Chinese Chemical Letters  2019, Vol. 30 Issue (6): 1307-1309   PDF    
Fluorescent and colorimetric immunoassay of nuclear matrix protein 22 enhanced by porous Pd nanoparticles
Wenyun Zhuge1, Xiaofeng Tan1, Ruyue Zhang, He Li*, Gengxiu Zheng*     
School of Chemistry and Chemical Engineering, University of Jinan, Ji'nan 250022, China
Abstract: A modified ELISA realizing fluorescent and colorimetric immunoassay of nuclear matrix protein 22 (NMP 22) was developed based on porous Pd nanoparticles. The unique structure and excellent enzyme mimetic activity of porous Pd nanoparticles favor to oxidize o-phenylenediamine (OPD) into 2, 3-phenazinediamine (oxOPD) by H2O2, producing colorimetric and fluorescence dual-readout signal for the detection of NMP 22. The developed immunoassay method will offer great potential in clinical research and diagnostic applications.
Keywords: Nuclear matrix protein 22     Porous Pd nanoparticles     ELISA     Colorimetric     Fluorescence    

Reliable and sensitive detection of nuclear matrix protein 22 (NMP 22) has significant meaning for the diagnosis and treatment of bladder cancer [1, 2], because NMP 22 has been already certificated as a golden tumor biomarker for the diagnose of bladder cancer by the United States Food and Drug Administration (FDA) [3, 4]. Enzyme-linked immunosorbent assay (ELISA) is the predominate method for clinical diagnosis of NMP 22 [5, 6]. However, ELISA is still facing some drawbacks, such as high cost, tedious labelling procedure and easy inactivation of natural enzymes [7-10]. Therefore, it is a high desire to develop an improved ELISA using natural enzyme alternatives. Nanozymes, emerged as highly stable and low-cost alternatives to natural enzymes, have been extensively used for advancing the development of ELISA [11-14]. After Dr. Yan first introduced the concept of nanozyme [15], her group presented a fascinating study using Fe3O4 magnetic nanoparticle with peroxidase activity for diagnosis of Ebola [16].

In this work, a newmethod realizing fluorescent and colorimetric immunoassay of NMP 22 has been presented. The signal transduction was enhanced by a new kind of nanozyme—porous Pd nanoparticles, which were prepared by a "one-pot" green approach through slow corrosion in the presence of weak reducing agent— hydroquinone. The unique porous structure and good crystallinity of Pd nanoparticles favor the transfer of substrates and electrons, offering excellent peroxidase-like activity. The dual-readout immunoassay for NMP 22 canproduce fluorescent and colorimetricoutput signals conveniently and rapidly, and will have merits such as high accuracy, high noise-to-signal ratios, and low environmental interference [17, 18]. As shown in Fig. 1, we construct a sandwich immunoassay platform for NMP 22 base on porous Pd nanoparticles (NPs). After introducing o-phenylenediamine (OPD) and H2O2 into the constructed immunosensor, porous Pd NPs can efficiently oxidize OPD to 2, 3-diaminophenazine (oxOPD) in the present of H2O2, resulting significant colorimetry and fluorescence signals for sensitive and reliable detection of NMP 22 using microplate reader.

Fig. 1. Schematic illustration of the preparation process of porous Pd nanoparticles (A) and the fabrication process of the duel-readout immunoassay platform (B).

The porous Pd NPs were synthesized via a wet chemical reduction method, polyvinylpyrrolidone (PVP) was used as surfactant for dispersion of nanoparticles, and hydroquinone (HQ) as a weak reductive agent for the preparation of well-defined porous nanoparticles, its relatively weak reducing power resulted in slow and continuous nucleation and epitaxial growth of porous Pd NPs [19-21].

