Dual-Mode Electrochemical Immunoassay for Insulin Based on Cu7S4

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Biological and Medical Applications of Materials and Interfaces

Dual Mode Electrochemical Immunoassay for Insulin Based on Cu7S4-Au as a Double Signal Indicator Yueyuan Li, Wenjuan Zhu, Qing Kang, Lei Yang, Yong Zhang, Yaoguang Wang, and Qin Wei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14908 • Publication Date (Web): 18 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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ACS Applied Materials & Interfaces

Dual Mode Electrochemical Immunoassay for Insulin Based on Cu7S4-Au as a Double Signal Indicator Yueyuan Li†, Wenjuan Zhu†, Qing Kang‡, Lei Yang†, Yong Zhang†, Yaoguang Wang†*, Qin Wei† †Key

Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong,

School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China ‡Institute

of Surface Analysis and Chemical Biology, University of Jinan, Jinan 250022,

China Corresponding author Dr. Yaoguang Wang Tel: 86-531-82767872; Fax: 86-531-82767367. E-mail addresses: [email protected] KEYWORDS: electrochemical immunosensor, insulin, dual mode, Cu7S4-Au, double signal indicator.

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ABSTRACT: The detection of insulin by electrochemical (EC) immunoassay is desirable but highly challenged due to the obstacle of improving accuracy, especially in single response system. In this work, based on Cu7S4-Au as a dual signal indicator, we fabricated a dual mode electrochemical immunoassay for insulin. Especially, Cu7S4 presents a strong differential pulse voltammetry (DPV) signal for the electron transfer between the Cu2+ and Cu+, without the addition of K3[Fe(CN)6] or other electron transfer mediators. Furthermore, Cu7S4 displays high sensitivity and high electrocatalytic activity towards the reduction of H2O2 through chronoamperometry (CA). The introduction of Au nanoparticles can not only link on the surface of Cu7S4 by the chemical bond of Au-SH, but also connect the second antibody (Ab2) by the chemical bond of Au-N. Due to the superior electroconductivity of Au nanoparticles and the synergistic effect between the Au nanoparticles and Cu7S4, a high sensitivity is achieved by means of DPV and CA. To improve loading capacity of antibodies, nanofibers polyaniline covalently-grafted graphene (GS-PANI) linked with Au NPs (GS-PANI-Au) as the matrix material was prepared. Based on Cu7S4-Au as a double signal indicator, the developed EC immunoassay for insulin exhibits a wide linear response for insulin detection in the range from 0.1 pg/mL to 50 ng/mL, with low detection limit of 35.8 fg/mL and 12.4 fg/mL through DPV and CA modes, respectively. Furthermore, the immunosensor performs an excellent analytical capability for insulin and promises the application for quantitative detection of other disease markers in clinical diagnosis. Page 2 ACS Paragon Plus Environment

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1 INTRODUCTION Insulin is a peptide hormone, which plays a major role in the glycometabolism.1 Insulin secretion deficiency is the one of the important reasons for diabetes. For example, the insulin of the type-1 is caused by the autoimmune-mediated destruction is under a low concentration whether the patients are empty stomach or after meal. As to the type-2 diabetes, the reason is insulopathic or insulin resistance. Furthermore, diabetes can cause many diseases, such as blindness, heart trouble, kidney failure and stroke, etc.2-3 Nevertheless, the severity of diabetes can be monitored form the concentration of insulin. Therefore, it is important and urgent to develop sensitive and rapid assays for the detection of insulin. The immunosensors have been used in the fields of food safety, environmental pollution detection and clinical medicine detection.4-8 For instance, enzyme linked immunosorbent assay (ELISA) have been developed more than 30 years for the detection of antibodies or the targeting potentially molecule with the advantages of easily assayed enzymatic amplification procedure and facility use. Compared with the ELISA, the electrochemical (EC) immunosensor is more desirable because it can provide a rapid, sensitive and low-cost detection method. In the past years, researchers have paid significant attention to EC immunoassay, that it has many merits for detection of insulin, such as no Page 3 ACS Paragon Plus Environment


