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A Sulfur-Doped Graphene-Based Immunological Biosensing Platform for Multianalysis of Cancer Biomarkers Xiang Ren, Hongmin Ma, Tong Zhang, Yong Zhang, Tao Yan, Bin Du, and Qin Wei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13416 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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ACS Applied Materials & Interfaces

A Sulfur-Doped Graphene-Based Immunological Biosensing Platform for Multianalysis of Cancer Biomarkers Xiang Ren,† Hongmin Ma,† Tong Zhang,† Yong Zhang,† Tao Yan,‡ Bin Du,‡ and 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 ‡ School of Resource and Environment, University of Jinan, Jinan 250022, China ABSTRACT: The accurate tumor marker detection at early stage can prevent people from getting cancer to a great extent. Herein, a novel tri-antibody dual-channel biosensing strategy is applied in multianalysis of carcino-embryonic antigen (CEA) and nuclear matrix protein 22 (NMP22). In this immunosensor fabrication process, graphene oxide/polyaniline nanostructures are used as matrix and mesoporous NKF-5-3 is used as labels. Two kind antigens can be obtained from the signals of neutral red and toluidine blue, respectively, which are modified on the labels. In this tri-antibody dual-channel biosensing platform, sulfur-doped graphene sheet (S-GS) is synthesized by click chemistry as framework structure. Majority of the incubations are conducted in individual steps, which ensure the surface-incubation more tightly. The detection limit of NMP22 and CEA are 25 fg/mL and 30 fg/mL respectively. The low detection limit and excellent stability can ascribe to the tri-antibody dual-channel strategy which makes the sensor platform from surface to the space. The clinical urine sample analysis achieves a good performance. The urine-based test can avoid the secondary injury on hemophilia or ischaemic patients, displaying a potential application in clinical diagnosis.

1. INTRODUCTION The detection of cancer biomarkers plays a vital role in basic medical research as well as in clinical diagnostics. Carcinoembryonic antigen (CEA) is a glycoprotein most often associated with colorectal carcinomas and commonly used as a clinical marker of tumors for clinical diagnosis of colorectal, pancreatic, gastric, bladder and cervical carcinomas.1 Nuclear Matrix Protein 22 (NMP22) is closely associated with bladder carcinoma2 and it can mainly indicated the patient physical condition. The CEA and NMP22 multianalysis can confirm the case of bladder carcinoma acquirement precisely. Recently, the morbidity of bladder carcinoma presents a growth trend in China and other countries. Thus, it is highly desired to develop simultaneous detection method can restrain the bladder carcinoma mortality rate to a great extent. Compared with the traditional single-analyte immunosensor,3− the simultaneous multianalysis is more accurate and precise in clinical application since it can quantitatively detect a panel of biomarkers in a single run with improved diagnostic specificity.12 Moreover, the multianalysis of cancer biomarkers can simplify the analytical procedure, enhance the detection throughput, and decrease the detection cost.13 Thus, some researches are focused on the simultaneous detection strategy. Ju and co-workers reported multiplexed immunoassay by combining alkaline phosphatase (ALP)-labeled antibody functionalized gold nanoparticles (ALPAb/Au NPs) and enzyme-Au NP catalyzed deposition of silver nanoparticles at a disposable immunosensor array on screenprinted carbon electrodes.12 Zhu and co-workers reported multianalyte electrochemical immunoassay based on metal ion functionalized titanium phosphate nanospheres as labels.14 All these methods can achieve good performance due to the usage of 11

nanomaterials in immunoassay,15−35 and biomolecules interaction can exhibit much tightly chemical bonds which is benefit to the immunoassay fabrication.36 On the basis of above considerations, biomolecule-interaction based immunological biosensing platform for multianalysis can achieve good performance, however, has not been reported before. Herein, sulfur-doped graphene (S-GS) is synthesized by click chemistry to stabilize the combination of biomolecules. A triantibody dual-channel biosensing platform fabrication strategy is first promoted to increase two kind antigen detection sensitivity by another pair immune. In this strategy, the base antibody (Human Immunoglobulin G) is used as the framework to amplify the capacity of other two antibodies like a modified-supersandwich model leading to a lower detection of the other two antigens. This fabrication may exhibit an excellent stability due to the strong and specific combination of the antigen-antibody complex than the interaction between inorganic materials and biomolecules. The incubations are proceeded in the pretreatment steps and S-GS is used in the fabrication, ensuring the tightly combination in this research, leading a reliable detection of CEA and NMP22. The detection limits of NMP22 and CEA are 25 fg/mL and 30 fg/mL, respectively. The results indicate that the multiplex immunoassay exhibits excellent applicability in protein detection.

