Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Tomtebodavägen 23B, 171 65 Solna, Sweden.
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského Sq. 2, 162 06 Prague 6, Czech Republic.
Mater Sci Eng C Mater Biol Appl. 2016 Apr 1;61:362-7. doi: 10.1016/j.msec.2015.12.061. Epub 2015 Dec 30.
Molecular diagnostics may provide tailored and cost efficient treatment for infectious disease and cancer. Rolling circle amplification (RCA) of padlock probes guarantees high specificity to identify nucleic acid targets down to single nucleotide resolution in a multiplex fashion. This makes the assay suitable for molecular analysis of various diseases, and interesting to integrate into automated devices for point-of-care analysis. A critical prerequisite for many molecular assays is (i) target-specific isolation from complex clinical samples and (ii) removal of reagents, inhibitors and contaminants between reaction steps. Efficient solid supports are therefore essential to enable multi-step, multi-analyte protocols. Superparamagnetic micro- and nanoparticles, with large surface area and rapid liquid-phase kinetics, are attractive for multi-step protocols. Recently, streptavidin-modified magnetic monodispersed poly(2-hydroxyethyl methacrylate) (STV-mag.PHEMA) microspheres were developed by multiple swelling polymerization. They are easily separated by a magnet and exhibit low non-specific protein sorption. In this study, the performance and the binding efficiency of STV-mag.PHEMA was addressed by circle-to-circle amplification (C2CA). A lower number of RCA products were detected as compared to the gold standard Dynabeads. Nevertheless, this study was the first to successfully adapt STV-mag.PHEMA microspheres as solid support in a DNA-based protocol, which is an important finding. The STV-mag.PHEMA microspheres were larger with about 16 times less surface area as compared to the Dynabeads, which might partly explain the lower rolling circle product (RCP) count obtained. Further research is currently ongoing comparing particles of similar sizes and optimizing reaction conditions to establish their full utility in the field. Ultimately, low cost and versatile particles are a great resource to facilitate future clinical molecular diagnostics.
分子诊断可以为传染病和癌症提供定制和具有成本效益的治疗方法。发夹探针的滚环扩增（RCA）保证了高特异性，可在多重方式下以单核苷酸分辨率识别核酸靶标。这使得该检测方法适用于各种疾病的分子分析，并且很有趣的是可以将其集成到用于即时检测的自动化设备中。许多分子检测的关键前提条件是（i）从复杂的临床样本中特异性分离靶标，（ii）在反应步骤之间去除试剂、抑制剂和污染物。因此，高效的固相载体对于实现多步、多分析物方案至关重要。具有大表面积和快速液相动力学的超顺磁性微球和纳米颗粒对于多步方案很有吸引力。最近，通过多次溶胀聚合开发了链霉亲和素修饰的磁性单分散聚（2-羟乙基甲基丙烯酸酯）（STV-mag.PHEMA）微球。它们可以通过磁铁轻松分离，并表现出低非特异性蛋白质吸附。在这项研究中，通过圈对圈扩增（C2CA）研究了 STV-mag.PHEMA 的性能和结合效率。与金标准 Dynabeads 相比，检测到的 RCA 产物数量较少。尽管如此，这项研究首次成功地将 STV-mag.PHEMA 微球作为基于 DNA 的方案中的固相载体进行了适配，这是一个重要的发现。STV-mag.PHEMA 微球比 Dynabeads 大 16 倍，表面积小，这可能部分解释了所得滚环产物（RCP）计数较低的原因。目前正在进行进一步的研究，比较类似大小的颗粒并优化反应条件，以确立它们在该领域的全部用途。最终，低成本和多功能的颗粒是促进未来临床分子诊断的宝贵资源。
