CN115452787A - Method for measuring streptomycin in milk by using fluorescence sensor constructed by silver nanoclusters and gold palladium nanoparticles - Google Patents
Method for measuring streptomycin in milk by using fluorescence sensor constructed by silver nanoclusters and gold palladium nanoparticles Download PDFInfo
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
Description
技术领域technical field
本发明提供一种检测牛奶中链霉素残留的适配体传感器的制备方法,属于食品安全检测技术领域。The invention provides a method for preparing an aptamer sensor for detecting streptomycin residue in milk, belonging to the technical field of food safety detection.
背景技术Background technique
链霉素(STR)是一种常见的氨基糖苷类抗生素,用于治疗奶牛的细菌感染引起的乳腺炎等疾病,不合理地使用会造成牛奶等乳制品中的STR残留,不仅影响乳制品的质量,而且会对人体造成严重的危害,包括过敏、破坏肠道菌群和肾脏的毒性。Streptomycin (STR) is a common aminoglycoside antibiotic used to treat mastitis and other diseases caused by bacterial infection in dairy cows. Improper use will cause STR residues in milk and other dairy products, which not only affects the quality of dairy products quality, and can cause serious harm to the human body, including allergies, destruction of intestinal flora and toxicity to the kidneys.
因此,开发灵敏和特异性技术来检测动物源性食品如牛奶中的STR是至关重要的,目前,毛细管电泳、高效液相色谱和酶联免疫等灵敏检测方法已被开发用于检测牛奶残留物中的STR,但由于周转时间长,步骤复杂,设备昂贵和技术要求高等缺点限制了它们的应用。Therefore, it is crucial to develop sensitive and specific techniques to detect STRs in foods of animal origin such as milk, and currently, sensitive assays such as capillary electrophoresis, high performance liquid chromatography, and enzyme-linked immunosorbent assays have been developed for the detection of milk residues However, their applications are limited by disadvantages such as long turnaround time, complicated steps, expensive equipment, and high technical requirements.
发明内容Contents of the invention
发明的目的在于建立一种快速、简单、高效的检测技术检测牛奶中的STR。The purpose of the invention is to establish a fast, simple and efficient detection technology for detecting STR in milk.
其技术方案为:适配体是通过数富集的配体系统进化技术(SELEX)筛选的高亲和力和特异性的RNA或ssDNA序列,近年来,因具有快速、灵敏和易于操作的优点,基于适配体的荧光传感器得到了广泛的应用。Its technical scheme is: aptamers are high-affinity and specific RNA or ssDNA sequences screened by the number-enriched ligand system evolution technique (SELEX). Aptamer-based fluorescent sensors have been widely used.
所述的一种检测牛奶中STR残留的荧光适配体传感器的制备方法(图1),其特征在于:在发夹式DNA-银纳米簇(DNA-AgNCs)环中加入G碱基,以实现对DNA-AgNCs荧光光谱的调节,DNA-AgNCs的模板链中含有STR适配体(STP)序列,在没有STR的情况下,DNA-AgNCs的适配体通过配位被吸附在金钯纳米粒子(Au@PdNPs)的表面,因此,能量从荧光供体DNA-AgNCs转移到Au@PdNPs上,导致了荧光淬灭,当加入STR时,STR与适配体的特异性结合导致适配体的构象发生变化,适配体和Au@PdNPs之间的相互作用减弱,导致荧光信号的恢复,基于荧光信号的变化,可以成功实现对牛奶中STR的检测。The method for preparing a fluorescent aptasensor for detecting STR residues in milk (Figure 1) is characterized in that: G bases are added to the hairpin DNA-silver nanocluster (DNA-AgNCs) ring to To achieve the adjustment of the fluorescence spectrum of DNA-AgNCs, the template strand of DNA-AgNCs contains STR aptamer (STP) sequence, in the absence of STR, the aptamer of DNA-AgNCs is adsorbed on the gold-palladium nanometer through coordination The surface of the particle (Au@PdNPs), therefore, the energy transfer from the fluorescent donor DNA-AgNCs to the Au@PdNPs leads to fluorescence quenching, and when STR is added, the specific binding of STR to the aptamer results in the aptamer The conformation of Au@PdNPs changed, and the interaction between the aptamer and Au@PdNPs weakened, resulting in the recovery of the fluorescent signal. Based on the change of the fluorescent signal, the detection of STR in milk could be successfully realized.
