目的: 建立基于热休克蛋白16.3（heat shock protein,Hsp16.3）核酸适体的 “竞争法”和“夹心法”两种可视化生物传感器检测结核分枝杆菌Hsp16.3,为辅助诊断潜伏性结核感染（LTBI）提供参考。方法: 利用含重组表达质粒pQE30-hsp16.3的大肠埃希菌BL21（DE3）诱导表达Hsp16.3并予金属螯合层析纯化。谷胱甘肽还原法制备金纳米簇（AuNCs）,偶联氨基修饰的核酸适体H66和核酸适体H63 SL-2 M6互补链。固定核酸适体H63 SL-2 M6于微孔板微孔内,以Hsp16.3为诊断抗原,加入AuNCs修饰的H63 SL-2 M6建立“竞争法”生物传感器,加入AuNCs修饰的H66建立“夹心法”生物传感器。柠檬酸三钠还原法制备金纳米颗粒（AuNPs）作为显色反应,以过氧化氢（H2O2）浓度高低引起AuNPs制备过程中的颜色变化作为输出信号,酶标仪测量A550,计算两种生物传感器的检测限。结果: 偶联后的AuNCs仍保持类过氧化物酶活性;与Hsp16.3反应后,“竞争法”生物传感器的显色反应呈红色;“夹心法”生物传感器的显色反应呈蓝色。所建立的两种可视化生物传感器线性响应范围为100~700 ng/mL,竞争法和夹心法的检测限分别为87 ng/mL和43 ng/mL。结论: 基于核酸适体的竞争法和夹心法可视化生物传感器可有效检测Hsp16.3。
Abstract
Objective To construct two visual biosensors named as “competitive method” and “sandwich method” based on heat shock protein 16.3(Hsp16.3) aptamer and gold nanoclusters (AuNCs) for detection of Mycobacterium tuberculosis Hsp16.3 and provide reference for diagnosis of latent tuberculosis infection (LTBI). Methods Recombinant Hsp16.3 was induced to express in Escherichia coli BL21(DE3) containing recombinant expression plasmid pQE30-hsp16.3 and purified by metal chelate chromatography. AuNCs were prepared by glutathione reduction and coupled with the amino modified aptamer H66 and aptamer complementary strand H63 SL-2 M6. aptamer H63 SL-2 M6 was fixed on the microplate, and Hsp16.3 was used as the diagnostic antigen. The “competitive” biosensor was established by adding aptamer complementary chain H63 SL-2 M6 modified with AuNCs, and the “sandwich” biosensor was established by adding aptamer H66 modified with AuNCs. The reaction of trisodium citrate reduction method to prepare gold nanoparticles (AuNPs) was introduced as color development reaction. The color change in the preparation process of AuNPs caused by the concentration of hydrogen peroxide (H2O2) was used as the output signal. The absorbance value at 550 nm was measured by microplate detector, and the detection limit of the two biosensors was calculated to evaluate the detection performance. Results The conjugated AuNCs retained peroxidase-like activity. After reacting with Hsp16.3, the color reaction of the “competitive method” biosensor appears red; The color reaction of the sandwich biosensor is blue. The linear response range of the two visual biosensors was 100-700 ng/mL, and the detection limit for competition method and sandwich method was 87 ng/mL and 43 ng/mL respectively. Conclusion The two visual biosensors based on aptamers can effectively detect Hsp16.3.
关键词
结核分枝杆菌 /
核酸适体 /
金纳米簇 /
热休克蛋白16.3
Key words
Mycobacterium tuberculosis /
aptamer /
Au nanoclusters /
heat shock protein 16.3
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] WORLD HEALTH ORGANIZATION.Global tuberculosis report 2024[M]. Geneva: World Health Organization, 2024.
[2] VASILIU A, MARTINEZ L, GUPTA R K, et al.Tuberculosis prevention: current strategies and future directions[J]. Clin Microbiol Infect, 2024, 30(9): 1123-1130.
[3] HUANG C, PAN L, SHEN X, et al.Hsp16.3 of mycobacterium tuberculosis in exosomes as a biomarker of tuberculosis[J]. Eur J Clin Microbiol Infect Dis, 2021, 40(11): 2427-2430.
