Electrochemical Assay of the Alpha Fetoprotein-L3 Isoform Ratio To

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Electrochemical Assay of the Alpha Fetoprotein-L3 Isoform Ratio to Improve the Diagnostic Accuracy of Hepatocellular Carcinoma Tianxiang Wei, Weiwei Zhang, Qian Tan, Xinwen Cui, and Zhihui Dai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04045 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Analytical Chemistry

Electrochemical Assay of the Alpha Fetoprotein-L3 Isoform Ratio to Improve the Diagnostic Accuracy of Hepatocellular Carcinoma Tianxiang Wei,a,b Weiwei Zhang,a Qian Tan,b Xinwen Cui,a Zhihui Daia,c,* a

Jiangsu Collaborative Innovation Centre of Biomedical Functional Materials and Jiangsu Key

Laboratory of Biofunctional Materials, School of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, P. R. China b

School of Environment, Nanjing Normal University, Nanjing, 210023, P. R. China

c

Nanjing Normal University Center for Analysis and Testing, Nanjing, 210023, P. R. China

ABSTRACT: Hepatocellular carcinoma (HCC) is now the major malignant diseases with high morbidity and mortality, which seriously endangers human lives and health. Alpha fetoprotein (AFP) assay is a commonly used serological biomarker for clinical diagnosis of HCC, but it lacks specificity. Analysis of its isoform AFP-L3, especially the AFP-L3 ratio in total AFP (AFP-L3%), can significantly improve the specificity for HCC identification. Herein, an electrochemical approach has been firstly proposed for simple, accurate and fast determination of AFP-L3% in clinical samples. Based on two independent electrochemical signals generated from the synthesized nanoparticles, 4-mercaptophenylboronic acid (MPA)-functionalized copper nanoparticles (MPA-CuNPs) and the lens culinaris agglutinin (LCA)functionalized silver nanoparticles (LCA-AgNPs), simultaneous quantification of the AFP-L3 and total AFP in serum sample has been achieved, thus achieving directly electrochemical assay of AFP-L3%. To be noted, both the assay time and the assay procedure have been significantly compressed when compared to that of available techniques in clinical use. Therefore, with the integration of electrochemical techniques, this new approach for AFP-L3% analysis would be promising for the accurate diagnosis of HCC.

Hepatocellular carcinoma (HCC) is the third leading cause of death from cancer.1,2 Development of the diagnostic method is thus crucial for early diagnosis of HCC, and is also essential for the treatment and prognosis determination of the suffered patients. Serological biomarker tests are frequently used in disease-related diagnosis due to its advantages of noninvasion, low-cost and informativeness.3-7 Alpha fetoprotein (AFP) is a

commonly used serological tumor biomarker for HCC.8-10 It has been shown that the concentration of total AFP is rapidly rising in the serum of HCC progressing patients. However, the serum content of total AFP has a limited relative specificity and sensitivity (25%-60%) to predict HCC.11,12 More recent studies have shown that different isoforms of AFP would appear along with different liver diseases. While under the condition of HCC, AFP-L3,

