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Supporting information

Ultrasensitive “signal-on” electrochemiluminescence immunosensor for prostate-specific antigen detection based on novel nanoprobe and poly(indole-6-carboxylic acid)/flower-like Au nanocomposite

Chaonan Yang, Qingfu Guo, Yan Lu, Bin Zhang, Guangming Nie*

Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, State Key Laboratory Base of Eco-chemical Engineering, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, 266042, PR China

*Corresponding author. Tel. +86-532-88959058, Fax. +86-532-88957187.

E-mail address: gnie@qust., gmnie@ (G. Nie)

Table of contents

Fig.S1. (Characterizations of AuNP) -------------------------------------------------------S5

Fig.S2. (Size distribution of FGNs) ----------------------------------------------------S6

Fig.S3. (Electrochemical performance testing of PICA) ---------------------------------S7

Fig.S4. (Electrochemical performance testing of PICA/FGNs nanocomposite and the CV comparation of AuNP/GQDs-PEI-GO, PICA/FGNs, PICA, AuNP/GQDs-PEI-GO/ PICA/FGNs coated electrode) ----------------------------------S8

Fig.S5. (ECL intensity stability of GQDs) -----------------------------------------------S10

Fig.S6. (Optimization of ECL detection conditions) ------------------------------------S11

Fig.S7. (ECL intensity of PICA/FGNs, AuNP/GQDs-PEI-GO and AuNP/GQDs-PEI-GO/PICA/FGNs--------------------------------------------------------S13

Fig.S8. (ECL intensity of designed different ECL sensor) -----------------------------S14

Table S1. (The recovery experiments of this ECL sensor in actual sample) --------S16

Reagents and instruments

HAuCl4·3H2O, indole-6-carboxylic acid (ICA, 98%), hexadecyl trimethyl ammonium bromide (CTAB) and chloroauric acid were purchased from aladdin and used as received. Graphene oxide (GO) and polyethyleneimine (PEI) both are obtained from Sigma-Aldrich, using directly. Tetrabutylammonium tetrafluoroborate (TBATFB, 98%) was purchased from Belling Technology Co., Ltd. Ethanol (AR. CH3CH2OH) was purchased from Yantai Far Eastern Fine Chemical Co. Ltd. Commercial HPLC grade acetonitrile (ACN) was purchased from Guangcheng Chemical Reagents Co. Ltd. (Tianjin, China) and use it as it is. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and bovine serum albumin (BSA, 96%) were purchased from Sigma-Aldric. Prostate antigen (PSA), primary antibody (Ab1), secondary antibody (Ab2), carcinoembryonic antigen (CEA), and embryonic protein (ATP) were all obtained from Shanghai Linc-Bio Science Co. LTD. Human serum samples were obtained from the Affiliated Hospital of Qingdao University, China. Phosphate buffer solution (PBS, 0.1 M) was prepared from NaH2PO4 (0.1 M), Na2HPO4(0.1 M) and NaCl (0.1 M). Ultrapure water (18.2 MΩ cm-1) was used throughout the experiment.

Electrochemiluminescence measurements were performed on an MPI-A ECL analyzer (Xi'an Remex Instrument Co., Ltd., China). Electrochemical testing was performed using a CHI660E electrochemical workstation (Shanghai CH Instruments). Transmission electron microscopy (TEM) images were obtained on a JEM-2100 electron microscope. UV-vis absorption spectra were obtained using a Cary 500 UV-vis-NIR spectrophotometer.

Preparation of AuNP

First, 1% HAuCl4 solution and 38.8 mM sodium citrate were prepared. Take 95.88 mL of ultrapure water and stir to boiling (120 °C), add 4.12 mL of 1% HAuCl4 solution, boil for 8 s, and immediately add 10 mL of 38.8 mM sodium citrate to the mixed solution. Return to liquid from light yellow to wine red (within 1 min), continue to reflux for 15 min, cool the solution to room temperature, and store in the dark. Through the characterization of dynamic light scattering (DLS), we can see that the average particle diameter of the prepared AuNP is about 10-15 nm (Fig. S1).

