NSP-SA-NHS is a chemiluminescent acridine ester compound with high stability, activity and sensitivity [1]. In alkaline H2O2 solution, the acridine ester is attacked by hydrogen peroxide ions, generating a tense and unstable diethylene oxide, which further decomposes into CO2 and electronically excited-state acridine ketone. When the acridine ketone returns to the ground state, it emits photons with a maximum absorption wavelength of 430nm [2]. NSP-SA-NHS can be used in chemiluminescence and immunoanalysis, receptor analysis, nucleic acid and peptide detection and other studies [3].
Product characteristics:
1. Before the formation of the electronically excited-state intermediate, the non-luminescent substituent part connected to the acridine ring separates from the luminescent part, so the luminescence efficiency is basically not affected by the substituent structure.
2. No catalyst or enhancer is required. It can emit light in a dilute alkaline solution with H2O2.
3. Its luminescence type is flash type. The luminescence intensity reaches the maximum in about 0.4s after adding the excitation solution, and the half-life is about 0.9s.
Product stability:
1. It is stable in acidic solutions (pH < 4.8). When coupled with protein conjugates and stored at room temperature for 4 weeks, the light quantum yield does not decrease. The freeze-dried product can be stored for more than one year at -20℃.
2. When pH > 4.8, especially in alkaline solutions, the acridine compound has reduced stability due to partial hydrolysis. The hydrolysis process is an inactive dark reaction process. The degree of hydrolysis increases with pH increase and temperature increase.
3. The stability of acridine amide is higher than that of acridine ester structure, and has a stronger anti-hydrolysis ability.
4. When there are electronegative groups such as methyl groups attached to the acridine ring, phenol ring or benzenesulfonamide ring, due to the large steric hindrance, the thermal stability increases; when there are electron-withdrawing groups, it is conducive to nucleophilic substitution reactions and its stability decreases.
5. If there is a decrease in luminescence after protein labeling, it is recommended:
The buffer solution used for storage after coupling should be as weakly acidic as possible. Blow nitrogen to remove the oxygen in it. Seal and store in a dark place at low temperature. If conditions permit, it can be made into freeze-dried for storage.
References:
[1] Yang F, Xu L, Liu X, et al. Chemiluminescence immunoassay approach to quantify Bisphenol S in canned beverage using a NSP-SA-labeled specific monoclonal antibody[J]. European Food Research and Technology, 2020, 246(9): 1857-1865.
[2] Zhang L, Wang D, Dai Y, et al. Machine Learning Reveals a Multipredictor Nomogram for Diagnosing the Alzheimer’s Disease Based on Chemiluminescence Immunoassay for Total Tau in Plasma[J]. Frontiers in Aging Neuroscience, 2022, 14: 863673.
[3] Du D, Wang J, Guo M, et al. Charge-dependent signal changes for label-free electrochemiluminescence immunoassays[J]. Analytical Chemistry, 2022, 94(47): 16436-16442.
NSP-SA-NHS是一种化学发光的吖啶酯化合物，具有较高的稳定性、活性和敏感性 [1]。在碱性H2O2溶液中，吖啶酯受到过氧化氢离子的进攻，生成一个有张力的不稳定的二氧乙烷，进一步分解成CO2和电子激发态的吖啶酮，当吖啶酮回到基态时发出最大吸收波长为430nm的光子 [2]。NSP-SA-NHS可用于化学发光及免疫分析、受体分析、核酸及多肽检测等研究 [3]。
产物特点:
1.发光反应在形成电子激发态中间体之前，联结于吖啶环上的不发光取代部分与发光部分分离，因而其发光效率基本不受取代基结构的影响。
2.不需催化剂，也不需要增强剂，在有H2O2的稀碱性溶液中即可发光。
3.其发光类型为闪光型，加入激发液后，0.4s左右发光强度达最大，半衰期0.9s左右。
产品稳定性：
1.在酸性溶液中（pH<4.8）都很稳定，与蛋白质偶联物在室温下保存4周，其光量子产率不降低，冻干品在-20℃下，可以保存一年以上。
2.当pH>4.8，尤其在碱性溶液中，吖啶化合物由于部分发生水解而稳定性降低，水解过程为不发光的暗反应过程；水解程度随pH增大而增大，随温度的升高而增大。
3.吖啶酰胺稳定性较高于吖啶酯结构，抗水解能力较强。
4.吖啶环或酚环或苯磺酰环上连有甲基等给电基团，由于空间位阻大，热稳定性增加；连有吸电子基团，有利于亲核取代反应而使其稳定性下降。
5.如出现蛋白标记后，过段时间发光量有所下降的情况，建议：
偶联后保存用的缓冲液尽量用弱酸性的缓冲液，鼓氮气除去其中的氧气。密封避光低温保存，有条件的可以制成冻干再保存。