Interaction of 3-animo phenylboronic acidwith ZnS:Cu quantum dots and glucose

Katarzyna KUR-KOWALSKA, Małgorzata PRZYBYT,Ewa MILLER

Keywords: 3-amino phenylboronic acid; quantum dots;fluorescence quenching

Abstract: This paper shows the preliminary results of studyof interaction between 3-amino phenylboronic acid and glucoseor ZnS:Cu quantum dots. QDs were obtained by a new proce-dure, using 49:1 ratio ZnSO4 to CuSO4 and mercaptopropionicacid as the capping agent. These QDs have interesting fluores-cence properties and high fluorescence intensity. 3-amino phenyl-boronic acid has a stable fluorescence intensity at pH 5.5 to 7.5and forms a reversible ester with glucose. The fluorescence inten-sity of 3-amino phenylboronic acid is quenched by glucose and byQDs. The Stern-Volmer constants were calculated. The obtainedvalue of Stern-Volmer constant at pH 7 was 24.72 ± 0.94 L/molfor quenching by glucose and 11.75 ± 0.57 mL/µg for quenchingby QDs.

1. Introduction

Because of the great need to find simple and rapid methods for measuring glucose,there are being developed methods for measuring glucose using quantum dots. Theyare good fluorescent probes because of their properties: photostability, broad absorp-tion spectrum and narrow fluorescence spectrum (Wu et al., 2010). To create a glucosesensor, glucose oxidase (GOx) is often used. This enzyme selectively oxidizes glucoseat the presence of oxygen (Egawa et al., 2011). However, this reaction is irreversible,and the use of the enzyme increases the cost significantly. Alternative sensors arenon-enzymatic (Sun et al., 2004; Wang et al., 2012) ones acting on the principle ofreverse reaction, keeping high stability and sensitivity. For this purpose, boronic acidderivatives which show good binding properties by creating an ester form with sugarsare used. The elaboration of two-component glucose sensor using phenylboronic acid(PBA) and quantum dots (QDs) is the goal of many research teams (Cordes et al.,2006; Freeman et al., 2009). This work presents a preliminary study using 3-aminophenylboronic (APBA) acid and ZnS QDs doped with Cu to check their potential toobtain such a sensor.

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2. Experimental section

2.1. Materials and methods

Sodium sulfide (Na2S · 9H2O p.a.) and copper sulfate (CuSO4· 5H2 O p.a.) werepurchased from POCH country-region S.A. (Poland). Zinc sulfate (ZnSO4· 7H2O,≥ 99.0%) and 3-amino phenylboronic acid (H2NC6H4B(OH)2· H2O, 98%)were pur-chased from SIGMA-ALDRICH (Germany). 3-Mercaptopropionic acid(MPA, ≥ 99.0%)was purchased from Fluka (Germany). Glucose (anhydrous pure p.a.) was purchasedfrom Chempur (Poland). Absorbance spectra were collected using spectrophotome-ter Nicolet Evolution 300 (Thermo Spectronic). Fluorescence spectra were measuredusing spectrofuorometer FluoroMax4 (JobinYvon). All experiments were performedat room temperature. Distillate water was used throughout.

2.2. Synthesis of ZnS:Cu quantum dots

ZnS:Cu quantum dots were prepared according to (Chen et al., 2012) with slightmodifications. 4.9 mL of ID0.1 M0.1 M ZnSO4, 0.1 mL of ID0.1 M0.1 M CuSO4 and0.17 mL of MPA were mixed together, added with water to obtain final volume of50 ml and adjusted to pH, 11.5. This mixture was heated for 30 minutes at 95◦C.Then the mixture was cooled to room temperature and QDs were precipitated with75 mL of ethanol. QDs were harvested by centrifugation, washed with ethanol anddried overnight at 40◦C. Stock solution was made by dissolving 10 mg of QDS in 2mL of ID0.01 M0.01 M phosphate buffer, pH7.

3. Results and discussion

3.1. Properties of QDs

Absorbance and fluorescence spectra of QDs are shown in Fig. 1A. The radius ofobtained QDs was evaluated as 1.7 nm from the absorbance spectrum (Khani et al.,2011). The absorbance spectrum shows a shoulder at 297 nm and fluorescence wasexcited at this wavelength. The emission fluorescence spectrum shows a narrow bandwith the maximum at 460 nm.

