Author's Accepted Manuscript
Detection of metronidazole and ronidazole from environmental Samples by surface enhanced Raman spectroscopy Caiqin Han, Jing Chen, Xiaomeng Wu, Yao-wen Huang, Yiping Zhao
www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(14)00361-0 http://dx.doi.org/10.1016/j.talanta.2014.04.083 TAL14755
To appear in:
Talanta
Received date: 21 March 2014 Revised date: 28 April 2014 Accepted date: 29 April 2014 Cite this article as: Caiqin Han, Jing Chen, Xiaomeng Wu, Yao-wen Huang, Yiping Zhao, Detection of metronidazole and ronidazole from environmental Samples by surface enhanced Raman spectroscopy, Talanta, http://dx.doi.org/ 10.1016/j.talanta.2014.04.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Detection of Metronidazole and Ronidazole from Environmental Samples by
2
Surface Enhanced Raman Spectroscopy
3 4
Caiqin Han1,2,3,4†*, Jing Chen2,5†*, Xiaomeng Wu2,5, Yao-wen Huang2,5, and Yiping Zhao1,2
5
1 Department of Physics and Astronomy, University of Georgia, USA
6
2 Nanoscale Science and Engineering Center, University of Georgia, USA
7
3 School of Physics and Electronic Engineering, Jiangsu Normal University, China
8
4 Jiangsu Province Key Laboratory of Advanced Laser Materials and Devices, China
9
5 Department of Food Science and Technology, University of Georgia, USA
10 11
* Corresponding authors:
12
Caiqin Han:
13
101 Shanghai Road, Tongshan District, Xuzhou, Jiangsu 221116, China
14
Email: [email protected]
15
Jing Chen:
16
220 Riverbend Rd, Athens, GA 30605, USA
17
Email: [email protected]
18 19
† Authors of equal contribution.
20
1
21
Abstract
22
In this study, the surface enhanced Raman spectra (SERS) of two prohibited veterinary
23
drugs, metronidazole (MNZ) and ronidazole (RNZ), have been acquired, and compared to the
24
theoretically calculated spectra using density function theory (DFT). The experimental Raman
25
and SERS spectra of MNZ and RNZ exhibit high resemblance with the DFT calculations. SERS
26
detection of MNZ and RNZ from standard solutions as well as real environmental samples (tap,
27
lake, swamp waters and soil) was performed on highly sensitive and reproducible silver nanorod
28
array substrates. The limits of detection for MNZ and RNZ are 10 and 1 μg/mL in methanol and
29
ultra-pure water, respectively, and 10-50 μg/mL in the environmental samples. The SERS-based
30
method demonstrates its potential as a rapid, simple, and inexpensive means for the onsite
31
screening of banned antibiotics from the aquatic and sediment environments, with minimal
32
requirement for sample pretreatment.
33
Keywords: surface enhanced Raman spectroscopy; antibiotics; nitroimidazoles; silver nanorods
2
34
Introduction
35
It is estimated that over 100,000 tons of antibiotics are being produced worldwide every
36
year.[1] Although antibiotics have been extensively used to treat infections for decades, their
37
negative impacts on human and animal health as well as on the environment have not been
38
widely recognized until the past two decades.[2, 3] The intense use of antibiotics is linked to the
39
emergence and spread of drug resistant or even multi-drug resistant pathogen strains, as in the
40
notorious examples of methicillin-resistant Staphylococcus aureus (MRSA), and carbapenem-
41
resistant Enterobacteriuaceae (CRE).[4] The induced antimicrobial resistance can in turn lead to
42
ineffective treatment of potentially deadly infections. In addition to the growing difficulty in
43
disease treatment, some antibiotics have also exhibited in vitro and in vivo carcinogenic
44
properties.[5] Based on these concerns, there is an increasing restriction on the access to
45
antimicrobial drugs through human medicine. Nonetheless, antibiotics are still capable of
46
entering the human diet through transference from animal feed or veterinary drugs to edible
47
tissues of farm animals, fish, and seafood.[5] Moreover, since current household water treatment
48
only aims at removing bacterial and particulate hazards, small, water-soluble antibiotics may still
49
well reside in potable water if the source has been contaminated. Consequently, their use in
50
animals, particularly the use of carcinogenic antibiotics, is also strictly controlled by regulatory
51
bodies.
