Development of a Novel Benzimidazole-Based Probe and Portable Fluorimeter for the Detection of Cysteine in Human Urine

Abstract

The measurement of cysteine in human urine and live cells is crucial for evaluating biological metabolism, monitoring and maintaining the immune system, preventing tissue/DNA damage caused by free radicals, preventing autoimmune diseases, and diagnosing disorders such as cystinuria and cancer. A method that uses a fluorescence turn-on probe and a portable fluorescence spectrometer device are crucial for highly sensitive, simple, rapid, and inexpensive cysteine detection. Herein, we present the synthesis and application of a benzimidazole-based fluorescent probe (ABIA) along with the design and development of a portable fluorescence spectrometer device (CysDDev) for detecting cysteine in simulated human urine. ABIA showed excellent selectivity and sensitivity in detecting cysteine over homocysteine, glutathione, and other amino acids with the response time of 1 min and demonstrated a detection limit of 16.3 nM using the developed CysDDev. Further, ABIA also demonstrated its utility in detecting intracellular cysteine, making it an excellent probe for bio-imaging assay.

Keywords: bio-imaging; biothiols; cancer; cysteine; cystinuria; fluorimeter; portable.

Scheme 1

Scheme for the synthesis of the probe ABIA.

Figure 1

(a) UV-vis and (b) fluorescence emission spectra of compound 3 (10 µM; λ_ex = 368 nm) and the probe ABIA (10 µM) in 90% DMSO:0.01 M HEPES buffer solution (λ_ex = 372 nm).

Figure 2

(a) Fluorescence spectra and (b) competitive study of ABIA (10 µM, λ_ex = 368 nm, λ_em = 455 nm) in the absence and presence of five equivalents of various analytes (50 µM). 1, Gly; 2, Ala; 3, Ser; 4, Pro; 5, Val; 6, Thr; 7, Ile; 8, Leu; 9, Asn; 10, Asp; 11, Gln; 12, Lys; 13, Glu; 14, Met; 15, His; 16, Phe; 17, Arg; 18, Tyr; 19, Typ; 20, Hcy; 21, Gsh; 22, Cys; 23, BSA; 24, Cl⁻; 25, Br⁻; 26, NO₃⁻; 27, AcO⁻; 28, SO₄²⁻; 29, PO₄³⁻; 30, Na⁺; 31, Cs⁺; 32, Ca²⁺; 33, Cu²⁺.

Figure 3

(a) Changes in ¹H NMR spectra of ABIA upon the addition of 0.5 and 1.0 equivalents of Cys in 90% DMSO-d₆:0.01 M HEPES buffer solution (D₂O); (b) proposed Cys detection mechanism by ABIA.

Figure 4

Schematic of various parts of the CysDDev (a): UV LED, sensor, and cuvette holder; (b) top lids for UV LED, sensor, cuvette holder; (c) the main case with top lid; (d) electronic components connected to the Arduino microcontroller; (e) assembly of CysDDev without the top lid; (f) CysDDev prototype connected to the laptop computer.

Figure 5

(a) Fluorescence spectra of compound 3 (10 µM, λ_ex = 370 nm) and ABIA (10 µM, λ_ex = 370 nm) recorded using CysDDev and commercial fluorescence spectrometer; (b) effect of pH on the detection of Cys (0, 50 µM) in simulated human urine at various pH using ABIA and CysDDev (λ_ex = 370 nm, λ_em = 455 nm).

Figure 6

(a) Changes in fluorescence spectra of ABIA (10 µM, λ_ex = 370 nm) recorded using CysDDev upon the successive addition Cys (0–150 µM) in urine at pH = 7.3 (b) Calibration curves for the detection of Cys in urine using ABIA and CysDDev (λ_ex = 370 nm, λ_em = 455 nm) Inset is the linear regression curve in the range of 0–20 µM of Cys.

Figure 7

(a) Cytotoxicity’s of ABIA, Cys, and ABIA + Cys on A549 cells at concentrations of 0.1, 10, 25, and 50 µM after 24 h; (b) Dark field, bright field, and merged images upon treatment of A549 cells with control, Cys, ABIA, Cys + ABIA, NEM + ABIA, and NEM + Cys + ABIA.