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which has strong relative ability with lens culinaris agglutinin (LCA), is mainly formed.13-15 AFP-L3 is a serological marker for the diagnosis of HCC with considerable high sensitivity. It can be detected in the serum 9-12 months before tissue lesions are found with imaging screenings. In addition, AFP-L3 can be used in the judgment of disease prognosis.16 The US Food and Drug Administration (FDA) has approved to apply the analysis of AFP-L3 in the early warning of HCC, with 10% of total AFP content as positive critical value as this ratio indicates an incidence of over 95% for HCC.17 Therefore, the assay for the ratio of LCA-reactive AFP to total AFP (AFP-L3%) has been an efficient HCC identification standard with high specificity. Nowadays, the AFP-L3% detection methods are mainly plant lectin affinity chromatography method,18 polyacrylamide gel electrophoresis method,19 and affinity imprinting method.20 These developed methods can meet the basic requirements of AFP-L3% assay, however, in many cases, they’re hard for the operators and too complex for subsequent processing, and is also time consuming in clinical practice. Therefore, it is necessary to construct a simple, efficient, and sensitive detection method for AFPL3% in HCC diagnostics. Benefited from the advantages of simple operation, high sensitivity and efficiency, electrochemical analysis has achieved rapid progress in the biological assay in many areas.21-23 Hence, we expect to develop an electrochemical assay method for AFP-L3% assay that could simply and fast obtain the information for accurate diagnosis of HCC. In this work, by taking advantages of the ultrasensitive and non-interfering electrochemical oxidation properties of the synthesized copper nanoparticles (CuNPs)24 and silver nanoparticles (AgNPs),25 we proposed a dual signal differential strategy to distinguish AFP-L3 from total AFP, achieving direct electrochemical assay for AFP-L3%. As shown in Scheme 1, at the electrode sensing surface, AFP antibodies capture the total AFP. To single out AFP-L3 from the base signal of AFP, two specific nanoparticle probes were synthesized: 4mercaptophenylboronic acid (MPA) functionalized CuNPs (MPA-CuNPs) and LCA functionalized AgNPs (LCAAgNPs). MPA-CuNPs were used to recognize the total AFP through the interaction of MPA with carbohydrates on AFP, serving as the base signal. More specific recognition for AFP-L3 in total AFP is achieved by LCA-AgNPs that can specifically bind to fucose of AFP-L3,26 serving as the specific signal. Combined with electrochemical oxidation technique, the concentrations of AFP-L3 can be singled out from total AFP, on the basis of independent oxidation signals of CuNPs and AgNPs. Therefore, simultaneously quantification of the AFP and AFP-L3 can be easily achieved to establish the ratio relationship. In this way, AFP-L3% can be easily calculated, for the fast and accurate diagnosis of HCC.

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Scheme 1. Schematic representation of the electrochemical biosensing platform for the determination of AFP-L3 in total AFP. LCAfunctionalized AgNP and MPA-functionalized CuNP are used to label AFP-L3 and total AFP at the electrode surface respectively, thus generating two independent signals from the two metal nanoparticles under electrochemical oxidation. EXPERIMENTAL SECTION Reagents and materials. BSA, branched polyethylene imine (BPEI), Tween 20, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES), tris (2carboxyethyl) phosphine hydrochloride (TCEP), MPA and 4-methylbenzeneboronic acid (MBBA) were purchased from Sigma-Aldrich (USA). Human AFP-L1, AFP-L3 and anti-AFP were purchased from Sino Biological Inc (China). LCA was purchased from Vectorlabs Co., Ltd (Switzerland). Other chemicals were all of analytical reagent grade. All solutions, unless otherwise indicated, were prepared with the ultrapure water (18.2 MΩ cm-1). Instruments. Electrochemical tests (Cyclic Voltammetry (CV) and Linear Sweep Voltammetry (LSV)) were conducted on an electrochemical workstation CHI 660D by a three-electrode system. A platinum electrode was used as counter electrode and an Ag/AgCl electrode was used as the reference electrode. Electrochemical Impedance Spectroscopy (EIS) was conducted on the Autolab electrochemical workstation PGSTAT302N with three electrode system. The voltage frequencies ranged from 105 Hz to 0.1 Hz and the AC voltage amplitude was 5 mV. The supporting electrolyte was 10 mM [Fe(CN)6]3-/4solution containing 0.1 M KCl. Ultraviolet and Visible Absorption Spectroscopy (UV-vis) was conducted on UVvis spectrophotometer Cary 50 with wavelength range from 200 nm to 800 nm. Preparation of the BPEI-RGO composite. Due to its own unique hexagonal planar two-dimensional structure, graphene shows excellent electron transfer ability, high specific surface area and good biocompatibility, and is the excellent material for the construction of a high sensitive electrochemical biosensor.27-30 In this work, graphene oxide (GO) modified on the gold electrode surface may provide lots of sites for the immobilization of AFP capture antibody (anti-AFP).