2. MATERIAL AND METHODS 2.1. Materials. Carcino-embryonic antibody (anti-CEA) and CEA, Nuclear Matrix Protein 22 antibody (anti-NMP22) and NMP22, Human Immunoglobulin G antibody (anti-HIgG) and HIgG are all purchased from Linc-Bio Science Company Limited (Shanghai, China). Bovine serum albumin (BSA, 96%-99%, m/m)

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is obtained from Sigma (USA). NKF-5-3 (SiO2/Al2O3 = 25) is purchased from Nankai University Catalyst Co., Ltd.. Chitosan hydrochloride CS-HCl is obtained from Lvshen Bioengineering Co., Ltd. (Nantong, China). All other chemical reagents (analytical grade) are purchased from Sinopharm Chemical Reagent Co., Ltd.. (Beijing, China). Phosphate buffered saline (PBS) is used as electrolyte which contains 0.1 M KCl for all electrochemistry measurements. Ultrapure water is used throughout the experiment. 2.2. Apparatus. All electrochemical measurements are performed on a CHI760D electrochemical workstation of Chenhua Co., Ltd (Shanghai, China). A three-electrode method is used for all electrochemical measurements: an Au electrode (AE, 4 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire electrode (PE) as the counter electrode. Scanning electron microscope (SEM) and Energy dispersive X-ray spectroscopy (EDS) images are obtained with a JEOL JSM-6700F microscope (Japan). Transmission electron microscope (TEM) images are recorded with a JEOL JEM-1400 microscope (Japan). The X-ray photoelectron spectra (XPS) are carried out on ESCALAB 250 electron energy spectrometer by Thermo Fisher Scientific (USA). Specific surface area is tested by BET equation under N2 gas on Quantachrome Micromeritics ASAP 2020 (USA). 2.3. Synthesis of graphene oxide/polyaniline nanostructures. Graphene oxide/polyaniline (GO-PANI) nanostructures was 37 synthesized by a reported method with some modifications. Specifically, GO (3.0 mg) and ultrapure water (1.5 mL) were added into aniline (300 mg) for 30 min ultrasonication, followed by little water addition and 1 h stirring. After cooling the mixture at 10 °C, (NH4)2S2O8 (APS) aqueous solution (solute: 750 mg) was added and the whole system was kept at low temperature for 24 h. The resultant precipitate was filtered and washed 6 times to remove the APS and oligoaniline. Last, the GO-PANI composite was obtained when it was dried under vacuum overnight. 2.4. Synthesis of Au nanoparticles. Au nanoparticles (Au NPs) were synthesized according to a classical way reported by Frens.38 The 1 mL HAuCl4 (1%, m/m) was added into ultrapure water (99 mL) and the solution was heated to boil under magnetic stirring. Then 2.5 mL sodium citrate (1%, m/m) was added into the solution until the color turned to red. After another 5 min heating, the solution was cooled to room temperature with tinfoil covered for storage in 4 °C refrigerator. The Au NPs were obtained. 2.5. Synthesis of S-GS by click chemistry method. In this research, S-GS was synthesized for the first time. GO (50 mg) and thiourea (50 mg) were added into docosanethiol (55 mL). The mixture was under ultrasonic for 30 min and kept at 150 °C for 3 h. After the reaction, the product was layered obviously. Then the mixture was handled by static separation in order to leave the supernate. The precipitate was washed in succession with water and ethanol, respectively. Finally, the S-GS was obtained. It not only can enlarge the specific surface area, but also can connect the Au NPs tightly due to the strong Au-S interaction which can combine antibodies leading the fabrication stability. In the S-GS synthetic process, the 1-docosanethiol is used to throw out ndodecyl on the surface, which can stabilize the framework. Besides, thiourea is used as a blasting fuse to accelerate the combination of small flake graphene oxide, which can enhance the immunosensor stability and biomolecules loading quantity.