influence by the volume used during measurement, the low expending and large-scale quantity production of electronic device.9 However, the EC immunosensor is still highly challenged due to the obstacle of improving accuracy, especially by the single response system. To conquer this challenge, it is reported that dual-mode EC immunosensor can reduce the background and is an effective way to boost the signals and increase the sensitivity.10-12 Copper sulfides nanocrystallites (CuxSy) contain numerous stable and metastable species due to 3d electrons. Especially, because of their excellent electrical conductivity, hypotoxicity, strong adsorptive ability and peroxidase mimicking activities, CuS and Cu2S nanoparticles have been employed in the design of immunosensor for quantitive determination of markers.13-16 Interestingly, the non-stoichiometric copper sulfides, Cu7S4, combining the advantages of CuS and Cu2S, has a stronger applied value in the field of EC immunosensor.17-19 Firstly, the Cu7S4 can perform strong differential pulse voltammetry (DPV) signal by the electron transfer between Cu2+ and Cu+. Usually, DPV detection method was utilized frequently with K3[Fe(CN)6] or electron transfer mediators (such as, methylene blue, toluidine blue and ferrocene et al.) as the signal indicators for the detection of target.20-24 However, these signal indicators are eco-friendly. Meanwhile, Cu7S4 can generate strong EC signals via the chronoamperometry (CA) detection owning to its peroxidase mimicking activities. These unique characteristics enlighten the design of dual mode EC immunoassay for insulin based on Cu7S4 as a double signal indicator. In addition, Page 4 ACS Paragon Plus Environment

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to further enhance the sensitivity of the immunosensor, Au nanoparticles (Au NPs) were introduced to the surface of Cu7S4 by the chemical bond of Au-SH. Au NPs not only can promote the electron transfer ability, but also connect with secondary antibodies (Ab2) by the bond of Au-NH2. Hence, the octadecahedron Cu7S4 loaded with Au nanoparticles (Cu7S4-Au) is a promising label to fabricate immunosensor. Graphene has been widely used as the electrode modification material due to its characteristics of electrical conductivity and large specific surface area.25 As is well known, polyaniline (PANI) is not only rich in amino groups, but also superior in electron transfer.26-28 In general, nanofibers PANI was covalently grafted on graphene, exhibiting synergistic effect of GS and PANI.29 Furthermore, GS-PANI performed large specific surface area as well as good connection with the Au NPs by the chemical bond of AuNH2.30 In this work, the formed GS-PANI-Au nanocomposites were used as the sensing platform to fabricate the immunosensor for its good electrical conductivity and large specific surface area. Cu7S4 was used as a double signal indicator and generated two signals via different measuring methods. Under this detection mode, the efficiency and precision improved significantly than the single signal mode. The fabricated immunosensor exhibited good performance for the insulin detection, which was also evaluated by comparing with ELISA method. At the same time, it can provide value guidance for the more precisely clinical diagnosis of tumor markers.



2 EXPERIMENTAL SECTION 2.1 Synthesis of GS-PANI-Au The preparation process of Au NPs and GS-NH2 were described in the Supporting Information.31-33 GS-PANI-Au was prepared by the following synthetic route. Typically, GS-PANI was prepared according to the report of Van Hoa Nguyen.34 10 mg of GS-NH2 was ultrasonically dispersed in 10 mL of 1 mol/L HCl solution in ice bath. Then 0.5 mL of aniline was added dropwise under magnetic stirring. Subsequently, the freshly prepared K2S2O8 (1.35 g, 10 mL) solution was added dropwise to the cold solution slowly and stirred in ice bath for 5 h. Then the mixture was washed with water and hexane. Finally, GS-PANI was obtained after dried in vacuum. To prepare GS-PANI-Au, 1 mL of 10 mg/mL GS-PANI was mixed with 40 mL of Au NPs solution with shaking for 5 h. The GS-PANI-Au was obtained after washed by centrifugation and dried in the vacuum. 2.2 Synthesis of Cu7S4-Au Octadecahedron Cu7S4 nanoparticles were synthesized following the Cao’s report.35 At first, 0.2 g of PVP (K30) was dissolved in the 100 mL of 0.01 mol/L CuSO4 solution at room temperature. Afterwards, the blue precipitates were produced with the addition of 25 mL of 1.5 mol/L NaOH solution. After stirring for 1 min, 25 mL of 0.1 mol/L ascorbic acid Page 6 ACS Paragon Plus Environment

