Chinese Chemical Letters  2014, Vol.25 Issue (01):77-79   PDF    
引用本文
Yan-Ping Li, Ling Jiang, Tao Zhang, Ming Lin, Dan-Bi Tian, He Huang.Colorimetric detection of glucose using a boronic acid derivative receptor attached to unmodified AuNPs[J]. Chin. Chem. Lett., 2014,25(01): 77-79   
Colorimetric detection of glucose using a boronic acid derivative receptor attached to unmodified AuNPs
Yan-Ping Lia,b, Ling Jianga,c, Tao Zhanga,b, Ming Lina, Dan-Bi Tianb, He Huanga     
* Corresponding authors at:a College of Biotechnology and Pharmaceutical Engineering, State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, Nanjing 210009, China;
b College of Science, Nanjing University of Technology, Nanjing 210009, China;
c College of Food Science and Light Industry, Nanjing University of Technology, Nanjing 210009, China
Received:2013-7-8, Revised:2013-10-8, online:2013-11-12.
E-mail addresses:biotech@njut.edu.cn
Abstract: A simple, cheap and non-enzymatic colorimetric strategy for glucose detection has been designed based on the interactions between a phenylboronic acid (PBA) derivative, which is coupled with gold nanoparticles (AuNPs) as the colorimetric reporters, and glucose. The PBA-AuNPs hybrid system proposed here exhibits ordered photochemistry behaviors upon the addition of glucose at different pH values. There are two linear regions of glucose concentration for the glucose sensor at different pH values, i.e., between 0.1 mmol/L and 9.8 mmol/L at pH 6 with the detection limit of 64 μmol/L and between 0 and 6.5 mmol/L with the detection limit of 48 μmol/L at pH 9, respectively. To test the practicality of the sensor system, we also applied this assay to detect a glucose sample in the artificial saliva.
Key words: Colorimetric assay     Glucose detection     Gold nanoparticles     Phenylboronic acid derivative    
1. Introduction

Glucose is considered not only a nutrient but also a biomarker correlated with certain diseases, such as renal glycosuria, cystic fibrosis, diabetes, human cancer, and so on. As an important regulatory signal, glucose levels also have a great impact on the hormone secretions. The current research of glucose relies mostly on the epidemics of obesity and the increased incidence of diabetes, which represent major threats for human health. At present, the detection of glucose based on glucose oxidase is very popular, but this method has several severe disadvantages, specially the potential instability during use or sterilization [1, 2]. Thus, there is a strong demand for the development of an efficient, cheap and stable glucose detecting strategies.

However, to achieve this goal, we must manipulate the characteristics of the recognized molecular. It has been demonstrated that a tetrahedral boronate ion can form stable complexes with carbohydrates with either 1,2- or 1,3-diol groups. Thus, phenylboronic acid derivatives can specifically recognize carbohydrate and have already been widely employed as promising components for glucose detection [3]. Herein, we used 3- aminophenylboronic acid (APBA) as the affinity selection part, since it is a cheap commercial compound that can stay stable for relatively long period of time.

In this study, we focused on the borate interactions with glucose at different pH ranges reported by gold nanoparticles (AuNPs). Colorimetric assays based on AuNPs have been widely used in a variety of research fields owing to the simple technology and flexible mechanical system. According to the rational surface/ interface design, AuNPs exhibit excellent recognition ability. The high extinction coefficient of AuNPs transduces the binding events into detectable optical signals that can be directly observed by naked eyes [4]. Comparing with the fluorescent methods, colorimetric assays need no tedious fabrication operation or any complicated instruments [5].

2. Experimental

HAuCl4⋅3H2O, 3-aminophenylboronic acid (APBA), α-amylase, and all metallic salts used in this study were obtained from Sigma Chemical Company. The glucose was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade. All solutions used in this study were prepared with ultrapure water (18.2 MΩ/cm) from an ultrapure water system (Sartorius, Germany). An artificial saliva matrix was prepared according to the ISO/TR 10271 standard [6].

AuNPs were prepared by using the usual citrate reduction method [7, 8] with some modifications. Briefly, trisodium citrate (3.2 mL 1 wt%) was rapidly added to a boiling solution of HAuCl4, the color of the solution changed from colorless to wine red after boiling for 10 min with stirring. The resulting AuNPs molar concentration was calculated to be 2.2 nmol/L. APBA (20 μL) was added to the AuNPs to produce a reaction mixture for the further glucose determination. UV-vis spectroscopy analysis was performed to determine the optimal detection conditions by a UV-vis absorption spectrophotometer Lambda 25 (Perkin Elemer). UV-vis extinction spectra of the standard solution of glucose with different concentrations and artificial saliva matrix were recorded. Transmission electron microscopy (TEM) analysis of the resulting AuNPs was obtained using a JEOL, JEM-200CX microscope (Japan).