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BPEI and L-ascorbic acid was used to resist the formation of irreversible agglomerates31,32 during the chemical reduction process of GO. Because GO was added to the BPEI solution with vigorous stirring, so GO was rapidly wrapped by BPEI molecules through electrostatic attractions resulted from carboxylic groups of GO and amino groups of BPEI and the reduction product was BPEIRGO composites. BPEI-RGO composite was synthesized referring to our previous synthetic method to prepare chitosan-RGO composite.33 In detail, 1.0 mL of GO suspension (2.5 mg mL-1) was gradually added into 2.0 mL of BPEI solution (5.58%), and meanwhile the mixture was stirred vigorously. Then L-ascorbic acid (23.6 mg) was added, and the mixture was held at 60 °C for 5 h. After cooling to room temperature, the BPEI-RGO was filtered over a cellulose membrane (0.22 m), washed with water, and then redispersed in 1.0 mL of water for further usage. Preparation of the LCA-AgNPs probe. AgNPs were prepared according to the two-step seed-mediated synthesis by citrate and ascorbic acid reduction.34 LCAAgNPs probe was prepared with the following steps: The pH of AgNPs colloidal solution was adjusted to 8.5 by adding 0.01 M sodium phosphate solution. Then, 100 L of LCA (2 mg mL-1 in H2O) was added into the AgNPs colloidal solution (1.0 mL) and stirred for 40 min. Afterwards, the mixture was performed with subsequent addition of 10 L of Tween 20 (1%) with stirring for 20 min. The product was obtained through centrifugation at 6000 rpm for 15 min and washed with water. The LCA-AgNPs probe was resuspended in 100 L of 10 mM phosphate buffer saline solution (PBS) (pH 7.4) and stored at 4 °C. Preparation of the MPA-CuNPs probe. CuNPs were synthesized according to the previous reported polyol method.35 MPA-CuNPs probe was prepared with the following steps: 80 L of 10 mM PBS (containing 10 mM TCEP, pH 8.0) and 1.0 mL of 50 M MPA were mixed at room temperature. The above mixture was described as solution A. Then 200 L of CuNPs (2 mg mL−1) was mixed with 10 L of solution A and stirred for 12 h. 100 L of 10 mM PBS (pH 7.4) was added and stirred for 6 h. Finally, 10 L of 3 M sodium chloride solution was added to the solution and the mixture was stirred for 20 h to strengthen the ionic strength of the MPA-CuNPs. The product was obtained through centrifugation at 6,000 rpm for 15 min and washed with water. The MPA-CuNPs probe was finally resuspended in 100 L of 10 mM PBS (pH 7.4). The product was stored at 4 °C for future use. Preparation of the electrochemical biosensing platform for AFP-L3% assay. The fabrication of the electrochemical AFP-L3% biosensor was illustrated in Scheme 1. Gold electrode was first polished at a metallographic sandpaper and then soaked in Piranha solution (98% sulfuric acid mixed with 30% hydrogen peroxide at a volume ratio of 3:1) for 5 min. After washing, it was polished to a mirror finish with alumina slurry (1.0, 0.3 and 0.05 m, respectively), followed by ultrasonication for 1 min to remove the alumina particles, and soaked in 50% nitric acid solution for 30 min. Then the gold electrode

was put into 0.5 M sulfuric acid solution for electrochemical activation. Finally, the electrode was rinsed with water and dried under nitrogen gas. Subsequently, 3 L of BPEI-RGO solution (2.5 mg mL-1) was cast on the gold electrode and then be vacuum dried at room temperature. After thoroughly washing with water, the electrode was immediately followed by incubation with glutaraldehyde (4 L, 2%) for 1 h at room temperature. After washing, anti-AFP (3 L, 4.55 g mL-1) was applied to the electrode and then incubated for 3 h at room temperature. After washing with 10 mM HEPES containing 0.1 M NaCl (pH 8.5), the electrode was incubated with BSA (4 L, 3%, in PBS) at 37 °C for 1 h to block excess active groups and nonspecific binding sites on the surface. After washing with HEPES solution, 5 mM MBBA was placed onto the electrode surface at 37 °C for 30 min to block the sugar chain sites of BSA and AFP antibodies. At last, the electrode was washed with HEPES solution for further usage. Process of electrochemical measurement. First, the MBBA/BSA/anti-AFP/glutaraldehyde/BPEI-RGO/Au electrode was incubated with 4 L of different concentrations of the mixture of AFP-L1 and AFP-L3 for 1 h at 37 °C via specific binding between AFP antibodies and AFP, followed by washing with HEPES solution. Subsequently, the electrode (AFP-L1, AFPL3/MBBA/BSA/anti-AFP/glutaraldehyde/BPEI-RGO/Au) was incubated with 4 L of MPA-CuNPs and LCA-AgNPs mixture for 45 min at 37 °C and washed for five times. Finally, the electrochemical detection was performed in 10 mM PBS.

