Sensitive Electrochemical Immunosensor for Detection of Nuclear Matrix Protein-22 based on NH2-SAPO-34 Supported Pd/Co Nanoparticles

A novel sandwich-type electrochemical immunosensor using the new amino group functionalized silicoaluminophosphates molecular sieves (NH2-SAPO-34) supported Pd/Co nanoparticles (NH2-SAPO-34-Pd/Co NPs) as labels for the detection of bladder cancer biomarker nuclear matrix protein-22 (NMP-22) was developed in this work. The reduced graphene oxide-NH (rGO-NH) with good conductivity and large surface area was used to immobilize primary antibody (Ab1). Due to the excellent catalytic activity toward hydrogen peroxide, NH2-SAPO-34-Pd/Co NPs were used as labels and immobilized secondary antibody (Ab2) through adsorption capacity of Pd/Co NPs to protein. The immunosensor displayed a wide linear range (0.001–20 ng/mL) and low detection limit (0.33 pg/mL). Good reproducibility and stability have showed satisfying results in the analysis of clinical urine samples. This novel and ultrasensitive immunosensor may have the potential application in the detection of different tumor markers.

In this work, a sandwich-type electrochemical immunosensor for the detection of NMP-22 was prepared by using NH 2 -SAPO-34-Pd/Co NPs as labels and rGO-NH as sensing platform for the signal amplification. The large surface area of rGO-NH could increase the loading of Ab 1 and the good conductivity of rGO-NH could promote the electron transfer. The high catalysis of NH 2 -SAPO-34-Pd/Co NPs toward the reduction of H 2 O 2 could improve the sensitivity of the immunosensor. Therefore, this simple, economic and sensitive immunosensor could be widely used in the clinical analysis.

Experimental
Materials and reagents. NMP-22 antigen and antibody were purchased from Guyan Biotech Co., Ltd.
Apparatus. All electrochemical measurements were performed on a CHI760D electrochemical workstation (Shanghai CH Instruments Co., China). Scanning electron microscope (SEM) and Energy Dispersive X-Ray Spectroscopy (EDS) were recorded by JEOL JSM-6700F microscope (Japan). A conventional three-electrode system was used for all electrochemical measurements: the modified glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and platinum wire electrode as the counter electrode.
Preparation of NH 2 -SAPO-34. 0.1 g of SAPO-34 powder, 0.1 mL of 3-ammonia propyl triethoxy silane and 10 mL of anhydrous ethanol were added into the three necked flask, the mixture was heated to and kept at 70 °C for 1.5 h. Then the product was cooled down to room temperature and collected by centrifugation (7000 rpm, 5 min). Finally, the product was collected after washing and drying in vacuum at 40 °C.

Synthesis of NH 2 -SAPO-34-Pd/Co NPs.
The NH 2 -SAPO-34-Pd/Co NPs were synthesized by a modified two-step reduction route under the protection of high-purity nitrogen in an ice bath 41 . Firstly, 4 mL of 0.2 mol/L Co(NO 3 ) 2 , 2.5 mL of 64 mmol/L sodium citrate, 30 mg of NH 2 -SAPO-34 and 25 mL of ultrapure water were mixed ultrasonically for 20 min. Then, 5 mL of 1.6 mol/L NaBH 4 solution was added into the above mixture at a rate of 20 mL/h under vigorous stirring to generate the Co NPs. Secondly, 10 mL of 40 mmol/L Na 2 PdCl 4 solution and 10 mL of 0.16 mol/L NaBH 4 solution were synchronously added into the solution of Co NPs at a rate of 20 mL/h. Finally, the resulting NH 2 -SAPO-34-Pd/Co NPs was filtrated, washed with ultra-pure water for more than three times and then dried in vacuum at 35 °C.

