Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 474–478

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Rapid monitoring of benzylpenicillin sodium using Raman and surface enhanced Raman spectroscopy Xin Jiang a,b, Xiaoyu Qin b, Di Yin b, Mengdi Gong b, Libin Yang b,⇑, Bing Zhao a,⇑, Weidong Ruan a a b

State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, People’s Republic of China College of Pharmacy, Jiamusi University, Jiamusi 154007, People’s Republic of China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The Ag NPs assembly was served as

a r t i c l e

i n f o

Article history: Received 28 September 2014 Received in revised form 27 December 2014 Accepted 10 January 2015 Available online 17 January 2015 Keywords: SERS Silver colloid Benzylpenicillin sodium (NaBP) Raman spectroscopy

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a b s t r a c t At present, fluorescence spectroscopy, ultraviolet spectroscopy and infrared spectroscopy are usually used to detect drug molecules, however the information about using Raman spectroscopy to detect drug molecules is very few. In this work normal Raman spectroscopy and surface-enhanced Raman spectroscopy were utilized to study benzylpenicillin sodium (NaBP). The results show that NaBP is close to the surface of silver substrate through the carboxyl group, and the detection limit of NaBP is reduced to 1  10 7 mol/L. Accordingly, the quantitative analysis of NaBP can be carried out in the range of 1  10 4–1  10 7 mol/L concentration. And it is proved that NaBP is not stable in acid and alkali conditions and the decomposition reaction is very complex. Ó 2015 Elsevier B.V. All rights reserved.

Introduction Penicillin is the first antibiotic used for clinical therapy, which is separated from culture medium of penicillium notatum. Penicillin belongs to the b-lactam antibiotics. Free benzylpenicillin is an organic acid (pKa = 2.65–2.70), insoluble in water and soluble in organic solvent (butyl acetate). Its sodium salt is commonly used in clinic to enhance their water solubility. The structure of benzylpenicillin sodium (NaBP) is mainly composed of b-lactam ring, thiazolidine ring and benzene ring. Because of its low cost, high ⇑ Corresponding authors. E-mail addresses: [email protected] (L. Yang), [email protected] (B. Zhao). http://dx.doi.org/10.1016/j.saa.2015.01.016 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.

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SERS substrate for detecting the penicillin drug.  NaBP is close to Ag surface by carboxyl. The detection limit arrives at 10 7 mol/L.  A quantitative detection of NaBP can be performed in the range of 10 4– 10 7 mol/L.  The stability of NaBP in acid and alkali conditions can be monitored by using SERS.

efficiency and other characteristics, penicillin antibiotic is widely used in clinical medicine, veterinary medicine and feed additive. But the irregularity and abuse exist during the use of antibiotics, causing drug residues in animal food, which bring harm to human health and the ecological environment. Currently, the main analytical methods used to detect penicillin antibiotics include high performance liquid chromatography (HPLC), capillary electrophoresis, microbiological method and so on. Zhu and Ghassempour et al. [1,2] used HPLC to study penicillin G (PG), the limit of detection (LOD) and linear concentration range were 1.1 lg/mL and 10–2400 lg/mL respectively. But it has some shortcomings such as expensive equipment and low sensitivity. Bailón-Pérez and Santos et al. [3,4] have proposed a very sensitive

