Sensitive fluorescence detection of saliva pepsin by a supramolecular tandem assay enables the diagnosis of gastroesophageal reflux disease

ABSTRACT

Gastroesophageal reflux disease (GERD) is a prevalent gastrointestinal disorder with substantial morbidity. However, the reliable, non-invasive, and specific diagnosis of GERD remains a challenge due to the lack of specific symptoms and imaging features. Herein, we report a supramolecular tandem assay through using a reporter pair of sulfonatocalix[4]arene and lucigenin for the sensitive and real-time detection of saliva pepsin, an ideal biomarker for GERD. Moreover, we achieve the detection of pepsin in commercial artificial saliva, demonstrating the potential of this supramolecular approach for point-of-care testing of GERD.

GRAPHICAL ABSTRACT

KEYWORDS:

• Supramolecular tandem assay
• calixarene
• lucigenin
• saliva pepsin
• gastroesophageal reflux disease

Acknowledgments

The authors gratefully thank National Natural Science Foundation of China (NSFC grants 31961143004 and 21672112) for financial support. Z. Zheng acknowledges funding support from the China Scholarship Council (grant 201806200075). All authors also thank Prof. Eric V. Anslyn at the University of Texas at Austin for his helpful discussions.

Disclosure statement

The authors declare no conflict of interest.

Experimental section

All solvents and reagents were commercially available and were used as received. LCG was obtained from Tokyo Chemical Industry. Human insulin was purchased from the Dalian Meilun Biotechnology Company. Human pepsin (3511 U mg^–1) was purchased from Sigma Aldrich. Artificial saliva was purchased from the Shanghai Yuanye Bio-Technology Company. p-Sulfonatocalix[4]arene tetrasodium was synthesized according to a reported method [46].

The PB buffer solution (pH 2.0) was prepared by dissolving phosphoric acid (0.98 g) in double-distilled water (900 mL). This solution was titrated to pH 2.0 using NaOH and made up to a volume of 1000 mL with distilled water. The pH value of the PB buffer was then verified using a calibrated pH meter. The artificial saliva was titrated to pH 2.0 with phosphoric acid prior to use.

Steady-state fluorescence spectra were collected using a Cary Eclipse equipped with a Cary single-cell Peltier accessory and a conventional quartz cell (light path, 10 mm). All mean values from the fluorescence titrations, limits of detection, and calibration lines were measured from at least three repeated experiments, and the errors are given as standard deviations (±1σ). The fluorescence data from the host–guest titrations were fitted in a nonlinear manner. The fitting pattern can be obtained from the website of Nau’s group (http://www.jacobs-university.de/ses/wnau). The enzyme assays were carried out in quartz cuvettes and the fluorescence intensity was monitored in the time-scan mode. The pepsin assays in PB buffer with the SC4A·LCG reporter pair were carried out in the presence of 1.0 μM LCG, 1.5 μM SC4A, 0.6–4.0 μM insulin, and 20 U mL^–1 pepsin at pH 2.0, 37°C, λ_ex = 368 nm, and λ_em = 510 nm. The pepsin assays in artificial saliva with the SC4A·LCG reporter pair were carried out in the presence of 2.0 μM LCG, 3.0 μM SC4A, 4.0 μM insulin, and 1.0–10.0 μg mL^–1 pepsin at pH 2.0, 37°C, λ_ex = 368 nm, and λ_em = 510 nm.

The K_M value of pepsin was obtained using the Michaelis–Menten model (Equation 1(1) ${\nu }_{\mathbf{0}}=\frac{k_{cat}\left[E_0\right]\left[S_0\right]}{K_M+\left[S_0\right]}$(1) ), where v₀ is the initial rate of the enzymatic reaction, k_cat is the catalytic rate constant, [E]₀ is the initial concentration of enzyme, [S]₀ is the initial concentration of substrate and K_M is the Michaelis–Menten constant. The value of K_M was obtained using the Lineweaver–Burk equation (Equation 2(2) $\frac{1}{{\left[v\right]}_0}=\frac{K_M}{k_{cat}\left[E_0\right]}\times \frac{1}{{\left[S\right]}_0}+\frac{1}{k_{cat}\left[E_0\right]}$(2) ), where 1/k_cat [E]₀ is the y-intercept, and K_M/k_cat[E]₀ is the slope of the curve.

(1) ${\nu }_{\mathbf{0}}=\frac{k_{cat}\left[E_0\right]\left[S_0\right]}{K_M+\left[S_0\right]}$(1)

(2) $\frac{1}{{\left[v\right]}_0}=\frac{K_M}{k_{cat}\left[E_0\right]}\times \frac{1}{{\left[S\right]}_0}+\frac{1}{k_{cat}\left[E_0\right]}$(2)

Additional information

Funding

This work was supported by the China Scholarship Council [201806200075]; National Natural Science Foundation of China [21672112,31961143004].