Preparation of NH 2 -SAPO-34-Pd/Co-Ab 2 .
The NH 2 -SAPO-34-Pd/Co-Ab 2 was synthesized by the following steps ( Fig. 1(a)). A solution of NH 2 -SAPO-34-Pd/Co NPs (2 mg/mL, 1 mL) was added into Ab 2 dispersion (10 μg/mL, 1 mL) and stirred for 12 h at 4 °C. After centrifugation, the resulting NH 2 -SAPO-34-Pd/Co-Ab 2 was dispersed in 1 mL of PBS at pH 7.0 and stored at 4 °C. Figure 1(b) showed the fabrication procedure of the immunosensor. A glassy carbon electrode was polished to a mirror-like finish with 1.0, 0.3 and 0.05 μm alumina powder and then thoroughly cleaned. Afterwards, 6.0 μL of rGO-NH solution (1.5 mg/mL) dispersed in chitosan (0.1 wt%) was dropped onto the electrode surface and then dried at room temperature. The utilization of chitosan could make rGO-NH forming a film on the electrode surface and the abundant amino groups in CS could provide active sites for Ab 1 immobilization. To immobilize the Ab 1 onto the electrode, 3.0 μL of GA (2.5%, v/v) was dropped onto the electrode surface and incubated until it was half-dry. Then, 6.0 μL of Ab 1 (10 μg/mL) was dropped onto the electrode surface and then incubated for 1 h. In this procedure, GA was used as cross-linking agent to link amino groups of antibody with amino groups of CS. After drying, the electrode was incubated in 1 wt% BSA solution for another 30 min to eliminate nonspecific binding sites. Subsequently, NMP-22 solution with different concentrations were dropped onto the electrode surface and incubated for 1 h, and the excess antigen was rinsed away with water. Finally, 6.0 μL of the prepared NH 2 -SAPO-34-Pd/Co-Ab 2 solution was dropped onto the electrode surface and bound to NMP-22 via the specific antibody-antigen interaction. After incubation, the electrode was then rinsed and stored at 4 °C before use. The label NH 2 -SAPO-34-Pd/Co could catalyze the reduction of H 2 O 2 , so that different current response could be generated in accordance with NMP-22 concentration when 10 μL of H 2 O 2 (5.0 mol/L) was added into 10 mL of PBS under magnetic stirring.

Results and Discussion
Characterization of rGO-NH and NH 2 -SAPO-34-Pd/Co NPs. The rGO-NH with large surface area was used to increase the amount of captured Ab 1 . The SEM images (Fig. 2a,b) of the rGO-NH showed that the rGO-NH had a wrinkle paper-like structure with irregular size, further indicating the large surface area of rGO-NH. The SEM image of NH 2 -SAPO-34 was shown in Fig. 2c, which showed the NH 2 -SAPO-34 possessed cubic structure. The NH 2 -SAPO-34 had a BET surface area of 536.6 m 2 /g. Due to the large surface area, more Pd/Co nanoparticles could be loaded on the surface of NH 2 -SAPO-34 and the SEM images (Fig. 2d,e) of NH 2 -SAPO-34-Pd/Co presented that the NH 2 -SAPO-34 was coated successfully. Elemental compositions of NH 2 -SAPO-34-Pd/Co NPs were analyzed by EDS (Fig. 2f). Signature peaks of Si, O, P, Al, Pd and Co were observed, indicating that the Pd/Co nanoparticles were formed successfully on the surface of NH 2 -SAPO-34.
Characterization of NH 2 -SAPO-34-Pd/Co NPs modified electrode. The performance of NH 2 -SAPO-34-Pd/Co toward the H 2 O 2 reduction was investigated (Fig. 3). As shown, the electrode modified by NH 2 -SAPO-34-Pd/Co-Ab 2 exhibited obvious current change toward H 2 O 2 . However, the electrode did not appear to be electroactive toward water. The NH 2 -SAPO-34-Pd/Co NPs showed the ability to promote the reduction of H 2 O 2 and resulted in the generation of electrochemical signals.
Characterization of the immunosensor. The stepwise modified process of electrode was characterized by cyclic voltammetry (CV). CV can also characterize the modification process of the immunosensor besides electrochemical impedance spectroscopy, and each immobilization step was shown in Fig. 4. It could be seen that  a pair of well-defined redox peak was observed on GCE (curve a), and this quasi-reversible one-electron redox peak was attributed to the transformation between Fe(CN) 6 4− and Fe(CN) 6 3− . The redox peak current increased strongly after rGO-NH was dropped onto the electrode surface (curve b), which suggested the rGO-NH had good conductivity and strong ability of electron transfer. The redox peak current decreased significantly after GA was dropped onto the rGO-NH modified electrode (curve c), which could be attributed to the large impedence of GA. The redox peak current decreased gradually when Ab 1 (curve d), BSA (curve e) and NMP-22 (curve f) as the non-conductive bioactive substances were modified layer by layer on the electrode. The results suggested that the non-conductive bioactive substances were immobilized onto the electrode successfully and blocked electron exchange between the redox probe and the electrode. The redox peak current decreased to the minimum (curve g) when NH 2 -SAPO-34-Pd/Co-Ab 2 were immobilized, indicated the formation of hydrophobic immunocomplex layer could embarrass electron transfer. As a result, the immunosensor was modified successfully.
Optimization of experimental conditions. To achieve an optimal electrochemical signal, the influence of the pH value of substrate solution to the immunosensor was investigated at first. Herein, 6.0 μL of rGO-NH solution (1.5 mg/mL) dispersed in chitosan (0.1 wt%) was dropped onto the electrode surface. Then the same amount of Ab 1 (6.0 μL, 10 μg/mL) and NMP-22 (6.0 μL, 10 ng/mL) were used to fabricate the immunosensors. As shown in Fig. 5(a), it could be found that the current response increased with increasing pH value from 5.8 to 7.0 to reach the maximum and decreased from 7.0 to 7.9. The reason is that the highly acidic or alkaline surroundings would damage the activity of immobilized protein 42 . Therefore, pH 7.0 PBS was selected for the test throughout this study.
Apart from the pH value of substrate solution, the concentration of rGO-NH was also an important parameter, which could affect not only the loading of captured antibody (Ab 1 ) but also the electrochemical behaviors of the rGO-NH modified electrode. In detail, different concentrations of rGO-NH from 0.5 mg/mL to 2.0 mg/mL with the same amount of Ab 1 (6.0 μL, 10 μg/mL) and NMP-22 (6.0 μL, 10 ng/mL) were employed to modify the electrodes. As seen in Fig. 5(b), with the increasing concentration of rGO-NH, the current response for detection of 10 ng/mL NMP-22 increased due to the enhanced loading of Ab 1 for bound more antigens and then  NH 2 -SAPO-34-Pd/Co-Ab 2 . However, when the concentration of the rGO-NH was more than 1.5 mg/mL, the current response decreased. Therefore, the optimal concentration of rGO-NH solution was 1.5 mg/mL.
Under the optimum conditions, the immunosensors using NH 2 -SAPO-34-Pd/Co NPs as labels were prepared for the detection of different concentrations of NMP-22 in pH 7.0 PBS at − 0.4 V. The relationship between the current response toward 5.0 mmol/L H 2 O 2 and NMP-22 concentration was shown in Fig. 6. As can be seen, the current response increased linearly with the increasing concentration of the NMP-22 in the range from 0.001 to 20 ng/mL, with a detection limit of 0.33 pg/mL based on S/N = 3. The detection limit of this immunosensor is significantly lower than other methods [43][44][45] , as shown in Table 1. The calibration curve was linear with a correlation coefficient of R 2 = 0.998 (ΔI = 0.434 c + 9.16).
To the further development of techniques, lower limit of detection is a major criterion of successful application. The low detection limit may be attributed to three factors: (1) The rGO-NH with large surface area could greatly increase the loading of Ab 1 and promote electrons transfer because of its good conductivity; (2) SAPO-34 molecular sieves with large surface area could increase the loading of Pd/Co nanoparticles, which means more Ab 2 could be loaded onto the label; (3) The good catalytic activity of NH 2 -SAPO-34-Pd/Co-Ab 2 toward H 2 O 2 could increase the sensitivity of the immunosensor. Hence, the proposed strategy could provide a stable immobilization and sensitized recognition platform for analytes such as micromolecules and possesses promising application in clinical sample.