3.2. Characteristics of 3-amino phenylboronic acid

3-amino phenylboronic acid is a strongly fluorescent compound; therefore, its 10−6 Msolution was used in further experiments. The absorbance and fluorescence spectraof APBA at pH 7 are shown on Fig. 1B. The emission fluorescence spectrum (excitedat 299 nm) shows a narrow band with the maximum at 460 nm. Its properties werechecked at pH range from 5 to 10. pH has no influence on the emission maximumbut only on intensity. Fig. 2 shows the dependence of fluorescence intensity on pH.Up to pH 7.5 fluorescence intensity is practically independent on pH and decreasedrapidly for higher values. APBA is Lewis acid and at neutral pH it forms the neutraltrigonal form, with the sp2-hybridization boron atom. In an alkaline environmentit is negatively charged with a higher sp3 electron state, which has a much lowerfluorescence (Egawa et al., 2011).

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3.3. Quenching of 3-a PBA fluorescence by glucose

Due to its structure (two hydroxyl groups) PBA binds diols by the covalent bondand therefore it has good properties to work with sugars, such as glucose, galactoseand fructose (Cannizzo et al., 2005). There are studies demonstrating the fluores-cence quenching of boronic acid derivatives in the presence of glucose (Fang et al.,2004). Fig. 4 shows the dependence of APBA fluorescence intensity on increasingglucose concentration at pH7. As it can be observed, a significant decrease of APBAfluorescence intensity with the increasing concentration of glucose occurred indicat-ing quenching. From these data the Stern-Volmer constant of APBA fluorescencequenching by glucose was calculated. Fluorescence quenching is described by theStern-Volmer equation:

I0I

= 1 +KSV [Q] (1)

Fig. 1. A)A- absorbance and F- fluorescence spectra of QDs at pH 7, B)A- absorbanceand F- fluorescence of APBA at pH 7, 10-6M

Fig. 2. . Structure of 3-aminophenylboronic acid

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1. I0- initial fluorescence intensity in the absence of a quencher

2. I - fluorescence intensity in the presence of a quencher

3. Ksv-static quenching constant

4. [Q] -quencher concentration

The obtained value of the Stern-Volmer constant was 24.72 ± 0.94 L/mol at pH7 (inset on Fig. 4). The obtained value is reasonable as compared with the valuesof glucose – AMPA binding values obtained by Torun and co-workers (Torun et al.,2009). Boronic acid binds compounds containing diols moieties with high affinitythrough reversible ester formation (Yan et al., 2004), which show weaker fluorescenceintensity.

Fig. 3. Dependence of fluorescence intensity APBA as a function of pH

Fig. 4. Quenching of AMPA fluorescence intensity by glucose at pH7, λexc=299 nm;concentration of glucose is varying from 0 to 0.012 mol/L by 0.001

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3.4. Quenching of APBA fluorescence by ZnS:Cu QDs

To check if AMPA interacts with ZnS:Cu QDs, 3 mL of a 10−6M solution of APBAat pH7 was added with a 3 µL of QDs stock solution. After addition of each portion,fluorescence of the solution was checked. An increase of the fluorescence intensity ofQDs and a decrease of the fluorescence intensity of APBA has been observed (Fig. 5)indicating quenching of APBA fluorescence by QDs.3-amino phenylboronic acid hasan amino group, which can interact with the carboxyl groups present on the surfaceof QDs covered with MPA. As the result of such interactions, quenching of APBAfluorescence at the presence of QDs can be observed. The Stern-Volmer constantwas evaluated as 11.75 ± 0.57 mL/µg. This phenomenon is probably characterizedby a static quenching which occurs as a result of the formation of a nonfluorescentground-state complex (Lakowicz, 2006) between APBA and QDs.

4. Conclusion

A new method of preparation of ZnS:Cu quantum dots is presented in this work.They are less toxic than the widely studied cadmium dots, which have a carcinogenicpotential. ZnS:Cu QD shave a high fluorescence intensity and similar λexc to 3-aminophenylboronic acid. Preliminary studies of APBA and QDs properties showed thatAPBA fluorescence is quenched by both glucoseand QDs at pH7. Due to this fact,3-amino phenylboronic acid which forms a reversible ester with glucose is potentiallya good component of the glucose sensor. Interactions between APBA, QDS andglucose could be very interesting, therefore research in this direction will be contin-ued. Another objective is the synthesis of other non-toxic quantum dots with goodfluorescence properties.

Fig. 5. Fluorescence emission spectra of APBA as a function of increasing QDs con-centration, λexc = 298 nm, pH=7

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