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was added into the mixture, and then the brick-red colour solution was obtained after stirring for 15 min. Subsequently, 5 mL of 0.2 mol/L thiourea was added and kept at 90 ºC for 6 h. The Cu7S4 was obtained after washing by centrifugation and dried in vacuum. Au NPs can be linked on the Cu7S4 by the chemical bond of Au-S. Hence, the Cu7S4-Au was prepared by shaking the prepared Au NPs solution (40 mL) with the Cu7S4 solution (1 mL, 10 mg/mL) for 5 h. The Cu7S4-Au was collected after washing and vacuum drying.

Figure 1. Preparing process of Cu7S4-Au-Ab2 (A) and schematic presentation for the fabrication of the immunosensor (B).

2.3 Preparation of the Cu7S4-Au-Ab2 Figure 1A shows the preparing steps of the Cu7S4-Au-Ab2. 1 mL of 10 μg/mL Ab2 Page 7 ACS Paragon Plus Environment


was added to 1 mL of 1.0 mg/mL Cu7S4-Au solution. The mixture was incubated overnight at 4 ºC. After centrifugation at low temperature, the precipitate was dispersed in 1 mL of PBS (pH=7.4). The obtained Cu7S4-Au-Ab2 was stored at 4 ºC until use. 2.4 The assembly of the EC immunosensor Figure 1B shows the fabricating process of the immunosensor. Glassy carbon electrode (GCE, Ф=4 mm) was polished with alumina powder and washed thoroughly. Afterwards, 6 μL of 1.2 mg/mL GS-PANI-Au was modified on the electrode. After drying, 6 μL of primary antibodies (Ab1, 10 μg/mL) was modified on the electrode and dried at room temperature for an hour. After washing, 3 μL of 1 wt% BSA was coated on the electrode to eliminate the nonspecific binding sites. After washing similarly, the electrode was modified with different concentration of insulin (6 μL) and incubated for an hour. Then the electrode was washed by PBS (pH=7.4). Finally, Cu7S4-Au-Ab2 (6 μL, 1.0 mg/mL) was dropped onto the electrode for an hour at room temperature and then the electrode was washed before measurement. 2.5 Experimental measurements The fabricated immunosensors tested through DPV are scanned from -0.2 V to 0.6 V directly in 10 mL of PBS and the peak currents were recorded at first. Subsequently, the CA method was scanned at the potential of -0.4 V (Figure S1). After the signal background remained stable, 10 μL of H2O2 (5 mol/L) was injected into PBS solution under mild Page 8 ACS Paragon Plus Environment

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stirring. 3 RESULTS AND DISCUSSION 3.1 Characterizations of the applied nanomaterials The morphology and composition of nanomaterials were characterized by scanning electron microscope (SEM) and fourier transform infrared spectroscopy (FTIR). Observed from Figure 2A, the GO presented the flake-like structure with the rippled and smooth surface. It was obvious that the nanofibers like PANI arranged densely on the surface of GS-NH2 in Figure 2B. The GS-PANI can provide large surface area to connect the Au NPs (Figure 2C). Meanwhile, the concentration of the prepared Au NPs solution was calculated as 3.057 nmol/L according to the report of Qun Huo (Figure S2).36 The Ab1 can be linked on the surface of GS-PANI-Au by the chemical bond of Au-NH2, indicating that the GSPANI-Au can be a good choice as the substrate of the immunosensor. The FTIR spectra of GO, GS-NH2 and GS-PANI composites were obtained to confirm the synthetic process of the GS-PANI (Figure 2D). The spectrum of GO (curve a) presented the representative peaks of O-H (3426 cm-1), C=O (1737 cm-1), C-OH (1420 cm-1) and C-O (1046 cm-1) chemical bonds. Compared with GO, the two new peaks of GS-NH2 (curve b) at 2919 cm-1 and 2854 cm-1 can be attributed to the stretching vibrations of C-H in the methylene and methyl groups from (3-aminopropyl)triethoxysilane (APTES).37 Another new peak appeared at 1099 cm-1 was the characteristic stretching vibration of C-O-Si bonding.37 Page 9 ACS Paragon Plus Environment


