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www.rsc.org/nanoscale Page 1 of 24 Nanoscale
1 Gold immunochromatographic assay for rapid and
2 simultaneous detection of fifteen β lactams
3 Yanni Chen 1, Yongwei Wang 2, Liqiang Liu 1, Xiaoling Wu 1, Liguang Xu 1, Hua
4 Kuang 1, Aike Li 2, Chuanlai Xu 1*
5 1State Key Lab of Food Science and Technology, School of Food Science 6 and Technology, Jiangnan University, Wuxi, JiangSu, 214122, PRC. 7 2Cereals & Oils Nutrition Research Group, Academy of Science & Technology of State 8 Administration of Grain, Beijing100037, PRC.
9
10 ABSTRACT
11 A novel gold immunochromatographic assay (GICA) based on anti β lactams
12 receptors was innovatively developed that successfully allowed rapid and Manuscript 13 simultaneous detection of fifteen β lactams in milk samples in 5 10 minutes. 14 Replacement of the antibodies used in traditional GICA with anti β lactam receptors 15 overcame the difficulty in producing broad specific antibodies against β lactams. 16 Conjugates of ampicillin with BSA and goat anti mouse immunoglobulin (IgG) were 17 immobilized onto the test and control lines on nitrocellulose membrane, respectively.
18 Since goat anti mouse IgG does not combine with receptors, negative serum from Accepted 19 mice labelled with gold nanoparticles (GNP) was mixed with GNP labelled receptors. 20 Results were obtained within 20 min using a paper based sensor. The utility of the 21 assay was confirmed with the analysis of milk samples. The limits of detection (LOD) 22 for amoxicillin, ampicillin, penicillin G, penicillin V, cloxacillin, dicloxacillin, 23 nafcillin, oxacillin, cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone, 24 cefathiamidie, and cefepime were 0.25, 0.5, 0.5, 0.5, 1, 5, 5, 10, 25, 10, 100, 10, 5, 5, Nanoscale 25 2 ng/mL, respectively, which satisfies the maximum residue limits (MRL) set by 26 European Union (EU). In conclusion, our newly developed GICA based 27 anti β lactams receptors assay provides a rapid and effective method for one site 28 detection of multiple β lactams in milk samples. 29 30 Keywords: β lactams, gold immunochromatographic assay, receptors, milk samples
E mail: [email protected]; [email protected]; Tel: 0510 85329076 1
Nanoscale Page 2 of 24
1 1 Introduction 2 In current veterinary, extensive administration of antibiotics to dairy cattle for
3 therapeutic purpose (for instance, treatment of bovine mastitis, pneumonia or bacterial
4 infections) 1 2 has triggered significant food safety issues because of antibiotics
5 resistance 3, which is transferred to humans through the ingestion of meat and milk
6 products 4, 5. Furthermore, allergic reaction to antibiotic residues and inhibition of
7 starter cultures used in the production of fermented milk products pose a significant
8 threat to our daily life 6, 7. At present, the β lactams, regarded as the oldest and the
9 most important group of antibiotics primarily composed of penicillins and
10 cephalosporins that possess a huge side chain attached to 6 aminopenicillanic acid
8, 9 11 and 7 aminocephaloporanic acid nuclei, respectively , are the most extensively Manuscript
12 characterized antimicrobial group in milk owing to their therapeutic efficacy 10, 11 .
13 To improve the quality of dairy products and ensure consumer safety, many
14 regulatory authorities have established maximum residue limits (MRL) of β lactams
15 in dairy products. The MRL of penicillin G in milk is 4, which is regulated by the Accepted 16 EU 12 . The EU additionally set MRLs of amoxicillin, ampicillin, cloxacillin,
17 dicloxacillin, nafcillin, oxacillin, cefacetrile, and cefoperazone as 4, 4, 30, 30, 30, 30,
18 125, and 50 µg/kg in milk, respectively 13 . Several analytical methods have been
19 developed to determine the antibiotic residues of β lactams in food. These procedures
20 have been classified into four main categories 13 : microbial inhibition, Nanoscale
21 chromatographic techniques, biosensors, immunochemical techniques. As the
22 traditional method for antibiotic detection, microbiological approaches based on
23 bacterial growth inhibition have been widely commercialized due to their reliability
24 and cost effectiveness 14, 15 . The LODs for β lactams of many commercial microbial
25 inhibition tests range from 2 to 100 µg/kg, which can meet the MRLs set by EU.
