Method for dual-mode detection of fluoroquinolone antibiotics by using aptamer-based test strip

Abstract

The invention relates to a method for dual-mode detection of fluoroquinolone antibiotics by using an aptamer-based test strip. The preparation method comprises the following steps: uniformly mixing AuNRs / PDA with a streptavidin solution, carrying out oscillation incubation, adding a biotin-modified aptamer Bio-Apt, and carrying out incubation again, so as to obtain an AuNRs / PDA-Apt signal probe; assembling a test strip comprising a sample pad, a nitrocellulose membrane, a water absorption pad and a polyvinyl chloride bottom plate; the nitrocellulose membrane is coated with a bovine serum albumin-target conjugate and SA-Bio-cDNA in advance to respectively form a test line T line and a control line C line; the method comprises the following steps: mixing a solution containing fluoroquinolone antibiotics with an Au NRs / PDA-Apt signal probe, incubating, dropping onto a sample pad of a test strip, reacting, detecting and observing the colorimetric intensity and the photo-thermal intensity, and carrying out qualitative or quantitative analysis on the fluoroquinolone antibiotics. According to the Au NRs / PDA mediated dual-mode detection constructed for the first time, the sensitivity and the practicability of antibiotic analysis are remarkably improved, and the flexibility, the reliability and the accuracy of the lateral flow chromatography test paper are remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for detecting fluoroquinolone antibiotics by utilizing an aptamer-based test strip in dual mode. Background Art

[0002] Antibiotics are a class of drugs that can inhibit or kill bacteria and are widely used to treat bacterial infections in animal farming. They include macrolides, aminoglycosides, tetracyclines, fluoroquinolones, and sulfonamides. Among them, fluoroquinolone antibiotics (FQs), as broad-spectrum antibacterial drugs, are particularly effective in inhibiting Gram-negative bacteria and have developed from the first generation to the fourth generation. Since FQs are difficult to degrade in the environment, they are considered to be one of the main antibiotic pollutants. Common ones include enrofloxacin (ENR), ciprofloxacin (CIP), norfloxacin (NOR), moxifloxacin (MOX), and levofloxacin (LVX). The World Health Organization report pointed out that FQs pose a serious threat to human health. Due to the excessive or unrestricted use of antibiotics in animal farming, antibiotics that are not completely metabolized before slaughter remain in foods such as milk, beef, and chicken. When humans consume these foods containing antibiotic residues, they may cause health problems such as intestinal flora disorders, increased bacterial resistance, and nephrotoxicity. Therefore, it is crucial to monitor antibiotic residues in animal-derived foods before they reach human tables.

[0003] Traditional detection methods mainly include high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Although these technologies can achieve accurate and reliable detection, they also have significant limitations. First, expensive large equipment is required, which is not conducive to detection work in laboratories with limited resources or in remote areas. In addition, these technologies require operators to have rich professional experience to ensure the accuracy of sample pretreatment and analysis processes. Sample pretreatment usually involves multiple steps of extraction, concentration and purification, and time-consuming operations reduce the detection efficiency. There is also enzyme-linked immunosorbent assay (ELISA) based on antibodies as recognition molecules, which achieves high specificity detection. However, the operation is relatively cumbersome, including multiple washing, incubation and color development steps, and is easily affected by human operation errors. False positive results are prone to occur in ELISA detection, affecting the accuracy of detection. Therefore, it is urgent to develop simple, low-cost and sensitive portable sensors for rapid detection of fluoroquinolone antibiotic residues in food.

