Mapping enzyme catalysis with metabolic biosensing

Xu, Linfeng; Chang, Kai‐Chun; Payne, Emory M.; Modavi, Cyrus; Liu, Leqian; Palmer, Claire M.; Tao, Nannan; Alper, Hal S.; Kennedy, Robert T.; Cornett, Dale S.; Abate, Adam R.

doi:10.1038/s41467-021-27185-9

Cited by 22 publications

(16 citation statements)

References 31 publications

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“…Notably, this crosstalk occurred with only 5.5 min of incubation time, which is substantially less than the hours or days of incubation time used in many high-throughput screens. 6,47 Analysis of the acetylpyridine crosstalk measurement shows an equal increase in signal for average blank droplet as a decrease in average analyte-containing droplet, demonstrating conservation of analyte between the analyte and blank droplets (Figure S1). The repeatability of this measurement was evaluated and is summarized in Table S1.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The crosstalk coefficient for the acetylpyridine test was 0.364 ± 0.009 ( n = 369 total droplets, details in Figure S1), or ∼75% carryover. Notably, this crosstalk occurred with only 5.5 min of incubation time, which is substantially less than the hours or days of incubation time used in many high-throughput screens. , …”

Section: Resultsmentioning

confidence: 99%

“…Droplet microfluidics has emerged as a promising tool for high-throughput experimentation (HTE) and screening. Droplets and segmented flow have been used successfully for screening enzyme variants, − rare genetic materials, − and reaction conditions , with higher reagent and time efficiency compared to traditional screening methods. Similarly, droplets have been applied to achieve high temporal analysis, for example, understanding the reaction kinetics − and in vivo sensing (e.g., neurotransmitters from microdialysis sampling). − Regardless of application, it is critical that droplets remain chemically distinct to achieve reliable results; however, chemical transport or crosstalk between droplets has been observed under some conditions. , Small molecule crosstalk has been identified as limiting the technology from broader use in industrial applications .…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of Analyte Transfer between Microfluidic Droplets by Mass Spectrometry

et al. 2023

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Droplet microfluidics enables high-throughput experimentation and screening by encapsulating chemical and biochemical samples in aqueous droplets segmented by an immiscible fluid. In such experiments, it is critical that each droplet remains chemically distinct. A common approach is to use fluorinated oils with surfactants to stabilize droplets. However, some small molecules have been observed to transport between droplets under these conditions. Attempts to study and mitigate this effect have relied on evaluating crosstalk using fluorescent molecules, which inherently limits the analyte scope and conclusions drawn about the mechanism of the effect. In this work, transport of low molecular weight compounds between droplets was investigated using electrospray ionization mass spectrometry (ESI-MS) for measurement. The use of ESI-MS significantly expands the scope of analytes that can be tested. We tested 36 structurally diverse analytes that were found to exhibit crosstalk ranging from negligible to complete transfer using HFE 7500 as the carrier fluid and 008-fluorosurfactant as a surfactant. Using this data set, we developed a predictive tool showing that high log P and log D values correlate with high crosstalk, and high polar surface area and log S correlate with low crosstalk. We then investigated several carrier fluids, surfactants, and flow conditions. It was discovered that transport is strongly dependent on all of these factors and that experimental design and surfactant tailoring can reduce carryover. We present evidence for mixed crosstalk mechanisms including both micellar and oil partitioning transfer. By understanding the driving mechanisms, surfactant and oil compositions can be designed to better reduce chemical transport for screening workflows.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of Analyte Transfer between Microfluidic Droplets by Mass Spectrometry

et al. 2023

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“…These screens can be used to optimize pathways or enzymes to enhance production of target analytes, or detect novel products through metabolic biosensing. [ 47 , 48 ] In addition, as we have shown, they provide a gravity well by which to bring cells into contact to ensure interaction, which is important for functional screens of B and T cells that comprise cell therapies. When combined with printed droplet microfluidics, every well can be loaded with an exact number of different cell types, allowing tens of thousands of interaction studies per square centimeter of the slide.…”