These data confirmed that the amine group was grafted to GO successfully. The representative peak of GO at 1737 cm-1 (C=O vibration) was almost impossible to see, indicating that the GO was reduced successfully. After polymerization, the curve c appeared a new peak at 796 cm-1, which was attributed to the N–H vibration of the secondary amine group. This phenomenon indicated that the PANI was grafted from the amino group of GS sheets. In addition, the peaks at 1566 cm-1 and 1487 cm-1 were attributed to the quinonoid band and benzenoid band. The results of FTIR bands were in good accordance with the SEM results.

Figure 2. SEM images of GO (A), GS-PANI (B) and GS-PANI-Au (C); FTIR spectra (D) of GO (a), GS-NH2 (b) and GS-PANI (c).


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Figure 3. The SEM images of Cu7S4 (A) and Cu7S4-Au (B); XRD pattern for Cu7S4 (C); XPS survey spectrum for Cu7S4 (D); XPS spectra of Cu7S4 in the Cu 2p (E) and S 2p (F) region.

In Figure 3A, the Cu7S4 with 18 facets and size of 500 nm was presented. As expected, abound of Au NPs were dispersed uniformly on the surface of Cu7S4 (Figure 3B). The elements of the Cu7S4 (Cu, S) and Cu7S4-Au (Cu, S and Au) were verified by the EDS (Figure S3), indicating that the Au NPs were bonded by the chemical bond of Au-SH. Beyond that, XRD in Figure 3C shows the crystalline diffraction peaks of the Cu7S4. Several prominent diffraction peaks at 32.1°, 37.9°, 46.4°, 48.8° and 54.6° were assigned to the (220), (302), (224), (323) and (206) crystallographic planes (JCPDS NO. 33-0489).38 Figure 3D shows the X-ray photoelectron spectrum (XPS) of Cu7S4, further confirming the presence of Cu and S elements. The Cu 2p spectrum is shown in Figure 3E and divided into four peaks, which was assigned to 2p3/2 of Cu2+ (931.7 eV) and Cu+ (933.2 eV), 2p1/2 Page 11 ACS Paragon Plus Environment


of Cu2+ (954.2 eV) and Cu+ (951.7 eV). The element S can be further analyzed as five different peaks, that was, 161.1 eV, 162.2 eV and 163.26 eV for bridging S2-. The peaks at 168.25 eV and 169.5 eV were associated with residual SO42- species, and its high intensity in Cu7S4 advocates the oxidation of surface sulfur.39 Resulting from SEM, EDS, XRD and XPS, the Cu7S4 was synthesized successfully and was appropriate for the fabrication of the immunosensor. The electrochemical performances of these nanoparticles were also characterized. GSNH2, PANI and GS-PANI (6 μL, 1.2 mg/mL) were modified on the GCE and scanned by DPV with the K3[Fe(CN)6] solution at pH=5.3 (Figure S4). Compared with GS-NH2 (curve a) and PANI (curve b), the GS-PANI showed larger current response (curve c). The nanofibers PANI grafted on graphene performed better electrical conductivity, indicating that there was synergistic effect between GS and PANI. At the same time, the electrochemical surface area (ESA) of 6 μL 1.2 mg/mL GS-NH2, GS-PANI and GS-PANIAu were calculated and the detailed process was shown in Figure S5. According to the Cottrell equation, the calculated result of ESA is 0.154 cm2, 0.536 cm2 and 0.720 cm2 for GS-NH2, GS-PANI and GS-PANI-Au, respectively. The ESA of the GS-PANI is almost 3.5 times of GS-NH2. And after the Au NPs modified the GS-PANI, the ESA became bigger, confirming that the GS-PANI-Au was a good candidate for the matrix of the immunosensor. The GS-PANI, GS-PANI-Au, Cu7S4 and Cu7S4-Au were modified on the GCE Page 12 ACS Paragon Plus Environment

