Department of Chemistry and Biochemistry, Auburn University, Auburn, AL, USA.
Department of Anatomy, Physiology, and Pharmacology, Auburn University, Auburn, AL, USA.
Lab Chip. 2024 Oct 22;24(21):5020-5031. doi: 10.1039/d4lc00664j.
Pathologies in adipose (fat) tissue function are linked with human diseases such as diabetes, obesity, metabolic syndrome, and cancer. Dynamic, rapid release of metabolites has been observed in adipocyte cells and tissue, yet higher temporal resolution is needed to adequately study this process. In this work, a microfluidic device with precise and regular valve-automated droplet sampling, termed a microfluidic analog-to-digital converter (μADC), was used to sample secretions from ∼0.75 mm diameter adipose explants from mice, and on-chip salt water electrodes were used to merge sampled droplets with reagent droplets from two different fluorometric coupled enzyme assays. By integrating sampling and assays on-chip, either glycerol or non-esterified fatty acids (NEFA), or both, were quantified optically within merged 12 nanoliter droplets using a fluorescence microscope with as high as 20 second temporal resolution. Limits of detection were 6 μM for glycerol (70 fmol) and 0.9 μM for NEFA (10 fmol). Multiple adipose tissue explants were analyzed with this system, all showing clear increases in lipolytic function after switching from feeding to fasting conditions. Enabled by high temporal resolution, lipolytic oscillations of both glycerol and NEFA were observed for the first time in the range of 0.2 to 1.6 min. Continuous wavelet transform (CWT) spectrograms and burst analyses (0.1 to 4.0 pmol bursts) revealed complex dynamics, with multiplexed assays (duplex for glycerol and NEFA) from the same explants showing mostly discordant bursts. These data support separate mechanisms of NEFA and glycerol release, although the connection to intracellular metabolic oscillations remains unknown. Overall, this device allowed automated and highly precise temporal sampling of tissue explants at high resolution and programmable downstream merging with multiple assay reagents, revealing unique biological information. Such device features should be applicable to various other tissue or spheroid types and to other assay formats.
脂肪组织功能的病变与人类疾病有关，如糖尿病、肥胖症、代谢综合征和癌症。在脂肪细胞和组织中观察到代谢物的动态、快速释放，但需要更高的时间分辨率来充分研究这个过程。在这项工作中，使用了一种具有精确和规则的阀自动液滴采样功能的微流控装置，称为微流控模拟-数字转换器（μADC），从小鼠约 0.75 毫米直径的脂肪外植体中采样分泌物，并在芯片上使用盐水电极将采样液滴与来自两种不同荧光偶联酶测定的试剂液滴合并。通过在芯片上集成采样和测定，使用荧光显微镜以高达 20 秒的时间分辨率，在合并的 12 纳升液滴中光学定量甘油或非酯化脂肪酸（NEFA）或两者。甘油的检测限为 6 μM（70 fmol），NEFA 的检测限为 0.9 μM（10 fmol）。该系统分析了多个脂肪组织外植体，所有外植体在从喂食切换到禁食条件后，脂解功能均明显增加。高时间分辨率使我们首次观察到甘油和 NEFA 的脂解振荡，范围在 0.2 到 1.6 分钟之间。连续小波变换（CWT）频谱和爆发分析（0.1 到 4.0 pmol 爆发）显示出复杂的动力学，来自同一外植体的多重测定（甘油和 NEFA 的双重测定）显示出大多不一致的爆发。这些数据支持 NEFA 和甘油释放的分离机制，尽管与细胞内代谢振荡的联系仍不清楚。总的来说，该装置允许以高分辨率自动且高度精确地对组织外植体进行时间采样，并可与多种测定试剂进行可编程的下游合并，从而揭示独特的生物学信息。这种装置功能应该适用于各种其他组织或球体类型以及其他测定格式。