2
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1 However, these protocols are time consuming and non specific, which are unsuitable
2 for rapid and high throughput detection. Chromatographic assays have traditionally
3 been employed as the reference method in antibiotic residue researches 16 19 . Although
4 this procedure is accurate and sensitive, expensive instruments and skilled operators
5 are needed. What’s more, the pretreatment of samples is complex. In recent years,
6 studies on biosensors have become increasingly prevalent. Various biosensors have
7 been developed based on different transduction: enzymes, proteins, and so on 11, 20 22 .
8 In general, the detection based on biosensors is sensitive and specific. Nevertheless, it
9 is unpractical for the one site detection and large batches. Immunochemical methods
10 mainly comprise enzyme linked immunoassay (EIA) 23 , gold
24
11 immunochromatographic assays (GICA) , fluorescence polarization immunoassay Manuscript
12 (FPIA) 25 and other immunosensors based on different transduction elements. To a
13 degree, the biosensors have always been utilized together with immunoassay 26 .
14 However, it is quite difficult to produce sensitive antibodies against β lactams, due to
15 the instability of the ring structure (the common structure of β lactams) 27, 28 . Even Accepted 16 though some papers reported that antibodies against one or several β lactams were
17 produced, they failed to develop the assay that can detect the both penicillins and
18 cephalosporins. Recently, the researches of β lactams detections based on receptors
19 have become the hot topic 8, 29, 30 . A receptor based enzyme linked immunosorbent
20 assay (ELISA) was developed to detect β lactams, but the total time of ELISA was Nanoscale 21 too long compared with GICA, which can be quickly achieved in 5 10 minutes. In
22 addition, there is no need for trained persons and specific apparatus for high
23 throughput one site determination by GICA.
24 In such a case, GICA based on receptors recognizing multi β lactams (both
25 penicillins and cephalosporins) can not only solve the bottleneck problem for
3
Nanoscale Page 4 of 24
1 traditional immunoassay, but also achieve the fast, sensitive, and high throughput
2 detection. To the best of our knowledge, we have established a novel GICA based on
3 class specific anti β lactam receptors that can simultaneously recognize fifteen
4 β lactams for the first time. This newly developed procedure which can be fulfilled in
5 5 10 minutes, effectively avoids the challenge of class specific antibodies, supporting
6 its utility in multiple β lactams identification.
7
8 2 Materials and methods
9 2.1 Reagents and apparatus
10 Ampicillin (AMP) sodium ， penicillin G potassium salt, nafcillin sodium salt Manuscript 11 monohydrate, and dicloxacillin sodium hydrate were purchased from J&K Scientific
12 Ltd. (Beijing, China). Penicillin V potassium salt, amoxicillin, cloxacillin sodium salt
13 monohydrate, oxacillin sodium salt were purchased from Aladdin Scientific Ltd.
14 (Shanghai, China). Cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone,
15 cefathiamidine, and cefepime were acquired from National Standard Substances Accepted
16 Center (Beijing, China). Goat anti mouse immunoglobulin (IgG) antibody was
17 purchased from Jackson ImmunoResearch Laboratories (PA, USA). Bovine serum
18 albumin (BSA), 1 (3 Dimethylaminopropyl) 3 ethylcarbodiimide (EDC), and
19 N hydroxysuccinimide (NHS) were purchased from Sigma Aldrich (St. Louis, MO,
20 USA). The 4 (2 Hydroxyethyl) 1 piperazineethanesulfonic acid (HEPES) and Nanoscale
21 2 (4 Morpholino) ethanesulfonic acid (MES) were purchased from J & K Scientific
