Graphene Oxide-Based Fluorescence Sensor for Betaxolol Hydrochloride Detection in Plasma. (2024)

Byline: Qiang Zhang, Jie Zhao, Wei Liu, Yue Yue, Kaili Yu, Dezhong Xuand Xin Ding

Summary: We introduce a fast, sensitive, label-free method for thedetection of Betaxolol hydrochloride (BH) based on fluorescentdye/graphene oxide (FD/GO) fluorescence sensor. Three fluorescent dyes(rhodamine 6G, rhodamine B and fluorescein) were chosen for BHdetection, where it has been found that the fluorescence recovery ratioof R6G was higher than the fluorescence recovery ratio of rhodamine Band fluorescein. Concentration, mixing order, detection time and someinterferents were also considered in the optimisation. The R6G/GOuorescent sensor has been successfully applied to the determination ofBH in fetal bovine serum (FBS). The fluorescence intensity ratio(I-I0)/I0 and concentrations of BH ranging from 0.0050 to 10.0 imol/L.The linear regression equation obtained was as follows: (I-I0)/I0 =0.980 + 1.480 x CBH with the correlation coefficient of 0.991.

Keywords: Graphene oxide; Rhodamine 6G; Betaxolol hydrochloride;Fluorescence recovery; Plasma.

Introduction

Betaxolol is a type of lipophilic and a selective [beta]1 receptorblocker used in the treatment of hypertension, arrhythmias, coronaryheart disease, glaucoma, and is also used to reduce non-fatal cardiacevents in patients with heart failure. The untoward effects of Betaxololinclude obvious bradycardia and hypotension [1-3] and intraocularpressure [4]. A variety of analytical methods have been established suchas chromatography [5], spectrophotometry [6-7], capillaryelectrophoresis [8] and electroanalytical [9] techniques. To establish anew rapid, simple, accurate and sensitive Betaxolol detection method hasimportant significance in clinical medicine, pharmacology, and eyedisease treatment etc.

Graphene oxide (GO) and reduced graphene oxide (rGO) with theirunique and excellent physical and chemical properties have been widelyused in diverse applications such as chemical sensors [10-15],biological detection [16-21], drug loading and drug delivery [22-30] ,attributing to I-I conjugated stacking and fluorescence resonance energytransfer (FRET) [31, 32]. As an ideal fluorophore acceptor, graphene hasshown superior quenching efficiency for a variety of fluorophores, withlow background and high signal-to-noise ratio [33, 34].

A common detection protocol is that the probe is labelled withfluorescent dye (FD), following mixed with graphene then analyte inorder. The fluorescence turn-off firstly, then recovered when analytereplaced FD labelled probe from the probe/graphene complex bycompetitive adsorption [16-17, 35]. Huang et al [36, 37] developed alabel-free mothed for the detection of tartrazine, methylene blue andsunset yellow. In this method, dye-labelled step was omitted, andfluorescent dye was adsorbed onto rGO or GO and then replaced by theanalyte.

In this research, we introduce a sensitive, rapid, label-free andgeneral uorescent method for the determination of Betaxolol bycompetitive binding to GO against a uorescent dye, and the uorescencerecovery upon uorescent dye desorption from GO provides a quantitativerelation for Betaxolol, giving a limit of detection (LOD) of 5.0 nmol/L.The presented method is a suitable one for measuring Betaxolol in fetalbovine serum (FBS).

Experimental

Materials and chemicals

Graphene Oxide powder (>99%) was obtained from Time NanoTechnology Co. Ltd (Chengdu, China). [beta]-CD (>96%) was purchasedfrom Aobox Biotechnology Co. Ltd (Beijing, China). Rhodamine B (RB) andRhodamine 6G (R6G) were supplied by Xiya Reagent (Shandong, China).Fluorescein (Fl) was purchased from Kaitong Chemical Reagent Co. Ltd(Tianjin, China). The stock solution of RB (1.0x10-5 mol/L), R6G(1.0x10-5 mol/L) and Fl (1.0x10-5 mol/L) were prepared by directlydissolving them in deionized water and their required concentration ofworking solutions were obtained by diluting the stock solution withdeionized water. Fetal bovine serum (FBS) was purchased from Gibco ofThermo Fisher Scientific Co. Ltd. Betaxolol hydrochloride was purchasedfrom Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The waterused in all the experiments had a resistivity higher than 18.2Ma|*cm-1.

