M NPs / M-TCPP nano composite material and preparation method and antibiotic sensing application thereof

Abstract

The invention discloses an M NPs / M-TCPP nano composite material and a preparation method and antibiotic sensing application thereof.The M NPs / M-TCPP nano composite material is formed by in-situ growth of metal M nano particles M NPs in a 2D M-TCPP MOF nano film, the MOF structure of the 2D M-TCPP MOF nano film is reserved, the M NPs are evenly loaded on the M-TCPP MOF nano film, the limitation that the structure of the M NPs is completely damaged by direct calcination of the MOF is overcome, and the application of the M NPs / M-TCPP nano composite material to antibiotic sensing is achieved. After calcination, the ultrathin 2D MOF structure is still effectively maintained; through calculation of a first principle, it is found that after in-situ growth of the loaded metal nanoparticles M NPs, the adsorption energy of active sites of the material to MNZ, LEV and TC is effectively improved. The electrochemical sensor constructed on the basis of the M NPs / M-TCPP nano composite material can be used for detecting MNZ, LEV and TC at the same time.

Description

Technical Field

[0001] The invention belongs to the field of sensor materials, and specifically relates to an M NPs / M-TCPP nanocomposite material and a preparation method thereof and antibiotic sensing application. Background Art

[0002] Antibiotics provide an effective method for preventing and controlling the proliferation of microorganisms and cells, but the widespread use of antibiotics has led to the emergence and widespread spread of drug-resistant bacteria, which makes antibiotics no longer show magical effects, and more and more people die from infections caused by antibiotic-resistant bacteria. However, antibiotics are still widely used in human medicine and many fields. The illegal abuse of antibiotics is directly reflected in the large amount of antibiotic residues in common food and the environment. The antibiotic resistance crisis has become the most pressing health issue of global concern. Therefore, it is of great significance to carry out sensitive monitoring of antibiotics in animal-derived foods, the environment and the human body.

[0003] At present, traditional strategies for antibiotic residue detection include: microbiological method, liquid chromatography-mass spectrometry, fluorescence spectroscopy, capillary electrophoresis and immunoassay, etc. Compared with these technologies which are limited by expensive instruments, time-consuming, cumbersome operating procedures and poor stability, electrochemical sensing has many significant advantages, such as low cost, simple operation, high sensitivity, rapid response and portability (see references 1, 2), and has become an excellent method for antibiotic residue monitoring. The emergence of functional nanomaterials provides great opportunities for the construction of high-performance electrochemical sensors.

[0004] Metal-organic framework (MOF) is a porous material with a network skeleton formed by the self-assembly of central metal ions and organic ligands. Thanks to its high specific surface area, high porosity, rich pore structure and confined metal sites, MOFs have become an ideal precursor for the preparation of various functional nanomaterials with complex compositions and structures. Functional nanomaterials prepared with MOFs as precursors have been shown to have high electrical conductivity and electrochemical activity, especially 2D MOF-derived functional nanomaterials with ultrathin nanosheet morphology, which can not only maximize the exposure of active metal sites on the surface, but also achieve rapid mass transfer processes, which has shown great potential in the field of electrochemical sensing. The strategy for preparing these functional nanomaterials is usually to calcine MOF precursors at high temperatures (see reference 3). However, the direct high-temperature calcination method usually leads to a sharp collapse of the rich pores inside MOFs and severe agglomeration of metal active sites, thereby reducing the contact between metal active sites and analytes, thereby affecting their performance (see references 4, 5). However, there are still few reports on the controlled calcination of MOFs to retain porous nanostructures.

[0005] Therefore, it is necessary to prepare a functional nanomaterial using 2D MOF as a precursor, and to construct an electrochemical sensor based on this material for the highly sensitive and rapid determination of antibiotics. In addition, the structure of 2D MOF should be retained during the preparation of the functional nanomaterial to achieve better performance applications of the material.

[0006] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field.