22 Ltd (Beijing, China). The anti β lactams receptors were purchased from Wuxi
23 Determine Bio Scientific Ltd (Wuxi, China).
24 All buffer solutions were prepared with pure water produced using the milli Q
25 ultrapure system (Bedford, MA). Polyvinylchloride (PVC) pads, absorbance pad 4
Page 5 of 24 Nanoscale
1 (H5079), and the sample pad (glass fiber membrane, GL b01) were obtained from
2 JieYi Biotechnology Co., Ltd. (Shanghai, China). The nitrocellulose (NC) membrane
3 (Unistart CN140) was obtained from Sartorius Stedim Biotech GmbH (Goettingen,
4 Germany). The CM4000 Guillotine Cutting Module and the Dispensing Platform
5 were purchased from Kinbio Tech Co., Ltd. (Shanghai, China). The electrophoresis
6 apparatus was supplied by Bio Rad Laboratories Co., Ltd (CA, USA).
7
8 2.2 Preparation of AMP BSA conjugates
9 Conjugation of AMP BSA was achieved by EDC/NHS method on account of
10 carboxyl group of AMP. The detailed procedure was based on several earlier reports,
31, 32
11 with some modifications . Briefly, 142 mg of EDC and 89 mg of NHS were added Manuscript
12 into100 mg of AMP dissolved in 5 mL of MES (0.01M, pH 6.5) and allowed to react
13 for 1 h under room temperature with constant stirring. The mixture solution was
14 slowly dropped into 330 mg of BSA dissolved in HEPES (0.01 M, pH 7.0) and
15 reacted for 4 h under room temperature with constant stirring. The final solution was Accepted 16 dialyzed for 3 days under 4 ℃ to obtain pure AMP BSA conjugates.
17 Electrophoretograms of AMP BSA and BSA (Figure S1) were used to confirm the
18 success of conjugates.
19
20 2.3 Preparation of anti β lactam receptors and negative serum labelled with Nanoscale
21 GNPs
22 The productions of receptors are as follows 29 : (1) the gene of receptors was cloned by
23 polymerase chain reaction (PCR); (2) the amplified gene was inserted into the carrier
24 in Escherichia coli (E. coli ); (3) the inserted gene expressed into receptor proteins;
25 and (4) the proteins were extracted via ultrasonic treatment and purified using the
5
Nanoscale Page 6 of 24
1 affinity column. Appropriate receptor generation was confirmed via electrophoresis.
2 GNPs were synthesized in our laboratory with the sodium citrate reduction
3 method as previously described with some modifications 31, 33, 34 . Briefly, 50 mL of
4 HAuCl 4·4H 2O (0.01%, w/v) was boiled thoroughly with constant stirring and rapidly
5 mixed with 2 mL of freshly prepared 1% (w/v) trisodium citrate. The mixture was
6 sequentially boiled for 6 min until the solution color changed to wine red. The
7 solution was cooled to room temperature and stored at 4 ℃ for future use. GNPs were
8 characterized by transmission electron microscopy (TEM) and UV vis spectrometry.
9 Anti β lactam receptors were characterized by SDS PAGE. Molecular weights of
10 receptors were determined as 67 96 kDa. The procedure of labelling anti β lactam
11 receptors with GNPs was similar to that of GNP labelling of monoclonal antibodies, Manuscript
35 12 with some modifications . Briefly, 80 L of K2CO 3 (0.1 M) was added into 20 mL of
13 GNPs for adjustment of pH to 9. Receptors (0.2 mg) were slowly added to the GNPs
14 solution under continuous stirring for 30 min at room temperature, followed by
15 treatment with BSA (50 mg) dissolved in 1 mL of ultrapure water under stirring for Accepted 16 30 min and centrifugation at 875 rpm for 40 min to remove free blocking agent and
17 excess receptor. The resulting precipitate was resuspended twice in borate buffer
18 (0.002 M, pH 8, containing 1% (w/v) sucrose and 0.01% Tween 20) to ~5% of its
19 original volume.