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2.6. Tri-antibody incubation. S-GS (10 mg) was added in a centrifuge tube and the Au collosol was added in it for 30 min ultrasonication. Then the mixture was centrifuged and separated. This process was conducted until the supernatant was exhibiting reddish color which indicates the Au NPs were adsorbed on S-GS sufficiently. The consumed Au collosol is about 27 mL. Au-S-GS was obtained. In this immunosensor fabrication strategy, the tri-antibody incubation was essential. It can connect anti-HIgG, anti-CEA and anti-NMP22 tightly through Au-NH2 interaction. Detailed, Au-SGS (2 mg) was put in 2 mL PBS with 30 min ultrasonication. Then, anti-HIgG, anti-CEA and anti-NMP22 were added into the Au-S-GS solution. The mixtures were shaken slightly at 4 °C for 24 h. The mixtures were centrifuged to remove the liquid supernatant which contains the unbonded three antibodies. The remained solid was redispersed in PBS containing 0.1 mL BSA (1%, m/m) solution which can eliminate the non-specific binding sites of Au-S-GS. The redispersed mixtures were centrifuged to remove the liquid supernatant. And the final precipitate was the tri-antibody incubation Au-S-GS|anti-HIgG|anti-CEA|antiNMP22. 2.7. NMP22 labels incubation. For the labels incubation, NKF-5-3 was used in the structure. In order to gain a tightly incubation, NKF-5-3 was amination by 3aminopropyltriethoxysilane (APTES). Typically, 2.0 mg NKF-5-3 was added into anhydrous toluene (50 mL) and heated to 70 °C. Afterwards, APTES (2 mL) was dropped into the solution rapidly and the reaction was refluent for 3 h at 70 °C. Finally, the amination NKF-5-3 (NKF-5-3-NH2) was obtained after washed three times with absolute alcohol and dried at 30 °C in vacuum. The NKF-5-3-NH2 was shown to contain-NH2 by ninhydrin test. To prepare Au-NKF-5-3 solution, NKF-5-3-NH2 (1 mg) was added into PBS (2 mL, pH 7.4) with 1 h ultrasonication. Au NPs were added into the solution for 5 min vibration. Then the mixture was centrifuged and separated. This process was carried on until the supernatant was exhibiting reddish color which indicates the Au NPs were adsorbed on NKF-5-3-NH2 sufficiently. The precipitation was dried in vacuum at 50 °C for 24 h. The AuNKF-5-3 was gained. To construct the NMP22 labels, 1 mg Au-NKF-5-3 was dispersed in 2 mL PBS (pH 7.4) solution under ultrasonication for 1 h. Then, anit-NMP22 (100 µL, 1 µg/mL) BSA (20µL, 1 wt%) and neutral red (NR, 1 mg) were added in the solution. The mixture was kept at 4 °C for 24 h mild shaking. The mixture was centrifuged and precipitation was redispersed in 2 mL PBS (pH 7.4) again. The obtained incubation Au-NKF-5-3|antiNMP22|NR|BSA was NMP22 labels (Scheme 1II). The Au-NKF5-3|anti-NMP22|NR|BSA (incubation II) were conjunct tightly due to the strong Au-NH2 interaction between Au NPs and antibody/NR which make it an excellent repeatability. 2.8. CEA labels incubation. In CEA labels incubation, the AuNKF-5-3 solution was prepared as before. 1 mg Au-NKF-5-3 was dispersed in 2 mL PBS (pH 7.4) solution under ultrasonication for 1 h. Then, anit-CEA (100 µL, 1 µg/mL) BSA (20µL, 1 wt%) and toluidine blue (TB, 1 mg) were added in the solution. The mixture was kept at 4 °C for 24 h mild shaking. The mixture was centrifuged and precipitation was redispersed in 2 mL PBS (pH 7.4) again. The obtained incubation Au-NKF-5-3|antiCEA|TB|BSA (incubation I) was CEA labels (Scheme 1I).


