SECTION Reagents and Chemicals. The antigen and antibody of cancer antigen 15-3 (CA15-3), carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) were come from Wanger Biotechnology Co., Ltd. (Beijing, China). GO was bought from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). HAuCl4 · 3H2O, tris(hydroxymethyl)aminomethane (Tris), bovine serum albumin (BSA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were supplied by Sigma–Aldrich. L-histidine, poly(ethylenimine) (PEI) and dopamine hydrochloride (DA · HCl), Glucose (Glc), Na2HPO4 and KH2PO4 were obtained from Xilong Chemical Co., Ltd. (Guangdong, China). All reagents and chemicals were of analytical grade and were not further purified for use. Ultra-pure water was employed throughout. Human serum samples were taken from Guilin Fifth People’s Hospital. Apparatus. An MPI-B ECL analyzer (Xi’an Remex Electronic Science-Tech Co., Ltd., Xi’an, China) was used to record the ECL responses. A voltage of 800 V was supplied to the photomultiplier tuber (PMT). A platinum wire, an Ag/AgCl (saturated KCl) electrode and a modified GCE electrode were used as counter electrode,

reference electrode and working electrode, respectively. A CHI660E electrochemical 4


Page 5 of 26 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

ACS Sensors

workstation (Shanghai Chenhua Instruments, Shanghai, China) was used to measure electrochemical impedance spectroscopy (EIS). The nanomaterial morphology was characterized using transmission electron microscope (TEM) of a Tecnai G2 F20 S-TWIN (FEI, USA) at 200 kV. The

Fourier transform infrared

(FT-IR) spectrophotometer (Perkin-Elmer, USA) was used to record the

FT-IR spectra. The

Ultraviolet-visible (UV–vis) absorption spectrum was gained from a Cary 60 UV–vis spectrophotometer (Agilent Technologies, USA). The RF-5301 fluorescence spectrometer (Shimadzu, Japan) was used to record the fluorescence spectra. Dialysis bags (U.S. Union Carbide Corp., molecular weight cut off 500 Da). Synthesis of CQDs. 0.7044 g L-histidine and 2 g citric acid were mixed with ultra-pure water (20 mL) via ultrasonic dissolution for 10 min. The mixture solution was heated at 180 °C for 4 h in a Teflon-lined autoclave. After the reaction was finished, the reactor was cooled to room temperature via air. The brown-yellow supernatant was collected after removing the big CQDs via centrifugation at 12,000 rpm for 30 min and was dialyzed for two days in a dialysis bag with a molecular weight cut off of 500 Da. The dialyzed solution was changed several times to obtain pure CQDs. The purified CQDs were preserved at 4 °C for later analysis and further use. Synthesis of PEI Functionalized GO. PEI-GO complexes were synthesized on the grounds of the literature with some modifications.24 15 mg GO were added into 50 mL ultrapure water. After the solution was sonicated for 10 h, 5 mg PEI were added into the solution. The mixture was stirred for 24 h. Next, the PEI-GO solution was centrifuged and dried at in an oven. Later, the PEI-GO thus obtained was re-dispersed in ultrapure water until further use. Synthesis of AuNPs/CQDs-PEI-GO Nanocomposite. The CQDs-PEI-GO nanocomposite was synthesized by adding 25 mL CQDs into PEI-GO solution and stirring at 80 °C for 3 h. The acquired CQDs-PEI-GO was separated by centrifugation, and then dispersed in ultrapure water. 200 μL of NHS and EDC (4:1) solution was mixed with 10 mL CQDs-PEI-GO solution via ultrasound for 30 min, then added 6 mL AuNPs and sonicated the mixture solution for 6 h. The obtained AuNPs/CQDs-PEI-GO solution was centrifuged and afterwards dispersed in PBS (pH 7.4). 29,30 Synthesis of Ab2-AuNPs/CQDs-PEI-GO Nanohybrids. The 25 μL mixture solution of NHS and EDC (4:1) were mixed with 1 mL of AuNPs/CQDs-PEI-GO solution. The mixture solution was sonicated for 5 min. Afterwards, 50 μL of 100 μg mL-1 Ab2 was added and sonicated for 5 minutes. Lastly, adding 10 μL BSA (0.5 wt%) to the solution eliminate nonspecific active sites. The solution was reacted at 4 °C for 12 h. AuNPs/CQDs-PEI-GO–anti-CA15-3 solution was centrifuged and re-dispersed in PBS at 4 °C for later use 5


