A colorimetric immunosensor based on self-linkable dual-nanozyme for ultrasensitive bladder cancer diagnosis and prognosis monitoring
Introduction
Bladder cancer (BC) is a tumor associated with high morbidity and mortality, and it is the fourth most common type of cancer (Siegel et al., 2018). The earlier that BC is found and treated, the better the outcome. Currently, cystoscopy is used as the gold standard for monitoring tumor diagnosis and progression as well as tumor recurrence; it is an invasive method associated with high costs and patient discomfort. Overall estimated costs for treatment and monitoring range between US$ 90,000 and US$ 200,000 per patient in the United States, because of the high recurrence rate and disease progression, which necessitates careful long-term monitoring (Yeung et al., 2014). Furthermore, it is the most expensive cancer to treat per patient. By contrast, urine cytology is specific and noninvasive for diagnosing BC, but it is limited by its poor sensitivity in low-grade tumors, which grow slowly and are less aggressive (Maas et al., 2018, Shao et al., 2017). Therefore, the invasiveness of cystoscopy and the limitations of cytology in detecting BC have generated the following critical goal for managing patients with BC: to replace cystoscopy with the examination of voided urine, to reduce the frequency of cystoscopy. Voided urine can be collected more easily than other bodily fluids, such as blood and cerebrospinal fluid. Approximately 70% of urinary proteins are estimated to derive from the kidney and urinary tract, and abundant plasma proteins are present at high concentrations in urine specimens (Thomas et al., 2016; Chen et al., 2012). Cancer-related biomarkers (including circulating tumor protein and circulating tumor DNA/RNA) not only enable the detection of early stage tumors with high accuracy, but also the monitoring of tumor recurrence and progression, as well as the prediction of tumor response to therapeutic approaches (Kang et al., 2017). In cancer patients, free DNA in serum or urine is increased to a mean of 180 ng/mL compared with 13 ng/mL in healthy individuals, which is too low (pM concentration range) to accurately detect (Pursey et al., 2017). To date, a high content of mutated genes has been reported in patients with BC compared with healthy individuals. These genes are known as a tumor suppressor gene (E. Cad), a mediator of cell death (DAPK) and of cell growth gene (RARβ). Detection of these genes simultaneously in voided urine has been reported to diagnose BC with an accuracy of 90.9% and with a detection limit of 10 ng/mL (Yokoi et al., 2017). Moreover, FGFR3 (S249C) and HRAS (G13R) mutations are associated with low-grade bladder tumors; these mutations are mutually exclusive and occur in over 80% of low-grade tumors (Kompier et al., 2010, Leiblich, 2017).
In addition, tumor-specific proteins are potential biomarkers for early BC diagnosis and prognosis monitoring, a variety of candidate bladder cancer biomarkers such as RT112 cell CDH1, FHIT, LAMC2, RASSF1A, TIMP3, SFRP1, SOX9, PMF1, and RUNX3, have been identified but require further validation (Roberts et al., 2013; Kandimalla et al., 2013). Among them, apolipoprotein A1 protein (ApoA1), apolipoprotein A2 protein (ApoA2), and nuclear matrix protein 22 (NMP22) have recently been identified as a new biomarker that occurs at highly elevated rates in pooled BC. The relative difference in the levels of the abovementioned proteins in urine is a highly significant factor. The urinary level of ApoA1 in patients with BC is much higher than in normal people, indicating its potential utility in the development of a reliable BC detection assay (Chen et al., 2013; Tsai et al., 2018; Pichler et al., 2017). To date, an enzyme-linked immunosorbent assay (ELISA) is a commonly used traditional method to quantify the level of ApoA1 molecules in biofluids (Zell et al., 2016); however, the cost is relatively high and the enzyme used to catalyze H2O2 for producing signals—horseradish peroxidase (HRP)—is environmentally unstable (Garg et al., 2015). Therefore, various types of enzyme-mimicking nanomaterial have been developed to overcome the drawbacks of enzymes, because of their superior H2O2 catalyzing efficiency, high stability, and low costs, such as iron oxide (IO) (Tian et al., 2018a, Tian et al., 2018b), Prussian blue (PB) (He et al., 2017, Farka et al., 2018), MoS2 (Hassanzadeh and Khataee, 2018), and G-quadruplex (G4) DNA-hemin complex (Li et al., 2018; Liu et al., 2018). Among them, the ferrous ions provided by IO or PB are widely utilized to catalyze H2O2-forming hydroxyl radical (OH·), which can oxidize 3,3′,5′,5′-tetramethylbenzidine (TMB) as peroxidase-mimic materials.
Herein, we prepare the ApoA1 antibody (AbApoA1)-functionalized glassy chip (biochipApoA1) and peroxidase-mimicking, PB-incorporated magnetic graphene oxide (PMGO) with excellent environmental stability and self-chain linking reaction ability to construct an enzyme-free ultrasensitive immunosensor for accurately determining ApoA1 concentration in urine. Our colorimetric immunosensor with self-linkable PMGOs for signal amplification exhibits a rapid and sensitive ApoA1 detection with a wide linear detection range, which has potential for easy clinical BC diagnosis and prognosis monitoring.
Section snippets
Preparation of ApoA1 antibody-modified glassy chip
To prepare the ApoA1 antibody (AbApoA1; chicken host)-modified glassy chip for capturing ApoA1, a glassy chip with a diameter of 5 mm was first cleaned using a mixture of tris-buffered saline and Tween 20 (TBST), acetone, ethanol, and DI-H2O. Subsequently, the cleaned chip was oxidized with HCl/methyl alcohol (volume ratio = 1:1) at 120 °C for 3 h to produce more hydroxide groups on the chip's surface. Next, the chip was soaked in APTES solution (10% in PBS buffer, pH 7.4) for 2 h to form a
Characterization and confirmation of prepared glassy biochips and PMGO
We envisioned a signal amplification approach to obtain a colorimetric immunosensor for determining ApoA1 concentration in patients’ urine. The ultrasensitive immunosensor was constructed with biochipApoA1 and self-linkable peroxidase-mimic PMGO. After the samples were incubated and ApoA1 was captured on the biochipApoA1, the PMGO-1 was functionalized with AbApoA1 and mouse IgG (PMGO-1), and then rabbit anti-mouse IgG antibody (PMGO-2) and goat anti-rabbit IgG antibody (PMGO-3) were added
Conclusion
We successfully developed self-linkable PMGO as a peroxidase-mimicking nanozyme for signal amplification to form an ultrasensitive immunosensor with biochipApoA1 for the simple and rapid diagnosis and prognosis monitoring of BC. As a result of this approach, our study demonstrated that the colorimetric immunosensor can accurately detect the ApoA1 concentration in urine as well as an ELISA assay can, and the self-signal amplification strategy can significantly widen the detection range of ApoA1,
Acknowledgments
This work was financially supported by the Ministry of Science and Technology (MOST107–2221-E-182-019, MOST106–2628-E-110-001-MY3, MOST106–2628-B-110-001-MY4), Chang Gung Memorial Hospital (CMRPG3D1091-3, CMRPG3D0501-3, CMRPG3D0103, CIRPG3E2041), and Formosa Plastics Corporation (FCRPD2H0011), Taiwan, R.O.C. We would also like to thank the Chang Gung Memorial Hospital Microscopy Core Laboratory for their assistance with TEM and EDS.
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C. Peng and M.Y. Hua contributed equally to this work.