所述的一种检测牛奶中STR残留的荧光适配体传感器的制备方法,其特征在于:发夹环中加入不同数量的G碱基,以实现发射光谱的红移和DNA-AgNCs荧光强度的增加,此外,合成具有优良淬灭特性的 Au@PdNPs复合材料,不仅解决了钯纳米粒子(PdNPs)在长波长范围内的弱淬灭能力问题,而且有效地扩大了金纳米粒子(AuNPs)的荧光淬灭范围。The preparation method of a fluorescent aptamer sensor for detecting STR residues in milk is characterized in that: different numbers of G bases are added to the hairpin loop to realize the red shift of the emission spectrum and the increase of the fluorescence intensity of DNA-AgNCs In addition, the synthesis of Au@PdNPs composites with excellent quenching properties not only solves the problem of weak quenching ability of palladium nanoparticles (PdNPs) in the long wavelength range, but also effectively expands the Fluorescence quenching range.
其制备原理为:在荧光共振能量转移(FRET)的基础上,我们设计了一种利用DNA-AgNCs和核壳金钯纳米粒子(Au@PdNPs)的荧光传感器来检测牛奶中的链霉素(STR),DNA-AgNCs的适配体通过配位被吸附在Au@PdNPs的表面。DNA-AgNCs和Au@PdNPs产生FRET,能量从荧光供体DNA-AgNCs转移到Au@PdNPs上,导致了荧光淬灭,当加入STR时,STR与适配体的特异性结合导致适配体的构象发生变化,适配体和Au@PdNPs之间的FRET被破坏,导致荧光信号的恢复。The preparation principle is as follows: Based on fluorescence resonance energy transfer (FRET), we designed a fluorescent sensor using DNA-AgNCs and core-shell gold-palladium nanoparticles (Au@PdNPs) to detect streptomycin in milk ( STR), the aptamers of DNA-AgNCs were adsorbed on the surface of Au@PdNPs through coordination. DNA-AgNCs and Au@PdNPs generate FRET, and the energy is transferred from the fluorescent donor DNA-AgNCs to Au@PdNPs, resulting in fluorescence quenching. When STR is added, the specific binding of STR to the aptamer leads to the aptamer A conformational change occurs and the FRET between the aptamer and Au@PdNPs is disrupted, resulting in the recovery of the fluorescent signal.
为达到以上目的,采取以下技术方案实现:银纳米簇的制备,首先用TE缓冲液制备所有的DNA模板溶液(100 μM),并在水浴中(95℃)保持10分钟,然后在冰水浴中将溶液冷却到室温,接着将500 μL的100 μM DNA溶液和300 μL的1 mM硝酸银(AgNO3)溶液分别加入到3.9 ml TE缓冲液中,充分混合和震荡后,将所有的混合溶液在冰浴中和黑暗条件下孵育30分钟,在混合溶液中分别加入300 μL的1 mM新鲜制备且冰冷的硼氢化钠(NaBH4)溶液,反应结束后,然后将混合溶液充分摇晃1分钟,最后,将得到的溶液避光存储在4℃处,Au@PdNPs的制备,在均匀搅拌的条件下,将100 mL0.01%(w/v)三水氯化金(HAuCl4·3H2O)溶液加热至沸腾,之后,迅速加入2.7 mL 1%(w/v)的柠檬酸三钠(Na3C6H5O7)溶液,连续加热和煮沸,直到金种子溶液的颜色没有变化,而后在不断搅拌下将金种子溶液冷却到室温,随后,在剧烈搅拌下,将2.0 mL金种子溶液和4 mL 1%(w/v)HAuCl4·3H2O溶液加入200 mL超纯水中,搅拌均匀后,将800 μL1%(w/v)的Na3C6H5O7溶液和400 μL30 mM的氢醌(C6H6O2)溶液迅速加入混合溶液中进行还原。将上述还原剂重复加入混合溶液中7次,时间间隔为10分钟,加入完毕后,混合溶液继续搅拌1小时,最后,用下面的方法合成1 mM的H2PdCl4溶液,将10.64 mg氯化钯(PdCl2)被溶解到6 mL 0.02 M HCl溶液中,然后在70℃的水浴中向该混合物中加入54mL超纯水,直到PdCl2完全溶解,得到1 mM的H2PdCl4溶液,通过钯的沉积得到Au@PdNPs,30mL 75 nm的AuNPs溶液分别与1 mL、4 mL、7 mL和10 mL的H2PdCl4溶液(1 mM)混合,混合后的溶液在冰浴中冷却,在剧烈的搅拌下,将1 mL、4 mL、7 mL和10 mL抗坏血酸溶液和18 mL、12mL、6 mL和0 mL超纯水分别慢慢加入混合溶液中,然后将混合溶液继续搅拌30分钟,搅拌后,将得到的溶液离心,用水-乙醇溶液(1:2,v/v)洗涤数次,最后,将Au@PdNPs溶解在C6H5Na3O7溶液(0.02%, w/v)中。