Linear range Detection limit Reference
Electrochemical immuno-biosensor 1. Reproducibility, selectivity and stability. To evaluate the reproducibility of the immunosensor, a series of five electrodes were prepared for the detection of NMP-22 (5 ng/mL). The results of the measurements were 11.7, 11.1, 11.9, 10.8 and 11.0 μA, respectively. The relative standard deviation (RSD) of the measurements for the five electrodes was 4.2%, suggesting the precision and reproducibility of the proposed immunosensor were good. The selectivity of the immunosensor was also investigated. Interferences study was performed by using bovine serum albumin (BSA), vitamin C, trioxypurine and glucose. NMP-22 (5 ng/mL) solutions containing 500 ng/mL of interfering substances were measured by the immunosensor. The results were shown in Fig. 7. The current variation due to the interfering substances was less than 5.0% of that without interferences, indicating that the selectivity of the immunosensor was acceptable.
Stability of the immunosensor is also a key factor in their application and development. The stability of the immunosensor for 5 ng/mL NMP-22 was examined by checking periodically its current response. When the immunosensor was not used, it was stored at 4 °C. After ten days, the current of the immunosensor retained about 97% of its initial value. The good long-term stability may be ascribed to the good stability of the NH 2 -SAPO-34-Pd/Co and rGO-NH.

Real sample analysis.
To evaluate the potential of this immunosensor for real sample analysis, the practical detection for two urine samples covered by the calibration curve is further conducted. As shown in Table 2, the RSD between 1.6% and 6.2% were obtained. The recovery was in the range from 99.5% to 101.2%. Therefore, the immunosensor could be used in the clinical analysis.

Conclusion
The large surface area of rGO-NH could increase the amount of Ab 1 immobilized on the electrode surface and the good conductivity of rGO-NH could promote the electrons transfer. The NH 2 -SAPO-34 supported Pd/Co nanoparticles showed high catalysis toward the reduction of H 2 O 2 , which improve the sensitivity of the immunosensor. The immunosensor has adequate sensitivity and precision, with wide linear range and low detection limit of 0.33 pg/mL. Due to the advantages of simplicity, high selectivity and good reproducibility, this new immunosensor may have the potential application in the detection of different cancer biomarkers.