[0004] Lateral flow analysis (LFA) has become the most user-friendly paper-based instant detection tool because it does not require a specific operating site and professional personnel to operate. With the advantages of rapidity, high efficiency, visual results, simple operation and low cost, it has been widely used in medical diagnosis, environmental monitoring and food safety. A survey of domestic and foreign literature found that the main recognition molecules in LFA are still antibodies, but antibodies have problems such as high batch-to-batch differences, low stability and high cost. In contrast, aptamers (Apt), as a nucleic acid molecule obtained by in vitro screening technology, have the advantages of specific binding to targets, high chemical stability, simple synthesis and easy modification. Therefore, the development of aptamer-based LFA is of great significance. However, there are still few related studies and further exploration is needed. In addition, the design of signal probes is crucial to the detection performance of LFA. However, the current aptamer-based LFA mainly relies on gold nanoparticles (AuNPs), and its single signal output and low color brightness make it difficult to meet the requirements of high-sensitivity detection. Therefore, it is particularly important to develop multimodal LFA with integrated intelligent devices, which will help improve detection sensitivity and applicability and meet more complex detection needs. At the same time, current Apt-LFA sensing strategies for detecting small molecule targets mostly rely on the design of complementary chains (cDNA), which is a time-consuming and labor-intensive process, increasing the difficulty of Apt-LFA development.

[0005] Therefore, in order to quickly monitor fluoroquinolone antibiotic residues in animal foods, this study is dedicated to constructing a dual-mode aptamer-based test strip mediated by a functional signal probe, in order to improve the sensitivity and stability of detection, overcome the limitations of existing technologies, and provide a more effective food safety monitoring solution. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for dual-mode detection of fluoroquinolone antibiotics using aptamer-based test strips. The aptamer-based lateral flow chromatography test strip based on polydopamine-coated gold nanorods constructed by the present invention realizes colorimetric-photothermal dual-mode detection of fluoroquinolone antibiotics. By preparing a signal probe (AuNRs / PDA-AntibioticApt) with excellent performance and assembling a test strip substrate, a sensitive response is achieved by using the competitive binding of the aptamer to the free antibiotics in the sample and the bovine serum albumin-antibiotic conjugate on the T line. For negative samples, a large number of signal probes bind to the bovine serum albumin-antibiotic conjugate on the T line to form a dark strip, while the C line only binds a very small amount of probes. For positive samples, the signal probe preferentially captures the free antibiotics to form a complex, resulting in a decrease in the amount of probe binding on the T line and a lighter color of the strip; when the complex migrates to the C line by capillary action, the complementary chain will compete for binding to the aptamer, making the strip color darker. Therefore, within a certain range, the signal intensity of the T line is inversely proportional to the target concentration, while the signal of the C line is directly proportional. In the colorimetric mode (CM-LFA), direct qualitative analysis can be performed by naked eyes, or the image of the test strip can be taken with a smartphone, and then grayscale analysis can be performed using Image J software. The colorimetric intensity of the T line is used as the ordinate and the antibiotic content in the sample is used as the abscissa to draw a colorimetric response curve. In the photothermal mode (PT-LFA), the T line is irradiated with an 808nm laser to produce a photothermal conversion effect, and then a handheld near-infrared camera is used to take thermal images and record the temperature change value. The temperature change generated by the T line is used as the ordinate and the antibiotic content in the sample is used as the abscissa to draw a photothermal response curve. The AuNRs / PDA-mediated dual-mode detection constructed for the first time significantly improves the sensitivity and practicality of antibiotic analysis, and is an effective instant analysis tool in the field of food safety testing. This dual-signal detection method combines the advantages of colorimetric and photothermal modes, significantly improving the flexibility, reliability and accuracy of lateral flow chromatography test strips.

[0007] The present invention is achieved through the following technical solutions:

[0008] The object of the present invention is to provide a method for dual-mode detection of fluoroquinolone antibiotics using an aptamer-based test strip, comprising the following steps:

[0009] (1) AuNRs / PDA and streptavidin solution were mixed evenly, incubated with shaking, and biotin-modified fluoroquinolone antibiotic aptamer was added and incubated again to obtain AuNRs / PDA-Apt signal probe;

[0010] (2) assembling a test strip comprising a sample pad, a nitrocellulose membrane, a water-absorbing pad and a polyvinyl chloride base; (3) mixing and incubating a solution containing fluoroquinolone antibiotics with the AuNRs / PDA-Apt signal probe, dropping the mixture onto the sample pad of the test strip obtained in step (2) to react, detecting and observing the colorimetric intensity and the photothermal intensity, and performing qualitative or quantitative analysis on the fluoroquinolone antibiotics.