20 Anti β lactams receptors produced via gene expression and could not conjugate Nanoscale
21 with goat anti mouse IgG, resulting in no color change in the control line to red. To
22 resolve this issue, negative serum was collected and labelled with GNPs using a
23 similar procedure to that described above.
24
25 2.4 Principle and assembly of GICA
6
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1 As shown in Figure 1, a gold immunochromatographic assay strip consist of four
2 sections (absorbent pad, conjugate pad, sample pad and NC membrane), which are
3 sequentially assembled onto a plastic backing sheet. The NC membrane is coated with
4 AMP BSA conjugate on the test line to capture the anti β lactams receptor labelled
5 with GNPs, which allowed colloidal gold to aggregate and form a visible line. The
6 control line was coated with goat anti mouse IgG for conjugation with GNP labelled
7 negative serum, allowing the aggregation of colloidal gold. One red band consistently
8 appears on the control line regardless of the presence of analytes, certifying the
9 validity of the test. In cases where visible red lines appear in both the control and test
10 zone, the sample is negative. Conversely, the presence of only one visible red band in
11 the control zone indicates a positive sample . Manuscript
12
13 (Figure 1)
14
15 In our study, the sample pad was firstly imbued with buffer solution (0.01M PBS Accepted 16 containing 0.2% BSA and 0.2% Tween 20) and air dried overnight for future use.
17 Next, the NC membrane was coated with two conjugate types (AMP BSA and goat
18 anti mouse IgG) on the test and control lines, respectively, using the rapid test
19 dispenser platform at a jetting rate of 1 L/cm. The NC membrane was dried at 37 0C
20 for 3 h and stored in a desiccator. Optimal volumetric ratios of receptor GNP and Nanoscale 21 negative serum GNP conjugates were added to a microwell and freeze dried in a
22 vacuum freeze dryer. The strips and freeze dried mixtures were stored in a desiccator
23 at room temperature until use.
24
25 2.5 Analysis of milk sample using GICA based on anti β lactam receptors
7
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1 Negative milk samples confirmed by LC/MS/MS, which were supported by Jiangsu
2 Entry Exit Inspection and Quarantine Bureau, were spiked with fifteen β lactams at a
3 range of concentrations. Amoxicillin, ampicillin, penicillin G, penicillin V, oxacillin,
4 dicloxacillin, cloxacillin, nafcillin, cefaclor, ceftezole, cefotaxime, ceftiofur,
5 cefoperazone, cefathiamidine, cefepime, cefalexin, cefadroxil, cefradine, cefuroxime,
6 and cefodizime were prepared at a concentration of 1 mg/mL using HEPES (0.01 M,
7 pH 7.0), and further diluted 100 times to a concentration of 1 g/mL to obtain stock
8 solution. Fortified milk was diluted 10 times with HEPES (0.01M, pH 7.0) to obtain
9 different final concentrations as follows: amoxicillin (0, 0.25, 0.5, 1, 2, and 5 ng/mL),
10 ampicillin (0, 0.5, 1, 2, 5, and 10 ng/mL), penicillin G (0, 0.25, 0.5, 1, 2, and 5 ng/mL),
11 penicillin V (0, 0.25, 0.5, 1, 2, and 5 ng/mL), cloxacillin (0, 1, 2, 5, 10, and 25 ng/mL), Manuscript
12 dicloxacillin (0, 1, 2, 5, 10, and 20 ng/mL), nafcillin (0, 2, 5, 10, 25, and 50 ng/mL),
13 oxacillin (0, 4, 10, 20, 50, and 100 ng/mL), cefaclor (0, 10, 25, 50, 100, and 200
14 ng/mL), ceftezole (0, 5, 10, 25, 50, and 100 ng/mL), cefotaxime (0, 50, 100, 250, 500,
15 and 1000 ng/mL), ceftiofur (0, 10, 25, 50, 200, and 200 ng/mL), cefoperazone (0, 1, 2, Accepted 16 5, 10, and 25 ng/mL), cefathiamidine (0, 5, 10, 25, 50, and 100 ng/mL) and cefepime
17 (0, 2, 5, 10, 25, and 50 ng/mL).