X. Jiang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 474–478

method based on off-line and on-line procedures for the simultaneous detection of seven b-lactam antibiotic residues in different kind of water samples, i.e., wastewater, river water and well water. Although it has high sensitivity, the preparation process is cumbersome. Ferrini et al. [5] proposed an improved high throughput microbial method for the simultaneous performance of first and second level screening for antibacterial residues in meat, the results could be obtained by 18–24 h. Obviously, it is timeconsuming. Therefore, it is important to set up a sensitive, reliable and simple method for detecting penicillin antibiotics. Surface-enhanced Raman scattering (SERS) effect is a phenomenon that the Raman signal intensity of molecules can be dramatically enhanced when the molecules are close to (or adsorb on) the nano-metal material surface. The noble metals (Ag, Au and Cu) nanometer materials are always served as typical SERS-active substrates [6,7]. Since SERS was found in 1974 [8], it has been widely used in areas of molecular detection [9–12] because of its advantages such as rich information content, high sensitivity, high selectivity, inhibiting fluorescence and so on. Many studies have shown that, SERS technique can reach 1014–1015 enhancement factor so as to achieve the single molecule level detection [13]. SERS is a rapid detection technique, and a powerful non-destructive and ultra-sensitive characterization method. In recent years, SERS has attracted tremendous attention and has been widely used as a powerful analytical tool [14–16]. Zhang et al. [16] reported a structure-selective hot-spot Raman enhancement method for the identification and detection of the trace penicilloic acid residue in penicillin, which focused on the rapid monitoring and tracking of allergen in penicillin for reducing the occurrence of allergic reactions. In this study, normal Raman spectroscopy and SERS spectroscopy of NaBP were mainly investigated. It is expected to be helpful for rapid detection of penicillin sodium as well as other b-lactam antibiotics by using SERS technology.

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H2O2:98% H2SO4 = 3:7, volume ratio), and the boiling state was kept until no bubbles were given out. After cooling, the glass slides were rinsed with water and stored in water for further experiments. These hydroxylated glass slides were soaked in 0.5 wt.% PDDA water solution for 1 h, then rinsed with water and dried under N2 stream. Finally the glass slides derivatized with PDDA were immersed in the prepared silver colloid for 4 h and dried by N2 gas. A layer of the silver NPs was assembled on the surface of the slides. Preparation of SERS samples 0.2564 g of NaBP was dissolved in 10 mL pure water, resulting in the 10 1 mol/L NaBP standard stock solution. The standard stock solution was sequentially diluted by the deionized water into series of concentrations: 1  10 2, 1  10 3, 1  10 4, 1  10 5, 1  10 6, 1  10 7 and 1  10 8 mol/L. In addition, the 1  10 2 mol/L NaBP solution was also adjusted to different pH value by HCl or NaOH. Then the glass slides with silver NPs were immersed in different concentration NaBP solutions and different pH value NaBP aqueous solutions for 10 h, respectively. Finally they were washed with water three times and dried naturally. Sample characterization

Experimental section

The surface morphology of the sample was measured on a JEOL JSM-6700F field emission scanning electron microscope (FE-SEM) operated at 5.0 kV. Ultraviolet–visible (UV–vis) absorption spectra were obtained on a Shimadzu UV-3600 spectrophotometer. Normal Raman and SERS spectra were obtained by using a LabRam Aramis Raman Microscope system (Horiba-Jobin Yvon) equipped with a multichannel air cooled charge-coupled device (CCD) detector and a temperature controlled charged coupled device detector ( 70 °C). The 633 nm radiation from a HeNe narrow bandwidth laser (Melles Griot) was used as exciting source. The exposure time for each measurement was 15 s with two times accumulation.

Chemicals

Results and discussion

Silver nitrate (AgNO3, 99%) and sodium citrate (Na3C6H5O72H2O, 99.8%) were purchased from Sigma–Aldrich Chemical Co. Poly(diallyldimethylammonium chloride) (PDDA) was obtained from Beijing Chemical Plant. Benzylpenicillin sodium (NaBP, 99%) was obtained from the Beijing Dingguochangsheng Biotechnology Co., Ltd. Ultrapure water (18.2 MX cm) was produced by using a Millipore water purification system (Millipore, Billerica, MA, USA). All other chemicals were analytical grade and used without further purification.