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room like the branches. TB and NR were used as the signals to quantify the CEA and NMP22. For square wave voltammetry (SWV) measurement of the immunosensor, -0.8 V - 0 V was selected as the detection potential range because the anodic peaks of the NR and TB are located in the area. With different CEA and NMP22 concentrations, different current intensity could be collected. All the measurements were performed at room temperature in PBS (pH 7.4).

Scheme 1. Incubation of the labels: (I) Au-NKF-5-3|anti-CEA|TB|BSA; (II) Au-NKF-5-3|anti-NMP22|NR|BSA.

2.9. Immunosensor fabrication. Scheme 2 illustrate the fabrication process of the proposed tri-antibody dual-channel biosensing platform. AE was polished with Al2O3 powders to a mirror-like surface. The AE was immersed into H2SO4 (0.1 M, aq) for two-time cyclic voltammetry (CV, -0.4 ~ 1.6 V) scanning: the first time is the 20 circles at the rate of 1 V/s and the second time is the 4 circles at the rate of 0.1 V/s. The pretreat AE was well for usage. GO-PANI was dispersed in CS-HCl to form the GOPANI–CS-HCl solution (0.7 mg/mL). 6 µL GO-PANI–CS-HCl solution was dropped on the AE surface to form a flat film followed by 6 µL Au NPs. Then each 6 µL of anti-HIgG, BSA, HIgG were dropped on the former layer like traditional label-free 39 immunosensor for the formation of framework.36 Afterwards, the tri-antibody incubation Au-S-GS|anti-HIgG|anti-CEA|antiNMP22 (6 µL) was dropped on the former layer by the conjunction between anti-HIgG and HIgG. And each 6 µL of two kinds of antibodies (anti-CEA, anti-NMP22), BSA, two antigens (CEA, NMP22) were dropped on layer-by-layer, respectively. Finally, the two kinds labels (Au-NKF-5-3|anti-NMP22|NR|BSA, Au-NKF-5-3|anti-CEA|TB|BSA) were incubated on the immunosensor. The tri-antibody dual-channel immunosensor was fabricated well. The SEM images of every layer are provided in Figure S1 (details in SI), and the overall morphology of this immunosensor (Figure S1G) indicates it is fabricated successfully. Although the novel strategy was a little complex, the stability is excellent due to three reasons. First, the immunosensor was constructed by a layer-by-layer method, and each of the layers were connect by a strong force like antibody-antigen, Au-NH2 interaction. Second, the complex incubations (tri-antibody incubation, NMP22 labels and CEA labels) proceeded by an individual step in a centrifuge tube not on the electrode surface, which can ensure the repeatability and the effective connection of electrode surface. The outside incubation can also guarantee the electrode surface without any dissociative substances which may affect the immunosensor property. Third, the biomolecule exhibits prominent hydrophilicity to form homogeneous solution than 40 nanomaterials. Therefore, the antibody-framework (tri-antibody) 36 can enhance the stability as a dual-antibody reported before. 2.10. Electrochemical measurements. The immunosensor can realize space amplification by the tri-antibody dual-channel strategy, which develops from the surface reaction to the space

Scheme 2. Fabrication of the tri-antibody dual-channel immunosensor.

3. RESULTS AND DISCUSSION 3.1. Characterization of GO-PANI and Au-NKF-5-3. The matrix material of the immunosensor is GO-PANI, which is an excellent candidate in the fabrication due to its good conductivity and large surface area. From Figure 1A, the PANI is covered on GO crisscross. The amplification of PANI is shown in Figure 1B, which exhibits nanowire morphology. NKF-5-3 is used as the label frame for loading more signal molecules because of its mesoporous structure. In Figure 1D, the mesoporous morphology can be observed clearly by the TEM image. When Au NPs are loaded on NKF-5-3, the combination Au-NKF-5-3 can grasp the biomolecules and dyes more tightly. From Figure 1C, the assynthesized Au-NKF-5-3 can be clearly noticed. The bright dots on the surface is the Au NPs which was distributed uniform. The specific surface area of the NKF-5-3 is characterized by Brunauer–Emmett–Teller (BET) analysis (Figure S2). Pore distributions and pore volume are calculated by using the adsorption branch of the N2 isotherms. The specific surface area by BET analysis is 337 m2/g. The most possible pore volume (p/p0 = 0.97) from adsorption curve is 0.22 cm3/g, and the pore diameter is 2.7 nm based on the BJH model. 3.2. Characterization of S-GS. S-GS is synthesized by the click chemistry for the first time. From Figure 1E and 1F, the paper-like S-GS can be observed with thin layer structure. The EDS analysis (Figure 1G) indicates the S-GS mainly consist of the elements of S, C and O. The thickness is about 4-7 nm with about 7-10 layer graphene (the AFM analysis in Figure 1H). Due to the S doping, the S-GS can adsorb more Au NPs without any chemical modification. Figure 1I and 1J can verify it. And the EDS analysis (Figure 1K) indicates the Au-S-GS is well synthesized. To illustrate the of structure of as-synthesized S-GS, XPS is conducted in this research. From the structure of S-GS in Figure 1L, we can see the S element exists in two kinds of