ACS Sensors 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 6 of 26

(Scheme 1a). Fabrication of the Immunosensor and ECL Detection. The fabrication procedure of the ECL immunosensor was shown in Scheme 1. First, the bare GCE was polished using 0.3 and 0.05 µm Al2O3 powders, cleaned with ethanol and ultrapure water, and dried with nitrogen. And then, 4 μL of 0.1 mg mL-1 DA containing 10 mmol L-1 Tris (pH 8.5) and 4 μL of 2 mg mL-1 AgNO3 were dropped onto GCE surface, and then dried at room temperature to form PDA-AgNPs/GCE. Subsequently, 6 μL of a primary anti-CA15-3 (Ab1) (50 μg mL-1) dropped onto the PDA-AgNPs/GCE electrode at 4 °C for 12 h to obtain Ab1/PDA-AgNPs/GCE. After rinsing with PBS (pH 7.4) to unbind the physically adsorbed Ab1, 4 μL BSA (0.5 wt%) dropped onto the modified electrode and incubated at 37 °C for 40 min to block the nonspecific coupling sites (BSA/Ab1/PDA-AgNPs/GCE). Next, the prepared electrode was immersed in different concentrations of CA15-3 at 37 °C for 40 min to capture CA15-3 (CA15-3/BSA/Ab1/PDA-AgNPs/GCE). Last, the sandwich-type immunosensor was constructed by incubating the electrode with 6 μL CA15-3-Ab2/CQDs-PEI-GO/AuNPs solution at 37 °C for 1 h (Scheme 1b). The acquired immunosensor was stored at 4 °C for following use. The ECL measurements of immunosensor were obtained in 1 mL PBS (pH 7.4) including 0.1 mol L-1 K2S2O8 and 0.1 mol L-1 KCl with the potential ranging from -1.8 to -0.2 V at 100 mV s-1 scan rate. The PMT voltage was set on 800 V. The synthesis of Au nanoparticles and quantum yield measurement were displayed in the Supporting Information.

RESULTS AND DISCUSSION Characterization of the Nanocomposite. The morphologies and sizes of AuNPs, CQDs, GO, and AuNPs/CQDs-PEI-GO were characterized using TEM. It can be observed the AuNPs are heterogeneous spherical shapes with approximately 30.84 nm diameter (Figure 1a). The prepared CQDs showed approximately a monodispersed spherical morphology (Figure 1b), and the statistical particle size distribution range of 5-10 nm with 7 nm average diameter. Graphene oxide is an irregular two-dimensional pleat shape (Figure 1c). The AuNPs/CQDs-PEI-GO morphology was revealed in Figure 1d. It can be observed that different size particles were decorated on the surface. The image of GO (Figure 1c) revealed the PEI functionalized GO was gained and the different size particles proved CQDs and AuNPs were successfully loaded on the PEI-GO surface. FT-IR spectroscopy was used to further study the surface functions of CQDs, AuNPs, GO, GO-PEI, GO-PEI-CQDs and GO-PEI-CQDS-AuNPs. The detailed 6


Page 7 of 26 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

ACS Sensors

explanation of FT-IR spectra was shown in the Supporting Information (Figure S1). Figure 1 UV absorption spectroscopy was performed for demonstrating the successful synthesis of AuNPs and CQDs. According to Figure 1e, the UV-visible absorption spectrum of GO was found at 233 nm, which belonged to the π-π* transition of the benzenoid rings.29,36 Whereas AuNPs at 533 nm has a strong absorption peak, which is its characteristic peak. The synthesis of CQDs revealed a characteristic absorption peak of 308 nm, which was ascribed to the aromatic sp2 structural domain transition.37 The fluorescent characteristics of the prepared CQDs were investigated. The excitation and emission spectra of the CQDs were shown in Figure 2a. There are two excitation peaks emerged about at 238 and 330 nm, respectively, and the emission peaks at 418 nm. Figure 2b shows the emission spectrum of CQDs at different excitation wavelengths. When the excitation wavelength is increased from 300 to 370 nm, the fluorescence of CQDs increases first and then decreases and the fluorescence peak position presents obvious red shift, from the original peak position of fluorescence spectrum at 418 nm and red shift to 435 nm.38 This red shift phenomenon is not only related to the uneven size distribution of the CQDs but also the surface emission points or defects caused by the surface passivation of the CQDs are different. The results are consistent with previous reports.39 Therefore, the carbon quantum dots prepared by hydrothermal method may cause the uneven distribution of particle size and emission site. The quantum yield of the synthesized CQDs was 27.25%. Figure 2 Characterization of ECL Immunosensor. Figure 3a showed ECL-potential curves of CQDs/GCE at scan rate from -1.8 to -0.2 V in 0.1 mol L-1 PBS (pH 7.4) including 0.1 mol L-1 K2S2O8. The optimal ECL signal arose around -1.2 V. Figure 3b appeared a good ECL repeatability of CQDs/GCE under consecutive 14 CV scan cycles. In accordance with the reported literatures,40,41 the possible ECL emission mechanism is described as below : CQDs + e- → CQDs-∙

(1)

S2O82 - + e- → S2O83-∙

(2)

S2O83-∙ → SO42- + SO4-∙

(3)

CQDs-∙ + SO4-∙ → CQDs* + SO42-

(4)

CQDs* → CQDs + hν

(5)