In order to achieve the above objectives, the following technical schemes are adopted: for the preparation of silver nanoclusters, first prepare all DNA template solutions (100 μM) with TE buffer, and keep them in a water bath (95°C) for 10 minutes, and then in an ice-water bath Cool the solution to room temperature, then add 500 μL of 100 μM DNA solution and 300 μL of 1 mM silver nitrate (AgNO 3 ) solution to 3.9 ml TE buffer, mix and shake thoroughly, and put all the mixed solutions in Incubate in an ice bath and in the dark for 30 minutes, add 300 μL of 1 mM freshly prepared and ice-cold sodium borohydride (NaBH 4 ) solution to the mixed solution, after the reaction is complete, shake the mixed solution for 1 minute, and finally , Store the resulting solution in the dark at 4°C. For the preparation of Au@PdNPs, 100 mL of 0.01% (w/v) gold chloride trihydrate (HAuCl 4 3H 2 O) The solution was heated to boiling, after which, 2.7 mL of 1% (w/v) trisodium citrate (Na 3 C 6 H 5 O 7 ) solution was quickly added, continuously heated and boiled until the color of the gold seed solution did not change, and then Cool the gold seed solution to room temperature with constant stirring, and subsequently, add 2.0 mL of gold seed solution and 4 mL of 1% (w/v) HAuCl 3H 2 O solution into 200 mL of ultrapure water with vigorous stirring, After stirring evenly, quickly add 800 μL of 1% (w/v) Na 3 C 6 H 5 O 7 solution and 400 μL of 30 mM hydroquinone (C 6 H 6 O 2 ) solution into the mixed solution for reduction. The above reducing agent was repeatedly added to the mixed
为达到以上目的,采取以下技术方案实现:在检测STR之前,在整个实验过程中,发夹式DNA-AgNCs的浓度被稀释到1 μM,激发波长为586 nm,而STR的检测结果是在最佳实验条件下获得的,将500 μl的DNA-AgNCs和500 μl的Au@PdNPs溶液混合15分钟,在DNA-AgNCs和Au@PdNPs的混合溶液中分别加入一系列500 μL不同浓度的STR溶液,反应持续了60分钟,然后测量荧光强度。In order to achieve the above purpose, the following technical scheme was adopted: before detecting STR, the concentration of hairpin DNA-AgNCs was diluted to 1 μM during the whole experiment, and the excitation wavelength was 586 nm, and the detection result of STR was at the most 500 μl of DNA-AgNCs and 500 μl of Au@PdNPs solution were mixed for 15 minutes, and a series of 500 μL of STR solutions of different concentrations were added to the mixed solution of DNA-AgNCs and Au@PdNPs respectively. The reaction was continued for 60 minutes, and then the fluorescence intensity was measured.