[0007] References: [1]Joshi, A., Kim, KH Recent advances in nanomaterial-basedelectrochemical detection of antibiotics: Challenges and future perspectives. Biosensors and Bioelectronics, 2020, 153, 112046; [2]Liu, [3]He, Y., Wang, Z., Wang, H., Wang, Z., Zeng, G., Xu, P., Huang, D., Chen, M., Song, B., Qin, H., Zhao, Y. Metal-organic framework-derivednanomaterials in environment related fields: Fundamentals, properties and applications. Coordination Chemistry Reviews, 2021, 429, 213618; [4]Hu, Q., Huang, X., Wang, Z., Li, G., Han, Z., Yang, H., Ren, 2140−2146; [5]Xu, B., Zhang, H., Mei, H., Sun, D. Recent progress in metal-organic framework-based supercapacitor electrode materials. CoordinationChemistry Reviews, 2020, 420, 213438. Summary of the invention

[0008] The purpose of the present invention is to provide an M NPs / M-TCPP nanocomposite material, a preparation method thereof and an electrochemical sensing application. The M NPs / M-TCPP nanocomposite material retains the structure of a 2D ultrathin MOF precursor, overcomes the limitation that direct calcination of MOF completely destroys its structure, and an electrochemical sensor constructed based on the M NPs / M-TCPP nanocomposite material can simultaneously detect three antibiotics.

[0009] In order to achieve the above-mentioned object, the present invention provides an M NPs / M-TCPP nanocomposite material, which is formed by in-situ growing metal M nanoparticles M NPs in a 2D M-TCPP MOF nanofilm, and the composite material retains the MOF structure of the 2D M-TCPP nanofilm, and the M NPs are uniformly loaded on the M-TCPP MOF nanofilm; wherein M=Cu, Co or Ni.

[0010] Preferably, in the M NPs / M-TCPP nanocomposite material, the particle size of the metal nanoparticles M NPs is 1-10 nm.

[0011] The second object of the present invention is to provide a method for preparing the M NPs / M-TCPP nanocomposite material, which comprises: subjecting a 2D M-TCPP MOF nanofilm to a solvent thermal reaction in an in-situ reaction process and calcining under an inert atmosphere to obtain the M NPs / M-TCPP nanocomposite material.

[0012] Preferably, the solvent thermal reaction has a reaction temperature of 180°C; or / and the calcination under the inert atmosphere has a temperature of 400°C.

[0013] Preferably, the solvent thermal reaction has a reaction time of 12 hours; or / and the calcination under an inert atmosphere has a reaction time of 1 hour.

[0014] Preferably, after the solvothermal reaction, the product is washed, centrifuged, freeze-dried, and then calcined under the inert atmosphere.

[0015] Preferably, the 2D M-TCPP MOF nanofilm is obtained by the following method: dispersing the nitrate of metal M and polyvinyl pyrrolidone in a mixture of DMF and ethanol, adding tetrakis(4-carboxyphenyl)porphyrin for ultrasonic treatment, transferring the obtained mixture to an autoclave, and reacting at 80°C; after the reaction is completed, cooling to room temperature to obtain a 2D M-TCPPMOF nanofilm.

[0016] The third object of the present invention is to provide a modified electrode based on the M NPs / M-TCPP nanocomposite material.

[0017] The fourth object of the present invention is to provide an electrochemical sensor constructed based on the modified electrode.

[0018] The fifth object of the present invention is to provide the use of the electrochemical sensor in detecting antibiotics, wherein the electrochemical sensor can simultaneously detect metronidazole, levofloxacin and tetracycline.

