组成图示
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传感器类型
全细胞生物传感器
检测对象
三磷酸腺苷（ATP）；样品基质：角质形成细胞与HEK293共培养微环境、小鼠阴囊皮肤灌流液
检测原理
热刺激（约40–42°C）激活角质形成细胞膜上的TRPV3/TRPV4阳离子通道，引起Ca2+内流并触发细胞释放ATP到细胞外微环境。ATP扩散至邻近表达P2X2受体的HEK293生物传感器细胞，与P2X2结合后打开阳离子通道，产生内向整流电流；局部ATP浓度越高，P2X2介导电流越大。全细胞patch-clamp记录该电流，从而将ATP浓度转换为电信号。ATP也可结合DRG神经元上的P2受体，引起Ca2+升高，并用fluo-4/fura-2荧光成像读出。该体系无核酸或酶催化放大，主要依赖离子通道电流和钙指示剂信号。
检测灵敏度
原文未报告LOD、线性范围、灵敏度斜率或相关系数。
效应效果
该体系具选择性：热刺激下仅P2X2表达HEK293细胞记录到ATP诱导电流，5HT3A或NR1/NR2B表达细胞未检测到5-HT或谷氨酸释放（各n=10）。TRPV3缺陷角质形成细胞热诱导P2X2电流显著降低（0.15±0.02 pA/pF，n=7），低于野生型（0.43±0.02 pA/pF，n=4）、TRPV1缺陷（0.36±0.04 pA/pF，n=5）和TRPV4缺陷（0.39±0.07 pA/pF，n=7），p<0.05；半定量估计ATP释放约1 µM，其他组约10 µM。皮肤灌流中约42°C热刺激诱导ATP释放，ruthenium red（50 µM）在40.3–41.9°C显著抑制，p<0.05。作者认为ATP是TRPV3介导皮肤热转导信使。
传感器的构成
- 培养基底：玻璃盖玻片（cover slip）/培养皿，承载HEK293细胞与角质形成细胞共培养
- 传感器细胞：HEK293细胞，作为全细胞生物传感器，表达离子型嘌呤受体
- 识别元件：P2X2受体（P2X2 cDNA转染），结合ATP并开放阳离子通道
- 信号换能：P2X2通道介导内向整流电流，电流幅度反映局部ATP浓度
- 换能电极：全细胞patch-clamp微电极（pipette），记录膜电流
中文摘要
瞬时受体电位香草酸亚型3（TRPV3）和TRPV4是角质形成细胞中热激活的阳离子通道。皮肤中TRPV3和/或TRPV4的热激活可能释放可扩散分子，进而激活邻近背根神经节（DRG）神经元末梢。本研究证明三磷酸腺苷（ATP）是加热后从角质形成细胞释放的候选信使分子。利用TRPV1缺陷型DRG神经元，作者发现加热引起的DRG神经元胞质Ca2+升高仅在与角质形成细胞共培养时出现，且可被P2嘌呤受体拮抗剂PPADS和suramin阻断。在角质形成细胞与转染P2X2 cDNA的HEK293细胞（作为生物传感器）共培养体系中，观察到热激活角质形成细胞分泌ATP，且TRPV3缺陷小鼠角质形成细胞的ATP释放明显受损。该研究为ATP是主要由TRPV3介导的皮肤热转导信使分子提供了证据。
英文摘要
Transient receptor potential V3 (TRPV3) and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here we show that adenosine triphosphate (ATP) is such a candidate molecule released from keratinocytes upon heating in the co-culture systems. Using TRPV1-deficient DRG neurons, we found that increase in cytosolic Ca(2+)-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by P2 purinoreceptor antagonists, PPADS and suramin. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X(2) cDNA to serve as a bio-sensor), we observed that heat-activated keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3-deficient mice. This study provides evidence that ATP is a messenger molecule for mainly TRPV3-mediated thermotransduction in skin.