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Figure 1. (A) (B) SEM images of GO-PANI. (C) SEM image of Au-NKF-5-3. (D) TEM image of Au-NKF-5-3. SEM (E) and TEM (F) images of S-GS. EDX analysis (G) and AFM image (H) of S-GS. (I) (J) SEM images of Au-S-GS. (K) EDX analysis of Au-S-GS. (L) XPS spectrogram of S-GS. (M) Schematic diagram of S-GS. (N) (O) (P) the elements of S, C and O XPS analysis in S-GS.

conditions. In Figure 1N, the peaks at 163.2 eV and 164.5 eV are consistent with the -C-S-C- structure (S 2p3/2 and S 2p1/2, the yellow annulus in Figure 1M). The peaks at 167.8 eV and 169 eV are consistent with the -C-SOX-C- structure (S 2p3/2 and S 2p1/2, the white annulus in Figure 1M). In Figure 1O, the binding energies (BEs) at 284.5 eV, 285 eV, 286.6 eV and 288.7 eV are ascribed to the C=C/C-C, C-O/C-S, O-C=O and C=O, respectively. And the O element analysis (Figure 1P) is attributed to the C-O interaction. The XPS further indicates the S-GS is well-synthesized. 3.3. Optimization of the experimental conditions. In order to get a good performance of the immunosensor, the experimental conditions are optimized. In this tri-antibody dual-channel biosensing platform, the ratio of antibodies is essential for the immunosensor property. Anit-HIgG is used as the framework to amply the capacity of other antibodies. From the optimized result (Figure 2A), 2:4:4 (anti-HIgG : anti-NMP22 : anti-CEA) is selected as the appropriate ratio of these three antibodies. The quantity of anti-HIgG is important. The captured anti-HIgG can

reduced if the anti-HIgG is too little, which cannot be incubated the immunosensor effectively. However, the detected CEA and NMP22 can reduced if the anti-HIgG is too much, which the antiHIgG can occupy much space without enough room for CEA/NMP22 immunoreaction. Therefore, the appropriate antibody ratio is necessary. Based on the 2:4:4 ratio, other conditions are optimized. PBS solution is applied as the acid-base environment for the experiment, and pH = 7.4 is the best condition (Figure 2B). The activity of the biomolecules and the dye property (TB, NR) are alterative if an overly basic or acidic surroundings exists. The base material (GO-PANI) is acted as an amplifier to enlarge the specific surface area. In Figure 2C, 1.3 mg/mL is the optimal concentration. This result can be explained as follows: at low concentrations, GO-PANI can increase the electro conductivity and increase the contact area, which may drive a positive correlation. However, at high concentrations, GOPANI electro conductivity is insufficient to significantly offset the layer resistance of the sheets. Thus, the optimum concentration is selected as 1.3 mg/mL. In order to improve the incubation efficiency and reduce a waste of time, incubation time is also


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tested. In Figure 2D, 50 min is the optimal time which owns the efficiency and effect.