Figure 3 7


ACS Sensors 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 8 of 26

As shown in Figure 4a to demonstrate the successful fabrication of immunosensor, cyclic voltammetry (CV) was performed. The bare GCE has a pair of [Fe(CN)6]3-/[Fe(CN)6]4- reversible redox peak

(Figure

4a, curve a) after adding PDA-AgNPs nanomaterials to the bare GCE.42 The oxidation and reduction peak currents decrease (Figure 4a, curve b). This may be due to the presence of PDA that weaken the electrical conductivity of the composite and decelerated the electron transfer rate between the probe and the PDA-AgNPs nanocomposite modified electrode.43 The redox currents decreased after continuing modifying Ab1 (Figure 4a, curve c), BSA (Figure 4a, curve d) and CA15-3 (Figure 4a, curve e). This is because of the hindrance effect of protein layer on electronic transmission. When the AuNPs-Ab2 is added (Figure 4a, curve f), the peak current of redox increased because of AuNPs increasing the conductivity. These facts indicated that bioactive substances are effectively captured on the electrode. The EIS was conducted to control the assembly procedure of the ECL immunosensor by using 5 mmol L-1 Fe(CN)63-/4- solution as the electrochemical probe. As seen in Figure 4b, when coated with PDA-AgNPs (curve b), the electrode revealed increased electron transfer resistance relative to that of bare GCE (curve a) due to two factors of PDA-AgNPs: (1) PDA is an organic semiconductor that can prominently impede the electron transfer from the redox probe of [Fe(CN)6]3-/4- to the electrode surface.43 (2) The repulsion between negatively charged AgNPs and [Fe(CN)6]3-/4- will result in high impedance. When Ab1, BSA and CA15-3 (curve c, d and e) bioconjugates were successively assembled onto the electrode, the electron transfer resistance increased distinctly, indicating the successful modification of insulated protein on the electrode surface. Finally, when the electrode coated with Ab2, a much lower impedance was obtained for AuNPs (curve f), resulting in an enhancement in the electron transfer kinetics of [Fe(CN)6]3-/4-. As described in curves e and f, AuNPs contributed similarly to improve the electron transfer efficiency. By and large, CV and EIS measurements all described the successful construction of the immunosensor. Figure 4 The ECL responses of different nanocomposites employed as ECL probes were revealed in Figure 5. When the AuNPs composite directly interacted with CA15-3-Ab2 to assemble the immunosensor, a weak ECL signal was obtained (curve a). In contrast, when the CQDs-AuNPs nanocomposite was employed as the ECL probe, the ECL intensity notably increased because the addition of CQDs can promote the ECL (curve b). Next, when the PEI-CQDs-AuNPs composite was used as ECL probe, the ECL intensity increased more than CQDs-AuNPs nanocomposite because the addition of PEI can improve the interaction of CQDs with AuNPs (curve c). Furthermore, the ECL signal further enhanced when the GO-PEI-CQDs-AuNPs nanocomposite was used (curve d). This was because the PEI-GO was used as a carrier for accommodating 8


Page 9 of 26 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

ACS Sensors

more CQDs nanoparticles and AuNPs. Hence, more ECL probes could be assembled on the electrode surface via the interaction between antigen and antibody to heighten the ECL signal. The results revealed that the GO-PEI-CQDs-AuNPs nanocomposite could be employed as a probe to fabricate an excellent ECL immnosensor. The optimization of the experimental conditions and effect of BSA on ECL intensity were described in the Supporting Information. The results were illustrated in Figure S2 and Figure S3. Figure 5 Detection of CA15-3. The ECL immunosensor was applied for detecting CA15-3 concentration under the optimal conditions. As exhibited in Figure 6a, ECL intensity as the CA15-3 concentrations within 0.005 to 500 U mL-1 is linear. The linear regression equation was y = 3753 + 928.4 lgC (CA15-3) (Figure 6b). The correlation coefficient was 0.9909 with detection limit of 0.0017 U mL-1 (S/N = 3). The ECL immunosensor can be used to detect CA15-3 quantitatively. Furthermore, as shown in Table S1, the detection limit of CA15-3 based on sandwich ECL immunosensor is lower than the reported methods and the linear range was wider than the reported methods. A detailed comparison was shown in Table S1. Figure 6 Stability, Selectivity and Reproducibility of the Sensor. To research the ECL analytical performance of the proposed sensor for CA15-3 detection, we devoted to study the stability, selectivity and reproducibility under above mentioned conditions. Therefore, stability assay was carried out at the presence of 100 U mL-1 CA15-3. The continuous cyclic voltammetry curves of 11 times were shown in Figure 6c using the immunosensor. The ECL signal nearly maintained a constant value during consecutive cyclic voltammetry scanning, indicating that the ECL emission of immunosensor was highly repeatable for analytical application. When the immunosensor was stored at 4 °C for one month, the ECL signal of CA15-3 sensor was decreased by 10.4%. In brief, the immunosensor was possessed of good stability. Significantly, the well-prepared ECL immunosensor exerted outstanding selectivity and specificity to different interference substances containing AFP, CEA, glucose, and human serum albumin (Figure 6d). What's more, the PBS solution alone was exploited as contrast. Hence, the results of interference experiment showed that the prepared ECL sensor manifested specific response to the testing samples including CA15-3, revealing that the fabricated immunosensor had receivable selectivity and specificity. The ECL immunosensor reproducibility was assessed by determining three different concentrations of CA15-3 using four electrodes under the same conditions (Figure 6e). The RSD of the prepared ECL sensor 9


