Battaglia Tina M, Masson Jean-Francois, Sierks Michael R, Beaudoin Stephen P, Rogers Joseph, Foster Kevin N, Holloway G Allen, Booksh Karl S
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
Anal Chem. 2005 Nov 1;77(21):7016-23. doi: 10.1021/ac050568w.
Sensing of three cytokines related to chronic wound healing, interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), with detection limits at or below 1 ng/mL in buffered saline solution and spiked cell culture medium (CCM) has been achieved. Fiber-optic surface plasmon resonance (SPR) sensors are coated with an antibody binding layer and antibodies specific to the cytokine of interest are covalently attached to this layer. To achieve such detection limits in a complex medium such as CCM, total protein content of 4 mg/mL, the use of a novel N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid (NHS-MHA) is necessary. A comparison of the detection limits for IL-6 using currently widely used CM-dextran and NHS-MHA shows an improvement by a factor of 3 using NHS-MHA. The detection limits for the monitoring of cytokines in spiked saline solutions and CCM were similar for TNF-alpha and slightly higher for IL-1 and IL-6. The detection of each cytokine in the presence of interfering agents resulted in concentration prediction well within the error of calibration. The SPR sensors are stable in CCM after 20 min of pretreatment in CCM, minimizing the reliance on a reference sensor to quantify the cytokines in complex media. This technique enables a major advancement in the field of real-time monitoring of biologically relevant molecules in complex biological fluids.
已实现对与慢性伤口愈合相关的三种细胞因子，即白细胞介素 -1（IL -1）、白细胞介素 -6（IL -6）和肿瘤坏死因子 -α（TNF -α）的传感，在缓冲盐溶液和加标细胞培养基（CCM）中的检测限达到或低于1 ng/mL。光纤表面等离子体共振（SPR）传感器涂有抗体结合层，并且将针对目标细胞因子的抗体共价连接到该层。为了在诸如CCM这种复杂培养基（总蛋白含量为4 mg/mL）中实现这样的检测限，需要使用新型的16 - 巯基十六烷酸N - 羟基琥珀酰亚胺酯（NHS - MHA）。使用当前广泛使用的CM - 葡聚糖和NHS - MHA对IL -6检测限的比较表明，使用NHS - MHA时检测限提高了3倍。在加标盐溶液和CCM中监测细胞因子时，TNF -α的检测限相似，而IL -1和IL -6的检测限略高。在存在干扰剂的情况下对每种细胞因子的检测，其浓度预测在校准误差范围内。SPR传感器在CCM中经过20分钟预处理后在CCM中稳定，从而最大限度地减少了在复杂介质中对参考传感器来定量细胞因子的依赖。这项技术在复杂生物流体中生物相关分子的实时监测领域实现了重大进展。