3.5. Selectivity, stability, and reproductivity of the immunosensor. Selectivity is an essential section in the immunosensor property test. In this research, it is tested with 10 times quantity (0.1 ng/mL) of alpha fetoprotein (AFP), BSA, HIgG, prostate specific antigen (PSA), and breast cancer susceptibility gene BRCA1 addition than NMP22 (0.01 ng/mL). The current change of CEA is less than 2.8% (Figure S4B) and the current change of NMP22 is less than 5.02% (Figure S4A), indicating an acceptable selectivity. Stability is conducted to evaluate the storage time of the fabricated immunosensor. Several well-fabricated immunosensors are stored in freezer at 4 °C before testing. The immunosensor is tested every five days. The result indicates the storage stability is about half a month within 88% of initial response (Figure S5). After 15 days, the current changes alter a lot, suggesting an unstable property which may be due to the biomolecule activity loss or the tri-antibody framework collapse.

Figure 2. Optimization of the experimental conditions: (A) Ratio of antibodies; (B) pH; (C) GO-PANI concentration; (D) Incubation time; Error bar = SD, n = 5; red line: CEA, black line: NMP22.

3.4. Analysis and detection. Under the optimal experimental conditions, the tri-antibody dual-channel immunosensor is used to detect different concentrations of CEA and NMP22. As shown in Scheme 2，NR and TB are used as the signals to quantify the antigens. SWV is used in the research. The detected two antigens can be distinguished by different SWV redox peaks. NR is around at -0.65 V which is related to the NMP22, and TB is about -0.3 V related to CEA concentration. The signal peaks are shown in Figure 3A. The linear ranges of CEA and NMP22 are all from 0.1 pg/mL - 0.3 ng/mL in Figure 3B. The NMP22 calibration curve is Y = 14.9 + 2.4logC (black line in Figure 3B), and the detection limit is 25 fg/mL. The CEA calibration curve is Y = 9.7 + 1.5logC (red line in Figure 3B), and the detection limit is 30 fg/mL. The results compares favourably to the behaviors of many recent reported multianalysis (Table S1). This new strategy exhibits low detection limit which can be attributed to four reasons: First, the base GO-PANI can provide a large specific surface area without too much resistance addition; Second, S-GS is synthesized successfully through click chemistry, and it is applied in the immunosensor fabrication appropriately; Third, NKF-5-3 is used in the labels to amplify the signal loading, leading to the accurate detection of two antigens; Last, tri-antibody dual-channel sensing platform is a new effective strategy in biomarker detection. In addition, electrochemical impedance spectroscopy (EIS) was also proceeded to verify the successful layer-by-layer assembly (Figure S3).

Reproductivity is to certify the precision of this strategy. The 5 well-prepared immunosensors were conducted. The result of this 5 immunosensor was acceptable with the RSD of 1.87% (CEA) and 2.98% (NMP22) in Figure S6. 3.6. Clinical analysis. To evaluate the performance of the triantibody dual-channel strategy, clinical analysis is conducted in this research in the Hospital of University of Jinan. Human urine samples are tested by standard addition method. The urine samples are diluted with ultrapure water to 100 times (Table 1). The RSD is 2.1% and 3.2% of CEA and NMP22. The recovery is 97.2% and 96.2% of CEA and NMP22, respectively. F-test is conducted to determine the accuracy whether there is a significant difference.41 Based on the F-test, the calculated F value (Equation 1) is less than the theoretical one (F = 6.39 at 95% confidence limits), demonstrating the precisions of these two methods are highly equivalent. Equation (1)

The t-test41 is also proceeded to evaluate the tri-antibody dualchannel strategy and ELISA method difference (Table 2). By ttest analysis (Equation 2), the mean values are not obviously different from the data gained from ELISA kit. The t value is less than 2.78 (P = 0.95, α = 0.05, f = 4). The results show the system error can be ignored. Equation (2) Matrix effect is the influence by other potential substances apart from the molecules to be tested based on CLSI (Clinical and laboratory Standards Institute). Matrix effect is evaluated based on calibration curve method and standard addition method.36 The calibration curve is an important method which cannot eliminate the matrix effect influence, however, standard addition method is opposite. Therefore, the influence of matrix effect can be ignored if these two methods exist no obvious difference.

Figure 3. (A) SWV curves of different antigen concentrations (a-g: 0.1, 0.5, 1, 10, 50, 100, 500 pg/mL): (B) Calibration curve of CEA (red line) and NMP22 (black line); Error bar = SD, n = 5.