ACS Sensors 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 10 of 26

is lower than 2.3%, verifying an excellent reproducibility in the aspect of measuring CA15-3 for clinical analysis. Real Sample Analysis. To further verify the practicability and reliability of the immunosensor at application to detecting real samples, under the optimum experiment conditions, we measured the samples collected from Guilin fifth People's Hospital (aged 39, 40, and 41; female). The human serum samples were collected using standard reclaiming test.44 The experimental results were seen in Table 1. The relative standard deviation of CA15-3 was between 1.05% and 1.80%, and the recovery of CA15-3 was between 100.8% and 101.3%. Table 1

CONCLUSIONS In this work, a new method for ultrasensitive sandwich ECL immunosensor of CA15-3 based on CQDs was developed. The linear range was from 0.005 to 500 U mL-1 CA15-3 with detection limit of 0.0017 U mL-1. Meanwhile, the immunosensor has been triumphantly employed for the detection of CA15-3 in human serum. The results showed that the prepared ECL immunesensor revealed good stability, high specificity and receivable repeatability. Therefore, the immunosensor offers promising potential for clinical diagnosis of CA15-3.

ASSOCIATED CONTENT Supporting Information Supporting Information is available free of charge on the ACS Publications website at DOI. Synthesis of Au nanoparticles, quantum yield measurement, FT-IR spectra characterization, optimization of the proposed ECL immunosensor, effect of BSA on ECL intensity and comparison of different methods for CA15-3 determination (PDF)

AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Telephone: +86-773-5845726; Fax: +86-773-2120958. Orcid Biyang Deng: 0000-0001-8083-589X 10


Page 11 of 26 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

ACS Sensors

Notes

The authors declare no competing financial interest

ACKNOWLEDGEMENTS We acknowledge the support from the National Natural Science Foundation of China (grant numbers 21765004

and

21365006),

the

Guangxi

Science

Foundation

of

China

(grant

numbers

2014GXNSFDA118004 and 1598025-4), the Innovation Project of Guangxi Graduate Education (YCSZ2013039), and the research fund of State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University) (CMEMR2017-A5).

REFERENCES (1) Giannetto, M.; Vittoria Bianchi, M.; Mattarozzi, M.; Careri, M. Competitive Amperometric Immunosensor for Determination of p53 Protein in Urine with Carbon Nanotubes/Gold Nanoparticles Screen Printed Electrodes: A Potential Rapid and Noninvasive Screening Tool for early Diagnosis of Urinary Tract Carcinoma. Anal. Chim. Acta 2017, 991, 133-141. (2) Tang, C. K., Vaze, A., Shen, M., Rusling, J. F. High-Throughput Electrochemical Microfluidic Immunoarray for Multiplexed Detection of Cancer Biomarker Proteins. ACS Sens. 2016, 1, 1036−1043. (3) Singh, V., Krishnan, S. Electrochemical and Surface Plasmon Insulin Assays on Clinical Samples. Analyst 2018, 143, 1544–1555. (4) Zhang, J.; Xiong, Z.; Chen, Z. Ultrasensitive Electrochemical Microcystin-LR Immunosensor Using Gold Nanoparticle Functional Polypyrrole Microsphere Catalyzed Silver Deposition for Signal Amplification. Sens. Actuators B 2017, 246, 623–630. (5) Mazloum-Ardakani, M.; Hosseinzadeh, L.; Taleat, Z. Synthesis and Electrocatalytic Effect of Ag@Pt Core–Shell Nanoparticles Supported on Reduced Graphene Oxide for Sensitive and Simple Label-Free Electrochemical Aptasensor. Biosens. Bioelectron. 2015, 74, 30–36. (6) Mazloum-Ardakani, M.; Hosseinzadeh, L. Highly-Sensitive Label-Free Immunosensor for Tumor Necrosis Factor Based on Ag@Pt Core–Shell Nanoparticles Supported on MWCNTs as an Efficient Electrocatalyst Nanocomposite. RSC Adv. 2015, 5, 70781–70786. (7) Khoshroo, A.; Mazloum-Ardakani, M.; Forat-Yazdi, M. Enhanced Performance of Label-Free Electrochemical Immunosensor for Carbohydrate Antigen 15-3 based on Catalytic Activity of Cobalt Sulfide/Graphene Nanocomposite. Sens. Actuators B 2018, 255, 580-587. 11


