有机长余辉纳米探针介导的 LFIA 胃蛋白酶原检测

Ultra-long organo-phosphorescent nanoprobe for detection of pepsinogen by lateral flow immunoassay

摘要

有机长余辉材料具有激发态性能丰富、成本低、柔韧性好、易于合成、发光寿命超长等优点，在背景荧光衰减后，其余辉的收集能有效排除背景光干扰。在此基础上，该文构建了一个基于有机长余辉纳米探针的侧流免疫层析（UOP-LFIAs）检测平台。首先，制备并合成长余辉晶体，然后通过在表面涂覆聚乙烯吡咯烷酮和羧甲基纤维素的步骤获得长余辉溶液。通过抗体偶联获得有机长余辉纳米探针，再设计制作出一种便携式 UOP-LFIAs 检测装置。最后，采用便携式 UOP-LFIAs 检测装置检测胃蛋白酶原（PG）Ⅰ和Ⅱ。结果表明，PG I 和 PG II 的检测限分别为 0.36 ng/mL 和 0.57 ng/mL，检测范围分别为 0.36～1 200 ng/mL 和 0.57～500 ng/mL。平均回收率分别为 101.30% 和 99.30%。该研究所构造的 UOP-LFIAs 检测平台不仅制备过程简便、快速、高效且经济，而且能有效地降低背景光信号的干扰，提升磷光化效率。此外，该平台实现了 PG I 和 PG II 的联合检测，仅需 5 min 即可快速获得结果，为即时检测提供了一个高效的疾病检测平台。

Abstract

Ultra-long organic phosphorescent materials have the advantages of rich excited state properties, low price and easy to synthesize, good flexibility, ultra-long luminescence lifetime. After the fluorescence decay of the background, the collection of the remaining light can effectively eliminate the background light interference. Based on this, a Lateral Flow Immunochromatographic Detection Platform Based on Ultra-Long Organo-Phosphorescent Nanoprobes (UOP-LFIAs) constructed by authors is presented in this paper. Firstly, long afterglow crystals were prepared and synthesized, and then the long afterglow solution was obtained by one-step coating polyvinylpyrrolidone and carboxymethyl cellulose on the surface. Next, it was coupled with antibodies to obtain ultra-long organic afterglow phosphorescent nanoprobes. A portable UOP-LFIAs test strip detection analyzer was then designed and manufactured. Finally, Pepsinogen (PG) I and II were detected by using a portable UOP-LFIAs detection device. The results show that the detection limits were 0.36 ng/mL and 0.57 ng/mL for PG I and PG II, with respective detection ranges of 0.36-1 200 ng/mL and 0.57-500 ng/mL. The average recoveries were 101.30% and 99.30%, respectively. It is concluded that the UOP-LFIAs assay platform established in this study is not only simple, rapid, cost-effective and suitable for macro-preparation, but also can effectively reduce the interference of background light signal and enhance the phosphorescence efficiency, thereby improving the detection sensitivity. Additionally, the platform enables the combined detection of PG I and PG II, providing results in just 5 minutes. This development offers a powerful tool for bedside disease detection.

关键词

有机长余辉纳米探针 / 即时检测 / 侧流免疫层析法 / 胃蛋白酶原检测

Key words

ultra-long organo-phosphorescent nanoprobe / point-of-care testing / lateral flow immunoassay / pepsinogen detection

参考文献

[1] KOHN J. A rapid method of estimating blood—glucose ranges[J]. Lancet, 1957, 273(6986): 119-121.

[2] LOU D D, FAN L, JIANG T, et al. Advances in nanoparticle—based lateral flow immunoassay for point—of—care testing[J]. View, 2022, 3(1): 20200125.

[3] BAHADıR E B, SEZGINTÜRK M K. Lateral flow assays: Principles, designs and labels[J]. TrAC Trends in Analytical Chemistry, 2016, 82: 286-306.

[4] 黄正壮 . 侧流免疫层析技术的研究进展[J]. 黑龙江科学, 2022, 13(20): 70-72.

[5] HUANG Z Z. Research progress of lateral flow immunochromatography[J]. Heilongjiang Science, 2022, 13(20): 70-72.

[6] 李娜, 杜汉宇, 王真真, 等. 侧流免疫层析技术在兽医诊断中的应用[J]. 动物医学进展, 2023, 44(4): 104-108.