52
Metronidazole (MNZ) and ronidazole (RNZ) are two antiprotozoal drugs from the
53
nitromidazole family, which were primarily labeled for the treatment of histomoniasis and
54
trichomoniasis in poultry and haemorrhagic enteritis in pigs, but were later banned by the
55
European Commission and the US food and drug administration (FDA) in food-producing
56
animals due to their suspected genotoxic, carcinogenic, and mutagenic properties.[6, 7]
57
Nonetheless, the drugs’ side effects on growth promotion and improvement in feed efficiency
58
have led to continued misuse in animals for short-term economic gain. In order to monitor such
59
illegal uses, a number of methods based on liquid (LC) or gas chromatography (GC) coupled
60
with mass spectroscopy (MS) or fluorescence detection have been developed for identifying
61
nitroimidazoles from water, animal tissues, food and feedstuffs, and clinical samples.[8-12] In
62
spite of high sensitivity, these methods have also encountered drawbacks from long assay
63
duration, complicated pre-treatment, low throughput, and high demands on instrumentation. 3
64
Apparently, the lack of rapid and inexpensive routine screening for these antibiotics has placed a
65
technical hurdle for effective enforcement of regulations in animals and animal farms. To
66
overcome this challenge, several alternative techniques are being developed. Immunoassays such
67
as enzyme-linked immunosorbent assay (ELISA) have been used for rapid sensing of
68
nitroimidazoles.[13] A surface plasmon resonance (SPR) method was recently developed and
69
validated for screening of nitromidazoles from animal tissues and serum samples.[14] These
70
immunocapture-based methods could achieve excellent detection sensitivity within a relatively
71
short time, but still face some challenges caused by the cross-reactivity and limited availability
72
of drug-specific antibodies.
73
Like SPR, surface enhanced Raman spectroscopy (SERS) has also been proposed as a
74
sensitive yet simple, rapid, and inexpensive alternative to GC or LC-based techniques. But unlike
75
SPR, SERS can directly reveal the characteristic vibrational modes of the drug molecule, thus it
76
does not rely on the use of antibodies. SERS takes advantage of the enhanced local
77
electromagnetic field due to the specific geometry of the nanostructures of noble metals (Au, Ag
78
and Cu) to amplify the Raman scattering signal by up to 106-1012 times, and has been used to
79
achieve single molecule detection.[15] A variety of antibiotics have been investigated using
80
SERS or surface enhanced resonance Raman spectroscopy (SERRS), including sulfa drugs
81
(sulfadiazine, sulfamerazine, and sulfamethazine),[16] penicillin and derivatives,[17, 18]
82
entrofloxacin, ciprofloxacin, chloramphenicol,[19] erythromycin,[20] nitrofuran antibiotics
83
(furadantin and furaltadone),[21] moxifloxacin,[22] and amphotericin and derivatives.[23]
84
Though considered as important targets for antibiotic surveillance, the SERS signature of any
85
nitroimidazole antibiotic is still lacking. Also, since antibiotic standards were detected from
86
aqueous solutions or organic solvents in most of the reported antibiotic SERS studies, but few
87
studies have involved detection from real or spiked samples of environmental, food safety, or
88
clinical relevance, so there is also a need to test the applicability of the SERS technique for real
89
sample detection.
90
In this study, we have acquired the Raman and SERS spectra of two representative
91
nitroimidazole antibiotics, MNZ and RNZ, and compared the experimental data with theoretical
92
calculation using density function theory (DFT). We have also demonstrated that rapid and facile
93
SERS screening of MNZ and RNZ from tap water, lake water, and swamp water samples and 4
94
real soil samples could be achieved using a highly uniform silver nanorod substrate prepared by
95
oblique angle deposition.
96 97
Materials and Methods
98
Materials and reagents
99
Metronidazole (MNZ) and Ronidazole (RNZ) were purchased from Sigma Aldrich (St
100
Louis, MO). Methanol, sulfuric acid, and hydrogen peroxide were obtained from J. T. Baker
101
(Phillipsburgm NJ). Silver (99.999%) and titanium (99.995%) pellets were obtained from Kurt J
102
Lesker (Clairton, PA).