所述适配体传感器的制备工艺如下:预处理前,STR被添加到牛奶中,有三个浓度水平(300 nM、600 nM和900 nM),随后,通过以下方法对牛奶样品进行预处理,20 mL牛奶-甲醇(1:4, v/v)混合物在-20℃下保存20分钟,然后将混合物在12500 rpm下离心25分钟,上清液通过膜过滤器(0.22 μm)过滤,过滤后,上清液被浓缩并在60℃的水浴中干燥,残余物用氮气干燥,然后用超纯水重构,重组液用薄膜过滤器(0.22 μm)过滤,最后,滤液的体积设定为4 mL。The preparation process of the aptasensor was as follows: before pretreatment, STR was added to milk at three concentration levels (300 nM, 600 nM, and 900 nM), and subsequently, milk samples were pretreated by the following method, 20 mL of milk-methanol (1:4, v/v) mixture was stored at -20°C for 20 minutes, then the mixture was centrifuged at 12,500 rpm for 25 minutes, and the supernatant was filtered through a membrane filter (0.22 μm). After filtration, The supernatant was concentrated and dried in a water bath at 60 °C, and the residue was dried with nitrogen, then reconstituted with ultrapure water, and the reconstituted solution was filtered with a membrane filter (0.22 μm), and finally, the volume of the filtrate was set to 4 mL .
附图说明Description of drawings
图1 荧光适配体传感器的构建过程。Fig. 1 Construction process of fluorescent aptasensor.
图2 不同模板链合成的DNA-AgNCs的激发和发射波长。Fig. 2 Excitation and emission wavelengths of DNA-AgNCs synthesized from different template strands.
图3 DNA-AgNCs的荧光光谱和荧光强度。Fig. 3 Fluorescence spectra and fluorescence intensity of DNA-AgNCs.
图4 纳米材料的电镜表征。Figure 4 Electron microscopy characterization of nanomaterials.
图5 钯壳厚度的优化。Fig. 5 Optimization of palladium shell thickness.
图6 试验条件优化。Figure 6 Optimization of experimental conditions.
图7 STR的荧光测定。Figure 7 Fluorescence measurement of STR.
图8 特异性的测试。Figure 8 Test of specificity.
图9 牛奶实际样品中的STR测试。Figure 9 STR test in real milk samples.
具体实施方式detailed description
实施例1:图3 A显示了不同模板链合成的DNA-AgNCs的荧光光谱,图2中列出了激发和发射波长,图3 B显示了荧光强度,然而,在DNA2、DNA3和DNA4、DNA5中,随着G碱基数量的增加,没有规律的红移,而当G碱基的数量达到10和12时,红移不再发生,当碱基数在0-6之间时,荧光强度变化不大,然而,当G碱基的数量增加到8时,发生了明显的增强和变化,由于DNA4-AgNCs、DNA5-AgNCs、DNA6-AgNCs和DNA7-AgNCs的发射光谱接近于近红外区域,这意味着它需要大尺寸的金属纳米粒子,但是较大的金属纳米颗粒可能会引起散射,导致荧光光谱的扭曲,DNA3-AgNCs的荧光强度在DNA1-AgNCs、DNA2-AgNCs和DNA3-AgNCs中最强,DNA3-AgNCs的最大发射峰的位置比DNA2-AgNCs短,所以所需的淬灭剂纳米粒子的直径较小,散射的影响也较弱,综上所述,选择DNA3-AgNCs作为本实验中使用的荧光材料。Example 1: Figure 3 A shows the fluorescence spectra of DNA-AgNCs synthesized with different template strands, the excitation and emission wavelengths are listed in Figure 2, and Figure 3 B shows the fluorescence intensity, however, in DNA 2 , DNA 3 and DNA 4. In DNA 5 , as the number of G bases increases, there is no regular red shift. When the number of G bases reaches 10 and 12, the red shift no longer occurs. When the number of bases is between 0-6 , the fluorescence intensity did not change much, however, when the number of G bases increased to 8 , a significant enhancement and change occurred , due to the The emission spectrum is close to the near-infrared region, which means that it requires large-sized metal nanoparticles, but larger metal nanoparticles may cause scattering, resulting in distortion of the fluorescence spectrum, and the fluorescence intensity of DNA 3 -AgNCs is lower than that of DNA 1 -AgNCs , DNA 2 -AgNCs and DNA 3 -AgNCs are the strongest, and the position of the maximum emission peak of DNA 3 -AgNCs is shorter than that of DNA 2 -AgNCs, so the diameter of the required quencher nanoparticles is smaller, and the influence of scattering is also smaller. Weak, in summary, DNA 3 -AgNCs were chosen as the fluorescent material used in this experiment.
