(K-427) Picoliter thin layer chromatography (pTLC)-based separation of phosphatidylinositol 4,5-bisphosphate (PIP₂) and diacylglycerol of single cells

Lipids are key components of cells forming membranes and acting as signaling agents.[1] Their mis-regulation can play a role in diseases such as cancer and neurologic dysfunction. Assay of lipid properties in single cells is increasingly important to understand how lipids can direct different physiologic outcomes for cells of identical genetic background and in seemingly identical microenvironments.[2] Due to the limited volume of a single cell as well as the large number of distinct lipids within a cell, current strategies for assays of single-cell lipid biology are complex and expensive and require significant expertise.[3] For this reason development of new, easy-to-use methods for assays of lipid signaling in single cells is of growing importance.[4] Traditional thin layer chromatography has been used for separating large quantities of lipids but with poor detection limits.[5] Mass spectrometry is growing in popularity for single cell assays but measurements of lipids in single cells remains technically challenging and requires significant expertise.[6] By applying scalable microfabrication techniques micron-scale channels can be fabricated to create miniaturized thin-layer chromatography bands. [7,8]  A silica precursor is loaded within a glass microtrough (60 µm wide and 15 µm deep) and cured to create a porous silica band that can be used to separate fluorescent lipids. For these assays, lipid reporters (enzyme substrates such as fluorescent PIP₂) are loaded into a single cell and the reporter and its various metabolites such as diacylglycerol can be detected when the contents of a cell are separated using pTLC.

The photoresist AZ4620 was spin-coated onto the soda lime glass slides (75 mm by 25 mm) that were treated with HMDS (0.5 ml in 50 ml PGMEA , hexamethyldisalazine). [9]    The slide was then illuminated with ultraviolet light through a photomask to reveal the locations where the channels were to be formed. Hydrofluoric acid was then used to etch the channels in the regions in which the photoresist was removed.  A poly(dimethyl siloxane) (PDMS) flat slab was placed onto the glass slide and silica precursormoved through the channels under a vacuum. The device was incubated for 24 h at 40 °C for sol–gel reaction followed by aging in urea solution at 120 °C for 1.5 h. The PDMS top  piece was removed to expose the silica bands. [7] The slide was then incubated in sulfuric acid for 24 h, rinse with deionized water, dry in air and followed by calcination at 330 °C for 24 h prior to use. Control lipids and cells were spotted onto the pTLC microbands using a piezoelectric dispenser. Extraction and separation of lipids by the addition of 1 ml of solvent (1 butanol/water/acetic acid, 18/4/4 v/v/v) to an absorbent pad on which the pTLC was placed in an inverted position. After separation, the microbands were dried under a stream of nitrogen. The pTLC chips were imaged by fluorescence microscopy to quantify the signal from the fluorescent lipids.[10]

Results

We used ANSYS simulation software  to simulate pTLC by varying and assessing the effect of various parameters like geometry, fluid density, viscosity, flow rates etc. on the separation mechanism. [11]By understanding the effect of these parameters, we can predict and optimize the scalability and production of these chips to optimize separation efficiency.

The separation of control fluorescent lipids (100-1000 pL, 20 µM Texas red DHPE (Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, [12]) and 10 µM 18:1 PE CF (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein), [13]) spotted onto the microbands was efficient and occurred with a resolution of >1 in < 1 min. The separation of DIO and DiD are shown in Figure 2. The separation of fluorescent phosphoinositides was also performed (possible figure). Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol-3,4,5-trisphosphate (PIP₃)were separated with a resolution of (1.28 ± 0.02, n = 4) in less than 10 min. This is shown in Figure 3. The PIP₂ was also loaded into  cells to reveal phospholipase C that hydrolyzes PIP2 to diacylglycerol. The fixed single cells were piezoelectrically spotted onto the microbands and their contents separated. Results from single cell separations with and without pharmacologic inhibition of phospholipase C will be presented.

Conclusion and Discussion

The pTLC chip demonstrated excellent separation of the control fluorescent lipids as well as lipid reporters loaded into cells. Metabolic products of the reporter PIP₂ such as diacylglycerol were readily detected from single cells.  pTLC shows promise for measurement of the activity of lipid modifying enzymes such as phospholipase C in single cells.[14] The method is simple, does not require expensive instrumentation and can be used by the nonexpert. Future steps will include the continued optimization of the microband and channel properties for separation of a diverse array fluorescent lipid reporters.[8]

References

1. Laouini A, Jaafar-Maalej C, Limayem-Blouza I, Sfar S, Charcosset C, Fessi H. Preparation, Characterization and Applications of Liposomes: State of the Art. Journal of Colloid Science and Biotechnology. 2012;1:147–68.

2. Dickinson AJ, Hunsucker SA, Armistead PM, Allbritton NL. Single-cell sphingosine kinase activity measurements in primary leukemia. Anal Bioanal Chem. 2014;406:7027–36.

3. Chul Lee W, Rigante S, P. Pisano A, A. Kuypers F. Large-scale arrays of picolitre chambers for single- cell analysis of large cell populations. Lab on a Chip. 2010;10:2952–8.

4. Listenberger LL, Brown DA. Fluorescent Detection of Lipid Droplets and Associated Proteins. Current Protocols in Cell Biology. 2007;35:24.2.1-24.2.11.

5. Bochner BR, Ames BN. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. Journal of Biological Chemistry. 1982;257:9759–69.

6. Yin L, Zhang Z, Liu Y, Gao Y, Gu J. Recent advances in single-cell analysis by mass spectrometry. Analyst. 2019;144:824–45.

7. Tanaka N, Kobayashi H, Ishizuka N, Minakuchi H, Nakanishi K, Hosoya K, et al. Monolithic silica columns for high-efficiency chromatographic separations. Journal of Chromatography A. 2002;965:35–49.

8. Wang Y, Yao M, Sims CE, Allbritton NL. Monolithic Silica Microbands Enable Thin-Layer Chromatography Analysis of Single Cells. Anal Chem. 2022;94:13489–97.

9. New Photoresist AZ P4620 Photoresists MicroChemicals GmbH [Internet]. [cited 2023 Jul 14]. Available from: https://www.microchemicals.com/products/photoresists/az_p4620.html

10. AX / AX R with NSPARC [Internet]. Nikon Instruments Inc. [cited 2023 Jul 14]. Available from: https://www.microscope.healthcare.nikon.com/products/confocal-microscopes/ax?gclid=CjwKCAjw5MOlBhBTEiwAAJ8e1v1wP9z65g5xvN14VhjSU1MW0Ow7bQG9Lb2YY9xsvc7WWn1c3oDQ5xoCaioQAvD_BwE

11. Predicting Species Separation During Chromatography using Ansys CFD | Ansys Webinar [Internet]. [cited 2023 Jul 17]. Available from: https://www.ansys.com/resource-center/webinar/predicting-species-separation-during-chromatography-using-ansys-cfd

12. Texas Red-DHPE *CAS 187099-99-6* | AAT Bioquest [Internet]. [cited 2023 Jul 17]. Available from: https://www.aatbio.com/products/texas-red-dhpe-cas-187099-99-6

13. 18:1 PE CF [Internet]. Avanti Polar Lipids (en-US). [cited 2023 Jul 17]. Available from: https://avantilipids.com/product/810332

14. Bunner B, Kromidas A, Kele M, Neue U. Simulation of Chromatographic Band Transport.