ACS Sensors 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 12 of 26

(8) Mittal, S.; Kaur, H.; Gautam, N.; Mantha, A. K. Biosensors for Breast Cancer Diagnosis: A Review of Bioreceptors, Biotransducers and Signal Amplification Strategies. Biosens. Bioelectron. 2017, 88, 217– 231. (9) Rheinbay, E.; Parasuraman, P.; Grimsby, J.; Tiao, G.; Engreitz, J. M.; Kim, J.; Lawrence, M. S.; Taylor-Weiner, A.; Rodriguez-Cuevas, S.; Rosenberg, M.; Hess,

J.; Stewart, C.; Stojanov, Y. E. P.; Cortes,

M. L.; Seepo, S.; Cibulskis, C.; Tracy, A.; Pugh, T. J.; Lee, J.; Zheng, Z.; Ellisen, L. M.; Iafrate, A. J.; Boehm, J. S.; Gabriel, S. B.; Meyerson, M.; Golub, T. R.; Baselga, J.; Hidalgo-Miranda, A.; Shioda, T.; Lander, A. E. S.; Getz, G. Recurrent and Functional Regulatory Mutations in Breast Cancer. Nature 2017, 547, 55–60. (10) Uliana, C.V.; Peverari, C. R.; Afonso, A. S.; Cominetti, M. R.; Faria, R. C. Fully Disposable Microfluidic Electrochemical Device for Detection of Estrogen Receptor Alpha Breast Cancer Biomarker. Biosens. Bioelectron. 2018, 99, 156-162. (11) Mohammadi, S.; Salimi, A.; Hamde-Qaddareh, S. Amplified FRET based CA15-3 Immunosensor Using Antibody Functionalized Luminescent Carbon-Dots and AuNPs-Dendrimer Aptamer as Donor-Acceptor. Anal. Biochem. 2018, 557, 18-26. (12) Slamon, D. J. Human Breast Cancer: Correlation of Relapse and Survival with Amplification of the HER-2/neu Oncogene. Science 1987, 235, 177–182. (13) Müller, V.; Stahmann, N.; Riethdorf, S.; Rau, T.; Zabel, T.; Goetz, A.; Jänicke, F.; Pantel, K. Circulating Tumor Cells in Breast Cancer: Correlation to Bone Marrow Micrometastases, Heterogeneous Response to Systemic Therapy and Low Proliferative Activity. Clin. Cancer Res. 2005, 11, 3678–3685. (14) Liu, A.; Zhao, F.; Zhao, Y.; Shangguan, L.; Liu, S. A Portable Chemiluminescence Imaging Immunoassay for Simultaneous Detection of Different Isoforms of Prostate Specific Antigen in Serum. Biosens. Bioelectron. 2016, 81, 97–102. (15) Li, H.; He, J.; Li, S.; Anthony, P. F. Turner Electrochemical Immunosensor with N-doped Graphene-Modified Electrode for Label-Free Detection of the Breast Cancer Biomarker CA 15-3. Biosens. Bioelectron. 2013, 43, 25-29. (16) Li, W.; Yuan, R.; Chai, Y.; Chen, S. Reagentless Amperometric Cancer Antigen 15-3 Immunosensor Based on Enzyme-Mediated Direct Electrochemistry. Biosens. Bioelectron. 2010, 25, 2548–2552. (17) Zhang, X.; Peng, X.; Jin, W. Scanning Electrochemical Microscopy with Enzyme Immunoassay of the Cancer-Related Antigen CA15-3. Anal. Chim. Acta 2006, 558, 10-114. (18) Zhang,

L.;

He,

Y.;

Wang,

H.;

Yuan,

Y.;

Yuan,

12


R.;

Chai,

Y.

