Expanded Multiplexing on Sensor-Constrained Microfluidic Partitioning Systems
Expanded Multiplexing on Sensor-Constrained Microfluidic Partitioning Systems
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DOI：
10.1021/acs.analchem.3c01176
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发表时间：
2023-11-16
影响因子：
7.4
通讯作者：
Drezek，Rebekah A.
中科院分区：
文献类型：
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作者：
Kota，Pavan K.;Vu，Hoang-Anh;Drezek，Rebekah A.
微流体可以将样品分成数千或数百万个分区，例如液滴或微细胞。分区根据泊松分布捕获分析物，并且在诊断中，分析物浓度通常通过最大似然估计（MLE）用封闭形式的解来推断。在这里，我们提出了一种新的可扩展的方法来复用分析物。我们通过微流体分区来概括MLE，并扩展我们之前开发的稀疏泊松恢复（SPoRe）推理算法。我们还提出了第一个在体外演示SPoRe与液滴数字PCR（ddPCR）对感染诊断。数字PCR本质上是高度敏感的，SPoRe通过规避其通道限制来帮助扩展其多路复用能力。我们广泛扩增细菌与16S ddPCR和分配条形码9个病原体属使用5个非特异性探针。鉴于我们的双通道ddPCR系统，我们在多组液滴中一次测量两个探针。虽然单个液滴的细菌含量不明确，但我们从合并的数据中恢复了样品中的细菌浓度。我们实现了稳定的定量，每个样品的16S基因的总拷贝数约为200个，从而实现了一系列的临床应用，给出了一个强大的上游微生物DNA提取程序。我们开发了一个新的理论，推广了这个框架的应用到许多现实的传感方式，我们证明了系统设计的缩放规则，以实现进一步扩展的复用。这里展示的核心原理可能会影响许多微流体分区的生物传感应用。
Microfluidics can split samples into thousands or millions of partitions, such as droplets or nanowells. Partitions capture analytes according to a Poisson distribution, and in diagnostics, the analyte concentration is commonly inferred with a closed-form solution via maximum likelihood estimation (MLE). Here, we present a new scalable approach to multiplexing analytes. We generalize MLE with microfluidic partitioning and extend our previously developed Sparse Poisson Recovery (SPoRe) inference algorithm. We also present the first in vitro demonstration of SPoRe with droplet digital PCR (ddPCR) toward infection diagnostics. Digital PCR is intrinsically highly sensitive, and SPoRe helps expand its multiplexing capacity by circumventing its channel limitations. We broadly amplify bacteria with 16S ddPCR and assign barcodes to nine pathogen genera by using five nonspecific probes. Given our two-channel ddPCR system, we measured two probes at a time in multiple groups of droplets. Although individual droplets are ambiguous in their bacterial contents, we recover the concentrations of bacteria in the sample from the pooled data. We achieve stable quantification down to approximately 200 total copies of the 16S gene per sample, enabling a suite of clinical applications given a robust upstream microbial DNA extraction procedure. We develop a new theory that generalizes the application of this framework to many realistic sensing modalities, and we prove scaling rules for system design to achieve further expanded multiplexing. The core principles demonstrated here could impact many biosensing applications with microfluidic partitioning.