Svojanovský Vilém, Máčala Jakub, Hlaváček Antonín, Čermák Aleš, Stráník Jaromír, Bouchal Pavel, Mašlaňová Ivana, Skládal Petr, Farka Zdeněk, Preisler Jan
Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno 625 00, Czech Republic.
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno 625 00, Czech Republic.
Anal Chem. 2025 Jul 8;97(26):13832-13839. doi: 10.1021/acs.analchem.5c00641. Epub 2025 Jun 24.
Single-particle (digital) immunoassays offer significantly lower limits of detection (LODs) than traditional immunoassays, making them suitable for the detection of low-abundance biomarkers. The most common approach for digital detection is based on counting individual labels. Here, we introduce a novel dot-blot particle-linked immunosorbent assay (PLISA) with digital readout utilizing laser ablation (LA) of photon upconversion nanoparticle (UCNP) labels from the nitrocellulose substrate. Compared to conventional LA, our approach allows desorption of intact nanoparticles and their precise counting by single-particle inductively coupled plasma mass spectrometry (SP ICP MS), thus counting individual UCNP-labeled immunocomplexes. Digital signal processing filters instrument noise and nanoparticle aggregates, minimizing potential errors. The immunoassay and LA SP ICP MS readout were optimized using human serum albumin, a kidney damage biomarker, as a model analyte, obtaining LODs of 0.18 and 0.12 ng/mL for the reference upconversion luminescence (UCL) and LA SP ICP MS readout, respectively. Building upon these optimized conditions, we developed PLISA for prostate-specific antigen, the key prostate cancer biomarker, with LODs of 2.4, 1.4, and 0.3 pg/mL for the UCL, analog, and digital LA SP ICP MS readout, respectively. The LOD in the sub-pg/mL range highlighted the advantage of particle counting and its ability to detect low-abundance biomarkers, as superior performance was achieved compared to the UCL and analog LA ICP MS readout. Finally, clinical serum samples of patients tested for prostate cancer were analyzed, and a strong correlation with the reference electrochemiluminescence method confirmed the potential of LA SP ICP MS for clinical diagnostics.
单颗粒（数字）免疫分析的检测限（LOD）比传统免疫分析低得多，使其适用于检测低丰度生物标志物。数字检测最常见的方法是基于对单个标记物进行计数。在此，我们介绍一种新型的斑点印迹颗粒连接免疫吸附测定法（PLISA），它利用从硝酸纤维素底物上激光烧蚀光子上转换纳米颗粒（UCNP）标记物进行数字读出。与传统激光烧蚀相比，我们的方法能够使完整的纳米颗粒解吸，并通过单颗粒电感耦合等离子体质谱（SP ICP MS）对其进行精确计数，从而对单个UCNP标记的免疫复合物进行计数。数字信号处理可过滤仪器噪声和纳米颗粒聚集体，将潜在误差降至最低。使用肾脏损伤生物标志物人血清白蛋白作为模型分析物，对免疫分析和激光烧蚀SP ICP MS读出进行了优化，参考上转换发光（UCL）和激光烧蚀SP ICP MS读出的检测限分别为0.18和0.12 ng/mL。基于这些优化条件，我们开发了用于前列腺特异性抗原（前列腺癌关键生物标志物）的PLISA，UCL、模拟和数字激光烧蚀SP ICP MS读出的检测限分别为2.4、1.4和0.3 pg/mL。亚皮克/毫升范围内的检测限突出了颗粒计数的优势及其检测低丰度生物标志物的能力，因为与UCL和模拟激光烧蚀ICP MS读出相比，实现了更优异的性能。最后，对前列腺癌患者的临床血清样本进行了分析，与参考电化学发光方法的强相关性证实了激光烧蚀SP ICP MS在临床诊断中的潜力。
