组成图示
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传感器类型
比色生物传感器
检测对象
mRNA（poly(A) mRNA）；样品基质：拟南芥（Arabidopsis thaliana）幼苗总RNA提取液、标准poly(A) RNA逆转录cDNA反应液
检测原理
该方法以poly(A) mRNA为检测对象。总RNA经oligo(dT)20引物逆转录，并在dNTPs中掺入DIG-11-dUTP，生成带DIG标记的第一链cDNA。cDNA与芯片上共价固定的oligo(dA)20探针杂交，洗涤去除未结合及错配分子后，anti-DIG-AP抗体结合DIG标记位点。碱性磷酸酶（AP）催化NBT/BCIP底物生成不溶性有色沉淀。cDNA和沉淀的质量沉积在氮化硅（Si3N4）光学薄膜表面，改变薄膜反射光波长，使颜色由紫红向蓝色变化。颜色深浅与cDNA沉积量正相关，从而反映原始mRNA量，并通过标准色板进行肉眼或显微镜比色定量。
检测灵敏度
定量范围: 1 ng–200 ng/100 μL；>200 ng/100 μL 颜色不再变化
效应效果
该方法对poly(A) mRNA具有特异性，oligo(dA)20探针只识别带poly(A)尾的mRNA逆转录产物，洗涤可去除错配DNA/DNA杂交体，降低非特异信号。实验显示可在100 μL反应液中检测并定量1–200 ng mRNA，超过200 ng时颜色不再增加，可通过稀释扩展上限。拟南芥幼苗总RNA样品中约8 ng和40 ng mRNA的检测颜色与标准色板一致，整个检测约30 min完成，可用肉眼或简单解剖显微镜判读。作者认为该方法快速、可行、省时，适用于多种生物mRNA水平检测，但未报告RSD、回收率及与ELISA/qPCR等方法的定量对比。
传感器的构成
- 基底/光学层：硅芯片表面沉积475 Å氮化硅（Si3N4）光学薄膜，作为光学换能层，表面质量沉积改变反射光波长
- 功能化层：涂覆多肽poly(Phe-Lys)，提供肼基（hydrazine）用于共价连接捕获探针
- 识别元件：5'端醛基修饰的oligo(dA)20捕获探针，与芯片肼基反应固定，特异性杂交poly(A)尾cDNA
- 信号标记物：DIG-11-dUTP掺入第一链cDNA，以及anti-DIG-AP（抗地高辛-碱性磷酸酶）偶联物识别DIG
- 显色底物：NBT/BCIP在AP催化下生成紫红至蓝色不溶性沉淀
- 封闭/杂交缓冲：5×SSC含5 mg/mL酸处理酪蛋白（ATC），用于杂交并降低非特异结合
中文摘要
mRNA定量是分子生物学研究的重要内容。传统分光光度法无法区分mRNA与DNA、rRNA和tRNA，Northern blot虽可用于mRNA半定量但耗时且存在放射性风险。为快速检测mRNA水平，作者开发了一种基于光学薄膜生物传感器芯片的mRNA定量方法。提取总RNA后，利用oligo(dT)20引物和掺有DIG-11-dUTP的dNTPs将带poly(A)尾的mRNA逆转录为第一链cDNA。该cDNA与点样在光学薄膜生物传感器芯片上的oligo(dA)20探针杂交，经洗涤去除过量cDNA、单链RNA及错配DNA/DNA杂交体后，用抗DIG-碱性磷酸酶（anti-DIG-AP）偶联物识别完全匹配的杂交体，再加入NBT/BCIP底物显色。芯片表面颜色由紫红色至蓝色随cDNA沉积量变化。拟南芥样品检测表明该方法可用于mRNA定量，并具有推广至多种生物mRNA定量应用的潜力。
英文摘要
mRNA quantification is very important in molecular biological researches. Traditional spectrophotometric method cannot distinguish DNA, rRNA and tRNA species from mRNA. Northern blot can be used for mRNA quantification but is known to be time consuming. To rapidly detect mRNA levels, we developed an optical thin-film biosensor chip based method, to quantify mRNA in samples. After total RNA was extracted, the mRNA with poly(A) tails was reverse transcribed with oligo(dT)(20) primers and dNTPs mixed with digoxigenin(DIG)-11-dUTP. The transcribed first strand cDNA was hybridized with oligo(dA)(20) nucleotide probes spotted on optical thin-film biosensor chips. Excess first strand cDNA, single-strand RNA, and mis-matched DNA/DNA hybrids were removed by washing. The perfect-matched DNA/DNA hybrid was detected with anti-DIG-AP (alkaline phosphatase) conjugate and then incubated with NBT/BCIP substrate for color development. The range of the color is from purplish red to blue, according to the cDNA mass deposited on chip surface. Detection of mRNA levels from Arabidopsis samples proved that this method is feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.