Fluolab【Angew.Chem.】四川大学李峰、廖红艳、宋涂润联合湖南大学邢航|仅需1小时！这种超稳“防弹”纳米传感器能从全血中直接揪出白血病基因
通讯作者： Turun Song, Hang Xing, Hongyan Liao, Feng Li
文章概要
引言
基因融合作为血液肿瘤和实体瘤的关键致病驱动因素及生物标志物，其在不同细胞克隆中的空间分布对于理解肿瘤进化、谱系可塑性及制定精准治疗方案至关重要。例如在急性淋巴细胞白血病（ALL） 中，若在髓系细胞中检测到BCR-ABL1融合转录本，则预示着这属于治疗策略截然不同的“多谱系ALL”。然而，现有的PCR或测序技术往往会丢失细胞谱系信息，而FISH技术则操作冗长且需要复杂的细胞固定与分选。虽然基于金纳米颗粒的球形核酸（SNA） 传感器具有进入活细胞检测mRNA的潜力，但传统SNA在面对血液中高浓度的生物硫醇时极易发生非特异性信号泄露，且其在复杂全血环境中的细胞选择性尚不明确，这严重限制了其临床转化。
Overall design of CRUSH. (a) Schematic illustration of CRUSH for analyzing gene fusion transcripts through toehold-mediated strand displacement on AuNPs. Both strands of the duplex probe were labeled with distinct fluorescent dyes to monitor both target-specific activation and nonspecific signal leakage of CRUSH. (b) CRUSH is highly resistant to high concentrations of biothiols in whole blood and living cells by forming perfect self-assembled monolayers of thiols on AuNP surfaces. (c) Schematic illustration of the cell selectivity of CRUSH in whole blood. (d) The workflow for one-step identification of multilineage ALL using CRUSH. This figure contains elements created using BioRender.com with permission.
主要实验及结论
为了攻克稳定性难题，研究团队开发了一种名为CRUSH的细胞解析超稳传感平台。研究人员通过密度泛函理论（DFT）计算和分子动力学模拟发现，传统SNA稳定性差的根源在于其表面硫醇密度过低，存在大量“缺陷位点”。为此，他们创新性地引入了化学计量控制的硫醇保护剂（MCH），与硫醇化DNA探针共同组装，在金纳米颗粒表面形成了无缺陷的自组装单层（SAM）。这种结构上的精密改进赋予了CRUSH前所未有的化学稳定性，其耐受二硫苏糖醇（DTT）的能力较传统SNA提升了1万倍，最高可抵御0.1 M DTT的冲击，这被认为是目前SNA能达到的最高稳定性记录。
Enhancing chemical stability of CRUSH via thiol protection. (a) Schematic illustration of conventional SNA nanoprobe with no thiol protection, where a parse layer of thiols (180 per AuNP) was formed on AuNP surface due to the steric hindrance and charge repulsion of thiolate DNA probes. (b) CRUSH prepared via the co-assembly of thiolate DNA probes and small molecular thiol protectors which creates defect-free thiol SAMs on AuNP (5,400 per AuNP). (c) DFT calculation of reaction energy for displacing a well-separated MCH from the (111) surface of a 16-Au-atom cluster by DTT. (d) DFT calculation for displacing one of two closely neighboring MCHs, which reveals a higher energy cost induced by a higher concentration of MCH. (e) The number of DNA remaining and the percentage of released DNA on AuNPs after treating CRUSH with DTT. CRUSH were prepared by adding MCH as thiol protectors at varying stoichiometric ratios, where δExperimental/δTheoretical of 1 represents 5,400 MCH molecules per 20 nm AuNP. (f) Experimentally determined the densities of MCH plotted against theoretical densities for CRUSH prepared using 13 nm, 20 nm, 30 nm, and 40 nm AuNPs. (g) Percentage released DNA upon DTT displacement for CRUSH prepared using thiol protectors with varying chain lengths from 5 to 8. h. Maximal concentrations of DTT withstood by CRUSH prepared by thiol protection combined with DNA anchors with varying valences. Each error bar represents one standard deviation from triplicate analyses.
Evaluating chemical stability of CRUSH in live cells and whole blood. (a) schematic illustration of the estimation of signal leakage for CRUSH and conventional SNA nanoprobe in live cells. (b) Flow cytometric analysis of MCF-7 cells treated with CRUSH or conventional nanoprobe. The fluorescence channel was set to monitor the nonspecific signal leakage of CRUSH or conventional nanoprobe. (c) Fold change of signal leakage of CRUSH or conventional nanoprobe against the background. (d) Schematic illustration of CRUSH in whole blood samples collected from 10 healthy participates. (e) Flow cytometric analysis of blood cells after treating whole blood with CRUSH or conventional nanoprobe. The fluorescence channel was set to monitor the nonspecific signal leakage of CRUSH or conventional nanoprobe. (f) Statistical analysis of nonspecific signal leakage of CRUSH or conventional nanoprobe in blood samples (n = 10). Unpaired two-tailed t-tests were used to evaluate statistical differences between samples. N.S.: not significant (p > 0.05); ***p < 0.001; ****p < 0.0001. Each error bar represents one standard deviation from triplicate analyses. This figure contains elements created using BioRender.com with permission.