A

Self-Enhanced

Page 13 of 26 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

ACS Sensors

Electrochemiluminescence Immunosensor Based on l-Lys-Ru Functionalized Porous Six Arises Column Nanorods for Detection of CA15-3. Biosens. Bioelectron. 2015, 74, 924–930. (19) Zhou, L.; Huang, J.; Yu, B.; Liu, Y.; You, T. A Novel Electrochemiluminescence Immunosensor for the Analysis of HIV-1 p24 Antigen Based on P-RGO@Au@Ru-SiO2 Composite. ACS Appl. Mater. Interfaces 2015, 7, 24438-24445. (20) Shi, L.; Li, X.; Zhu, W.; Wang, Y.; Du, B.; Cao, W.; Wei, Q.; Pang, X. Sandwich-Type Electrochemiluminescence Sensor for Detection of NT-proBNP by Using High Efficiency Quench Strategy of Fe3O4@PDA toward Ru(bpy)32+ Coordinated with Silver Oxalate. ACS Sens. 2017, 2, 1774-1778. (21) Zhang, X.; Ke, H.; Wang, Z.; Guo, W.; Zhang, A.; Huang, C.; Jia, N. An Ultrasensitive Multi-Walled Carbon Nanotube–Platinum–Luminol Nanocomposite-Based Electrochemiluminescence Immunosensor. Analyst 2017, 142, 2253-2260. (22) Ke, H.; Zhang, X.; Huang, C.; Jia, N. Electrochemiluminescence Evaluation for Carbohydrate Antigen 15-3 based on the Dual-Amplification of Ferrocene Derivative and Pt/BSA Core/Shell Nanospheres. Biosens. Bioelectron. 2018, 103, 62-68. (23) Zhang, A.; Xiang, H.; Zhang, X.; Guo, W.; Yuan, E.; Huang, C.; Jia, N. A Novel Sandwich Electrochemiluminescence Immunosensor for Ultrasensitive Detection of Carbohydrate Antigen 19-9 Based on Immobilizing Luminol on Ag@BSA Core/Shell Microspheres. Biosens. Bioelectron. 2016, 75, 206-212. (24) Li, X.; Yu, S.; Yan, T.; Zhang, Y.; Du, B.; Wu, D.; Wei, Q. A Sensitive Electrochemiluminescence Immunosensor Based on Ru(bpy)32+ in 3D CuNi Oxalate as Luminophores and Graphene Oxide-Polyethylenimine as Released Ru(bpy)32+ Initiator. Biosens. Bioelectron. 2017, 89, 1020–1025. (25) Shan, J.; Ma, Z. A Review on Amperometric Immunoassays for Tumor Markers Based on the Use of Hybrid Materials Consisting of Conducting Polymers and Noble Metal Nanomaterials. Microchim. Acta 2017, 148, 969–979. (26) Yuan, Y.; Zhang, L.; Wang, H.; Chai, Y.; Yuan, R. Self-Enhanced PEI-Ru(II) Complex with Polyamino Acid as Booster to Construct Ultrasensitive Electrochemiluminescence Immunosensor for Carcinoembryonic Antigen Detection. Anal. Chim. Acta 2018, 1001, 112-118. (27) Sun, Y.; Zhou, B.; Lin, Y.; Wang, W.; Fernando, K. S.; Pathak, P.; Meziani, M. J.; Harruff, B. A.; Wang, X.; Wang, H.; Luo, P.; Yang, H.; Kose, M. E.; Chen, B.; Veca, L. M.; Xie, S. Quantum-Sized Carbon Dots for Bright and Colorful Photoluminescence. J. Am. Chem. Soc. 2006, 128, 7756-7757. (28) Geim, A. K.; Novoselov, K. S. The Rise of Grapheme. Nat. Mater. 2007, 6, 183–191. (29) Wei,

Y.;

Wang,

H.;

Sun,

S.;

Tang,

L.;

Cao,

13


Y.;

Deng,

B.

An

Ultrasensitive

ACS Sensors 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 14 of 26

Electrochemiluminescence Sensor Based on Reduced Graphene Oxide-Copper Sulfide Composite Coupled with Capillary Electrophoresis for Determination of Amlodipine Besylate in Mice Plasma. Biosens. Bioelectron. 2016, 86, 714-719. (30) Li, N.; Jia, L.; Ma, R.; Jia, W.; Lu, Y.; Shi, S.; Wang, H. A Novel Sandwiched Electrochemiluminescence Immunosensor for the detection of Carcinoembryonic Antigen Based on Carbon Quantum Dots and Signal Amplification. Biosens. Bioelectron. 2017, 89, 453–460. (31) Kong, D.; Bi, S.; Wang, Z.; Xia, J.; Zhang, F. In Situ Growth of Three-Dimensional Graphene Films for Signal-on Electrochemical Biosensing of Various Analytes. Anal. Chem. 2016, 88, 10667-10674. (32) Wang, Z.; Zhao, C.; Gui, R.; Jin, H.; Xia, J.; Zhang, F.; Xia, Y. Synthetic Methods and Potential Applications of Transition Metal Dichalcogenide/Graphene Nanocomposites. Coord. Chem. Rev. 2016, 326, 86–110. (33) Guo, H.; Jin, H.; Gui, R.; Wang, Z.; Xia, J.; Zhang, F. Electrodeposition One-Step Preparation of Silver Nanoparticles/Carbon Dots/Reduced Graphene Oxide Ternary Dendritic Nanocomposites for Sensitive Detection of Doxorubicin. Sens. Actuators B 2017, 253, 50–57. (34) Wang, Z.; Yu, J.; Gui, R.; Jin, H.; Xia, Y. Carbon Nanomaterials-Based Electrochemical Aptasensors. Biosens. Bioelectron. 2016, 79, 136–149. (35) Xing, B.; Zhu, W.; Zheng, X.; Zhu, Y.; Wei, Q.; Wu, D. Electrochemiluminescence Immunosensor Based on Quenching Effect of SiO2@PDA on SnO2/rGO/Au NPs-Luminol for Insulin Detection. Sens. Actuators B 2018, 265, 403–411. (36) Wei, H.; Ni, S.; Cao, C.; Yang, G.; Liu, G. Graphene Oxide Signal Reporter Based Multifunctional Immunosensing Platform for Amperometric Profiling of Multiple Cytokines in Serum. ACS Sens. 2018, 3, 1553-1561. (37) Gao, Z.; Lin, Z.; Chen, X.; Zhong, H.; Huang, Z. A Fluorescent Probe Based on N-doped Carbon Dots for Highly Sensitive Detection of Hg2+ in Aqueous Solutions. Anal. Methods 2016, 8, 2297-2304. (38) Fang, Q.; Dong, Y.; Chen, Y.; Lu, C.-H.; Chi, Y.; Yang, H.-H.; Yu, T. Luminescence Origin of Carbon Based Dots obtained from Citric Acid and Amino Group-Containing Molecules. Carbon. 2017, 118, 319-326. (39) Chowdhury, D.; Gogoia, N.; Majumdar, G. Fluorescent Carbon Dots Obtained from Chitosan Gel. RSC Adv. 2012, 2, 12156-12159. (40) Zheng, L.; Chi, Y.; Dong, Y.; Lin, J.; Wang, B. Electrochemiluminescence of Water-Soluble Carbon Nanocrystals Released Electrochemically from Graphite. J. Am. Chem. Soc. 2009, 131, 4564-4565. 14