RESULTS AND DISCUSSION Characterization of the BPEI-RGO composite. UV-vis absorption spectroscopy was used to confirm the reduction of GO (Figure 1). From the changes of the visible colors, yellowish-brown GO suspension was reacted with BPEI solution and L-ascorbic acid, to create reduced GO which can disperse in water phase well. As shown in Figure 1, the maximum absorption of the GO dispersion shifted from 230 (curve a) to 270 nm (curve b) after its reduction. In addition, the absorption intensity of RGO was higher than GO from 300 to 800 nm, indicating the restoration of the electronic conjugations36 due to the reduction. Fabrication and characterization of the electrochemical AFP-L3% biosensor. EIS was an effective method to characterize the modification process of the electrochemical biosensor. The semicircle diameter equals to the electron-transfer resistance (Ret). As shown in Figure 2, after modification with BPEI-RGO on bare Au electrode, the curve changed from a small semicircle (curve a) to almost a straight line (curve b), reflecting good conductivity and low electron-transfer resistance on electrode surface, indicating the excellent electron transfer capability of RGO. After further modification of anti-AFP (curve c), MBBA/BSA (curve d), AFP + AFP-L3 (curve e),



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MPA-CuNPs + LCA-AgNPs (curve f), the gradual increase in charge transfer resistance was observed, this was due to the rise of resistivity from the electrode surface to the probes. These results further confirmed that the biosensor was successfully fabricated.

Figure 1. UV-vis spectra and photographs of (a) GO, (b) BPEI, and the (c) BPEI-RGO composite.

Figure 2. Nyquist diagrams of (a) bare Au electrode, (b) BPEI-RGO/bare Au, (c) anti-AFP /glutaraldehyde/BPEI-RGO/Au, (d) MBBA/BSA/anti-AFP/glutaraldehyde/BPEI-RGO/Au, (e) AFP+AFP-L3/MBBA/BSA/anti-AFP/glutaraldehyde/BPEI-RGO/Au, (f) MPA-CuNPs+LCA-AgNPs/AFP+AFP-L3/MBBA/BSA/anti-AFP/glutaraldehyde/BPEI-RGO/Au in 10 mM [Fe(CN)6]3-/4- containing 0.1 M KCl.

Feasibility of the fabricated electrochemical biosensor. To investigate the stepwise modification process of the biosensor, CV measurements were performed. The CV spectra of the each step of construction processes were shown in Figure 3. As the negative control, in the absence of AFP, a mixture of the two probes (MPACuNPs and LCA-AgNPs) was incubated with the constructed electrochemical sensor (curve a). No obvious oxidative peaks of CuNPs and AgNPs were observed when scanning from -0.15 V to 0.25 V. However, when only the AFP-L1 existed, a peak at -100 mV was detected, corresponding to the electrochemical oxidation of CuNPs (curve b). In the presence of sole AFP-L3, CV spectrum showed two peaks at -100 mV and +200 mV corresponding to the electrochemical oxidation of CuNPs and AgNPs respectively (curve c), which is owing to the fact that AFPL3 is a subunit of AFP, it can both bind with MPA-CuNPs and Ag-LCA. When AFP-L1 and AFP-L3 both existed in the test sample, the electrode showed the two peaks at -100 mV

and +200 mV in the curve d, ascribed to the direct oxidation of CuNPs and AgNPs. These results indicated that an electrochemical detection platform has been proposed for AFP-L3 assay with good feasibility. Optimization of detection conditions. Some factors can affect the electrochemical response of the biosensor. Herein, we explored the major four factors, including the amount of BPEI-RGO (Figure 4A), the MBBA concentration (Figure 4B), the incubation temperature of labeling nanoprobes (MPA-CuNPs + LCA-AgNPs) (Figure 4C), and the incubation time of these nanoprobes (Figure 4D). As shown in Figure 4A, with increasing the amount of BPEI-RGO from 1 to 12.5 g, the current response of the electrochemical sensor toward AFP or AFP-L3 increased to a maximum at 7.5 g of BPEI-RGO, this phenomenon was due to the fact that the BPEI-RGO nanomaterials with good conductivity coated on the electrode surface increased the electrode conductive area. Then the current


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response tended to decrease from 7.5 to 12.5 g of BPEIRGO coated on the surface of gold electrode, which was probably owing to the limited electron transfer from a thicker film. Therefore, the amount of 7.5 g of BPEI-RGO was chosen to modify the gold electrode.