[0011] In one embodiment of the present invention, in step (1), the AuNRs / PDA solution is prepared by the following method:

[0012] S1, stirring the polyethylene glycol methyl ether thiol solution and the Au NRs solution to obtain Au NRs / PEG, and dissolving them in a buffer to obtain an AuNRs / PEG solution;

[0013] S2. The Au NRs / PEG solution obtained in step S1 is mixed with the dopamine solution to obtain Au NRs / PDA.

[0014] In one embodiment of the present invention, in step S2, the concentration of the dopamine solution is 0.1 mg / mL-0.3 mg / mL.

[0015] In one embodiment of the present invention, in step (1), the concentration of the streptavidin solution is 0.5 mg / mL-1.5 mg / mL.

[0016] In one embodiment of the present invention, in step (1), the shaking incubation conditions are: shaking incubation at 25°C-37°C for 1h-2h.

[0017] In one embodiment of the present invention, in step (1), the conditions for the second incubation are: incubation at 25°C-37°C for 10 min-40 min.

[0018] In one embodiment of the present invention, in step (2), the nitrocellulose membrane is pre-coated with bovine serum albumin-target binder and SA-Bio-cDNA to form a test line T line and a control line C line, respectively.

[0019] In one embodiment of the present invention, the SA-Bio-cDNA is obtained by mixing a streptavidin solution and a biotin-modified complementary chain cDNA solution.

[0020] In one embodiment of the present invention, in step (3), the concentration of the solution containing fluoroquinolone antibiotics is 0.5 ng / mL-500 ng / mL.

[0021] In one embodiment of the present invention, in step (3), the incubation conditions are: incubation at 25°C-37°C for 20min-50min.

[0022] In one embodiment of the present invention, in step (3), the fluoroquinolone antibiotics are selected from one or more of enrofloxacin, norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, etc. The detection of different fluoroquinolone antibiotics is achieved by regulating the corresponding aptamer and the BSA coupling-target on the T line.

[0023] In one embodiment of the present invention, the sequence of the biotin-modified enrofloxacin aptamer Bio-Apt (SEQ ID NO. 1) is: 5'-bio GCT GTG TGA CTC CTG CAA GTC CGA CAT ACC TTA GTG CCCTGA TATAAT GTAACA CTA TTG AGC AGC TGT ATC TTG TCT CC-3';

[0024] The sequence of the biotin-modified complementary chain cDNA (SEQ ID NO. 2) is: 5'-bio GGA GAC AAGATA CAG CTG CTC AATAGT GTTACA TTATAT CAG GGC ACT AAG GTATGT CGGACT TGCAGGAGTCACACAGC-3'.

[0025] The above technical solution of the present invention has the following advantages compared with the prior art:

[0026] (1) The present invention prepares a bifunctional signal probe-aptamer-modified gold nanorods / polydopamine (Au NRs / PDA-Apt), assembles a lateral flow chromatography (LFA) test strip with a test line (T line) and a control line (C line), and uses the aptamer's specific recognition of the target to construct a highly responsive, sensitive and rapid test strip detection method for monitoring antibiotics.

[0027] (2) The present invention uses Au NRs / PDA, which, compared with Au NPs, AuNRs and PDANPs, has multiple advantages, such as excellent dispersibility in solution, better stability, larger reaction surface area and convenient surface functionalization, by virtue of its dual-component material advantages and unique core-shell structure. More importantly, it significantly enhances the color contrast and photothermal conversion efficiency on the fiber membrane. Therefore, this nanocomposite material with excellent performance can be used as a dual-function signal probe for test strips.

[0028] (3) In the present invention, a bovine serum albumin-target conjugate (BSA-Antibiotic) is sprayed on the test line (T line) of the test strip, and the competitive reaction between the target and the conjugate is detected by the aptamer. Compared with the competitive test strip based on complementary chains, the time-consuming design of complementary chains is avoided, and a simple and effective new strategy for antibiotic detection is provided.