Characterization of the Ag NPs and Ag-NaBP assemblies The dispersibility and morphology of the prepared Ag NPs assembly were characterized by SEM (as shown in Fig. 1). The Ag NPs were assembled on the PDDA derivatized glass slide by the electrostatic attractive interaction between the positively charged PDDA molecules and the negatively charged Ag NPs. Because of the electrostatic repulsion between the negatively charged Ag NPs, the assembled Ag NPs should be isolated from each other. It can be clearly seen from Fig. 1 that Ag NPs form a submonolayer

Sample preparation Preparation of Ag nanoparticles The classical Ag nanoparticles (NPs) were prepared by using a chemical reduction method according to Lee and Meisel [17]. 36 mg of AgNO3 was dissolved in 200 mL of deionized water and heated to boiling ( 98 °C). 4 mL of 1% sodium citrate aqueous solution was added into the boiling AgNO3 solution with vigorous stirring. The mixed solution was kept at boiling for 40 min. After the flask was cooled, green-gray and transparent colloid solution was obtained. The diameter of Ag NPs was about 60 nm. Finally, the classical Ag NPs solution was stored in refrigerator at 4 °C for further experiments. Preparation of Ag NPs assembly Glass slides washed with organic solvent (ethanol, acetone and chloroform) were first placed in the boiling mixed solution (30%

Fig. 1. SEM image of the silver NPs assembly.

X. Jiang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 140 (2015) 474–478

structure on the derivatized glass slide surface. Most of the particles are isolated and uniformly distributed on the surface. Furthermore, no remarkable change in the assembly structure was observed after the adsorption of the NaBP molecules on the Ag NPs assembly. From the UV–vis spectra (Fig. 2), a maximum absorption peak at 458 nm can be observed. This optical absorption corresponds to the monodisperse colloidal Ag NPs with the diameter in the range of 50–60 nm, which is attributed to the absorption of the localized surface plasmon resonance (LSPR) of Ag NPs. The result is the same as that reported in the literature [17]. For a given metal system, the SERS enhancement will depend on the uniform distributions and the size of nanostructure [18]. For Ag NPs system, the ideal nanostructure size is in the range of 40–80 nm for 633 nm excitation. It can be seen from Fig. 2 that, compared with the Ag NPs assembly, the LSPR maximum absorption of the Ag-NaBP assembly takes place a red-shifted from 458 to 483 nm. This should be resulted from the interaction between the Ag NPs and NaBP molecules due to the result of charge transfer from the Ag NPs to the NaBP molecules [19], indicating that the NaBP molecules have been assembled on the Ag NPs substrate. Raman and SERS spectra of NaBP

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Fig. 3. Raman spectra of solid NaBP (a) and 1  10 1 mol/L NaBP aqueous solution (b), SERS spectrum of 1  10 1 mol/L NaBP adsorbed onto the silver colloid (c) and Ag NPs assembly (d) and Raman spectrum of Ag NPs assembly (e).

proving that NaBP is close to the silver surface through the carboxyl group. The peak at 583 cm 1 is very weak and the peak at 1000 cm 1 has a shift of 2 cm 1 compared with the normal Raman spectra of NaBP. These changes are attributed to the electromagnetic field and chemical or electronic enhancement effect of Ag NPs on the b-lactam ring and thiazolidine ring of NaBP molecules, which are commonly recognized as the active ingredient of NaBP drug.

Quantitative analysis of NaBP Raman spectroscopy is one of the most important detection tools that can provide a lot of information associated with the molecule [22], but the most lethal bottleneck is that the conventional Raman spectroscopy signal is very weak and cannot be used in the high sensitive qualitative and quantitative detection. In recent years, SERS as a new method has broken this bottleneck. In this study, we conducted a quantitative analysis on NaBP by SERS. Fig. 4 shows the SERS spectra of NaBP aqueous solution with different concentrations from 1  10 1 mol/L to 1  10 8 mol/L adsorbed on Ag NPs substrate. It can be clearly seen that SERS spectral intensity enhanced with the increase of NaBP concentration. When the concentration of NaBP decreases to 1  10 8 mol/L, it is difficult to obtain the apparent SERS signals. Therefore, the minimum detectable concentration of NaBP is 1  10 7 mol/L.