[0019] The M NPs / M-TCPP nanocomposite material of the present invention, its preparation method and antibiotic sensing application have the following advantages: (1) The present invention overcomes the limitation of direct calcination of MOF that completely destroys its structure through two steps: solvothermal reaction and calcination, so that the 2D M-TCPP MOF still maintains its ultra-thin 2D MOF structure after calcination, and in situ grows MNPs on the surface of M-TCPP MOF, successfully preparing M NPs / M-TCPP nanocomposites. On the one hand, MNPs are grown in situ from the M active sites on the surface of M-TCPPMOF. Compared with the single M-TCPP MOF, the appearance of MNPs adds new antibiotic catalytic active sites; on the other hand, since the prepared M NPs / M-TCPP nanocomposites retain the 2D M-TCPPMOF structure, the appearance of MNPs can effectively increase the adsorption capacity of the M active sites in M-TCPP for antibiotics. The above two reasons can greatly enhance the catalytic properties of the material for antibiotics;

[0020] (2) The present invention constructed a new electrochemical sensing platform for metronidazole (MNZ), levofloxacin (LEV) and tetracycline (TC) based on MNPs / M-TCPP. Among them, NiNPs / Ni-TCPP showed the best catalytic activity for MNZ and LEV, with detection limits of 0.2 nM and 30 nM, respectively; while CuNPs / Cu-TCPP showed the best catalytic activity for TC, with a detection limit of 0.3 μM. More importantly, in the potential range of -1.0~1.2 V, MNZ, LEV and TC obtained response peaks at -0.8, 0.7 and 0.83 V, respectively, indicating that the prepared sensor can achieve simultaneous detection of the above three analytes. The sensor of the present invention has been successfully used to detect MNZ, LEV and TC in actual honey, milk and human serum samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The Cu-TCPP MOF prepared in Example 1 of the present invention and the characterization results of the materials during the solvothermal reaction and calcination process; in the figure, A~D are the characterization results of Cu-TCPP MOF; E~H are the characterization results of Cu-TCPP MOF after solvothermal reaction at 180°C for 12h; I~L are the characterization results after freeze-drying; M~P are the characterization results of Cu NPs / Cu-TCPP after calcination.

[0022] Figure 2 The Co-TCPP MOF prepared in Example 1 of the present invention and the characterization results of the materials during the solvothermal reaction and calcination process; in the figure, A~D are the characterization results of Co-TCPP MOF; E~H are the characterization results of Co-TCPP solvothermal reaction at 180°C for 12 h; I~L are the characterization results of Co NPs / Co-TCPP after calcination.

[0023] Figure 3 The Ni-TCPP MOF prepared in Example 1 of the present invention and the characterization results of the materials during the solvothermal reaction and calcination process; in the figure, A~D are the characterization results of Ni-TCPP MOF; E~H are the characterization results of Ni-TCPP solvothermal reaction at 180°C for 12 h; I~L are the characterization results of Ni NPs / Ni-TCPP after calcination.

[0024] Figure 4 This is a graph showing the differential pulse voltammetric response results of the electrochemical sensors constructed with Cu NPs / Cu-TCPP (A), Co NPs / Co-TCPP (B) and Ni NPs / Ni-TCPP (C) nanocomposites to MNZ, TC and LEV in Experimental Example 2 of the present invention.

[0025] Figure 5 These are the test results of the electrochemical sensor prepared in Example 3 of Experimental Example 2 of the present invention in actual samples; in the figure, A~D are the test results of Co / Co-TCPP / SPE in honey samples; E~H are the test results of Ni / Ni-TCPP / SPE in milk samples; I~L are the test results of Cu / Cu-TCPP / SPE in human serum. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] It should be noted that if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased from the market.

[0028] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be deemed to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0029] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features, all possible combinations should be considered as the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0030] The invention provides an M NPs / M-TCPP nanocomposite material, wherein the composite material is formed by in-situ growing metal M nanoparticles M NPs in a 2D M-TCPP MOF nanofilm, the composite material retains the MOF structure of the 2D M-TCPP nanofilm, and the M NPs are uniformly loaded on the M-TCPP MOF nanofilm; wherein M=Cu, Co or Ni.