Page 15 of 26 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

ACS Sensors

(41) Dong, Y.; Zhou, N.; Lin, X.; Lin, J.; Chi, Y.; Chen, G. Extraction of Electrochemiluminescent Oxidized Carbon Quantum Dots from Activated Carbon. Chem. Mater. 2010, 22, 5895-5899. (42) Jiang, L.; Han, J.; Li, F.; Gao, J.; Li, Y.; Dong, Y.; Wei, Q. A Sandwich-Type Electrochemical Immunosensor Based on Multiple Signal Amplification for α-fetoprotein Labeled by Platinum Hybrid Multiwalled Carbon Nanotubes Adhered Copper Oxide. Electrochim. Acta 2015, 160, 7–14. (43) Mcginness, J.; Corry, P.; Proctor, P. Amorphous Semiconductor Switching in Melanins. Science 1974, 183, 853-835. (44) Zhang, H.; Zuo, F.; Tan, X.; Xu, S.; Yuan, R.; Chen, S. A Novel Electrochemiluminescent Biosensor Based

on

Resonance

Energy

Transfer

between

Poly(9,9-di-n-octylfluorenyl-2,7-diyl)

3,4,9,10-Perylenetetracarboxylic Acid for Insulin Detection. Biosens. Bioelectron. 2018, 104, 65–71.

15


and

ACS Sensors 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 16 of 26

List of Tables and Figures Table 1 Analytical results of human serum samples（n=6）

Scheme 1. (a) The immobilization of AuNPs, CQDs and Ab2 on the PEI-GO matrix. (b) The fabrication process of proposed ECL immunosensor.

Figure 1. The TEM of AuNPs (a), CQDs (b), GO (c) and AuNPs-CQDs-GO-PEI (d). UV–vis spectra of GO, AuNPs and CQDs (e).

Figure 2. (a) FL emission and excitation spectra of the CQDs, (b) FL emission spectra at different excitation wavelengths. Inset showed the relationship between FL emission intensity and excitation wavelength.

Figure 3. (a) ECL intensity-potential curves of CQDs/GCE. (b) Repeatability of the ECL signals of CQDs/GCE, continuously scanning 14 times from -1.8 to -0.2 V in 0.1 mol L-1 PBS (pH 7.4) containing 0.1 mol L-1 K2S2O8.

Figure 4. (a) CV curves and (b) Nyquist diagrams of EIS recorded from 0.1 to 10-5 Hz in 5.0 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 mol L-1 KCl. a: bare GCE, b: PDA-AgNPs/GCE, c: Ab1/PDA-AgNPs/GCE, d: BSA/Ab1/PDA-AgNPs/GCE, e: CA15-3/BSA/Ab1/PDA-AgNPs/GCE, f: AuNPs/CQDs-GO-PEI/Ab2/CA15-3/BSA/Ab1/PDA-AgNPs/GCE.

Figure 5. ECL−time profiles of CA15-3/BSA/Ab1/PDA-AgNPs/GCE with (a) AuNPs, (b) CQDs-AuNPs, (c) PEI-CQDs-AuNPs and (d) GO-PEI-CQDs-AuNPs as an ECL probe incubated with 100 U mL−1 of CA15-3. Electrolyte: 0.1 mol L-1 PBS (pH 7.4) containing of 0.1 mol L-1 K2S2O8 and 0.1 mol L-1 KCl; scan rate: 100 mV s−1; scan potential: -1.8 to -0.2 V. The voltage of the photomultiplier tube was set at 800 V.