103
SERS substrate preparation
104
SERS-active silver nanorod (AgNR) array substrates were fabricated using oblique angle
105
deposition (OAD) in a custom-built electron beam evaporation system as described in our
106
previous studies.[24, 25] Briefly, glass slides were cleaned with Piranha solution (80% sulfuric
107
acid, 20% hydrogen peroxide), rinsed with deionized (DI) water, dried with nitrogen gas, and
108
loaded into the deposition chamber. Films of 20 nm of titanium and 200 nm of silver were
109
sequentially deposited onto the glass slides at a normal incidence angle at the rates of 0.2 nm/s
110
and 0.3 nm/s, respectively. In the final step, the substrate surface normal was rotated to an angle
111
of 86° with respect to the vapor incident direction, and silver continued to be deposited at a rate
112
of 0.3 nm/s. The chamber was maintained at a pressure of
Detection of metronidazole and ronidazole from environmental Samples by surface enhanced Raman spectroscopy Caiqin Han, Jing Chen, Xiaomeng Wu, Yao-wen Huang, Yiping Zhao
www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(14)00361-0 http://dx.doi.org/10.1016/j.talanta.2014.04.083 TAL14755
To appear in:
Talanta
Received date: 21 March 2014 Revised date: 28 April 2014 Accepted date: 29 April 2014 Cite this article as: Caiqin Han, Jing Chen, Xiaomeng Wu, Yao-wen Huang, Yiping Zhao, Detection of metronidazole and ronidazole from environmental Samples by surface enhanced Raman spectroscopy, Talanta, http://dx.doi.org/ 10.1016/j.talanta.2014.04.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Detection of Metronidazole and Ronidazole from Environmental Samples by
2
Surface Enhanced Raman Spectroscopy
3 4
Caiqin Han1,2,3,4†*, Jing Chen2,5†*, Xiaomeng Wu2,5, Yao-wen Huang2,5, and Yiping Zhao1,2
5
1 Department of Physics and Astronomy, University of Georgia, USA
6
2 Nanoscale Science and Engineering Center, University of Georgia, USA
7
3 School of Physics and Electronic Engineering, Jiangsu Normal University, China
8
4 Jiangsu Province Key Laboratory of Advanced Laser Materials and Devices, China
9
5 Department of Food Science and Technology, University of Georgia, USA
10 11
* Corresponding authors:
12
Caiqin Han:
13
101 Shanghai Road, Tongshan District, Xuzhou, Jiangsu 221116, China
14
Email: [email protected]
15
Jing Chen:
16
220 Riverbend Rd, Athens, GA 30605, USA
17
Email: [email protected]
18 19
† Authors of equal contribution.
20
1
21
Abstract
22
In this study, the surface enhanced Raman spectra (SERS) of two prohibited veterinary
23
drugs, metronidazole (MNZ) and ronidazole (RNZ), have been acquired, and compared to the
24
theoretically calculated spectra using density function theory (DFT). The experimental Raman
25
and SERS spectra of MNZ and RNZ exhibit high resemblance with the DFT calculations. SERS
26
detection of MNZ and RNZ from standard solutions as well as real environmental samples (tap,
27
lake, swamp waters and soil) was performed on highly sensitive and reproducible silver nanorod
28
array substrates. The limits of detection for MNZ and RNZ are 10 and 1 μg/mL in methanol and
29
ultra-pure water, respectively, and 10-50 μg/mL in the environmental samples. The SERS-based
30
method demonstrates its potential as a rapid, simple, and inexpensive means for the onsite
31
screening of banned antibiotics from the aquatic and sediment environments, with minimal
32
requirement for sample pretreatment.
33
Keywords: surface enhanced Raman spectroscopy; antibiotics; nitroimidazoles; silver nanorods
2
34
Introduction
35
It is estimated that over 100,000 tons of antibiotics are being produced worldwide every
36
year.[1] Although antibiotics have been extensively used to treat infections for decades, their
37
negative impacts on human and animal health as well as on the environment have not been
38
widely recognized until the past two decades.[2, 3] The intense use of antibiotics is linked to the
39
emergence and spread of drug resistant or even multi-drug resistant pathogen strains, as in the
40
notorious examples of methicillin-resistant Staphylococcus aureus (MRSA), and carbapenem-
41
resistant Enterobacteriuaceae (CRE).[4] The induced antimicrobial resistance can in turn lead to
42
ineffective treatment of potentially deadly infections. In addition to the growing difficulty in
43
disease treatment, some antibiotics have also exhibited in vitro and in vivo carcinogenic
44
properties.[5] Based on these concerns, there is an increasing restriction on the access to
45
antimicrobial drugs through human medicine. Nonetheless, antibiotics are still capable of
46
entering the human diet through transference from animal feed or veterinary drugs to edible
47
tissues of farm animals, fish, and seafood.[5] Moreover, since current household water treatment
48
only aims at removing bacterial and particulate hazards, small, water-soluble antibiotics may still
49
well reside in potable water if the source has been contaminated. Consequently, their use in
50
animals, particularly the use of carcinogenic antibiotics, is also strictly controlled by regulatory
51
bodies.