[0031] The present invention overcomes the limitation of direct calcination of MOF that completely destroys its structure through two steps of solvent thermal reaction and calcination, so that the 2D M-TCPP MOF still maintains its ultra-thin 2D MOF structure after calcination, and in situ grows MNPs on the surface of M-TCPP MOF, successfully preparing M NPs / M-TCPP nanocomposite materials. On the one hand, MNPs are grown in situ from the M active sites on the surface of M-TCPP MOF. Compared with the single M-TCPP MOF, the appearance of MNPs adds new antibiotic catalytic active sites; on the other hand, since the prepared M NPs / M-TCPP nanocomposite material retains the 2D M-TCPP MOF structure, the appearance of MNPs can effectively increase the adsorption capacity of the M active sites in M-TCPP for antibiotics. The above two reasons can greatly enhance the catalytic properties of the material for antibiotics.

[0032] Moreover, the inventors of the present invention found through first-principles calculations that the adsorption energy of the active sites of the material for MNZ, LEV and TC was effectively improved after in-situ growth of loaded metal nanoparticles MNPs.

[0033] The M NPs / M-TCPP nanocomposite material provided by the present invention, its preparation method and antibiotic sensing application are described in detail below through embodiments, experimental examples and application examples.

[0034] Example 1 A M NPs / M-TCPP nanocomposite material, the preparation method of which is as follows: (1) Synthesis of 2D M-TCPP MOF (M = Cu, Co or Ni) nanofilms The surfactant-assisted method was used as follows: In a 25 mL beaker, 5.0 mg Cu(NO3)2•3H2O or 15.0 mg Co(NO3)3•6H2O or 15.0 mg Ni(NO3)4•6H2O and 20.0 mg PVP (polyvinyl pyrrolidone) were dispersed in a mixture of 12 mL DMF and ethanol (V: V=3: 1). Subsequently, 12 mg TCPP (tetrakis(4-carboxyphenyl)porphyrin) was added to the beaker and further sonicated for 10 min. After that, the obtained mixture was transferred to a 20 mL Teflon-lined autoclave and kept at 80 °C for 4 h (24 h for Co-TCPP and Ni-TCPP nanofilms), and then cooled to room temperature. The resulting product was washed 3 times with ethanol and collected to obtain 2D M-TCPP MOF (M=Cu, Co, or Ni).

[0035] (2) Synthesis of M NPs / M-TCPP (M = Cu, Co and Ni) MNP / M-TCPP was prepared by two steps of solvothermal reaction and calcination in an in situ reaction process as follows: The prepared 2D M-TCPP (M = Cu, Co or Ni) was dispersed in 10 mL of ethanol, and the dispersion was transferred to a 20 mL autoclave and reacted at 180 ° C for 12 h. After washing with ethanol and centrifugation, the product was freeze-dried for 24 h. Then, the product was in situ calcined at 400 ° C for 1 h under an argon atmosphere to obtain the final M NPs / M-TCPP. The 2D M-TCPP still effectively maintained its ultrathin 2D nanofilm structure after calcination, and abundant ultrasmall metal nanoparticles (Cu NPs: about 6~10 nm, Co NPs: about 1.5 nm, Ni NPs: about 1.5~5 nm) were prepared in the ultrathin 2D M-TCPP nanofilm.

[0036] Example 2 An M NPs / M-TCPP / SPE modified electrode, the preparation method of which is as follows: 1 mg of each of the three MNP / M-TCPP nanocomposites prepared in Example 1 was dispersed in 1 mL of 5% perfluorosulfonic acid solution, and 7.5 μL of the dispersed solution was dropwise coated on the cleaned surface of the screen-printed electrode (SPE) to prepare MNPs / M-TCPP / SPE modified electrodes.

[0037] Example 3 An electrochemical sensor based on M NPs / M-TCPP / SPE modified electrode, the preparation method of which is as follows: The modified MNPs / M-TCPP / SPE was used as a three-electrode system, paired with the Wuhan Coster CS100 portable electrochemical analyzer, and a mobile phone Bluetooth connected to the electrochemical analyzer as the signal terminal to construct a portable antibiotic electrochemical sensor.

