Positive dielectrophoresis-based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo

Abstract

The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis-based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. Screening of yeasts for triacylglycerol (TAG) content demonstrated near-theoretical-limit accuracy, ~120 cells min^-1 throughput and full-vitality preservation, while sorting fatty acid degree of unsaturation (FA-DU) featured ~82% accuracy at ~40 cells min^-1. From a yeast library expressing algal diacylglycerol acyltransferases (DGATs), a pDEP-RADS run revealed all reported TAG-synthetic variants and distinguished FA-DUs of enzyme products. Furthermore, two previously unknown DGATs producing low levels of monounsaturated fatty acid-rich TAG were discovered. This first demonstration of RACS for enzyme discovery represents hundred-fold saving in time consumables and labor versus culture-based approaches. The ability to automatically flow-sort resonance Raman-independent phenotypes greatly expands RACS' application.

Fig. 1. The system set of pDEP-RADS.

(A) General applicability and throughput of the existing RACS platforms. However, few RACS platforms that are both generally applicable to nonresonance Raman bands and of high throughput were available. (B) Strategy to develop a generally applicable and higher-throughput RACS platform in this study. (C) Diagram of pDEP-RADS system setup. EMCCD, electron-multiplying charge-coupled device. (D) Schematic of chip design. The red spheres represent target cells, and the green triangle indicates position of the 532-nm laser beam. The inset is a bright-field image of the main functional units. The dashed box indicates the ITO electrode array. Scale bar, 200 μm.

Fig. 2. Operation of the pDEP-RADS system.

(A) System operation. Step 1, cell suspension was loaded and focused into single-cell flow by tow pinch flow. Step 2, single cells were delivered to the laser detection point via pDEP-based trap and release. Step 3, SCRS were acquired and analyzed. Step 4, the cells were encapsulated into droplets individually after SCRS acquisition. Step 5, the nontarget droplet flowed into the “Waste” channel automatically. Step 6, the target droplet was sorted into the “Collect” channel by triggering DEP. (B) Single cells were trapped and released by pDEP at 5 Hz. (C) Accurate immobilization of single cells at the angle of final electrode. (D) Averaged SCRS of 30 cells at 2800 to 3050 cm⁻¹ under the acquisition time of 50 ms. a.u., arbitrary units. (E) Percentage of detectable target cells under various acquisition times. The slight decrease in detection efficiency is mainly caused by the reduction of acquisition time. (F) Without DEP, nontarget droplet flowed into the waste channel. (G) With DEP, target droplets were sorted into the collect channel.

Fig. 3. The pDEP-RADS of TAG-synthetic yeast cells.

(A) GC-MS–based quantification of TAG levels extracted from bulk amounts of control cells (transformed with empty vector) or ScDGA1 cells. The total amount of TAG was normalized on the basis of that of total lipids. (B) Averaged SCRS of 60 control and 60 ScDGA1 cells. (C) Distribution of “I₂₈₆₇ - I₂₈₇₉” of control and ScDGA1 cells separately. n > 100 in each of the three groups. (D) Sorting efficiency of pDEP-RADS, by comparison of relative abundance of target cells between “Sorted” and Waste pools. (E) Evaluation of viability of post-sorting cells. CFU, colony-forming units. (F) Evaluation of sorting accuracy under a series of dilution of target cells using nontarget cells. The average number of collected cells for one experiment was ~300, ~300, ~100, and ~ 10 for dilutions of 2, 10¹, 10², and 10³ separately. (G) Polymerase chain reaction (PCR) validation of sorting accuracy. PC, positive control; NC, negative control. (H) Performance of pDEP-RADS under various dilution factors in three replicate experiments. Unless specified, error bars indicate the SD of three independent experiments. **P < 0.01; n.s., no significant difference.

Fig. 4. The pDEP-RADS of PUFA (EPA)–accumulating yeast cells.

(A) Profiles of SFA, MUFA, and PUFA in yeast expression of NoDGAT2A and 2C cells. (B) Representative SCRS at 1400 to 1700 cm⁻¹ of 2A and 2C cells. (C) Linear correlation between “(I₁₆₆₅ - I₁₈₀₀) / (I₁₄₄₅-I₁₈₀₀)” (represents the DU value measured via Raman) and the FA-DU value [measured via GC-MS from the bulk yeast cells expressing NoDGAT2A, 2C (supplemented with PUFA of 18:2), 2C (supplemented with PUFA of 20:4), and 2D], with R² of 0.995. Error bars represent the SD of >30 cells. (D) Distribution of (I₁₆₆₅ - I₁₈₀₀) / (I₁₄₄₅-I₁₈₀₀) (positively correlated with DU) of 2A and 2C cells separately. n > 100 in in each of the three groups. (E) Enrichment of target cells by pDEP-RADS, representing approximately fivefold enrichment. (F) PCR validation of sorting accuracy. Unless specified, error bars indicate the SD of three independent experiments. *P < 0.05; **P < 0.01. bp, base pair.