18 2.6 Live subject statement
19 This article does not contain any studies involving human subjects. All animal studies
20 were carried out under the guidance of animal welfare committee of Jiangnan Nanoscale
21 University.
22
23 3 Results and Discussion
24 3.1 Preparation of AMP BSA conjugates
25 Successful generation of AMP BSA conjugates was confirmed via SDS PAGE. As
8
Page 9 of 24 Nanoscale
1 shown in Figure S2, the apparent shift band between the same concentration of BSA
2 and AMP BSA was indicative of successful conjugation.
3
4 3.2 Preparation of anti β lactam receptors labelled with GNPs
5 The charge and stability of conjugates are determined by solution pH. Therefore, pH
6 plays a crucial role in the preparation of receptor GNP conjugates. Colloidal gold
7 carries a negative charge and therefore combines efficiently with positively charged
8 proteins through electrostatic bonds. Receptor GNP conjugates are formed through
9 electrostatic interactions between IgG and negatively charged GNPs. Colloidal gold
10 must be stabilized with BSA or polyethylene glycol (PEG) after conjugation with
11 receptors. The conjugation between negative serum and GNPs was similar to that Manuscript
12 between receptors and GNPs. K2CO 3 was used to adjust the pH of colloidal gold. The
13 K2CO 3 volume and final receptor concentrations were optimized. Our results indicate
14 that 6 L of 0.1 M K 2CO 3 for each mL GNP solution is optimal for the mixture of
15 receptor GNP and negative serum GNP conjugates. Accepted 16 In total, 50 L of 10% BSA (used as a stabilizer) was added dropwise with
17 constant stirring to 1 mL of conjugates to reduce non specific reactions.
18
19 3.3 Optimization of the GICA
20 Different concentrations of anti β lactam receptors (0.1, 0.2, 0.4 and 0.8 µg/mL), Nanoscale 21 AMP BSA (0.05, 0.1, 0.2, and 0.4 µg/mL), and GNPs (2, 4, and 8 nM) were
22 examined, with a view to optimizing the results. The optimal conditions were
23 determined as 0.2 µg/mL anti β lactam receptor, 0.1 µg/mL AMP BSA, and 4 nM
24 GNPs. Under these conditions, receptor GNP and negative serum GNP solutions were
25 stable after centrifuging twice and resuspension.
9
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1
2 3.4 Validation of GICA based on anti β lactam receptors with milk samples
3 Spiked milk samples were pretreated with simple dilution, with a view to eliminating
4 matrix interference. As shown in Figures S3 9, the strip performed well at a sequence
5 of concentrations. This biosensor can therefore be applied for one site detection for
6 fifteen β lactams by untrained persons with no need for specific apparatus. The test
7 line becomes less intense or colorless relative to the control line depending on the
8 concentration, which can be judged by the naked eye. Figures 2 and 3 depict four
9 representative penicillin and cephalosporin compounds in milk samples, respectively.
10 For detection of amoxicillin, the LOD was 0.25 ng/mL, which was at the advanced
11 level. Analyses of the remaining seven compounds are shown in Figures S3 9. A Manuscript
12 summarized image for all 15 chemicals is shown in Figure 4. The test line of each
13 strip is obviously less intense in color than the control line. The concentration is
14 defined as visual LOD. The cut off values for the GICA, defined as the concentration
15 producing no color on the test line, present another parameter for semi quantitative Accepted 16 assessment. Cut off values and LOD obtained for β lactams in the milk sample
17 confirm that the method meets the MRLs set by EU (Table 1). Moreover, 20
18 independent milk samples were analyzed to ascertain the reproducibility of the
19 method. Stability experiments indicate that neither reaction intensity nor sensitivity
20 are influenced during the course of storage for six months. Nanoscale 21
22 (Figure 2)
23 (Figure 3)
24 (Figure 4)
25 (Table 1)
10
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1
2 4 Conclusions
3 In this paper, a novel biosensor was developed that allowed successful detection of
4 fifteen β lactams in milk samples for the first time. Rapid, semi quantitative detection
5 by the naked eye was achieved using simple GICA based on anti β lactam receptors
6 in 5 10 minutes, facilitating high throughput testing for β lactams in milk samples. In
7 recent years, the usage of β lactams in animal husbandry and dairy products has
8 drastically increased leading to an urgent requirement for rapid, simple, and
9 high throughput detection methods that assist in company surveillance and
10 safeguarding consumer rights.