[0029] (4) Compared with the traditional Au NPs-LFA, the Au NRs / PDA-LFA constructed in the present invention significantly improves the detection performance, shows a stronger response ability to low-concentration targets, has a higher visual detection sensitivity, and meets the needs of high-sensitivity detection.

[0030] (5) Compared with enzyme-linked immunosorbent assay (ELISA) and enzyme-linked aptamer assay (ELAA), the Au NRs / PDA-LFA constructed in the present invention simplifies the detection process, shortens the detection time, reduces the construction cost, and improves the convenience of user operation.

[0031] (6) The present invention utilizes a colorimetric-photothermal functional probe to provide an amplified colorimetric signal and a sensitive photothermal signal, which can be used in conjunction with a smart phone and a thermal imager to achieve sensitive quantitative detection of antibiotics, and is particularly suitable for application in remote areas with scarce resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0033] Figure 1 It is the transmission electron microscope characterization image (TEM) and the enlarged image of AuNRs (AB) and AuNRs / PDA (CD) in Example 1 of the present invention;

[0034] Figure 2 is an assembly diagram of the test strip in Example 1 of the present invention;

[0035] Figure 3 It is a colorimetric-photothermal detection method of ENR using an aptamer-based test strip based on polydopamine-coated gold nanorods in Example 1 of the present invention;

[0036] Figure 4 is the calibration curve and dynamic linear relationship between the colorimetric intensity (AB) and photothermal intensity (CD) of the T line and the ENR concentration in Example 1 of the present invention;

[0037] Figure 5 It is the specific analysis diagram in Example 1 of the present invention; wherein A is a colorimetric intensity analysis diagram; and B is a photothermal intensity analysis diagram. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0040] In order to further verify the sensitivity of the detection method prepared by the present invention to the detection of antibiotics in food, the present invention takes enrofloxacin (ENR) as an example, and the specific operation steps are as follows:

[0041] Example 1

[0042] Step 1) Preparation of AuNRs / PDA:

[0043] Au NRs: Gold nanorods were prepared by seed-mediated growth. Seed solution: Freshly prepared sodium borohydride solution was added to a mixture of hexadecyltrimethylammonium bromide (0.1M, 9.75mL) and chloroauric acid (0.01M, 0.25mL). After reacting for 30s under magnetic stirring, the solution was allowed to stand at room temperature for 2h. Growth solution: 4mL of chloroauric acid (0.01M), 0.8mL of silver nitrate (0.01M), 1.6mL of hydrochloric acid (1.0M) and 0.64mL of ascorbic acid (0.1M) were added to 80mL of hexadecyltrimethylammonium bromide solution (0.1M) in sequence, and the solution turned colorless. Subsequently, 0.04mL of seed solution was added, the mixture was stirred evenly, reacted in a water bath at 30℃ for 24h, and the color of the solution turned reddish brown. Finally, the reaction product was purified by centrifugation to obtain AuNRs.

[0044] AuNRs / PDA: Using the ligand exchange method, polyethylene glycol methyl ether thiol solution (SH-PEG) was added to the above AuNRs solution and gently stirred at room temperature for 20 hours. After the reaction was completed, the product (Au NRs / PEG) was washed twice by centrifugation, redispersed in Tris-HCl buffer, and ultrasonicated for 5 minutes. Then dopamine solution was added to react for 30 minutes, and the excess reagent was removed by centrifugation to obtain the AuNRs / PDA product with the thickness of the PDA shell, which was stored at 4°C.

[0045] Figure 1 The transmission electron microscopy (TEM) and magnified images of AuNRs (AB) and AuNRs / PDA (CD) are shown in Figure 1. Figure 1It can be seen that Au NRs are well-dispersed rod-like structures with an average length and width of approximately 67.67±6.12nm and 19.70±1.76nm; after surface coating with PDA coating, a stable core-shell rod-like structure is formed, and the particle size of the composite material is further increased, with an average length and width of approximately 74.97±6.96nm and 28.18±2.48nm, proving the successful preparation of the material.