在复杂生物环境的实测中，CRUSH展现了卓越的抗干扰能力。在具有高水平生物硫醇的MCF-7细胞以及10名健康志愿者的全血样本测试中，CRUSH均保持了极低的背景信号，而传统SNA则出现了严重的假阳性泄露。更有趣的是，研究团队揭示了纳米探针在全血中的运动规律：由于髓系细胞（如单核细胞） 比淋巴系细胞具有更强的吞噬偏好，CRUSH能特异性地被髓系细胞摄取。通过高分辨率流式细胞术和ICP-MS分析证实，超过90% 的有荧光信号细胞均为髓系细胞，这种天然的髓系选择性为直接在全血中区分白血病亚型提供了可能。
Cell selectivity of CRUSH in whole blood. (a) Schematic illustration of the CRUSH design for determining the cell selectivity in whole blood, where a house keeping RPL13a mRNA was chosen as a specific intracellular target to induce target-specific activation of CRUSH. (b) Flow cytometric analysis of THP1 cells as a representative myeloid cell line treated with CRUSH at varying time points. (c) Flow cytometric analysis of Jurkat cells as a representative lymphoid cell line treated with CRUSH at varying time points. (d) Kinetic curves showing target-specific activation of CRUSH in both THP1 and Jurkat cells at varying time points. Each error bar represents one standard deviation from triplicate analyses. (e) Schematic illustration of assay design for determining cell selectivity of CRUSH in whole blood using both flow cytometry and ICP-MS analyses. (f) Percentage cell uptake determined in platelet, myeloid, and lymphoid cells in blood samples collected from healthy participants (n = 14). g. Number of AuNPs per cell determined in myeloid and lymphoid cells in blood samples collected from healthy participants (n = 6) treated with CRUSH. Unpaired two-tailed t-tests were used to evaluate statistical differences between samples. ****p < 0.0001. Each error bar represents one standard deviation from triplicate analyses. This figure contains elements created using BioRender.com with permission.
最后，研究团队将CRUSH制备成性质稳定的冻干粉剂，可在室温下保存2年以上。在针对7名白血病患者的临床样本验证中，该系统实现了“加样即读”的简便流程。CRUSH不仅成功在CML患者样本中检测到融合基因，更关键的是，它在短短1小时内就精准识别出了3例具有多谱系特征的ALL病例（表现为髓系细胞中BCR-ABL1阳性）。这些病例随后被证实携带IKZF1缺失，进一步验证了CRUSH在复杂临床样本诊断中的高度准确性和鲁棒性。
One-step identification of multilineage ALL in unprocessed whole blood samples using CRUSH. (a) Schematic illustration of CRUSH design for the detection of BCR-ABL1 fusions in clinical settings. (b) Schematic illustration of gene fusion distribution in typical ALL and multilineage ALL. (c) To enable one-step clinical test and long-term storage, CRUSH was prepared as lyophilized powders that demonstrated no significant nonspecific release or loss of function during 2-year storage at room temperature. Each error bar represents one standard deviation from triplicate analyses. (d) Workflow for the one-step identification of multilineage ALL in whole blood samples using CRUSH and flow cytometry analysis. (e.-j) Flow cytometry analysis of clinical whole blood samples collected from ALL patients, the positive test result (p210) for the CML case (e), negative test results for the two BCR-ABL1 negative ALL cases (f), lymphoid only ALL with no detectable BCR-ABL1 fusion in myeloid cells (g) and three multilineage cases with one p210 positive (h) and two p190 positives (i, j). This figure contains elements created using BioRender.com with permission.
总结及展望
这项研究通过解决金-硫界面SAM结构的完整性问题，打破了SNA技术在临床全血检测中的应用瓶颈。CRUSH平台不仅在化学稳定性上实现了数量级的跨越，更巧妙地利用细胞内源性的吞噬差异实现了无需分选的谱系特异性检测。这种无需扩增、快速便捷的检测模式，能够完美适配现有的临床流式细胞分析流程，为血液肿瘤的精准分型、疗效监测以及个性化治疗提供了强有力的技术支撑，也为未来开发更多基于纳米技术的精密诊断工具开辟了新路径。