11 Manuscript
12 Acknowledgements
13 This work is financially supported by the National Natural Science Foundation of
14 China (21471068 ，31400848), the Key Programs from MOST (2013AA065501).
15 Accepted
16 Appendix A. Supplementary material
17 Supplementary data associated with this article was provided.
18
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16
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1 Captions
2 Fig. 1. The principle of GICA based on anti β lactams receptors
3 Fig. 2. The images of GICA based anti β lactams receptors for four kinds of representative
4 penicillins in milk samples: (A) ammocillin: 0, 0.25, 0.5, 1, 2 and 5 ng/mL; (B)
5 ampicillin: 0, 0.5, 1, 2, 5, and 10 ng/mL; (C) penicillin G: 0, 0.25, 0.5, 1, 2, and 5 ng/mL;
6 (D) penicillin V: 0, 0.25, 0.5, 1, 2, and 5 ng/mL, respectively.
7 Fig. 3. The images of GICA based anti β lactams receptors for four kinds of representative
8 cephalosporins in milk samples: (A) cefaclor: 0, 10, 25, 50, 100, and 200 ng/mL; (B)
9 ceftezole: 0, 5, 10, 25, 50, and 100 ng/mL; (C) cefoperazone: 0, 1, 2, 5, 10, and 25
10 ng/mL; (D) cefathiamidine: 0, 5, 10, 25, 50, and 100 ng/mL, respectively.
11 Fig. 4. The detection of fifteen β lactams in milk sample using GICA based on anti β lactams
12 receptors. The final concentrations of fifteen β lactams in ten time diluted milk sample: Manuscript
13 (1) 0 ng/mL, (2) 1 ng/mL of amoxicillin, (3) 2 ng/mL of amoxicillin, (4) 2 ng/mL
14 ampicillin, (5) 5 ng/mL ampicillin, (6) 0.5 ng/mL penicillin G, (7) 1 ng/mL penicillin G,
15 (8) 1 ng/mL penicillin V, (9) 2 ng/mL penicillin V, (10) 5 ng/mL cloxacillin (11) 10
16 ng/mL cloxacillin, (12) 10 ng/mL dicloxacillin, (13) 20 ng/mL dicloxacillin, (14) 5
ng/mL nafcillin (15) 10 ng/mL nafcillin, (16) 20 ng/mL oxacillin, (17) 50 ng/mL
17 Accepted
18 oxacillin, (18) 50 ng/mL cefaclor, (19) 100 ng/mL cefaclor, (20) 10 ng/mL ceftezole, (21)
19 25 ng/mL ceftezole, (22) 250 ng/mL cefotaxime, (23) 500 ng/mL cefotaxime, (24) 25
20 ng/mL ceftiofur, (25) 50 ng/mL ceftiofur, (26) 5 ng/mL cefoperazone , (27) 10 ng/mL
21 cefoperazone, (28) 25 ng/mL cefathiamidine, (29) 50 ng/mL cefathiamidine, (30) 10
22 ng/mL cefepime, (31) 25 ng/mL cefepime, respectively.
23 Nanoscale
24 Table 1. Cut off values of β lactams in milk sample using GICA based on anti β lactams
25 receptors.
