Detection of residual rifampicin in urine via fluorescence quenching of gold nanoclusters on paper

Abstract

Background: Rifampicin or rifampin (R) is a common drug used to treat inactive meningitis, cholestatic pruritus and tuberculosis (TB), and it is generally prescribed for long-term administration under regulated dosages. Constant monitoring of rifampicin is important for controlling the side effects and preventing overdose caused by chronic medication. In this study, we present an easy to use, effective and less costly method for detecting residual rifampicin in urine samples using protein (bovine serum albumin, BSA)-stabilized gold nanoclusters (BSA-Au NCs) adsorbed on a paper substrate in which the concentration of rifampicin in urine can be detected via fluorescence quenching. The intensity of the colorimetric assay performed on the paper-based platforms can be easily captured using a digital camera and subsequently analyzed.

Results: The decreased fluorescence intensity of BSA-Au NCs in the presence of rifampicin allows for the sensitive detection of rifampicin in a range from 0.5 to 823 µg/mL. The detection limit for rifampicin was measured as 70 ng/mL. The BSA-Au NCs were immobilized on a wax-printed paper-based platform and used to conduct real-time monitoring of rifampicin in urine.

Conclusion: We have developed a robust, cost-effective, and portable point-of-care medical diagnostic platform for the detection of rifampicin in urine based on the ability of rifampicin to quench the fluorescence of immobilized BSA-Au NCs on wax-printed papers. The paper-based assay can be further used for the detection of other specific analytes via surface modification of the BSA in BSA-Au NCs and offers a useful tool for monitoring other diseases.

Figure 1

Schematic illustration of the synthesis, BSA-Au NC immobilization on paper and application to detect rifampicin.

Figure 2

a Absorption spectra of BSA-Au NCs (0.1× dilution) in the absence and presence of 10 µM rifampicin. b Fluorescence emission spectra (excitation wavelength at 480 nm) of BSA-Au NCs (0.1× dilution) in the absence and presence of 10 µM rifampicin.

Figure 3

a Concentration-dependent quenching of BSA-Au NCs (0.1× dilution) by rifampicin. From higher to lower concentrations: normalized quenching at 823 µg/mL [(F₀ − F)/F ~ 22.6 ± 0.68], 411 µg/mL [12.78 ± 0.5], 82 µg/mL [2.8 ± 1.93], 41 µg/mL [1.68 ± 0.16], 8 µg/mL [0.25 ± 0.6], 4 µg/mL [0.23 ± 0.008], 0.8 µg/mL [0.016 ± 0.009], 0.4 µg/mL [0.046 ± 0.031], 0.08 µg/mL [0.017 ± 0.016], and 0.004 µg/mL [0.06 ± 0.04] (each data point represents the average of three separate studies (n = 3), and the error bars denote the standard error of measurements within each experiment). b Plot of the linear region of the normalized decrease in fluorescence intensity of BSA-Au NCs (0.1× dilution) versus rifampicin concentration (each data point represents an average of three separate studies (n = 3); the error bars denote the standard error of measurements within each experiment). The excitation wavelength was set at 480 nm, and the emission wavelength was 640 nm.

Figure 4

a Fluorescence emission spectra of BSA-Au NCs (0.1× dilution) in the presence of primary TB drugs (100 µM each in the final concentration). At 640 nm (emission wavelength): BSA-Au NCs (fluorescence intensity = 123.74 ± 1.56), NCs + rifampicin (83 µg/mL) (13.67 ± 0.33), NCs + pyrazinamide (12.3 µg/mL) (121.95 ± 0.69), NCs + ethambutol (27.7 µg/mL) (114.27 ± 1.28) and NCs + izoniazid (13.7 µg/mL) (127 ± 0.69) (each data point represents the average of three separate studies (n = 3), and the error bars denote the standard error of measurements within each experiment). b Comparison of the normalized decrease in fluorescence intensity of BSA-Au NCs (0.1× dilution) in the presence of primary TB drugs (100 µM each in final concentration). At 640 nm (emission wavelength): normalized quenching of NCs + rifampicin [(F₀ − F)/F₀ ~ 0.89 ± 0.006], NCs + pyrazinamide [0.013 ± 0.005], NCs + ethambutol [0.065 ± 0.008] and NCs + izoniazid [0.028 ± 0.007] (each data point represents the average of three separate studies (n = 3), and the error bars denote the standard error of measurements within each experiment). [In this work, (F₀ − F)/F₀ = 1 indicates complete quenching and (F₀ − F)/F₀ = 0 indicates no quenching]. The excitation wavelength was set at 480 nm, and the emission wavelength was 640 nm.

Figure 5

a Test paper for the detection of rifampicin after modification (A) with BSA-Au NCs under UV light. The tenfold diluted urine samples with original rifampicin concentrations are as follows: (B) 0.5 µg/mL (fluorescence quenching ratio = 91% ± 1); (C) 5 µg/mL (82% ± 1.1); (D) 10 µg/mL (81% ± 1.8); (E) 30 µg/mL (79% ± 3.4); (F) 50 µg/mL (78% ± 1.7); (G) 100 µg/mL (75% ± 0.3); (H) 500 µg/mL(73% ± 1.9); and (I) 1000 µg/mL(69% ± 1.1). b Change in the fluorescence quenching ratio of the embedded BSA-Au NCs versus rifampicin concentration on the paper sensor (each data point represents an average of three separate studies (n = 3) in three different wax-printed 96-microplate paper platforms; three measurements were taken in each micro-well, and the error bars denote the standard deviation of the reading).