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respectively and characterized by CA to illustrate the electrocatalytic effect for the H2O2 reduction and the results were shown in Figure 4A. Compared with GS-PANI (curve a), GS-PANI-Au (curve b) had a little current change when H2O2 was added into the PBS. The current response of Cu7S4 (curve c) and Cu7S4-Au (curve d) were significantly greater than the GS-PANI-Au. Simultaneously, the current response of Cu7S4-Au was bigger than Cu7S4, indicated that Cu7S4 have a good electrocatalytic effect for the H2O2 reduction and the Au NPs linked with Cu7S4 can perform the synergistic effect to increase the current response of the immunosensor. 3.2 Characterization of immunosensor The Nyquist plots of the EC impedance spectroscopy (EIS) was shown in Figure 4B, which is the characterization for the process of the modified electrode. The equivalent circuit is consisted of the solution resistance (Rs), the Warburg impedance (Zw), the constant phase angle element related to capacitance (Q) and the charge transfer resistance (Ret). The values of them were fitted by ZSimWin software and listed in Table S1. Ret value implied the blocking behavior of the assembled layer on the GCE surface. Observed from the Figure 4B and Table S1, the GCE performed a small Ret value (curve a, 64.85 Ω). After the GS-PANI-Au (cure b) was modified on the GCE, a bigger Ret value (230.7 Ω) can be obtained. When Ab1 (curve c, 584.2 Ω), BSA (curve d, 1404 Ω), insulin (curve e, 2006 Ω) and Cu7S4-Au-Ab2 (curve f, 2567 Ω) were modified onto the electrode, the Ret value further increased, revealing that the immunosensor has been fabricated successfully. Page 13 ACS Paragon Plus Environment


In order to further confirm the modified process. The immunosensor was fabricated with the same concentration of PANI-GO-Au (1.0 mg/mL) under the same detection condition. As shown in Figure 4C, the current response of CA or DPV detection method in the absence of Ab1 or insulin was far less than the unabridged immunosensor. These results indicated that the immunosensor cannot work without the Ab1 or insulin during the fabricated process. 3.3 Optimization of working conditions for the immunosensor In order to achieve the sensitive insulin detection, experimental conditions such as pH, the concentrations of GS-PANI-Au and Cu7S4-Au-Ab2 were optimized. The electrical conductivity of PANI and the activity of the antibodies or antigens were affected by the pH.40 During the optimization experiment of pH, 1.0 mg/mL of PANI-GOAu and 1.0 ng/mL of insulin were used during the fabrication of the immunosensor. The current of the two detection modes in different pH were shown in Figure 4D and the maximum value was at pH=5.3. From this result, the optimum pH of the analytical experiments was chosen at 5.3.


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Figure 4. (A) Amperometric response of the immunosensors with different labels: GS-PANI (a), GSPANI-Au (b), Cu7S4 (c) and Cu7S4-Au (d). (B) EIS obtained for different modified electrodes in Fe(CN)63-/4- containing 0.1 mmol/L KCl solution, GCE (a), GS-PANI-Au/GCE (b), Ab1/GS-PANIAu/GCE (c), BSA/Ab1/GS-PANI-Au/GCE (d), Insulin/BSA/Ab1/GS-PANI-Au/GCE (e) and Cu7S4Au-Ab2/Insulin/BSA/Ab1/GS-PANI-Au/GCE (f), the inset is the equivalent circuit for EIS (Ⅱ). (C) The current response comparison of the fabricated immunosensor (insulin: 1 ng/mL). And the optimization of experimental conditions with (D) pH, (E) the concentration GS-PANI-Au and (F) Cu7S4-Au-Ab2 on the response of the immunosensor to 1.0 ng/mL insulin. Error bar = SD (n= 5)

The matrix of the immunosensor, GS-PANI-Au, with the unique morphology had good conductive ability and bonded abundant Ab1. Under this condition, the concentration of GS-PANI-Au was selected and the best concentration was 1.2 mg/mL (Figure 4E). As the signal label of the immunosensor, the concentration of Cu7S4-Au is very important and the optimal experimental conditions were used 1.2 mg/mL of GS-PANI-Au, 1.0 ng/mL of insulin and the PBS with pH=5.3. As shown in Figure 4F, with the increase of the concentration of Cu7S4-Au-Ab2, the signals increased rapidly from 0.4 mg/mL to 1.0 Page 15 ACS Paragon Plus Environment


mg/mL, and from 1.0 mg/mL to 1.6 mg/mL the signals were plateau. Therefore, the best concentration of Cu7S4-Au-Ab2 was chosen as 1.0 mg/mL.