Figure 3. CVs obtained at the fabricated electrochemical sensors that were respectively incubated with (a) MPA-CuNPs and LCA-AgNPs, (b) AFP-L1, MPA-CuNPs and LCA-AgNPs, (c) AFP-L3, MPA-CuNPs and LCA-AgNPs, (d) AFP-L1, APF-L3, MPA-CuNPs and LCA-AgNPs. The electroyte was 10 mM PBS. CVs were scanned from -0.15 V to 0.25 V, with a scan rate at 100 mV s-1. The AFP-L1 concentrations were (a) 0, (b) 50, (c) 0, (d) 45 ng mL-1 respectively. The AFP-L3 concentrations were (a) 0, (b) 0, (c) 50, (d) 5 ng mL-1 respectively.

In the construction process of this AFP-L3% electrochemical platform, non-specific adsorption was avoided through the following two approaches: i) bovine serum albumin (BSA) was loaded onto electrode surface to occupy excess aldehyde reaction sites, ii) 4methylbenzeneboronic acid (MBBA) was used to further block the sugar chain sites of BSA and AFP antibodies, so that the following modification steps could not interfere the quantitative determination. To avoid the possible competitive reaction from MPA and the sugar chain sites of BSA and AFP antibodies, the concentration of MBBA

was optimized. As shown in Figure 4B, with increasing the concentration of MBBA from 2.0 to 5.0 mM, the current response towards AFP obviously decreased to a low value and then the peak currents had little change from 5.0 to 7.0 mM MBBA, while the current response towards AFP-L3 showed little change from 2.0 to 7.0 mM MBBA. This was because MBBA effectively blocked the nonspecific adsorption between MPA and the sugar chain sites of BSA and AFP antibodies, and the sugar chain sites of BSA and AFP antibodies had no inteference toward LCA. So, 5.0 mM MBBA was chosen as the optimized concentration. The incubation temperature of signal nanoprobes (MPACuNPs + LCA-AgNPs) for binding AFP and AFP-L3 antigen was another important parameter. Figure 4C showed that the current responses toward both AFP and AFP-L3 increased with increasing incubation temperature of nanoprobes and tended to level off above 37 °C. Hence, 37 °C was selected as the incubation temperature of nanoprobes. The electrochemical current response was also related to the incubation time of the two signal nanoprobes. As shown in Figure 4D, the current responses toward AFP and AFP-L3 both reached a maximum value at 45 min of incubation time of the mixed nanoprobes, which was owning to the fact that 50 min was enough to reach a saturated binding rate between AFP (AFP-L3) and MPA (LCA). Therefore, the optimal incubation time was 45 min. Performance of the fabricated electrochemical biosensor for AFP and AFP-L3. Under the optimized conditions, a series of were measured by using this electrochemical assay method, in order to confirm the ability to detect AFP and AFP-L3 simultaneously. First, the AFP was detected with this electrochemical platform. The result was shown in Figure 5, the LSV peaks increased with the increasing concentrations of total AFP. We can obtain a detection range of 0.4–1000 ng mL-1 and a calculated detection limit of 0.01 ng mL-1. In terms of the testing of AFP-L3, as shown in Figure 6, the current signals intensely increased with the increase of AFP-L3 concentration. The detection range for AFP-L3 was from 50 pg mL-1 to 100 ng mL-1, and the detection limit was 40 pg mL-1.



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Figure 4. Effects of A) the amount of BPEI-RGO coated on the surface of gold electrode, B) MBBA concentration, C) the incubation temperature of nanoprobes, and D) the incubation time of nanoprobes on the current responses of the immunosensor toward 500 ng mL-1 AFP (solid line) and 100 ng mL-1 AFP-L3 (dashed line).

