Ultrasensitive and specific fluorescence detection of a cancer biomarker via nanomolar binding to a guanidinium-modified calixarene

Abstract

We designed a water-soluble guanidinium-modified calix[5]arene to target lysophosphatidic acid (LPA), an ideal biomarker for early diagnosis of ovarian and other gynecologic cancers, achieving binding on the nanomolar level. An indicator displacement assay, coupled with differential sensing, enabled ultrasensitive and specific detection of LPA. Moreover, we show that using a calibration line, the LPA concentration in untreated serum can be quantified in the biologically relevant low μM range with a detection limit of 1.7 μM. The reported approach is feasible for diagnosing ovarian and other gynecologic cancers, particularly at their early stages.

Scheme 1. Schematic illustration of the binding between LPA and GC5A and the operating IDA principle of fluorescence “switch-on” sensing of LPA by the GC5A·Fl reporter pair.

Scheme 2. Synthetic route for GC5A. (a) K₂CO₃, RBr, CH₃CN, reflux, 72%; (b) HNO₃, AcOH, dry CH₂Cl₂, r.t., 46%; (c) SnCl₂·2H₂O, C₂H₅OH/AcOEt (1 : 1, v/v), reflux, 52%; (d) N,N′-bis-tert-butoxycarbonylthiourea, Et₃N, AgNO₃, dry CH₂Cl₂, r.t., 32%; (e) SnCl₄, AcOEt, r.t., 65%.

Fig. 1. (a) Direct fluorescence titration of Fl (1.0 μM) with GC5A (up to 3.0 μM), λ_ex = 500 nm. (Inset) the associated titration curve at λ_em = 513 nm and the fit according to a 1 : 1 binding stoichiometry. (b) Competitive titration of GC5A·Fl (0.4/0.5 μM) with LPA (up to 1.9 μM). (Inset) fit of the titration data to a 1 : 1 competitive binding model. All experiments are in HEPES buffer (10 mM, pH = 7.4) at 25 °C.

Fig. 2. ¹H NMR spectra of (a) 6:0 LPA (1 mM), (b) 6:0 LPA (1 mM) with addition of GC5A (1 mM), and (c) GC5A (1 mM) in D₂O at 25 °C. (d) Optimized structure of the GC5A·6:0 LPA complex at the B3LYP-D3(BJ)/6-31G(d)/SMD(water) level of theory. Hydrogen atoms are omitted for clarity. (e) ESP-mapped molecular vdW surface of GC5A, 6:0 LPA and GC5A·6:0 LPA.

Fig. 3. Fluorescence responses of (a) GC5A·Fl (0.8/1.0 μM) at 513 nm (λ_ex = 500 nm) and (b) GC4A·AlPcS₄ (0.8/1.0 μM) at 680 nm (λ_ex = 608 nm) upon the addition of LPA and various biological co-existing species (0.4 μM for small species and 0.15 mg L^–1 for ctDNA, RNA and BSA) in HEPES buffer. (c) Score plot of the first two principal components obtained by PCA of analytes. The percent of total variance is given in brackets for each principal component. Ellipsoids on the scatter plot are drawn at 95% confidence. (d) The set-up calibration line of the fluorescence intensity for quantitatively determining the LPA concentrations in serum. Error bars could not be shown if less than 0.005.