[0038] Experimental Example 1 Material Structure Characterization The material prepared in Example 1 was characterized using an atomic force microscope (AFM), a scanning electron microscope (SEM) and a transmission electron microscope (TEM), and the results are as follows: like Figure 1 As shown, the characterization results of the Cu-TCPP MOF prepared in Example 1 of the present invention and the materials thereof during the solvothermal reaction and calcination process, wherein A~D are the characterization results of Cu-TCPP MOF, E~H are the characterization results of the solvothermal reaction of Cu-TCPP MOF at 180°C for 12 h, I~L are the characterization results after freeze-drying, M~P are the characterization results of Cu NPs / Cu-TCPP after calcination, A, E, I, M, B, F, J and N are scanning electron microscope SEM photos, C, G, K, O, D, H, L and P are transmission electron microscope TEM photos, Q is the atomic force microscope AFM characterization result of Cu-TCPP MOF, and R and S are the atomic force microscope AFM characterization results of CuNPs / Cu-TCPP after calcination.

[0039] like Figure 2 As shown, the characterization results of the Co-TCPP MOF prepared in Example 1 of the present invention and the materials thereof during the solvothermal reaction and calcination process, wherein A~D are the characterization results of Co-TCPP MOF, E~H are the characterization results of the solvothermal reaction of Co-TCPP at 180°C for 12 h, I~L are the characterization results of Co NPs / Co-TCPP after calcination, A, E, I, B, F and J are scanning electron microscope SEM photos, C, G, K, D, H and L are transmission electron microscope TEM photos, M is the atomic force microscope AFM characterization result of Co-TCPP MOF, and N and O are the atomic force microscope AFM characterization results of CoNPs / Co-TCPP after calcination.

[0040] like Figure 3As shown, the characterization results of the Ni-TCPP MOF prepared in Example 1 of the present invention and the materials during the solvothermal reaction and calcination process thereof, wherein A~D are the characterization results of Ni-TCPP MOF, E~H are the characterization results of Ni-TCPP solvothermal reaction at 180°C for 12 h, I~L are the characterization results of Ni NPs / Ni-TCPP after calcination, A, E, I, B, F and J are scanning electron microscope SEM photos, C, G, K, D, H and L are transmission electron microscope TEM photos, M is the atomic force microscope AFM characterization result of Ni-TCPP MOF, and N and O are the atomic force microscope AFM characterization results of NiNPs / Ni-TCPP after calcination.

[0041] Experimental Example 2 Performance test of the electrochemical sensor prepared in Example 3 1. Experimental process Different concentrations of MNZ, TC and LEV antibiotic test solutions were prepared with 0.1M PBS solution with pH=7.4. 0.05mL of antibiotic solutions with concentrations from low to high were dripped onto the working electrode of M NPs / M-TCPP / SPE in sequence, and electrochemical detection was performed using differential pulse voltammetry (DPV). The linear relationship between antibiotic concentration and DPV peak current response value was plotted, and the detection limit, linear range and sensitivity were calculated based on the linear equation.

[0042] 2. Experimental results Table 1 shows the performance results of the electrochemical sensor of Example 3

[0043] The electrochemical sensor prepared by the present invention can simultaneously measure three antibiotics (metronidazole, levofloxacin and tetracycline). It is important to separate the peak potentials of the analytes to avoid interference. The feasibility of DPV in simultaneously detecting MNZ, TC and LEV in 0.1 M PBS (pH 7.4) containing the three analytes was studied in the potential window of -1.0~1.2 V. The results are shown in Figure 2. Figure 4 As shown, the characteristic oxidation peaks of MNZ, TC, and LEV obtained from MNP / Co-TCPP / GCE appear at −0.8, 0.7, and 0.83 V, respectively. The distance between the peak potentials of MNZ, TC, and LEV is above 0.13 V, which is satisfactory for the simultaneous determination of these compounds.

[0044] Application Example 1 1. Application process The sensors constructed by the above three MNPs / M-TCPP / SPE were used for the determination of antibiotics in honey, milk and human serum, respectively, and the recovery rates were calculated by the standard addition method, as follows: Honey and pure milk samples were purchased from local supermarkets, and human serum was obtained from healthy volunteers at the Northwest University Hospital. Antibiotics of different concentrations were first added to the actual samples. Honey samples were diluted 2 times with 0.10 M PBS (pH = 7.4); pure milk samples were diluted 2 times with 0.10 M PBS (pH = 7.4), centrifuged at 9000 rpm for 5 minutes, and the supernatant was taken; human serum samples were measured directly without any treatment. Take 0.05 mL of the above-treated sample and drop it directly on the working electrode of the sensor, and use DPV for electrochemical detection.