[7] LI N, DU H Y, WANG Z Z, et al. Application of lateral flow immunochromatography in veterinary diagnosis[J]. Progress in Veterinary Medicine, 2023, 44(4): 104-108.

[8] HUANG X L, AGUILAR Z P, XU H Y, et al. Membrane—based lateral flow immunochromatographic strip with nanoparticles as reporters for detection: A review[J]. Biosensors and Bioelectronics, 2016, 75: 166-180.

[9] TANAKA R, YUHI T, NAGATANI N, et al. A novel enhancement assay for immunochromatographic test strips using gold nanoparticles[J]. Analytical and Bioanalytical Chemistry, 2006, 385(8): 1414-1420.

[10] HE X Y, HAO T J, GENG H X, et al. Sensitization strategies of lateral flow immunochromatography for gold modified nanomaterials in biosensor development[J]. International Journal of Nanomedicine, 2023, 18: 7847-7863.

[11] GUO J C, CHEN S Q, GUO J H, et al. Nanomaterial labels in lateral flow immunoassays for point—of—care—testing[J]. Journal of Materials Science & Technology, 2021, 60: 90-104.

[12] BOCK S, KIM H M, KIM J, et al. Lateral flow immunoassay with quantum—dot—embedded silica nanoparticles for prostate—specific antigen detection[J]. Nanomaterials, 2021, 12(1): 33.

[13] ZHANG H Y, LUO J X, BELOGLAZOVA N, et al. Portable multiplex immunochromatographic assay for quantitation of two typical algae toxins based on dual—color fluorescence microspheres[J]. Journal of Agricultural and Food Chemistry, 2019, 67(21): 6041-6047.

[14] KRIZKOVA S, NGUYEN H V, STANISAVLJEVIC M, et al. Microchip capillary electrophoresis: Quantum dots and paramagnetic particles for bacteria immunoseparation: Rapid superparamagnetic—beads—based automated immunoseparation of Zn—proteins from staphylococcus aureus with nanogram yield[J]. Methods in Molecular Biology, 2015, 1274: 67-79.

[15] LI Q R, YANG Y X, HU F, et al. Rapid detection of escherichia coli O157: H7 by a fluorescent microsphere—based immunochromatographic assay and immunomagnetic separation[J]. Analytical Biochemistry, 2019, 564: 32-39.

[16] QU Q, ZHU Z, WANG Y, et al. Rapid and quantitative detection of Brucella by up—converting phosphor technology—based lateral—flow assay[J]. Journal of Microbiological Methods, 2009, 79(1): 121-123.

[17] LI L P, ZHOU L, YU Y, et al. Development of up—converting phosphor technology—based lateral—flow assay for rapidly quantitative detection of hepatitis B surface antibody[J]. Diagnostic Microbiology and Infectious Disease, 2009, 63(2): 165-172.

[18] XU Y, LIU Y H, WU Y, et al. Fluorescent probe—based lateral flow assay for multiplex nucleic acid detection[J]. Analytical Chemistry, 2014, 86(12): 5611-5614.

[19] QIU W W, BARYEH K, TAKALKAR S, et al. Carbon nanotube—based lateral flow immunoassay for ultrasensitive detection of proteins: Application to the determination of IgG[J]. Mikrochimica Acta, 2019, 186(7): 436.

[20] HUANG Q Q, GAO H Q, YANG S M, et al. Ultrastable and colorful afterglow from organic luminophores in amorphous nanocomposites: Advanced anti—counterfeiting and in vivo imaging application[J]. Nano Research, 2020, 13(4): 1035-1043.

[21] CAI S Z, SHI H F, TIAN D, et al. Enhancing ultralong organic phosphorescence by effective π—type halogen bonding[J]. Advanced Functional Materials, 2018, 28(9): 1705045.

[22] BOLTON O, LEE K, KIM H J, et al. Activating efficient phosphorescence from purely organic materials by crystal design[J]. Nature Chemistry, 2011, 3(3): 205-210.

[23] WANG Y S, GAO H Q, YANG J, et al. High performance of simple organic phosphorescence host—guest materials and their application in time—resolved bioimaging[J]. Advanced Materials, 2021, 33(18): e2007811.

[24] 王伟光, 张鑫, 贺飞, 等. 激基复合物有机长余辉材料的研究进展[J]. 科学通报, 2024(26): 3849-3863.