Fig. 5. The pDEP-RADS versus the culture-based methods in screening in vivo function of DGAT genes.

(A) Traditional strategy to screening the in vivo activities of TAG-synthetic genes. pYES2.0 and NoDGAT genes (2A-2I) from N. oceanica were transformed separately into the yeast strain H1246, followed by plating, clone picking, PCR, and sequencing to identify the successfully transformed clones. The clones were then separately cultured, and then each underwent a period of induction to synthesize TAG. In the end, the TAG profile of each clone was analyzed by the tedious and time-consuming TLC and GC/LC-MS from the cultured biomass. (B) pDEP-RADS strategy. pYES2.0 and NoDGAT genes (2A-2I) were transformed simultaneously into strain H1246, and then induction and functional sorting ensued directly from the yeast library (without any plating). The two strategies were compared for time (C), consumable cost (D) and labor (E). In (C) and (D), the steps are (i) transformation, (ii) plating, (iii) liquid cultivation, (iv) PCR and Sanger sequencing, (v) induction, (vi) lipid extraction, (vii) TLC-based TAG isolation, and (viii) GC-MS analysis for the culture-based strategy, whereas (1) transformation, (2) induction, (3) pDEP-RADS, and (4) high-throughput sequencing (HT-seq) for the pDEP-RADS strategy. In (E), the steps are (i) transformation, (ii) plasmid extraction, PCR and electrophoresis-based target gene recovery, (iii) lipid extraction, (iv) TLC-based TAG isolation, and (v) GC-MS analysis for the culture-based strategy, whereas (1) transformation and (2) the pDEP-RADS procedure in the pDEP-RADS strategy.

Fig. 6. Screening in vivo TAG-synthetic activities of N. oceanica DGATs via pDEP-RADS.

Compositions of the yeast library (profiled by HT-seq) before (A) and after (B) pDEP-RADS were compared. (C) Composition of the post–pDEP-RADS cells [profiled via plating, random picking, and Sanger sequencing (Sanger-seq)]. (D) Distribution of “I₂₈₆₇-I₂₈₇₉” of the sorted NoDGAT2F and 2H cells. n > 1000 for each group. I₂₈₆₇-I₂₈₇₉ can be negative; as for control cell (i.e., non–TAG producing) or mutants with no or low TAG, 2867 cm⁻¹ is at a continuously rising slope (Fig. 3B). (E) Percentage of TAG-synthetic cells in the sorted 2D, 2F, and 2H cells (showing those with I₂₈₆₇ - I₂₈₇₉ > 0). (F) GC-MS–based quantification of TAG content via extraction from bulk cultures of sorted 2F and 2H cells. “Control” in (D to F): empty vector–transformed cells. Positive control is the 2D-transformed cells. (G) Linear correlation between I₂₈₆₇ - I₂₈₇₉ (the TAG content measured via SCRS) and the %TAG per total lipid measured via GC-MS from bulk cells, with R² of 0.97. Notably, on the x axis, despite the negative averaged I₂₈₆₇-I₂₈₇₉ values for 2H, 2F, 2C, and 2D, all are higher than the control, consistent with their higher overall TAG content than the control (designated as zero). (H) Averaged SCRS at 1400 to 1700 cm⁻¹ of the NoDAGT2-expressing yeasts. (I) (I₁₆₆₅ - I₁₈₀₀) / (I₁₄₄₅-I₁₈₀₀) of SCRS, which is positively correlated with DU of FAs for the NoDGAT2-expressing cells. (J) GC-MS–based quantification of DU of FAs in the cells. (K) Linear correlation between (I₁₆₆₅ - I₁₈₀₀) / (I₁₄₄₅-I₁₈₀₀) (i.e., the DU measured via SCRS) and the DU measured via GC-MS of bulk cells, with R² of 0.99. In (F to K), all cells in (D) are shown. Error bars: SD of three replicates. *P < 0.05; **P < 0.01; n.s., not significant.