Five different concentrations (10 pg/mL, 20 pg/mL, 30 pg/mL, 40 pg/mL, 50 pg/mL) CEA and NMP22 are added in the diluted urine sample (1:100). For CEA, five current values obtained (11.32 µA, 11.48 µA, 11.66 µA, 11.73 µA, 11.84 µA). Based on



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Table 1 Results for the detection of CEA and NMP22 in human urine sample Sample content CEA NMP22

Found in diluted urine (pg/mL) 46.5, 44.2, 45.7, 44.7, 47.6 13.7, 12.9, 13.4, 11.8, 12.5

Mean (pg/mL) 45.7

Added ApoE (pg/mL) 100

12.9

100

Found (pg/mL) 145.7, 140.1, 146.2, 143.4, 148.0 115.8, 108.2, 112.6, 109.8, 116.4

Mean (pg/mL) 144.7

RSD (%) 2.1

Recovery (%)

112.6

3.2

96.2

97.2

Table 2 F-test and t-test about the human urine analysis ELISA Mean s RSD This methoda Mean s RSD Fb tb (pg/mL) (pg/mL) (%) (pg/mL) (pg/mL) (%) value test 46.2, 44.1, 45.1 0.97 2.1 46.5, 44.2, 45.7, 45.7 1.37 3.0 2.0 0.98 44.7 44.5, 44.7, 47.6 46.1 NMP22 12.8, 12.5, 12.5 0.65 5.2 13.7, 12.9, 13.4, 12.9 0.75 5.8 1.3 1.2 12.1, 13.4, 11.8, 12.5 11.7 a The method is standard addition method above mentioned; b The t- and F- values refer to the proposed ELISA kit. The theoretical values at 95% confidence limits: F = 6.39, t = 2.57 Sample content CEA

the “logarithm-type” Gran graphing method, to get the graph in rectangular coordinate system, a modified formula similar to Nernst equation was applied in this research. That is according to φ = k + S · logC (Equation 3). The equation 4 is a transformed one on immunosensor calibration curve. Here, y is the current change; b is the intercept of the CEA calibration curve, k is the slope of the CEA calibration curve, respectively. C = 10-k/S • 10φ/S = K • 10φ/S

Equation (3)

C = 10-y/b • 10y/k = K • 10y/k

Equation (4)

The tri-antibody dual-channel fabrication platform for bladder carcinoma biomarkers multianalysis is constructed based on S-GS by click chemistry. The S-GS was synthesized for the first time and it is an all-right candidate in framework fabrication. The detection limit of NMP22 and CEA are 25 fg/mL and 30 fg/mL, respectively. The immunosensor was analyzed by clinical analysis, including F-test, t-test, ELISA, and the influence of matrix effect, revealing an acceptable result. It can be developed into a promising tool in clinical diagnosis.

■ ASSOCIATED CONTENT Supporting Information Figure S1 ~ S7; Table S1; the influence of NMP22 matrix effect; This material is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected] (Q. Wei) ORCID Qin Wei: 0000-0002-3034-8046

Notes The authors declare no competing financial interest. Figure 4. Gran graph about the matrix influence of CEA

Based on Equation 4, five ordinate values can be calculated. According to the five points (10, 428391), (20, 514568), (30, 632405), (40, 685206), and (50, 777226), the Gran graph was drawn (Figure 4). The calibration curve is Y* = 372207 + 8596 X*, r = 0.99. In terms of standard addition method, the extension of the curve with abscissa can generate a point (Figure 4, point A). The abscissa value (point A) is the urine sample content of CEA. Thus, X* = – 43.3 (Y* = 0). Here, RSD = (43.3 – 45.1)/45.1 × 100% = – 4.0%, indicating the matrix effect of CEA can be ignored in this research. The matrix effect of NMP22 is in the same way (SI, Figure S7).

■ ACKNOWLEDGMENTS This work was supported by the Special Foundation for Taishan Scholar Professorship of Shandong Province (No.ts20130937); the National Key Scientific Instrument and Equipment Development Project of China (No.21627809); National Natural Science Foundation of China (Nos.21375047, 21377046, 21405059, 21575050, 21505051); the China Postdoctoral Science Foundation (2017M612170); the Natural Science Foundation of Shandong Province (No.ZR2017MB027); Graduate Innovation Foundation of UJN (YCXB15004).

■ REFERENCES

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