[0046] Step 2) Preparation of AuNRs / PDA-ENRApt:

[0047] 1 mL of Au NRs / PDA solution prepared in step 1) and 20 μL of streptavidin solution (1 mg / mL) were mixed evenly, incubated at 37°C with shaking for 2 h, and the excess streptavidin was removed by centrifugation. Subsequently, 50 μM of biotin-modified aptamer (Bio-apt) was added and incubated at 37°C for 30 min to achieve rapid coupling of the aptamer on AuNRs / PDA. Unbound Bio-apt was removed by multiple centrifugation, and the obtained AuNRs / PDA-ENRApt signal probe was stored at 4°C.

[0048] Bio-ENRapt:5'-bio GCT GTG TGA CTC CTG CAA GTC CGA CAT ACC TTA GTG CCCTGA TAT AAT GTAACA CTA TTG AGC AGC TGT ATC TTG TCT CC-3'(SEQ ID NO.1)

[0049] Step 3) Assemble the test strips (Assembly diagram as shown Figure 2 shown):

[0050] The test strip consists of four parts: sample pad, nitrocellulose membrane, absorbent pad and polyvinyl chloride bottom plate. The sample pad was soaked in buffer (10mM PBS, 2% BSA, 1% sucrose and 0.05% Tween-20) for 2h, dried at 40℃ for 4h, and stored in a drying bag away from light. For the conjugate of C line, 1mg / mL SA solution and 100μM Bio-cDNA solution were fully mixed in a volume ratio of 1:1, reacted at 4℃ for 2h, and the SA-Bio-cDNA conjugate was collected with a 30Kd ultrafiltration tube. Subsequently, BSA-ENR and SA-Bio-cDNA were sprayed on the T line (test line) and C line (control line) on the NC membrane at a uniform speed. The test strip was assembled in a way that the components overlapped 2mm to ensure the smooth circulation of the solution. Finally, the test strip was automatically cut into 3mm width and stored in an aluminum foil bag containing silica gel desiccant for later use.

[0051] Bio-cDNA:5'-bio GGA GAC AAG ATA CAG CTG CTC AAT AGT GTT ACATTATAT CAGGGCACTAAG GTATGT CGGACT TGCAGGAGT CAC ACAGC-3'(SEQ ID NO.2)

[0052] Step 4) Establish a linear curve for monitoring antibiotics:

[0053] ENR standard solution was mixed with AuNRs / PDA-Apt probe at different final concentrations (0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL and 500 ng / mL) and incubated at 37 °C for 40 min to promote full binding of the probe and target. Then, it was dropped onto the sample pad and passed through the nitrocellulose membrane to the absorbent pad by capillary action. In the CM-LFA detection, the colorimetric signal was observed by naked eye and the image was recorded with a smartphone. The signal intensity was quantified by Image J software. In the PT-LFA detection, the photothermal signal was recorded in real time with a portable infrared camera after irradiation of the T line with 808 nm laser. A linear curve was established with ENR concentration as the abscissa and colorimetric intensity / photothermal intensity as the ordinate (colorimetric-photothermal detection of ENR by aptamer-based test strips based on polydopamine-coated gold nanorods as shown in Figure 2). Figure 3 The calibration curve and dynamic linear relationship between the colorimetric intensity (AB) and photothermal intensity (CD) of the T line and the ENR concentration are shown in Figure 4 As shown. Figure 4 It can be seen that the colorimetric mode-ENR concentration and the colorimetric intensity of the T line show a good linear relationship, and the corresponding regression equation is Y=-22.66X+52.63(R 2 =0.992), the detection limit was 0.30 ng / mL; the photothermal mode-ENR concentration and the photothermal intensity of the T line showed a good linear relationship, and the corresponding regression equation was Y=-15.84X+35.48(R 2 =0.993), and the detection limit was as low as 0.12 ng / mL, which was 2.5 times higher than that of the colorimetric mode. The visual detection limit of LFA in dual mode (5 ng / mL) was 20 times higher than that of traditional Au NPs-LFA, which verified that Au NRs / PDA-LFA can achieve dual-mode sensitive detection of ENR.