Fluorescence measurements were monitored on a Shimadzu RF-5301 PCspectrofluorophotometer. JEM-2100 type transmission electron microscope(TEM, JEOL, Japan) was operated at an accelerating voltage of 200 kV toanalyze the morphology of GO.

Preparation of GO

GO was synthesized from graphite powder according to the modifiedHummer's method. Before being used, GO (5.0 mg) was dissolved in10.0 ml of deionized water and then sonicated for 1 h (60 W). GOdispersion (0.5 mg/mL) was used as a working solution.

General procedure

An appropriate volume of the stock solution of GO was introducedinto a vial and mixed with R6G solution. Then a series of differentconcentrations of analyte were added and diluted to 10.0 ml usingdeionized water. All the working solutions were thoroughly allowed tostand for 5 min at room temperature. The fluorescence spectra wererecorded in the emission wavelength range of 510-650 nm with theexcitation of 480 nm.

Pretreatment of sample

Fetal bovine serum (FBS) was stored at -20 AdegC until it was used.Freeze-thawed serum was centrifuged at 12000 rpm for 5 min at 4 AdegC.Then, 0.5 ml of supernatant fluid mixed with 1.0 ml of acetonitrile inthe centrifuge tube and immediately shaken for 10 s and then placed inthe shaker for 30 min (60 rpm, 37 AdegC). Subsequently, the tube wascentrifuged at 12000 rpm for 15 min at 4 AdegC. Precipitated protein wasremoved by centrifugation (12000 rpm, 4 AdegC, 15 min) and thesupernatant fluid stored at 4 AdegC.

Results and Discussion

Design of the sensor

The underlying assay principle (Scheme-1) is based on the reversibleinteraction (adsorption or desorption) between fluorescent dye and GO.Fluorescent dye can been bound onto GO to form FD/GO complex for the I-Istacking and the following fluorescence resonance energy transfer (FRET)results in high-efficiency fluorescence quenching ('turn-off'). Competitive adsorption exists between fluorescent dyes andanalyte (Betaxolol hydrochloride). When the binding intensity offluorescent dyes is weaker than BH, fluorescent dyes could be replacedby BH from GO, therefore obvious fluorescence recovery is observed. Thecompetitive interaction between fluorescent molecules and BH on GO is asshown in the scheme 1.

The morphology and structure of GO were characterized by TEM. Fig. 1demonstrated a typical sheet-like structure of GO with the largethickness, smooth surface, and wrinkled edge. The obtained GO displaylayered structures and become very thin.

To prove our design, we monitored the fluorescence'turn-off' after the adsorption of fluorescent molecules on GO(Fig. 2). Fig. 2(a-c) showed the uorescence spectra of R6G/GO/BH,RB/GO/BH and Fl/GO/BH systems, respectively. Owing to the existent ofFRET, fluorescence of R6G, RB and Fl were decreased firstly. With the BHbeing added, fluorescent molecules were replaced from GO surface and thefluorescence recovered ('turn-on'). However, the adsorptioncapacity of GO is different with R6G, RB, Fl and BH, and so thefluorescence 'turn-on' were different. As shown in Fig. 2d, Iand I0 were fluorescence intensity when Betaxolol hydrochloride waspresent and absent in FD/GO solution. Fluorescence recovery ratio(I-I0)/I0 of Fl was less than others and hardly enhanced with anincrease in the concentration of BH, indicating that GO adsorptioncapacity of Fl is similar to BH.

Fluorescence recovery ratio (I-I0)/I0 of R6G and RB increasedobviously. The fluorescence recovery ratio of RB level off when BHconcentration is greater than 10 umol/L, however the fluorescencerecovery ratio of R6G still increase rapidly until BH concentration30imol/L. Meanwhile, fluorescence recovery ratio of R6G is 2.03 times asmuch as RB at BH concentration 10imol/L, and 3.28 times at BHconcentration 30imol/, suggesting that GO has a stronger adsorptioncapacity for BH than R6G and RB. The signicant uorescence recovery (bluein Fig. 2a) was observed when a lower concentration of BH (4.0 umol/L)was added into R6G/GO system. Meanwhile, quenching effects andfluorescence recovery ratio of RB and Fl were lower than R6G (Fig. 2d),and thus R6G was chosen for the detection of Betaxololhydrochloride.