实施例2:通过TEM观察DNA3-AgNCs的形态,如图4 A所示,合成的DNA3-AgNCs表现出均匀的分散性,几乎是圆形的,平均尺寸为2.47 nm,测量了DNA3-AgNCs在120小时内的稳定性,结果显示,DNA3-AgNCs在12-24小时内是稳定的,因此,实验是在DNA3-AgNCs合成后的12-24小时内进行的,经测量,DNA3-AgNCs的绝对量子产率为10.76%,金种子的TEM图像显示在图4 B中,从图中可以看出,金种子是球形颗粒,其直径为19.9 nm,图4 E显示了通过种子生长方法合成的大尺寸金纳米粒子,其直径为75纳米,从图4 F-I可以看出,用不同量的H2PdCl4和抗坏血酸在AuNPs的表面沉积了钯,可以看出,钯壳的厚度分别约为4 nm、7 nm、11nm和16 nm,图4 J-M显示了直径为75+16 nm的Au@PdNPs的EDX,可以看出,钯分散在金核的外层,金/钯的重量比为33.36/66.64,图4 C,D显示了直径为75+16 nm的Au@PdNPs的SEM,根据图像,Au@PdNPs几乎是球形颗粒,直径为107.11 nm,上述分析表明,Au@PdNPs和DNA3-AgNCs的合成效果良好。Example 2: Observation of the morphology of DNA 3 -AgNCs by TEM, as shown in Figure 4A, the synthesized DNA 3 -AgNCs showed uniform dispersion, almost circular, with an average size of 2.47 nm, and the DNA 3 -AgNCs were measured - Stability of AgNCs within 120 hours, the results showed that DNA 3 -AgNCs were stable within 12-24 hours, therefore, the experiment was carried out within 12-24 hours after the synthesis of DNA 3 -AgNCs, and it was measured that The absolute quantum yield of DNA 3 -AgNCs is 10.76%. The TEM image of gold seeds is shown in Fig. 4B. It can be seen from the figure that the gold seeds are spherical particles with a diameter of 19.9 nm. Fig. 4E shows that the gold seeds passed The large-sized gold nanoparticles synthesized by the seed growth method have a diameter of 75 nm. It can be seen from Fig. 4 FI that palladium is deposited on the surface of AuNPs with different amounts of H 2 PdCl 4 and ascorbic acid. It can be seen that the palladium shell The thicknesses are about 4 nm, 7 nm, 11 nm, and 16 nm, respectively. Figure 4 JM shows the EDX of Au@PdNPs with a diameter of 75+16 nm. It can be seen that palladium is dispersed in the outer layer of the gold core, and the gold/palladium The weight ratio is 33.36/66.64. Figure 4 C, D shows the SEM of Au@PdNPs with a diameter of 75+16 nm. According to the image, Au@PdNPs are almost spherical particles with a diameter of 107.11 nm. The above analysis shows that Au@PdNPs And the synthesis effect of DNA 3 -AgNCs is good.
实施例3:钯壳厚度的优化(图5),75+4 nm、75+7 nm、75+11 nm和75+16 nm Au@PdNPs的能量转移效率(E)分别为26.7%、46.6%、56.7%和62.8%,可以发现能量转移效率随着钯壳厚度的增加而逐渐增加,75+16 nm的Au@PdNPs具有很高的能量传递效率,足以满足实验要求,同时考虑到进一步增加颗粒尺寸会增加金属纳米颗粒散射的干扰,因此,选择淬灭效率最高的75 + 16 nm Au@PdNPs作为实验的淬灭剂。Example 3: Optimization of palladium shell thickness (Figure 5), the energy transfer efficiencies (E) of 75+4 nm, 75+7 nm, 75+11 nm and 75+16 nm Au@PdNPs are 26.7% and 46.6%, respectively , 56.7% and 62.8%, it can be found that the energy transfer efficiency gradually increases with the increase of the thickness of the palladium shell, and the Au@PdNPs with 75+16 nm have high energy transfer efficiency, which is enough to meet the experimental requirements. The size will increase the interference of metal nanoparticle scattering, therefore, the 75 + 16 nm Au@PdNPs with the highest quenching efficiency were selected as the quencher for the experiment.