Figure 5. A) LSV curves for the detection of different concentrations of AFP (0.4, 2, 10, 40, 200 ng mL-1) by using the proposed electrochemical biosensor, and B) Linear calibration plots for AFP detection.


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Figure 6. A) LSV curves for detection of different concentrations of AFP-L3 (0.1, 0.2, 10, 60, 90 ng mL-1) by using the proposed electrochemical biosensor and B) Linear calibration plots for AFP-L3 detection.

The above results obviously showed that the two LSV current signals were not overlapped (Figure 5A and Figure 6A), with centered potentials at -100 mV and +200 mV, respectively. Thus, the cross interference has been avoided. Based on the two independent electrochemical signals, the quantification of AFP and AFP-L3 showed acceptable recovery rate and relative standard deviation (Table 1), which certified the ability of this electrochemical assay for detection of these two liver cancer markers simultaneously. Furthermore, to demonstrate the

electrochemical assay was robust for the analysis of AFPL3% in real samples, serum samples were tested. Two serum samples were serially dilutes by PBS, and our electrochemical analysis strategy was used for the determination of total AFP concentration and AFP-L3%. As shown in Figure 7, AFP-L3% changed a little for different dilution ratios. Thus, AFP-L3% can be easily and accurately obtained via the proposed electrochemical biosensor for the accurate diagnosis of HCC.

Table 1. The detection of AFP-L1 and AFP-L3 mixture solution. Experimental concentration (ng mL-1)

Spiked concentration (ng mL-1)

Recovery (%)

Standard deviation (%)

AFP-

Sample AFP

AFP-L3

AFP-L1

AFP-L3

AFP

a

77.45

5.55×10-3

75.00

5.0×10-3

103.2

b

108.3

3.687

105.0

4.00

c

36.08

12.57

25.00

12.00

AFP-

L3% AFP

AFP-L3

111.0

5.67

7.86

0.007%

99.36

92.18

4.55

4.32

3.4%

97.51

104.8

4.73

6.60

35%


L3


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Figure 7. Linearity of the analysis. A) Serum sample A with an AFP concentration of 102.8 ng mL-1 and AFP-L3% of 41.2%. B) Serum sample B with an AFP concentration of 52.3 ng mL-1 and AFP-L3% of 6.9%.

Figure 8. A) Correlations of AFP-L3% determined by LCA affinity electrophoresis method and our proposed differential signal based on the proposed electrochemical method, and B) comparison of AFP-L3% of five selected serum samples measured by the hospital with our method.

Correlation with LCA affinity electrophoresis. Twenty serum samples were measured by our electrochemical method, and the determined AFP-L3% was compared with the LCA affinity electrophoresis method. The results (Figure 8A) showed a good correlation with LCA affinity electrophoresis (R = 0.995). The corresponding regression formula is y = 0.921x + 0.895. On the other hand, we selected five serum samples of them to compare the measured results of AFP-L3% with the given value from the hospital (measured by the LCA-affinity electrophoresis method). The result also showed a good correlation (Figure 8B), which further confirmed the high practical usability of our electrochemical method. CONCLUSIONS The assay for the determination of AFP-L3% is an efficient HCC identification standard with high specificity. However, most of the present detection methods are difficult for the operators and too complex for processing, and is also time consuming in clinical practice. Therefore,

a simple, efficient, sensitive detection method for AFP-L3% determination is in urgent need. In this study, we developed an electrochemical analysis method based on the non-interfering electrochemical oxidation signals of two nanoprobes (MPA-CuNPs and LCA-AgNPs) for AFPL3% assay, which has realized the simple and fast detection. This work provides an effective strategy to improve the diagnostic accuracy of HCC, and offers a new idea for differential detection based on the nano-labelled electrochemical potentiometric stripping technique with high sensitivity, and shows a great potential in being developed to a practical device through further combination with integrated design and miniaturization technology due to the intrinsic advantages of electrochemical method (automation and highthroughput processing).

AUTHOR INFORMATION Corresponding Author *Tel./Fax: +86-25-85891051. E-mail: [email protected].


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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (21625502, 21475062 and 21705079), the Natural Science Foundation of Jiangsu Province (BK20171033), and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (17KJB150026). We appreciate the financial support from the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Program for Jiangsu Collaborative Innovation Center of Biomedical Functional Materials.

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