[0045] 2. Experimental results Table 2 shows the results of the electrochemical sensor of Example 3 in actual sample detection

[0046] Note: MNZ in the table stands for metronidazole; LEV stands for levofloxacin; TC stands for tetracycline.

[0047] The three antibiotics in the actual sample were determined simultaneously, and the results were as follows Figure 5 As shown in the figure, A~D are the detection results of Co / Co-TCPP / SPE in honey samples, E~H are the detection results of Ni / Ni-TCPP / SPE in milk samples, and I~L are the detection results of Cu / Cu-TCPP / SPE in human serum, among which A, E, and I are the simultaneous determination of different concentrations of MNZ (0~400μM), TC (0~500μM), and LEV (0~80μM); B, F, and J are the detection of different concentrations of MNZ in the presence of a certain amount of TC and LEV; C, G, and K are the detection of different concentrations of TC in the presence of a certain amount of MNZ and LEV; D, H, and L are the detection of different concentrations of LEV in the presence of a certain amount of TC and MNZ.

[0048] In the actual sample detection of honey, milk, and human serum, the distance between the peak potentials of MNZ, TC, and LEV was also above 0.13 V, further demonstrating the feasibility of the proposed sensor for simultaneous detection of MNZ, TC, and LEV.

[0049] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A M NPs / M-TCPP nanocomposite material, characterized in that: The composite material is formed by in-situ growth of metal M nanoparticles MNPs in a 2D M-TCPP MOF nanofilm. The composite material retains the MOF structure of the 2D M-TCPP nanofilm, and the M NPs are uniformly loaded on the M-TCPP MOF nanofilm. Wherein, M=Cu, Co or Ni.

2. The M NPs / M-TCPP nanocomposite material according to claim 1, characterized in that: In the M NPs / M-TCPP nanocomposite material, the particle size of the metal nanoparticles M NPs is 1-10 nm.

3. The method for preparing the M NPs / M-TCPP nanocomposite material according to claim 1 or 2, characterized in that: The method includes: The 2D M-TCPP MOF nanofilm is subjected to two steps of solvent thermal reaction in an in-situ reaction process and calcination under an inert atmosphere to obtain the M NPs / M-TCPP nanocomposite material.

4. The preparation method according to claim 3, characterized in that: The solvent thermal reaction, the reaction temperature is 180 ° C; Or / and, calcining under the inert atmosphere at a temperature of 400°C.

5. The preparation method according to claim 4, characterized in that: The solvent thermal reaction has a reaction time of 12 hours; Or / and, the calcination time under the inert atmosphere is 1 hour.

6. The preparation method according to claim 3, characterized in that: After the solvent thermal reaction, the product is washed, centrifuged, freeze-dried, and then calcined under the inert atmosphere.

7. The preparation method according to claim 3, characterized in that: The 2D M-TCPP MOF nanofilm is obtained by the following method: The nitrate of metal M and polyvinyl pyrrolidone are dispersed in a mixture of DMF and ethanol, tetrakis(4-carboxyphenyl)porphyrin is added for ultrasonic treatment, and the obtained mixture is transferred to an autoclave and reacted at 80°C; after the reaction is completed, it is cooled to room temperature to obtain a 2D M-TCPP MOF nanofilm.

8. A modified electrode based on the M NPs / M-TCPP nanocomposite material as claimed in claim 1 or 2.

9. An electrochemical sensor constructed based on the modified electrode as claimed in claim 8.

10. Use of the electrochemical sensor according to claim 9 in detecting antibiotics, characterized in that: The electrochemical sensor can simultaneously detect metronidazole, levofloxacin and tetracycline.

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