实施例4:对试验中的条件进行优化(图6), (A)DNA-AgNCs合成中pH的优化(6.5-9.0),最佳pH为7.5,(B)将浓度为0-24.8 pm的Au@PdNPs加入到1 μM的DNA3-AgNCs中,最佳Au@PdNPs浓度为17.2 pM,(C)荧光猝灭的孵育时间(0-30min),最佳淬灭时间为15min,(D)荧光恢复的孵育时间(0-90min),最佳恢复时间为60min。Example 4: Optimizing the conditions in the experiment (Figure 6), (A) pH optimization (6.5-9.0) in the synthesis of DNA-AgNCs, the optimal pH is 7.5, (B) the concentration of 0-24.8 pm Au@PdNPs were added to 1 μM DNA 3 -AgNCs, the optimum concentration of Au@PdNPs was 17.2 pM, (C) the incubation time of fluorescence quenching (0-30min), the optimum quenching time was 15min, (D) Incubation time for fluorescence recovery (0-90min), the best recovery time is 60min.
实施例5:通过测量不同浓度的STR,评估该传感器测定分析物的性能,如图7所示,在50~2800 nM范围内,荧光强度随着STR浓度的增加而逐渐增加,在50~1250 nM范围内,荧光强度与STR浓度呈正的线性关系,线性校准曲线的方程式如下:I=0.43314CSTR +102.94683,相关系数(R2)为0.99258,根据3σ/k,STR荧光测定的检测极限(LOD)为18.7 nM,其中σ是校准标准空白的测量的标准偏差(n=10),k是校准曲线的斜率。Example 5: By measuring different concentrations of STR, evaluate the performance of the sensor for measuring analytes, as shown in Figure 7, in the range of 50-2800 nM, the fluorescence intensity gradually increases with the increase of STR concentration, and between 50-1250 nM In the nM range, the fluorescence intensity has a positive linear relationship with the STR concentration. The equation of the linear calibration curve is as follows: I=0.43314C STR +102.94683, the correlation coefficient (R 2 ) is 0.99258, and according to 3σ/k, the detection limit of STR fluorescence measurement ( LOD) is 18.7 nM, where σ is the standard deviation of the measurements of the calibration standard blank (n = 10) and k is the slope of the calibration curve.
实施例6:为了评价该传感器的特异性,通过与其他抗生素如特拉霉素(TER)、氯霉素(CHL)、四环素(TET)、红霉素(ERY)、盐酸金霉素(CHH)和青霉素(PEN)进行比较,确定了基于DNA-AgNCs的荧光适配体的特异性,其他抗生素的浓度为STR的10倍,如图8所示,I和I0分别表示不同类型抗生素存在和无抗生素存在下的荧光强度,且STR对荧光适增剂的反应高于其他抗生素,这些结果表明,荧光适应器对检测STR具有很强的特异性,表明所开发的荧光适应器可用于检测STR。Example 6: In order to evaluate the specificity of the sensor, by combining with other antibiotics such as telarmycin (TER), chloramphenicol (CHL), tetracycline (TET), erythromycin (ERY), aureomycin hydrochloride (CHH ) and penicillin (PEN) to determine the specificity of fluorescent aptamers based on DNA-AgNCs, the concentration of other antibiotics is 10 times that of STR, as shown in Figure 8, I and I 0 respectively indicate the presence of different types of antibiotics and fluorescence intensities in the absence of antibiotics, and the response of STR to fluorescent adapters was higher than that of other antibiotics. These results indicated that the fluorescent adapters had strong specificity for detecting STRs, indicating that the developed fluorescent adapters could be used to detect str.
实施例7:为了评估该发明在牛奶样品中检测STR的可行性,我们将三种不同浓度的链霉素加入到实际牛奶中,回收率见图9,可以看出,卡那霉素检测的峰值回收率在97.45%-105.34%之间,相对标准差(RSD)在1.05%-3.04%之间,这些优良的性能表明了我们提出的链霉素检测方法在该应用中的可行性。Example 7: In order to evaluate the feasibility of the invention for detecting STR in milk samples, we added three different concentrations of streptomycin to actual milk, and the recovery rate is shown in Figure 9. It can be seen that the kanamycin detection The peak recovery was between 97.45%-105.34%, and the relative standard deviation (RSD) was between 1.05%-3.04%. These excellent performances indicated the feasibility of our proposed streptomycin detection method in this application.
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