Figure 5. The current responses of DPV (A) and CA (B), (a~i: 0.0001 ng/mL, 0.001 ng/mL, 0.01 ng/mL, 0.05 ng/mL, 0.1 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL and 25 ng/mL); Calibration curve of the immunosensor under DPV (C) and CA (D) toward different concentrations of insulin.

3.4 Immunoassay performance Under optimized conditions, the record signals increased with the concentrations of insulin increasing from 0.1 pg/mL to 50 ng/mL, the current response of DPV and CA were shown in Figure 5A and Figure 5B. The linear regression equations of DPV (Figure 5C) and CA (Figure 5D) were ΔI (μA) = 12.4+ 2.57 logc (ng/mL, r = 0.996) and ΔI (μA) = 104.1 + 19.0 logc (ng/mL, r = 0.995), and the detection limit are 35.8 fg/mL and 12.4 fg/mL, respectively.41 The good analytical performance and application potential for other Page 16 ACS Paragon Plus Environment

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biomarkers of the technique can be attributed to the following factors. At first, the matrix material of GS-PANI-Au nanoparticle perform large specific surface area which have the superior load capacity of antibodies and a good electron transfer ability. Secondly, the double signal indicator of Cu7S4-Au presents a strong DPV signal and highly sensitive of CA response. The DPV signal is generated by Cu7S4 itself and without the addition of K3[Fe(CN)6], the electron transfer mediators, even the further process of acid dissolution and enzyme-label. At the same time, the Cu7S4-Au displays high electrocatalytic activity towards the reduction of H2O2 and without peroxidase. At last, the double signal can mutual authenticate to get the accuracy detection result. Hence, the developed strategy can provide a stable immobilization and accuracy method for the analogical biomarkers detection and potential application in clinical sample. 3.5 Selectivity, reproducibility and stability

Figure 6. The DPV (A) and CA (B) methods response of the immunosensor to 10 ng/mL PSA (1), 10 ng/mL BNP (2), 10 ng/mL CEA (3), 10 ng/mL AFP (4), 10 ng/mL glucose (5), 10 ng/mL uric acid (6), 10 ng/mL ascorbic acid (7), 10 ng/mL dopamine (8), 10 ng/mL insulin glargine (9), 10 ng/mL insulin aspart (10), 0.1 ng/mL insulin (11) and 10 ng/mL of interferences (PSA + BNP + CEA + AFP



+ glucose + uric acid + ascorbic acid + dopamine + insulin glargine + insulin aspart) + 0.1 ng/mL insulin (12). Error bar = SD (n = 5).

The selectivity of the immunosensor is important for this analytical technique. This examination was estimated with other biomarkers: prostate-specific antigen (PSA), brain natriuretic peptide (BNP), carcino-embryonic antigen (CEA) and alpha fetoprotein (AFP), the traditional components contained in the blood (glucose, uric acid, ascorbic acid and dopamine) and the insulin analogues (insulin glargine and insulin aspart). As shown in Figure 6A and Figure 6B (samples 1-10), the samples just had different interferents (10 ng/mL). The sample 12 had 0.1 ng/mL of insulin and 10 ng/mL of interfering substances. The current value of sample 12 was almost equal to the sample 11 (0.1 ng/mL insulin). The relative standard deviations (RSD) of the detection results are less than 5.0%, indicating that the selectivity of the proposed immunosensor is acceptable. To study the reproducibility of the fabricated immunosensor, 1.0 ng/mL of insulin sample was applied. The RSD of the two detection methods were less than 5.0% and the F-test and t-test were evaluated (Table S2). Through the calculation by the formulas, the F value was 1.11, when the confidence coefficient α=0.05, F9.975

Dual-Mode Electrochemical Immunoassay for Insulin Based on Cu7S4

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