Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range.
Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range.
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DOI：
10.1021/ac103161b
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发表时间：
2011-03-15
影响因子：
7.4
通讯作者：
Duffy, David C.
中科院分区：
文献类型：
--
作者：
Rissin, David M.;Fournier, David R.;Piech, Tomasz;Kan, Cheuk W.;Campbell, Todd G.;Song, Linan;Chang, Lei;Rivnak, Andrew J.;Patel, Purvish P.;Provuncher, Gail K.;Ferrell, Evan P.;Howes, Stuart C.;Pink, Brian A.;Minnehan, Kaitlin A.;Wilson, David H.;Duffy, David C.
我们报道了一种将单分子(数字)和分子集合(模拟)检测相结合的方法，该方法能够检测从10−19M到10−13M的酶标记，用于高灵敏度的酶联免疫吸附试验(ELISA)。该方法的工作原理是捕获微珠上的蛋白质，使用传统的多步免疫和夹心方法用酶标记蛋白质，在50飞升阵列(单分子阵列，SiMoA)中分离微珠，并使用荧光成像检测与微珠相关的酶活性。在低蛋白质浓度时，当酶标记与珠子的比例小于∼1.2时，珠子携带的酶数量为零或较少，蛋白质浓度通过计数“开”或“关”珠子的存在来量化(数字区域)。在较高的蛋白质浓度下，每个珠子通常带有多个酶标记，每个珠子上存在的酶标记的平均数是根据平均荧光强度的测量来量化的(模拟区域)。数字和模拟浓度范围都由一个共同的单位来量化，即每珠的平均酶标记数(AEB)。通过对单珠的数字和模拟检测相结合，实现了酶标记物6个数量级以上的线性动态范围。利用这种方法，建立了前列腺特异性抗原(PSA)的免疫分析方法。数字和模拟结合的PSA检测在大约四个对数的浓度范围内([PSA]从8fg/m l-100 pg/m l或250 am-3.3 pm)提供线性响应。这种方法在一次测量中将酶联免疫吸附试验的动态范围从微微摩尔水平扩展到亚毫微摩尔水平。
We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10−19 M to 10−13 M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multi-step immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ∼1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of “on” or “off” beads (digital regime). At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL – 100 pg/mL or 250 aM – 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to sub-femtomolar levels in a single measurement.
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影响因子：
15
作者：
Craig, DB;Arriaga, EA;Dovichi, NJ
通讯作者：
Dovichi, NJ
影响因子：
8.6
作者：
Pantano, P;Walt, DR
通讯作者：
Walt, DR
影响因子：
9.3
作者：
Todd, John;Freese, Bob;Goix, Philippe
通讯作者：
Goix, Philippe
影响因子：
64.8
作者：
XUE, QF;YEUNG, ES
通讯作者：
YEUNG, ES
影响因子：
10.8
作者：
Rissin, DM;Walt, DR
通讯作者：
Walt, DR