[4] ZHANG Y, LI S, LIU Q, et al.Mycobacterium tuberculosis Heat-Shock Protein 16.3 Induces Macrophage M2 Polarization Through CCRL2/CX3CR1[J]. Inflammation, 2020, 43(2): 487-506.
[5] BARIK S, PANDA A K, BISWAS V K, et al.Lysine acetylation of Hsp16.3: Effect on its structure, chaperone function and influence towards the growth of Mycobacterium tuberculosis[J]. Int J Biol Macromol, 2024, 268: 131763.
[6] PORNPROM T, PHUSI N, THONGDEE P, et al.Toward the early diagnosis of tuberculosis: A gold particle-decorated graphene-modified paper-based electrochemical biosensor for Hsp16.3 detection[J]. Talanta, 2024, 267: 125210.
[7] MOR P, DAHIYA B, SHARMA S, et al.Diagnosis of peritoneal tuberculosis by real-time immuno-PCR assay based on detection of a cocktail of Mycobacterium tuberculosis CFP-10 and HspX proteins[J]. Expert Rev Gastroenterol Hepatol, 2022, 16(6): 577-586.
[8] DHIMAN A, HALDAR S, MISHRA S K, et al.Generation and application of DNA aptamers against HspX for accurate diagnosis of tuberculous meningitis[J]. Tuberculosis (Edinb), 2018, 112: 27-36.
[9] ROTUNDO S, TASSONE M T, SERAPIDE F, et al.Incipient tuberculosis: a comprehensive overview[J]. Infection, 2024, 52(4): 1215-1222.
[10] HAMADA Y, CIRILLO D M, MATTEELLI A, et al.Tests for tuberculosis infection: landscape analysis[J]. Eur Respir J, 2021, 58(5): 2100167.
[11] ALHUSAIN F.HspX-mediated survival pathways of pathogenic mycobacteria[J]. Saudi Med J, 2021, 42(7): 721-727.
[12] LI Y, TAM W W, YU Y, et al.The application of Aptamer in biomarker discovery[J]. Biomark Res, 2023, 11(1): 70-87.
[13] BETHU R, MITTAL H G, SHARMA T K, et al.Rapid diagnosis of TB using Aptamer-based assays for Mycobacterium tuberculosis antigens in children and adolescents[J]. Lung India, 2023, 40(5): 434-439.
[14] KUMARI P, DHIMAN A, LAVANIA S, et al.Assessment of DNA aptamers targeting GlcB and HspX antigens for application in the diagnosis of abdominal tuberculosis[J]. Tuberculosis (Edinb), 2022, 134: 102206.
[15] YANG J, WANG X, SUN Y, et al.Recent Advances in Colorimetric Sensors Based on Gold Nanoparticles for Pathogen Detection[J]. Biosensors (Basel), 2022, 13(1): 29-41.
[16] LIU D-M, DONG C.Gold nanoparticles as colorimetric probes in food analysis: Progress and challenges[J]. Food Chem, 2023, 429: 136887.
[17] CHEN Q, GAO R, JIA L.Enhancement of the peroxidase-like activity of aptamers modified gold nanoclusters by bacteria for colorimetric detection of Salmonella typhimurium[J]. Talanta, 2021, 221: 121476.
[18] 李茜雯, 李民, 刘苗, 等. 金纳米簇可视化免疫传感器检测弓形虫GRA7抗体的研究[J]. 中国病原生物学杂志, 2022, 17(11): 1256-1260.
[19] XIAO Y, HUANG N, WEN J, et al.Detecting uric acid base on the dual inner filter effect using BSA@Au nanoclusters as both peroxidase mimics and fluorescent reporters[J]. Spectrochim Acta A Mol Biomol Spectrosc, 2023, 293: 122504.
[20] ANTÓNIO M, VITORINO R, DANIEL-DA-SILVA A L. LSPR-Based Aptasensor for Rapid Urinary Detection of NT-proBNP[J]. Biosensors (Basel), 2023, 13(7): 736-748.
基金
长沙市科技项目“基于核酸适体的金纳米簇可视化免疫传感技术诊断潜伏性结核感染”（kq2208173）
PDF(2986 KB)