Immobilization of Ab2 on AuNP/GQDs-PEI-GO substrate

20 μL EDC and NHS (4:1) mixed solution was added to 1 mL AuNP/GQDs-GO-PEI solution and stirred for 5 min. Then, 200 μL of Ab2 (50 μg mL-1) was added, and then shaken it for 5 min. Finally, 20μL of BSA was added to the above solution to block the active site. The resulting solution was stored at 4 °C overnight. After centrifugation, it was dispersed in PBS (pH 7.4) for next use.

Preparation of actual serum samples

30 uL real serum samples were added to 0.5 mL serum, shake and mix to obtain diluted samples. In this state, the concentration of the diluted samples was unknown. Then, the sample was detected by the current clinically applied ELISA method, and the concentration was found to be 0.27 ng mL-1. After that, 0.1, 1, 5, 10 ng mL-1 standard samples were added by standard addition method to detect the signal of the designed ECL sensor.

Characterizations of AuNP

[pic]

Fig. S1 TEM images of AuNP (inset: size distribution of AuNPs).

Size distribution of FGNs

[pic]

Fig. S2 Size distribution of FGNs.

Electrochemical performance testing of PICA

[pic]

Fig. S3 Cyclic voltammograms of PICA film in monomer free ACN and 0.1 M TBATFB at different potential scan rates. Potential scan rate: 250 (a), 200 (b), 150 (c), 100 (d), 50 (e), 25 (f) mV s-1.

As shown in Fig. S3, the cyclic voltammetric scan of the PICA film showed significant redox peaks (O1, R1) and (O2, R2), indicating that the material has two electron transfer processes and good electrochemical activity. And as the sweep speed increases, the density of the redox peak current gradually increases, indicating that the film has good redox activity. As can be seen from the illustration, the current density of the redox peak shows a good linear relationship with the sweep speed. When the sweep speed changes, the number and position of the redox peaks are basically unchanged. This also indicates that the redox activity in the ACN solution is stable.

Electrochemical performance testing of PICA/FGNs nanocomposite and the CV comparation of AuNP/GQDs-PEI-GO, PICA/FGNs, PICA, AuNP/GQDs-PEI-GO/ PICA/FGNs coated electrode

[pic]

Fig. S4 CV of (A) PICA/FGNs complexes in monomer-free ACN solutions. Scan rate: 250 (a)，200 (b)，150 (c)，100 (d)，50 (e)，25 (f) mV s-1, and the comparison chart of (B) AuNP/GQDs-PEI-GO, PICA/FGNs, PICA, AuNP/GQDs-PEI-GO/ PICA/FGNs coated electrode in monomer-free ACN solutions at the scan rates of 100 mV s-1.

Fig. S4(A) is CV of the PICA/FGNs complex in monomer-free ACN solution. The PICA/FGNs nanocomposite exhibits two pairs redox peaks, (O1, R1) and (O2, R2), respectively, and the redox peaks are relatively broad. This indicates that the PICA/FGNs complex has good electrochemical activity.

To further investigate the electrochemical activity of PICA/FGNs nanocomposite, the CV curves of PICA/FGNs nanocomposite and PICA in monomer-free ACN solutions were studied with scan rate of 100 mV s-1. It can be seen from Fig. S4 (B) that the redox peak current density of the PICA/FGNs composite film is larger than PICA, indicating the good electrochemical activity. For comparation, the CV of AuNP/GQDs-PEI-GO was also studied (Fig. S4 B). However, the AuNP/GQDs-PEI-GO showed no redox activity. After AuNP/GQDs-PEI-GO was coated on the surface of PICA/FGNs composites, AuNP/GQDs-PEI-GO/ PICA/FGNs electrode showed larger peak current density compared with PICA/FGNs composites (Fig. S4 B). This result may be attributed to the good conductivity of AuNP and GO, which accelerates electron migration during redox process.