Fig. 3 shows the relationship between the efficiency of fluorescencerecovery ratio (I-I0)/I0 and the concentration of R6G added to GOsolution. It could be seen that the fluorescence recovery ratio(I-I0)/I0 increased with an increase in the concentration of R6G up to0.080 umol/L. A small quantity of R6G could not occupy all the GOsurface, so that the added BH was adsorbed on the GO surface at firstwithout replacing R6G to bulk solution. However, the fluorescencerecovery ratio (I-I0)/I0 decreased when the concentration of GO exceeded0.080 umol/L. As more R6G was added, there was excess free R6G in thebulk solution and R6G was more difficult to be displaced by BH from GOsurface due to the adsorption equilibrium between R6G, BH and GOsurface. Therefore, we chose 0.080 umol/L as the optimum concentrationof R6G in the subsequent experiments.

Furthermore, the effects of BH on the fluorescence of R6G also havebeen investigated as shown in Fig. 4. Excitation wavelength(I>>ex) and emission wavelength (I>>em) of R6G were 480 nmand 551 nm, respectively. Whereas, the excitation wavelength andemission wavelength of BH were 227 nm and 301 nm, respectively. Itclearly shows that there were no spectroscopic overlaps between BH andR6G when the mixed solution of BH and R6G was placed under theexcitation wavelength of 480 nm. Meanwhile, as shown in the insert ofFig. 4, a change in the concentration of BH had little influence on thefluorescence intensity of R6G.

Optimization of the sensor

The effect of order of reagent addition on the fluorescence recoveryratio (I-I0)/I0 was investigated by six approaches with differentreagent addition orders (Fig. 5). The result showed that mixing GO, R6G,buffer solution and then adding BH could result in the highestfluorescence recovery ratio than any other addition sequences of thereagents (Fig. 5).

A kinetic study was carried out to record the inuence of theincubation time on the uorescence immediately after mixing differentamounts of BH with R6G/GO (Fig. 6). Adding different concentrations ofBH, the uorescence intensity increased rapidly and reached a plateau inless than 1 min, indicating that most of the R6G molecules in the R6G/GOcomplex had been promptly replaced by BH in the initial stage.

Application of the sensor

To further investigate the selectivity of the established R6G/GOsystem, some of the inorganic and organic interferents were tested (Fig.7). Of these tested substances, NaCl, KCl, glucose, sucrose, citricacid, NaH2PO4, CaSO4, Cr(VI), NaIO4, CaCl2, CuCl2, KI and MgCl2exhibited lower fluorescence response as compared to BH, while SDS,isatin and FeCl2 had a little obvious effect on the fluorescencerecovery ratio (I-I0)/I0. These observed results suggested that theproposed strategy demonstrates good selectivity for the determination ofBH.

To test the practicality of the proposed approach, we detected theconcentration of BH in FBS using the proposed method after thepretreatment procedures. The fluorescence spectra of the mixture in thepresence of various amounts of BH are shown in Fig. 8. It could be seenthat the fluorescence intensity ratio dramatically increases with theincreasing concentration of BH, then dropped rapidly when theconcentration of BH exceeded 10.0 imol/L. A good linear relationshipcould be noted between the fluorescence intensity ratio andconcentration of BH ranging from 0.0050 to 10.0 imol/L. The linearregression equation was as follows: (I-I0)/I0 = 0.980 + 1.480 x CBH witha correlation coefficient of 0.991.

Table-1: Analytical results for BH in FBS sample (n= 5).

###Sample###initial value (imol/L)###added value (imol/L)###measurevalue (imol/L)###Recovery (%)###RSD (%)

###1###Blank###0.0250###0.0259###104###1.67

###2###Blank###0.0800###0.0773###97.1###1.37

###3###Blank###0.200###0.193###100###2.44

To demonstrate the accuracy of the proposed method, a recoveryexperiment was tested and the results showed that the recovery was inthe range of 97.1-104% with the RSD of 1.37-2.44% (Table-1).

Conclusions

We introduce a fast, sensitive, label-free, and general assayprinciple for the detection of BH based on R6G/GO complex. Under optimumconditions, BH can be rapidly and sensitively detected. The R6G/GOuorescent sensor has been successfully applied to the determination ofBH in fetal bovine serum with the LOD of 5.0 nmol/L. This design issimply based on the competitive adsorption of GO between BH and R6G, andthus it can be potentially applied to other drugs of a broader range forthe label-free detection.

Acknowledgements

This work was supported in part by the Natural Science ResearchProjects of the Education Department of Henan Province (14A150010) andthe Science Research Cultivation Fund of Xinxiang Medical University(2014ZD110).

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