Uniformed Pd NPs with porous structure can be easily observed by the transmission electron microscopy (TEM) image (Fig. 2A), and the average diameter of NPs was approximately 44.76 nm (Fig. 2B). The high-resolution TEM (HRTEM) image indicated the porous Pd NPs were polycrystalline, where distinct lattice spacing was measured to be 0.22 nm, which can be ascribed to the (111) plane of fcc Pd structure (Fig. 2C). Moreover, selected area electron diffraction (SAED) patterns exhibited four bright rings, which were corresponding to (111), (200), (220) and (311) lattice planes of Pd, respectively. The crystallographic properties were also investigated by power X-ray diffraction (PXRD), it is can be seen from Fig. S1 (Supporting information) were also confirmed the good polycrystallinity of porous Pd NPs.

Fig. 2. (A) TEM image, (B) size distribution, (C) HRTEM image and (D) SAED pattern of porous Pd nanoparticles.

To investigate the mimetic peroxidase activity of porous Pd NPs, OPD as a classic nonfluorescent substrate, can be oxidized into the fluorescent product of oxOPD by H2O2 in the presence of peroxidase-like catalysts. As revealed in Fig. 3, after porous Pd NPs were introduced into OPD/H2O2 system, a stable fluorescence emission at 580 nm and an absorption peak at 450 nm attributed to oxOPD were appeared, indicating porous Pd NPs possess good peroxidase-like activity.

Fig. 3. Peroxidase-like activity of porous Pd NPs using OPD as substrate (180 mL OPD (5 mmol/L) + 10 mL Pd solution (200 mg/mL) + 10 mL H2O2 (40 mmol/L)) presenting by UV–vis spectra (A) and fluorescence spectra (B) for the OPD, OPD + H2O2 and OPD + H2O2 + porous Pd.

Under the optimized conditions (Fig. S4 in Supporting information), porous Pd NPs were utilized as signal labels for sensitive immunoassay of NMP 22 by colorimetric and fluorescence dual-modal signal readout. As shown in Fig. 4, with the increase of concentration of NMP 22, the signal intensity of either absorption or fluorescence was enhanced gradually. A linear relation between the dual signal and NMP 22 concentration in the range of 1–500 pg/mL was determined, respectively. For colorimetric detection, the linear calibration curve was fitted to be Y = 0.1288X + 0.6226 (R2 = 0.99), and the limit of detection was calculated to be 0.35 pg/mL (S/N = 3, Fig. 4B). And for fluorescence detection, the linear regression equation was fitted to be Y = 3517X + 17, 653 (R2 = 0.98), and the limit of detection was calculated to be 0.31 pg/mL (S/N = 3, Fig. 4D), the dual-modal immunoassay platform exhibited excellent analytical performance for the detection of NMP 22, which was comparable or better than those recently reported work (Table S1 in Supporting information).

Fig. 4. Dual-modal immunoassay of NMP 22 range from 1 pg/mL to 5 ng/mL: Absorbance response (A) and the corresponding calibration curve (B) (top of A: photograph of the color change in the well), fluorescence intensity (C) and the corresponding calibration curve (D). Error bar = RSD (n = 5)

Moreover, the developed dual-modal immunoassay platform presented good reproducibility and high selectivity for the detection of NMP 22 (Fig. S5 in Supporting information). The reliability of dual-mode immunoassay platform was investigated by standard addition method, different concentrations of NMP 22 were added into blank human serum. As shown in Table S2 (Supporting information), the recovery ranges from 95.2% to 105.1%, and relative standard deviation (RSD) range from 2.2% to 8.4%, which indicated that the proposed platform were reliable for clinical analysis of NMP 22.

In summary, a new modified ELISA enhanced by porous Pd nanoparticles for colorimetric and fluorescence dual-modal immunoassay of NMP 22 has been demonstrated. Benefited from different signal-readout and independent signal amplified mechanism, the improved ELISA will afford more reliable detection performance, which will bring high promising for clinical diagnosis.


The authors would like to thank the Natural Science Foundation of Shandong Province (No. ZR2017MB017) for the financial support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at

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