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Fig. 3 shows normal Raman spectra of NaBP in the solid state and in 1  10 1 mol/L aqueous solution, and SERS spectrum of NaBP (1  10 1 mol/L) adsorbed onto the silver colloid and Ag NPs assembly. As shown in Fig. 3, the dominating characteristic bands of the normal Raman and SERS spectra of NaBP, ranging from 300 to 3000 cm 1, are distinguishable clearly. SERS spectrum of NaBP adsorbed on the Ag NPs assembly is significantly different from the normal Raman spectra of NaBP aqueous solution and solid powder in not only Raman intensity but also Raman shift. These changes show that there are some interactions between NaBP molecules and silver nanoparticles assembly. It is clear that the Raman signals of NaBP adsorbed on the Ag NPs assembly are remarkably enhanced relative to those in solution and solid state. In normal Raman spectrum of solid NaBP, the typical Raman band at 1002 cm 1 is identified as the vibration of b-lactam ring and thiazolidine ring, involving the stretching vibration of b-lactam ring and twisting vibration of the two methyls (CH3) [16]. Another strong peak at 583 cm 1 is assigned to the stretching vibration of C–C (alkyl chain between the benzene ring and the b-lactam ring) and the wagging vibration of NH [20]. The other weak bands at 1584 and 1602 cm 1 can be attributed to the stretching vibration of C–C (phenyl ring) [16]. Compared to the normal Raman spectra of NaBP, the SERS spectrum has a strong broad peak at 1590 cm 1, which is thought to come from the stretching vibration of C–O in the carboxyl [21],

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Fig. 4. SERS spectra of NaBP aqueous solution with different concentrations adsorbed on Ag NPs substrate. The spectra correspond to the following concentrations in the order from top to bottom: (a) 1  10 1 mol/L; (b) 1  10 2 mol/L; (c) 1  10 3 mol/L; (d) 1  10 4 mol/L; (e) 1  10 5 mol/L; (f) 1  10 6 mol/L; (g) 1  10 7 mol/L and (h) 1  10 8 mol/L. The insert is the intensities of the SERS peaks at 1000 cm 1 and 1590 cm 1 versus the logarithm of NaBP concentrations.

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The influence of pH value on NaBP and its SERS spectrum Penicillin is not stable in acidic and alkaline conditions [23,24] and prone to bring b-lactam ring opening, which will result in inactivation of NaBP drug. In this part, the effects of pH value on NaBP and its SERS spectrum were studied. Fig. 6 shows the SERS spectra of 1  10 2 mol/L NaBP aqueous solution with different pH values adsorbed on Ag NPs substrate. For the natural state NaBP sample with pH = 6 (i.e. no regulating pH value), the intensity of the SERS signal at 1000 cm 1 (the characteristic band of b-lactam ring) is a little higher than that at 1590 cm 1. However, it can be seen clearly that the intensity of the SERS signal at 1000 cm 1 (the characteristic band of b-lactam ring) is obviously lower than that at 1590 cm 1 under the conditions of strong acid (Fig. 6a) and alkali (Fig. 6d and e). Even there are some new Raman peaks appearing at 1283 and 1155 cm 1 under strong acidic (pH = 2) and alkaline (pH = 12) conditions, which can be assigned to the scissoring vibration of C–N–C and twisting vibration of CH in thiazolidine ring, and the bending vibration of CH in phenyl ring, respectively [16]. These changes must be derived from the structural alterations of benzylpenicillin. It is because that NaBP easily opens its b-lactam ring to produce penicilloic acid and penilloaldehyde in acidic condition and generate penicilloic acid and penilloic acid in alkaline

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It also can be observed that, with the decrease of NaBP concentration, the decrease degree in intensity of SERS signal at 1000 cm 1 is more evident compared with that at 1590 cm 1, illuminating that the Raman band at 1000 cm 1 on Ag NPs substrate is more sensitive to the NaBP concentration (as shown in Fig. 4). And, the Raman band at 1000 cm 1 is identified as the characteristic vibration of active ingredients of NaBP drug (i.e. b-lactam ring and thiazolidine ring). As this is the case, it can be used as the characteristic peak to monitor the concentration of NaBP drug. Fig. 5 shows the quantitative relationship between the concentration of NaBP and the intensity of SERS signal at 1000 cm 1. Each datum point represents an average intensity at 1000 cm 1 from five measurements and the error bars indicate standard deviation. From Fig. 5 it can be obviously seen that the SERS signal intensity becomes weaker as the concentration of NaBP decrease and there is a good linear relationship in the low concentration range (from 1  10 4 mol/L to 1  10 7 mol/L), which can be described by the equation y = 122.42 + 11.13  lg C with a correlation coefficient (R2) of 0.9631. This shows that SERS can be used for the quantitative analysis of NaBP in an appropriate concentration range, and also maybe have some promising applications in the quantitative detection of other b-lactam antibiotics besides their qualitative detection.

