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江南大學(xué)采用我司鏈霉親和素磁珠進(jìn)行MB-SELEX通過12輪篩選篩選出阿奇霉素特異性適配體

In vitro selection and engineering azithromycin-specific aptamers and construction of a ratiometric fluorescent aptasensor for sensitive detection of azithromycin

Tianyu Huang, Xin Chen, Jinri Chen, Yuting Zhang, Xiaoli Wang, Zhimeng Wu, Nandi Zhou
The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Received 12 December 2023, Revised 7 April 2024, Accepted 7 April 2024, Available online 9 April 2024, Version of Record 9 April 2024.

Sensors and Actuators B: Chemical
Volume 411, 15 July 2024, 135789
https://doi.org/10.1016/j.snb.2024.135789


Highlights
? DNA library-immobilized MB-SELEX was used to screen azithromycin-specific aptamers.
? Truncating non-binding segments of aptamers was based on structural prediction and rational design.
? Specific single-base mutations were based on molecular docking analysis.
? The aptamer was used in a ratiometric fluorescent aptasensor for azithromycin.
? The aptasensor has been successfully utilized for detection of azithromycin in actual samples.

Abstract
Azithromycin can effectively inhibit bacterial protein synthesis and reduce biofilm formation and quorum sensing, and thus has been preferentially used in the treatment of various infections. However, azithromycin is also among the most highly concentrated antibiotics present in wastewater due to the inappropriate use and treatment. In order to sensitively and rapidly monitor the azithromycin residues, aptamers specific to azithromycin were selected through 12 rounds of screening by using magnetic beads-SELEX (MB-SELEX). The screened aptamers were assessed for their affinity and specificity using fluorescent assay. Apt3 emerged as the optimal candidate aptamer with the dissociation constant (Kd) of 235.07?nM. After secondary structure analysis and molecular docking, Apt3 was systematically truncated and subjected to single-base mutations. The optimal aptamer Apt3–27?T was identified, exhibiting the Kd value of 215.84?nM and the improved specificity compared to the original aptamer. Then Apt3–27?T was utilized to construct a ratiometric fluorescent aptasensor, which exhibited the linear dynamic range of 25–400?nM and low detection limit of 9.78?nM. More importantly, the ratiometric fluorescence mechanism further improved the specificity of the biosensor, which finally can discriminate azithromycin from structurally similar erythromycin. Evaluation experiments using actual samples spiked with azithromycin showed the recovery ranging from 97.1% to 106% and the relative standard deviation ranging from 4.29% to 8.61%. The screened aptamer and constructed aptasensor offer the advantages of high affinity, high effectiveness and ease of use in actual sample detection, thereby displaying promising applications in the monitoring of azithromycin residues.
阿奇霉素能有效抑制細(xì)菌蛋白質(zhì)合成，減少生物膜形成和群體感應(yīng)，因此被優(yōu)先用于各種感染的治療。然而，由于使用和處理不當(dāng)，阿奇霉素也是廢水中濃度最高的抗生素之一。為了靈敏、快速地監(jiān)測(cè)阿奇霉素殘留，采用磁珠-SELEX（MB-SELEX）通過12輪篩選篩選出阿奇霉素特異性適配體。使用熒光測(cè)定法評(píng)估篩選的適配體的親和力和特異性。Apt3 成為最佳候選適配體，解離常數(shù) （Kd）的235.07 nM。經(jīng)過二級(jí)結(jié)構(gòu)分析和分子對(duì)接，Apt3被系統(tǒng)地截?cái)嗖l(fā)生單堿基突變。確定了最佳適配體 Apt3–27 T，表現(xiàn)出 Kd值為 215.84 nM，與原始適配體相比，特異性有所提高。然后利用Apt3–27 T構(gòu)建了比例式熒光適配體傳感器，其線性動(dòng)態(tài)范圍為25–400 nM，檢出限低至9.78 nM。更重要的是，比例熒光機(jī)制進(jìn)一步提高了生物傳感器的特異性，最終可以區(qū)分阿奇霉素和結(jié)構(gòu)相似的紅霉素。使用阿奇霉素加標(biāo)的實(shí)際樣品的評(píng)估實(shí)驗(yàn)顯示，回收率在97.1%至106%之間，相對(duì)標(biāo)準(zhǔn)偏差在4.29%至8.61%之間。篩選的適配體和構(gòu)建的適配體傳感器在實(shí)際樣品檢測(cè)中具有親和力高、有效性和易用性等優(yōu)點(diǎn)，在阿奇霉素殘留監(jiān)測(cè)中具有廣闊的應(yīng)用前景。


Graphical Abstract

In vitro selection and engineering azithromycin-specific aptamers and construction of a ratiometric fluorescent aptasensor for sensitive detection of azithromycin

2. Experimental section
2.1. Materials and reagents
Azithromycin, erythromycin, roxithromycin, clarithromycin, streptomycin, penicillin and chloramphenicol were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). DNA oligonucleotides were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). The sequences are listed in Table S1. PCR and electrophoresis reagents were obtained from TaKaRa Biotechnology Co., Ltd (Dalian, China). Streptavidin-modified magnetic beads were purchased from PuriMag Biotechnology Co., Ltd (Xiamen, China). GO (20?mg/mL) was obtained from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). SYBR Green I (SGI, 10000×) was from Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals were purchased from Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China). Ultrapure water (18.2?MΩ cm) obtained from a Millipore water purification system (Gradient A10, Merck Millipore, Burlington, USA) was used in all experiments. Lake water was collected from Taihu Lake, Wuxi, China. Milk was purchased from local market. All antibiotics and sequences were diluted with binding buffer (8?mM Na2HPO4, 1.5?mM KH2PO4, 137?mM NaCl, 2.5?mM KCl, 1?mM CaCl2, 0.5?mM MgCl2, pH 7.4).

2.2. Preparation of the immobilized dsDNA library
The initial ssDNA library modified with 6-FAM (1?μM) was amplified using the forward primer modified with 6-FAM fluorophore and the reverse primer modified with biotin (The PCR reaction system is detailed in Table S2). The PCR cycling conditions were as follow: initial denaturation at 95 ℃ for 5?min, followed by 4 cycles of denaturation, annealing and extension at 95 ℃ for 15?s, 55 ℃ for 15?s, and 72 ℃ for 15?s, respectively. Subsequently, 95?μL PCR product was mixed with 105?μL binding buffer. 0.5?mg streptavidin-modified magnetic beads were then added to the mixture and incubated at 25 ℃ for 1?h with continuous shaking. dsDNA was immobilized onto magnetic bead surface through the specific interaction between biotin and streptavidin. Finally, the mixture was washed three times with binding buffer to remove unbound oligonucleotides.

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