ECL intensity stability of GQDs

[pic]Fig. S5 The ECL intensity stability of GQDs.

It can be seen from Fig. S5 that the luminous intensity is very high (up to 16,000). Moreover, the luminescence stability of GQDs is particularly good. The luminescence intensity is not significantly attenuated after 300 s. It can be used as an excellent luminescent material in ECL sensors.

Optimization of ECL detection conditions

[pic]Fig. S6 Effects of (A) incubation time and (B) incubation temperature on ECL signal intensity.

In order to achieve the best performance of the sensor, this experiment mainly studied the effects of incubation time and incubation temperature on ECL immunosensors. Incubation time is an important parameter that affects the performance of immunosensors. As shown in Fig. S6A, in the same state of other experimental conditions, the ECL response signal shows an upward trend as the increase of incubation time. After 50 min, the ECL intensity reaches to a stable value, indicating that the optimal incubation time of PSA is 50 min. After 50 min, the process of specific binding of Ab1 to PSA has reached saturation. In addition, incubation temperatures have a significant impact on maintaining optimal activity of antigens and antibodies. In order to obtain the best ECL signal, the effect of incubation temperature on the constructed sensor performance was also studied. Fig. S6B shows the effect of incubation temperature in the experiment. It can be seen that between 20 and 37 °C, the ECL intensity gradually increases. After 37 °C, the activity of the antibody and antigen decreases due to the temperature rise. Therefore, 37 °C is chosen as the optimal incubation temperature.

ECL intensity of PICA/FGNs, AuNP/GQDs-PEI-GO and AuNP/GQDs-PEI-GO/PICA/FGNs.

[pic]

Fig. S7 The ECL intensity of PICA/FGNs nanocomposite, AuNP/GQDs-PEI-GO composite and AuNP/GQDs-PEI-GO/PICA/FGNs.

ECL intensity of designed different ECL sensors

[pic]

Fig. S8 The ECL intensity of (a) the complete ECL sensor of components. (b) AuNP-free ECL sensor constructed by GQDs-PEI-GO and PICA/FGNs. (c) FGNs-free ECL sensor constructed by AuNP/GQDs-PEI-GO and PICA. (d) PICA-free ECL sensor constructed by AuNP/GQDs-PEI-GO and FGNs.

In order to evaluate the signal amplification effect of AuNP/GQDs-PEI-GO as Ab2 carrier, control experiments were studied by the different constructed ECL sensors. These ECL sensors were tested with the same PSA concentration. As can be seen from Fig. S8, the signal strength of the ECL sensor (b, c) is significantly reduced compared to the signal intensity of the complete component sensor (a). This is because both AuNP, FGNs are good conductive materials. When the ECL sensor lacks AuNP and FGNs, the conductivity of the ECL sensor will decrease and the ECL sensor signal will decrease. The sensor lack of PICA (d) has almost no ECL signal. This is because the lack of PICA will not provide a carboxyl group that can be connected to Ab1. Hence, it cannot form a sandwich immunological structure for PSA detection, and eventually the ECL signal cannot be generated. These results indicated that the AuNP/GQDs-PEI-GO as Ab2 carrier showed good signal amplification effect. And the signal amplification effect mainly attributed to the synergistic effect of PICA, FGNs, AuNP, GQDs, PEI, and GO. This synergistic effect is beneficial to enhance the signal of the ECL sensor and improve the sensitivity of the designed sensor.

Table S1. The recovery experiments of this ECL sensor in actual sample.

Concentration (ng mL-1) |Amount added

(ng mL-1) |actual detection

(ng mL-1) |RSD

|recovery

(%) | |0.27 |0.1 |0.38, 0.36, 0.41, 0.44, 0.42 |3.2 |108.6 | | |1 |1.25, 1.29, 1.33, 1.26, 1.32 |3.5 |101.6 | | |5 |5.15, 5.21, 5.25, 5.19, 5.24 |4.0 |98.8 | | |10 |10.17, 10.23, 10.19, 10.25, 10.24 |3.4 |99.5 | |

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