52
Metronidazole (MNZ) and ronidazole (RNZ) are two antiprotozoal drugs from the
53
nitromidazole family, which were primarily labeled for the treatment of histomoniasis and
54
trichomoniasis in poultry and haemorrhagic enteritis in pigs, but were later banned by the
55
European Commission and the US food and drug administration (FDA) in food-producing
56
animals due to their suspected genotoxic, carcinogenic, and mutagenic properties.[6, 7]
57
Nonetheless, the drugs’ side effects on growth promotion and improvement in feed efficiency
58
have led to continued misuse in animals for short-term economic gain. In order to monitor such
59
illegal uses, a number of methods based on liquid (LC) or gas chromatography (GC) coupled
60
with mass spectroscopy (MS) or fluorescence detection have been developed for identifying
61
nitroimidazoles from water, animal tissues, food and feedstuffs, and clinical samples.[8-12] In
62
spite of high sensitivity, these methods have also encountered drawbacks from long assay
63
duration, complicated pre-treatment, low throughput, and high demands on instrumentation. 3
64
Apparently, the lack of rapid and inexpensive routine screening for these antibiotics has placed a
65
technical hurdle for effective enforcement of regulations in animals and animal farms. To
66
overcome this challenge, several alternative techniques are being developed. Immunoassays such
67
as enzyme-linked immunosorbent assay (ELISA) have been used for rapid sensing of
68
nitroimidazoles.[13] A surface plasmon resonance (SPR) method was recently developed and
69
validated for screening of nitromidazoles from animal tissues and serum samples.[14] These
70
immunocapture-based methods could achieve excellent detection sensitivity within a relatively
71
short time, but still face some challenges caused by the cross-reactivity and limited availability
72
of drug-specific antibodies.
73
Like SPR, surface enhanced Raman spectroscopy (SERS) has also been proposed as a
74
sensitive yet simple, rapid, and inexpensive alternative to GC or LC-based techniques. But unlike
75
SPR, SERS can directly reveal the characteristic vibrational modes of the drug molecule, thus it
76
does not rely on the use of antibodies. SERS takes advantage of the enhanced local
77
electromagnetic field due to the specific geometry of the nanostructures of noble metals (Au, Ag
78
and Cu) to amplify the Raman scattering signal by up to 106-1012 times, and has been used to
79
achieve single molecule detection.[15] A variety of antibiotics have been investigated using
80
SERS or surface enhanced resonance Raman spectroscopy (SERRS), including sulfa drugs
81
(sulfadiazine, sulfamerazine, and sulfamethazine),[16] penicillin and derivatives,[17, 18]
82
entrofloxacin, ciprofloxacin, chloramphenicol,[19] erythromycin,[20] nitrofuran antibiotics
83
(furadantin and furaltadone),[21] moxifloxacin,[22] and amphotericin and derivatives.[23]
84
Though considered as important targets for antibiotic surveillance, the SERS signature of any
85
nitroimidazole antibiotic is still lacking. Also, since antibiotic standards were detected from
86
aqueous solutions or organic solvents in most of the reported antibiotic SERS studies, but few
87
studies have involved detection from real or spiked samples of environmental, food safety, or
88
clinical relevance, so there is also a need to test the applicability of the SERS technique for real
89
sample detection.
90
In this study, we have acquired the Raman and SERS spectra of two representative
91
nitroimidazole antibiotics, MNZ and RNZ, and compared the experimental data with theoretical
92
calculation using density function theory (DFT). We have also demonstrated that rapid and facile
93
SERS screening of MNZ and RNZ from tap water, lake water, and swamp water samples and 4
94
real soil samples could be achieved using a highly uniform silver nanorod substrate prepared by
95
oblique angle deposition.
96 97
Materials and Methods
98
Materials and reagents
99
Metronidazole (MNZ) and Ronidazole (RNZ) were purchased from Sigma Aldrich (St
100
Louis, MO). Methanol, sulfuric acid, and hydrogen peroxide were obtained from J. T. Baker
101
(Phillipsburgm NJ). Silver (99.999%) and titanium (99.995%) pellets were obtained from Kurt J
102
Lesker (Clairton, PA).
103
SERS substrate preparation
104
SERS-active silver nanorod (AgNR) array substrates were fabricated using oblique angle
105
deposition (OAD) in a custom-built electron beam evaporation system as described in our
106
previous studies.[24, 25] Briefly, glass slides were cleaned with Piranha solution (80% sulfuric
107
acid, 20% hydrogen peroxide), rinsed with deionized (DI) water, dried with nitrogen gas, and
108
loaded into the deposition chamber. Films of 20 nm of titanium and 200 nm of silver were
109
sequentially deposited onto the glass slides at a normal incidence angle at the rates of 0.2 nm/s
110
and 0.3 nm/s, respectively. In the final step, the substrate surface normal was rotated to an angle
111
of 86° with respect to the vapor incident direction, and silver continued to be deposited at a rate
112
of 0.3 nm/s. The chamber was maintained at a pressure of
