基於核酸適體探針之心肌鈣蛋白與D-二聚體免疫檢測探究

Abstract

研究探討了心肌梗塞和血栓形成的兩個關鍵生物標誌物：心肌肌鈣蛋白 I (cTnI) 和D-二聚體的診斷能力。研究重點在於使用胎牛血清模擬真實樣本中cTnI的檢測效能，以及對D-二聚體進行基於適配體的感測開發，後者尚未被充分探索。首先，表面電漿共振（SPR）評估了cTnI單株抗體與Tro4適體之間的結合位點重疊情況，確認了非干擾的結合位點，支持了使用抗體-適體三明治法的可行性。開發的抗體-適體三明治示出了對cTnI的劑量依賴效應，並在SPR中略微提高了3.27%的靈敏度，儘管檢測限度（LOD）也有所提高。為了評估真實樣本的影響，使用了間接酶聯免疫吸附試驗（ELISA）和間接酶聯寡核苷酸吸附試驗（ELONA）進行檢測，儘管存在20% 的胎牛血清干擾，抗體-適體三明治ELISA顯示出強大的靈敏度和14.2 nM的檢測限度。在間接ELONA中使用的通用polyA-生物素手柄達到了與傳統方法可比的結果，靈敏度為0.0254，LOD為20.0 nM。 對於D-二聚體，研究採用了基於單珠玻璃珠的系統性配位子指數增益演繹技術（single-bead SELEX），並使用人血清白蛋白（HSA）進行反向篩選以增強選擇性。選定的SB3R3和HB3R4 DNA序列池進行了進一步分析。SPR序列和親和力測試顯示，SB3R3-9和HB3R4-5具有劑量依賴的結合，其解離常數（KD）分別為78 nM和408 nM，但其結合反應弱且低於理論水平，使它們不適合用於開發有效的D-二聚體適體傳感器。而通過ELONA進行的SB3R3-9和HB3R4-5的親和力評估顯示，HB3R4-5對D-二聚體顯示出更好的劑量反應性和選擇性，並有效區分了包括空白組、HSA和凝血酶在內的對照組。相反，SB3R3-9既無劑量依賴性也無選擇性。此外，HB3R4-5的結合信號是SB3R3-9的兩倍，這與之前基於SPR的親和力結果相矛盾，這種差異突顯了需要進一步研究以闡明潛在原因的必要性。

This study explores the diagnostic capabilities of cardiac troponin I (cTnI) and D-dimer, essential biomarkers for myocardial infarction and thrombosis, respectively. The research focuses on the efficacy of cTnI detection in simulated real samples using fetal bovine serum and the development of aptamer-based sensing for D-dimer, which remains underexplored. Initially, surface plasmon resonance (SPR) assessed the overlap of binding sites between cTnI monoclonal antibody and Tro4 aptamer, confirming non-interfering binding sites and supporting the feasibility of a sandwich detection method. The developed antibody-aptamer sandwich demonstrated a dose-dependent effect on cTnI with a slight sensitivity increase of 3.27% in SPR, although the limit of detection (LOD) also increased. To evaluate the impact of real samples, indirect enzyme-linked immunosorbent assay (ELISA) and indirect enzyme-linked oligonucleotide adsorption assay (ELONA) were used, with the antibody-aptamer sandwich ELISA showing robust sensitivity and a detection limit of 14.2 nM, despite 20% fetal bovine serum interference. A universal polyA-biotin handle in indirect ELONA achieved comparable results to conventional methods, with a sensitivity of 0.0254 and LOD of 20.0 nM. For D-dimer, the study applied single-bead SELEX with reverse screening using human serum albumin (HSA) to enhance selectivity. The selected SB3R3 and HB3R4 DNA sequence pools underwent further analysis. SPR sequencing and affinity tests revealed that SB3R3-9 and HB3R4-5 had dose-dependent binding with KDs of 78 nM and 408 nM, respectively, but their binding responses were weak and below theoretical levels, making them unsuitable for effective D-dimer aptamer sensors. Furthermore, affinity assessments of SB3R3-9 and HB3R4-5 through ELONA reveal that HB3R4-5 displays superior dose responsiveness and selectivity towards D-dimer, and differentiates effectively from controls including blank, HSA, and thrombin. Conversely, SB3R3-9 shows neither dose dependency nor selectivity. Moreover, HB3R4-5's binding signals are notably double those of SB3R3-9, presenting a conflict with previous SPR-based affinity results. This discrepancy highlights the need for additional research to elucidate the underlying causes.