Figure 6. (a) ECL signals of the immunosensor for the detection of CA15-3 at different concentrations (a-g: 0.005, 0.01, 0.1, 1, 10, 100, 500 U mL-1). (b) The calibration plot between ECL intensity and logCCA15-3. (c) ECL-time curves of 100 U mL-1 CA15-3 in PBS (0.1 mol L-1 pH 7.4) containing 0.1 mol L-1 16


Page 17 of 26 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

ACS Sensors

K2S2O8 and 0.1 mol L-1 KCl by continuous cyclic voltammetry from -1.8 V to -0.2 V for 11 cycles at a scan rate of 100 mV s-1. (d) Specificity and selectivity of the as-prepared ECL immunosensor with various interferences: blank, CEA （100 ng mL-1), AFP （50 ng mL-1 ）, Glucose (20 mg mL-1), CA15-3 (100 U mL-1) and mixture. (e) Reproducibility of the designed ECL immunosensor with four electrodes in the presence of three different concentrations, which is 100 U mL-1 CA15-3, 10 U mL-1 CA15-3, 1 U mL-1 CA15-3.(Error bar = SD, n = 4.)

17


ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 Table 1. Analytical results of human serum samples（n=6） 13 14 S Serum sample No. Original Added Found Recovery 15 16 (U mL-1) (U mL-1) (U mL-1) (%) 17 18 Cancer patient 1 159.0 160.0 321.0±5.8 101.3 19 20 Cancer patient 2 56.67 57.00 114.1±1.2 100.8 21 22 Cancer patient 3 32.10 33.00 65.41±0.86 100.9 23 24 [Found] ― [Original] 25 × 100% Note: Recovery = [Added] 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 18 of 26

RSD (%)

Hospital provides reference value (U mL-1)

1.80

161.2

1.05

57.00

1.32

31.80

18


Page 19 of 26 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

ACS Sensors

Scheme 1

19


ACS Sensors

Figure 1

1.5

Absorbance (a.u)

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 20 of 26

(e)

1.0

AuNPs 0.5 GO

0.0 200

CQDs

300

400

500

600

Wavelengh (nm)

20


700

800

Page 21 of 26

Figure 2

1000

(a)

Em

Ex

800 600 400 200 0 200

250

300

350

400

450

500

550

1000

(b)

800

600

FL Intensity (a.u)

Wavelengh (nm)

FL Intensity (a.u)

FL Intensity (a.u)

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

ACS Sensors

900 800 700 600 500 400 300 290 300 310 320 330 340 350 360 370 380

Em (nm)

400

200

0 400

450

500 Em(nm)

21


550

300 310 320 330 340 350 360 370

600

ACS Sensors

Figure 3 (b)

1200

(a)

1200

1000

1000

ECL Intensity (a.u.)


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 22 of 26

800 600 400

800 600 400

200

200

0

0

-1.8

-1.5

-1.2

-0.9

-0.6

-0.3

0

Potential (V)

100

200

Time (s)

22


300

400

Page 23 of 26

Figure 4

7000

(a)

50

(b)

6000

a b c d e f

a b c

d

5000

-Z''(ohm)

100

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

ACS Sensors

0

e f

4000 3000 2000

-50

1000 -100

0 -0.2

0.0

0.2

0.4

0.6

0

Potential (V)

4000

8000

12000

Z' (ohm)

23


16000

20000

ACS Sensors

Figure 5 6000 5000

ECL intensity ( a.u.)

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 24 of 26

a b c d

4000 3000 2000 1000 0 -5

0

5

10

15

20

25

30

Time (s)

24


35

40

Page 25 of 26

Figure 6 8000

5000 4000 3000 2000 1000

a

0 -1000

(b)

6000

g

6000


ECL Intensity (a.u)

7000

(a)

7000

y = 3753 + 928.4 lgC (CA15-3)

5000

R2=0.9909

4000 3000 2000 1000

0

6

12

18

24

30

-3

-2

Time (s)

6000

7000


(c)

6000 5000 4000 3000 2000 1000

5000

-1

0

1

-1 log(CCA15-3 U mL )

2

3

(d)

4000 3000 2000 1000

0

0 0

50

100

150

200

250

300

350

Blank

Time (s)

8000

ECL intensity (a.u.)

ECL intensity ( a.u.)

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

ACS Sensors

(e)

-1

100 U mL R.S.D.=2.3% -1 10 U mL R.S.D.=1.5% -1 1 U mL R.S.D.=1.8%

6000

4000

2000

0

1

2

3

Electrode Number

25


4

CEA

AFP Glucose BSA CA15-3 Mixture

ACS Sensors 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

For TOC only

26


Page 26 of 26

A Novel Carbon Quantum Dots Signal Amplification Strategy Coupled

Recommend Documents