[0054] Example 2 Specificity

[0055] Kanamycin (KAN), tetracycline (TC), gentamicin (GM), ofloxacin (OFX) and vancomycin (VCM) were selected as potential interferents with a concentration of 100 ng / mL. The specificity of the method was evaluated by the above detection procedure. Figure 5 As shown in the figure, only when ENR was present, the T line signal intensity in the dual mode decreased significantly, indicating a positive result; while in the presence of other interfering substances, the T line signal did not change significantly, verifying the good specificity of the AuNRs / PDA-LFA platform for detecting ENR.

[0056] Example 3 Determination of the content of enrofloxacin in real samples

[0057] Milk was selected as the actual sample. The pretreatment process was as follows: 1 mL of milk was mixed with 5 mL of acetonitrile, rotated for 5 min, centrifuged at 10000 rpm for 10 min, the supernatant was collected, and impurities were removed by 0.22 μm membrane filtration. After spiking with ENR standard solution, AuNRs / PDA-LFA was used for recovery rate experiment. The results are shown in Table 1:

[0058] Table 1 Recovery experiment of Au NRs / PDA-LFA in dual mode for detecting ENR in milk.

[0059]

[0060] As can be seen from Table 1, good recoveries ranging from 93.5% to 109.5% were obtained in milk, and the relative standard deviation values ​​were lower than 11.7%. Therefore, the Au NRs / PDA-mediated CM-LFA and PT-LFA methods have good practical applicability in food.

[0061] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for dual-mode detection of fluoroquinolone antibiotics using aptamer-based test strips, characterized in that: The following steps are involved: (1) AuNRs / PDA and streptavidin solution were mixed evenly, incubated with shaking, and biotin-modified fluoroquinolone antibiotic aptamer was added and incubated again to obtain Au NRs / PDA-Apt signal probe; (2) assembling a test strip comprising a sample pad, a nitrocellulose membrane, an absorbent pad, and a polyvinyl chloride base; (3) Mixing and incubating the solution containing fluoroquinolone antibiotics with the AuNRs / PDA-Apt signal probe, dropping it onto the sample pad of the test strip obtained in step (2) to react, detecting and observing the colorimetric intensity and photothermal intensity, and performing qualitative or quantitative analysis on the fluoroquinolone antibiotics.

2. The method according to claim 1, characterized in that In step (1), the AuNRs / PDA solution is prepared by the following method: S1, stirring the polyethylene glycol methyl ether thiol solution and the Au NRs solution to obtain Au NRs / PEG, and dissolving them in a buffer to obtain an AuNRs / PEG solution; S2. The Au NRs / PEG solution obtained in step S1 is mixed with the dopamine solution to obtain Au NRs / PDA.

3. The method according to claim 2, characterized in that In step S2, the concentration of the dopamine solution is 0.1 mg / mL-0.3 mg / mL.

4. The method according to claim 1, characterized in that: In step (1), the concentration of the streptavidin solution is 0.5 mg / mL-1.5 mg / mL.

5. The method according to claim 1, characterized in that In step (1), the shaking incubation conditions are: shaking incubation at 25°C-37°C for 1h-2h; And / or, the conditions for the second incubation are: incubation at 25°C-37°C for 10min-40min.

6. The method according to claim 1, characterized in that In step (2), the nitrocellulose membrane is pre-coated with bovine serum albumin-target binding substance and SA-Bio-cDNA to form a test line T line and a control line C line, respectively.

7. The method according to claim 6, characterized in that The SA-Bio-cDNA is obtained by mixing a streptavidin solution and a biotin-modified complementary chain cDNA solution.

8. The method according to claim 1, characterized in that In step (3), the concentration of the solution containing fluoroquinolone antibiotics is 0.5 ng / mL-500 ng / mL.

9. The method according to claim 1, characterized in that: In step (3), the incubation conditions are: incubation at 25°C-37°C for 20min-50min.

10. The method according to claim 1, characterized in that In step (3), the fluoroquinolone antibiotic is selected from one or more of enrofloxacin, norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin and gatifloxacin.

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