[25] WANG W G, ZHANG X, HE F, et al. Research progress of exciplex organic long afterglow materials[J]. China Industrial Economics, 2024(26): 3849-3863.

[26] 苏艳 . 有机聚合物长余辉发光材料制备及防伪技术研究[D]. 重庆: 重庆理工大学, 2019.

[27] SU Y. Study on preparation and anti—counterfeiting technology of organic polymer long afterglow luminescent materials[D]. Chongqing: Chongqing University of Technology, 2019.

[28] ZHOU Z H, ZHENG W, KONG J T, et al. Rechargeable and LED—activated ZnGa ₂O ₄: Cr ³⁺ near—infrared persistent luminescence nanoprobes for background—free biodetection [J]. Nanoscale, 2017, 9(20): 6846-6853.

[29] ZHOU Y D, LU S, ZHI J H, et al. Microscopic afterglow bioimaging by ultralong organic phosphorescent nanoparticles in living cells and zebrafish[J]. Analytical Chemistry, 2021, 93(16): 6516-6522.

[30] PATERSON A S, RAJA B, GARVEY G, et al. Persistent luminescence strontium aluminate nanoparticles[EB/OL]. [ 2023—10—11]. https://pubs.acs.org/doi/pdf/10.1021/ac502624.

[31] HAI O, PEI M K, YANG E L, et al. Exploration of long afterglow luminescence materials work as round—the—clock photocatalysts[J]. Journal of Alloys and Compounds, 2021, 866: 158752.

[32] 王嘉森, 李进让, 吴慕坤 . 胃蛋白酶试纸条检测诊断咽喉反流性疾病的可行性分析[J]. 中华耳鼻咽喉头颈外科杂志, 2019, 54(7): 501-505.

[33] WANG J S, LI J R, WU M K. Feasibility analysis of pepsin strip test in diagnosis of laryngopharyngeal reflux disease[J]. Chinese Journal of Otorhinolaryngology Head and Neck Surgery, 2019, 54(7): 501-505.

[34] 赵水娣, 李彬, 贾宁人 . 血清胃蛋白酶原临床检测中的稳定性分析[J]. 国际检验医学杂志, 2011, 32(20): 2395-2397.

[35] ZHAO S D, LI B, JIA N R. Stability analysis of serum gastric proenzyme in clinical detection[J]. International Journal of Laboratory Medicine, 2011, 32(20): 2395-2397.

[36] 李小溪, 李筱涵, 陈澎, 等. 四种胃蛋白酶原检测试剂的性能比较[J]. 现代生物医学进展, 2017, 17(7): 1270-1273.

[37] LI X X, LI X H, CHEN P, et al. Comparative analysis of four reagents in detection of serum pepsinogen[J]. Progress in Modern Biomedicine, 2017, 17(7): 1270-1273.

[38] CAO Y, CHEN Z H, LI X J, et al. Dual—color quantum dot—loaded nanoparticles based lateral flow biosensor for the simultaneous detection of gastric cancer markers in a single test line[J]. Analytica Chimica Acta, 2022, 1218: 339998.

[39] GUO J C, LI Y, WANG B, et al. Self—propelled Janus nanomotor as active probe for detection of pepsinogen by lateral flow immunoassay[J]. Mikrochimica Acta, 2022, 189(12): 468.

[40] MENG X M, ZUO W C, WU P C, et al. Bimetallic nanozyme: A credible tag for in situ—catalyzed reporter deposition in the lateral flow immunoassay for ultrasensitive cancer diagnosis[J]. Nano Letters, 2024, 24(1): 51-60.

[41] LI K J, LI X Q, FAN Y L, et al. Simultaneous detection of gastric cancer screening biomarkers plasma pepsinogen I/II using fluorescent immunochromatographic strip coupled with a miniature analytical device[J]. Sensors and Actuators B: Chemical, 2019, 286: 272-281.

[42] HAJ—SHEYKHOLESLAMI A, RAKHSHANI N, AMIRZARGAR A, et al. Serum pepsinogen I, pepsinogen II, and gastrin 17 in relatives of gastric cancer patients: Comparative study with type and severity of gastritis[J]. Clinical Gastroenterology and Hepatology, 2008, 6(2): 174-179.

[43] FANG Z J, ZHANG H, GUO J C, et al. Overview of therapeutic drug monitoring and clinical practice[J]. Talanta, 2024, 266: 124996.