26
17
Nanoscale Page 18 of 24 Manuscript
1
2 Fig. 1. The principle of GICA based on anti β lactams receptors Accepted
3 Nanoscale
18
Page 19 of 24 Nanoscale Manuscript
1
2 Fig. 2 The images of GICA based anti β lactams receptors for four kinds of
3 representative penicillins in milk samples: (A) ammocillin: 0, 0.25, 0.5, 1, 2 and 5 Accepted
4 ng/mL; (B) ampicillin: 0, 0.5, 1, 2, 5, and 10 ng/mL; (C) penicillin G: 0, 0.25, 0.5, 1, 2,
5 and 5 ng/mL; (D) penicillin V: 0, 0.25, 0.5, 1, 2, and 5 ng/mL, respectively.
6 Nanoscale
19
Nanoscale Page 20 of 24 Manuscript Accepted
1
2 Fig. 3 The images of GICA based anti β lactams receptors for four kinds of
3 representative cephalosporins in milk samples: (A) cefaclor: 0, 10, 25, 50, 100, and Nanoscale
4 200 ng/mL; (B) ceftezole: 0, 5, 10, 25, 50, and 100 ng/mL; (C) cefoperazone: 0, 1, 2,
5 5, 10, and 25 ng/mL; (D) cefathiamidine: 0, 5, 10, 25, 50, and 100 ng/mL,
6 respectively.
7
20
Page 21 of 24 Nanoscale
1
2 Fig. 4 The detection of fifteen β lactams in milk sample using GICA based on
3 anti β lactams receptors. The final concentrations of fifteen β lactams in ten time Manuscript 4 diluted milk sample: (1) 0 ng/mL, (2) 1 ng/mL of amoxicillin, (3) 2 ng/mL of
5 amoxicillin, (4) 2 ng/mL ampicillin, (5) 5 ng/mL ampicillin, (6) 0.5 ng/mL penicillin
6 G, (7) 1 ng/mL penicillin G, (8) 1 ng/mL penicillin V, (9) 2 ng/mL penicillin V, (10) 5
7 ng/mL cloxacillin (11) 10 ng/mL cloxacillin, (12) 10 ng/mL dicloxacillin, (13) 20
8 ng/mL dicloxacillin, (14) 5 ng/mL nafcillin (15) 10 ng/mL nafcillin, (16) 20 ng/mL Accepted
9 oxacillin, (17) 50 ng/mL oxacillin, (18) 50 ng/mL cefaclor, (19) 100 ng/mL cefaclor,
10 (20) 10 ng/mL ceftezole, (21) 25 ng/mL ceftezole, (22) 250 ng/mL cefotaxime, (23)
11 500 ng/mL cefotaxime, (24) 25 ng/mL ceftiofur, (25) 50 ng/mL ceftiofur, (26) 5
12 ng/mL cefoperazone , (27) 10 ng/mL cefoperazone, (28) 25 ng/mL cefathiamidine, (29)
13 50 ng/mL cefathiamidine, (30) 10 ng/mL cefepime, (31) 25 ng/mL cefepime, Nanoscale
14 respectively.
15
16
21
Nanoscale Page 22 of 24
1 Table 1. Cut off values of β lactams in milk samples using GICA based on
2 anti β lactams receptors
Cut off values LOD Compounds Structures (ng/mL) (ng/mL)
Amoxicillin 5 0.25
Ampicillin 10 0.5
Manuscript
Penicillin G 5 0.5
Accepted
Penicillin V 5 0.5
Cloxacillin 100 1 Nanoscale
22
Page 23 of 24 Nanoscale
Dicloxacillin 20 5
Nafcillin 50 5
Manuscript
Oxacillin 25 10
cefaclor 200 25 Accepted
ceftezole 100 10
Nanoscale cefotaxime 1000 100
23
Nanoscale Page 24 of 24
ceftiofur 200 10
cefoperazone 25 5
cefathiamidie 100 5
Manuscript
cefepime 25 2
1
2 Accepted 3 Nanoscale
24