Section: Discussionmentioning

confidence: 99%

Microbowls with Controlled Concavity for Accurate Microscale Mass Spectrometry

et al. 2022

Advanced Materials

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The properties of the substrates, including their chemistry, conductivity, and micropatterning impact sample ionization efficiency and, thus, measurement sensitivity. [8][9][10][11] For example, micrometer-scale wells are useful for segregating samples of distinct compositions so they can be separately analyzed. [12][13][14] Well arrays are also compatible with active [15,16] or passive loading techniques, [12,17] to simplify the preparation of samples for analysis. However, MALDI-MS requires samples to be dried prior to analysis. When droplets are dried on flat surfaces, they tend to distribute their analytes about the perimeter due to the coffee ring effect. [18,19] Similar processes occur in cylindrical wells, leading to precipitation along the periphery [20,21] where the signal is inhibited due to laser occlusion by the walls. The result in both cases is lowered sensitivity and increased measurement variability due to inhomogeneity of the sample spots. [18,22] Bowl-shaped wells with curved bases are advantageous because, upon drying, precipitated analytes concentrate at the center in a more uniform fashion, [23] where they are efficiently ionized. [24] These wells, however, are difficult to fabricate, requiring micromachining, subtractive etching methods, embossing of plastics or specialized deposition methods. [23,[25][26][27][28] Additive fabrication approaches are superior because they use photolithographic techniques that are simple, inexpensive, and ubiquitous. However, photolithographic fabrication of wells with controlled curvature is challenging, requiring complex mask and lens systems to modulate light intensity with micrometer resolution across the array. [29,30] Consequently, simpler but inferior methods with stamping or backfilling of sharp features with polymer are more common, even though they are tedious and provide limited control. [6,[31][32][33] A superior approach would use simple photolithographic techniques without sacrificing control over well shape and curvature.In this paper, we describe a simple method to photolithographically fabricate wells with controlled curvature in SU8. This photoresist has several properties useful for mass spectrometry, including chemical robustness, precision control of surface features, and scalable fabrication. Using the approach, we fabricate curved-bottom wells at a density of 100 000 per square centimeter on a glass slide. To demonstrate the utility of these wells, we show they yield enhanced sensitivity in microscale mass spectrometry compared to cylindrical wells. Several approaches, such as the AnchorChip [34] and the μFocus plate, [35] use surface rather than physical modification to control drying and enhance MALDI MS, but do not Patterned surfaces can enhance the sensitivity of laser desorption ionization mass spectrometry by segregating and concentrating analytes, but their fabrication can be challenging. Here, a simple method to fabricate substrates patterned with micrometer-scale wells that yield more accurate and sensitive mass spectrometry mea...

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“…We recently reported the development of MALDI-time-of-flight (TOF)-based approaches for screening microbial products directly from large numbers of colonies in a high-throughput manner (∼2 s per colony). − As is typical for MALDI approaches, the analytes remain on the surface, and the analyte vaporization and ionization processes depend on both the sample and matrix crystallization, making absolute quantitation problematic even when using standards. Other approaches involve microfluidics coupled to ESI–MS or MALDI–MS, − the integration of self-assembled monolayers with matrix-assisted laser desorption ionization (SAMDI) , and the use of desorption ESI (DESI) …”

Section: Introductionmentioning

confidence: 99%

Mass Spectrometry-Based High-Throughput Quantification of Bioproducts in Liquid Culture

et al. 2023

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To meet the ever-increasing need for high-throughput screening in metabolic engineering, information-rich, fast screening methods are needed. Mass spectrometry (MS) provides an efficient and general approach for metabolite screening and offers the capability of characterizing a broad range of analytes in a label-free manner, but often requires a range of sample clean-up and extraction steps. Liquid extraction surface analysis (LESA) coupled MS is an image-guided MS surface analysis approach that directly samples and introduces metabolites from a surface to MS. Here, we combined the advantages of LESA−MS and an acoustic liquid handler with stable isotope-labeled internal standards. This approach provides absolute quantitation of target chemicals from liquid culture-dried droplets and enables high-throughput quantitative screening for microbial metabolites. In this study, LESA−MS was successfully applied to quantify several different metabolites (itaconic acid, triacetic acid lactone, and palmitic acid) from different yeast strains in different mediums, demonstrating its versatility, accuracy, and efficiency across a range of microbial engineering applications.

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Mapping enzyme catalysis with metabolic biosensing

Cited by 22 publications

References 31 publications

Evaluation of Analyte Transfer between Microfluidic Droplets by Mass Spectrometry

Evaluation of Analyte Transfer between Microfluidic Droplets by Mass Spectrometry

Microbowls with Controlled Concavity for Accurate Microscale Mass Spectrometry

Mass Spectrometry-Based High-Throughput Quantification of Bioproducts in Liquid Culture

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