3. Results and discussion

Sensing mechanism employed in this study was shown in Scheme 1. AuNPs can be triggered to aggregate by the electric charge transition after the addition of APBA. The amine group in the APBA plays an important role in this strategy due to its strong binding to the AuNPs interface through both covalent and electrostatic attractions [9, 10]. Also, the amine group lowers the pseudo pKa-value of the boronic acid. The charged forms of APBA can form a stable complex with glucose through reversible covalent binding whereas the neutral forms are highly susceptible to hydrolysis. When the pH-value is higher than the pKa of the APBA, the high density charges caused by the boronic cyclic ester adsorbed around the AuNPs would largely amplify the electrostatic repulsions and keep them from coming close as shown in Scheme 1(A). Here, glucose acts just like a protector, which successfully prevents the formation of bis-bidentate complexes and effectively maintains the AuNPs dispersion [11, 12]. However, as described in Scheme 1(B) when the pH-value of the detection system went down below the pKa of APBA, the presence of positive charges minimized the electrostatic repulsions between the negatively charged boronates, inducing cross-linking of the borate moieties upon bis-bidentate complex formation with glucose. The mechanism illustrates the AuNPs-aggregation involves one glucose molecule bound simultaneously to two boronates and brings the gold nanoparticle closer, eventually causes a color change.

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Scheme 1.Detection of glucose at pH-value upon pKa (A), and below pKa (B). The pKa of the APBA is 8.86.
Glucose prefers to bind with PhB(OH)3 through reversible covalent binding, while the PhB(OH)2 form is highly susceptible to hydrolysis [13]. The interactions between phenylboronic and glucose produce negative charges, which enhance the electrostatic repulsion and strongly inhibit AuNPs aggregations caused by the positive charge of APBA. Gradually increasing the amount of glucose led to a decrease in absorbance at 650 nm and AuNPs appeared to change from the aggregation state to the monodispersed state (Fig. 1B to A). The degree of inhibited aggregation was associated with glucose concentration (Fig. 2A). Here, we used the ratio of the extinction value at 650 nm and 520 nm to quantify the aggregation process or color change, because it is less vulnerable to fluctuation in sampling and monitoring conditions. The inset image was the plot of the response curve for the glucose sensor system. Their linear equation is Y = -0.1477x + 1.09488 with R2 = 0.995. However, the interesting results appeared by reducing the pH values (Fig. 2B). Upon the addition of various amounts of glucose, the intensity of the UV-vis absorption peak at 520 nm concomitantly decreased along with the increase in the extinction at 650 nm, indicating the AuNP aggregation was suppressed with the increase of glucose concentration, which could be supported by TEM measurements (Fig. 1B to C). The linear equation is Y = 0.05137x + 0.11233 with R2 = 0.983.

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Fig. 1.TEM images of AuNPs with APBA and glucose addition at pH 9 (A) AuNPs with APBA addition only (B) AuNPs with APBA and glucose addition at pH 6 (C).

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Fig. 2.UV–vis spectra of solutions of 15 nm AuNPs upon the addition of 30 μmol/L APBA and glucose at pH 9 (A); and AuNPs upon the addition of 10 μmol/L APBA and glucose at pH 6 (B).
To test the practicality of our detection system, we applied this assay to detect the glucose in artificial saliva. Fig. 3 illustrated the visual color of AuNPs solutions upon adding 10 mmol/L of APBA and different concentrations of glucose in artificial saliva. As the concentration increased, the color progression of the AuNPs-APBA system changed from red to blue and could be easily distinguished by naked eyes. We also determined the recovery of glucose that was spiked into artificial saliva samples and the results were listed in Table 1. The good recovery indicated that our sensor system might be suitable for the detection of glucose levels in real samples.

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Fig. 3.Visual color changes of 15 nmAuNPs solutions after addition of 8 μmol/L APBA and different concentrations of glucose in artificial saliva. (a) 0 mmol/L, (b) 0.25 mmol/L, (c) 1.5 mmol/L, (d) 2.75 mmol/L, (e) 4.5 mmol/L, and (f) 6.75 mmol/L.

Table 1
The results of recovery test.
4. Conclusion

In this work, we managed to design a novel strategy for the effective detection of glucose that exhibited dual behaviors in response to glucose concentrations depending on the operating pH-value. This work provided a new thought for the development of simple, inexpensive assays thatmay be highly useful for practical glucose monitoring. We anticipate that more improvements can be achieved in such systems for the development of glucose sensors and more information can be gathered for the studies on the interactions between boronic acids and glycoproteins on cell surfaces.

Acknowledgments

This work was supported by the National Natural Science Foundation of China for Young Scholars (No. 21106064), the National Basic Research Program of China (No. 2012CB725204), the National Science Foundation for Distinguished Young Scholars of China (No. 21225626), the Natural Science Foundation of Jiangsu Province (Nos. BK2012822 and BK20131406).

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