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Fig. 6. SERS spectra of 1  10 2 mol/L NaBP aqueous solution with different pH values adsorbed on Ag NPs substrate. The spectra correspond to the following pH values in the order from bottom to top: (a) pH = 2; (b) pH = 5; (c) pH = 6 (in natural state, i.e. no regulating); (d) pH = 9; (e) pH = 12.

environment [25], which result in the decrease of Raman intensity at 1000 cm 1 and the appearing of new Raman bands. The chemical structures of penicilloic acid, penilloaldehyde and penilloic acid are shown in Fig. 7. Penicillin is not stable either in acidic or alkaline environments and the decomposition reaction is very complicated due to the diversification of products and the incompleteness of reactions. Penicilloic acid is a major allergen causing the fatal immune responses [25–28]. It is obvious that SERS can be used to detect whether there are other substances presenting in the penicillin (or whether there are ring-opening reactions occurring), which is helpful to reduce the occurrence of allergic reactions. In addition, compared with the natural state NaBP sample with pH = 6, it also can be seen in Fig. 6 that the intensity of the SERS signal at 1590 cm 1 can be further enhanced under strong acidic and alkaline conditions. This should be attributed to the more aggregation of drug molecules on Ag NPs substrate surface after b-lactam ring opening due to their structure changes. The results are consistent with the previous report by Zhang et al. [16]. Zhang’s report showed that silver-coated gold (Au@Ag) NPs exhibit a structure-selective hot-spot Raman enhancement for penicilloic acid in penicillin. It has been demonstrated that penicilloic acid can very easily link Au@Ag NPs together by its two carboxyl groups, locating itself spontaneously at the interparticle of Au@Ag NPs to form strong Raman hot-spot. The Ramanenhanced effect of penicilloic acid is higher than that of penicillin. In particular, the selective Raman enhancement to the two carboxyl groups makes the Raman peak of carboxyl group become stronger.

Conclusions

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Equation: y = 122.42 + 11.13 x lgC R-Square: 0.9631

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In this paper, the silver NPs assembly was served as SERS substrates for detecting the penicillin drug. The results show that NaBP is close to the silver surface through the carboxyl group, the detection limit can be reduced to 1  10 7 mol/L by SERS method. Importantly, there is a good linear relationship in the range of 1  10 4–1  10 7 mol/L concentration (R2 = 0.9631), and a quantitative detection method of benzylpenicillin sodium can be established. In addition, it is proved that NaBP is not stable under the conditions of strong acid and alkali, which can be identified whether there are degradation products by using SERS technology. SERS technique is extremely simple, inexpensive and rapid compared with other methods and each test only needs a very little amount of sample. This work is expected to be helpful for rapid detection of b-lactam antibiotic drugs.

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COOH

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Fig. 7. The chemical structures of penicilloic acid (a), penilloaldehyde (b) and penilloic acid (c).

Acknowledgments The research was supported by National Natural Science Foundation (Grant Nos. 21003063, 21473078, 21273091, 21327803) of People’s Republic of China, Natural Science Foundation of Heilongjiang Province of China (Grant No. B201418), Program for New Century Excellent Talents in Heilongjiang Provincial University (Grant No. 1253-NCET-023), National Undergraduate Training Programs for Innovation and Entrepreneurship (Grant No. 201410222021), Postgraduate Scientific Research Innovation Project of Jiamusi University (Grant No. LM2014_067).

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Rapid monitoring of benzylpenicillin sodium using Raman and surface enhanced Raman spectroscopy.

At present, fluorescence spectroscopy, ultraviolet spectroscopy and infrared spectroscopy are usually used to detect drug molecules, however the infor...
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