Picoscale thin-layer chromatography for the analysis of single cells and microsamples

Abstract

A thin layer chromatography (TLC) device for the analysis of picoscale samples, methods of using the device, and methods of making the device.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 158,047, filed March 8, 2021, the entirety of which is expressly incorporated herein by reference.

[0002] Government License Rights Statement This invention was made with Government support under Grant No. R01 CA233811 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] 2. Background of the Invention Thin-layer chromatography (TLC) is an analytical technique commonly used to separate non-volatile compounds by differential migration in a thin layer of an adsorbent matrix. A conventional TLC plate consists of an adsorbent layer (such as silica gel, aluminum oxide or cellulose) formed on a sheet of glass plate, a plastic sheet or an aluminum foil. The performance of TLC consists of three steps: spotting, developing and detection. A solution of the compound to be analyzed is first spotted on the starting line of the TLC plate. The sample's solvent is evaporated to leave the compounds to be separated. The TLC plate is then developed in a chamber by contacting its lower end with a solvent (eluent). The solvent is drawn into the adsorbent matrix by capillary action, moving the multiple compounds of the sample vertically at various speeds due to their different interactions with the matrix and different solubilities in the developing solvent. When the solvent front reaches a point (usually about 1 cm from the edge of the upper plate), the plate is removed from contact with the eluent and dried. The location of compounds on the plate can be visualized using ultraviolet (UV) light, chemical dyes, fluorescence, mass spectrometry, infrared spectroscopy, or a wide range of other methods (almost always with very low sensitivity or high signal to noise). Conventional TLC is widely used in synthetic chemistry, food, pharmaceutical, and environmental analysis because of its simplicity, low cost, and speed of separation.

[0004] TLC technology has advanced significantly since the 1950s, when standardized procedures for conventional TLC were established. Conventional TLC has a typical adsorbent layer thickness of 250 μm, an average particle size of 10-12 μm, and a particle size distribution of 5-20 μm. High performance thin layer chromatography (HPTLC) was developed in the 1970s by utilizing adsorbent particles with smaller size (5-6 μm), particle size distribution (4-8 μm), and layer thickness (100 μm), resulting in improved sensitivity, resolution, and analytical speed compared to conventional TLC. Further improvements led to the introduction of ultra-thin layer chromatography (UTLC) by Merck in 2001. UTLC has a 10 μm thick monolithic silica adsorbent layer bonded directly to a glass plate. UTLC has further improved sensitivity and reduced analysis time and separation distance compared to HPTLC. In addition to monolithic silica, other materials have been used to construct the adsorbent layers of UTLCs, including electrospun nanofibers, carbon-nanotube-templated forests, porous polymer monolithic layers, and nanostructured thin films.

[0005] In the past decades, the majority of TLC research has focused on optimizing the adsorbent layer and detection methods with the aim of improving sensitivity, resolution and analytical speed, but still focuses on large sample sizes due to the severe challenges posed by ultra-trace assays. To date, TLC technology has not been seen as a preferred method for single-cell assays due to the insufficient sensitivity of TLC, the large sample loading required, and the moderate separation performance. Indeed, previous attempts have demonstrated that single-cell or ultra-trace TLC is simply not possible, despite the great efforts and significant resource expenditure.

[0006] Single-cell analysis is a rapidly growing field of biomedical research, further gaining high prominence for clinical disease diagnosis and prognosis due to the fast deployment of highly sensitive and integrated technologies such as micro / nanofluidic devices. It is now well recognized that cellular heterogeneity within a population is both a disease driver and fundamentally important to understanding almost all aspects of normal physiological function. Furthermore, these micro / nanofluidic and array-based devices can be easily scaled to match the dimensions and volume of single cells to achieve assay success and achieve adequate signal-to-noise detection. As mentioned previously, one of the main obstacles to using TLC for single-cell analysis is that the dimensions of the TLC adsorptive matrix (thickness: 10->100 μm, width: >1 cm) significantly exceed those of a single cell (typical diameter and thickness: 10-20 μm, volume: 1-2 pL). As a result, compounds eluted from single cells are laterally and vertically diluted within the matrix and rapidly reach concentrations below the detection limit. To address this challenge, the thickness and width of the TLC porous matrix must be miniaturized into microbands with thickness / width comparable to the size of a single cell, which is expected to reduce lateral diffusion and confine compound movement along the microbands.

[0007] The sample loading zone and workflow must also be suitable for that of single cells. Several previous publications have generated TLC bands as separation lanes, but the band width exceeds 200 μm, and none of them have miniaturized the TLC bands for single cell analysis. Furthermore, the lane thickness (≧200 μm) is much too large to support small sample assays. Although these methods are interesting, they do not demonstrate any benefit compared to standard TLC for very small sample assays.

[0008] A wide range of detection strategies have been coupled with TLC, including absorbance, fluorescence, colorimetric development, mass spectrometry, and various spectroscopic strategies. Despite intensive efforts, detection limits remain relatively poor compared to other separation methods, with limits of detection (LODs) typically in the range of 10-14 ~10 -10 moles / spot. This is due to sample dilution, matrix noise, and low or zero off rate analyte adsorption. Efforts to optimize or eliminate these undesirable matrix challenges have been unsuccessful. Sample deposition typically utilizes glass capillaries to spot liquid volumes of 0.1-0.5 μL (for HPTLC) with poor spatial precision (i.e., typically one spot per cm wide plate). Thus, conventional TLC, HPTLC, and UTLC cannot achieve the tight specifications required for trace analysis. Summary of the Invention [Problem to be solved by the invention]

[0009] There exists a need for improved TLC instruments capable of trace analysis, and methods of making TLC instruments having improved performance, particularly trace analysis. The present invention seeks to fulfill this need, and further provides related advantages. [Means for solving the problem]

[0010] Summary of the Invention The present invention provides a chromatography device (thin layer chromatography (TLC) device), methods of making the device, and methods of using the device.

[0011] In one aspect, the invention provides a thin layer chromatography (TLC) device comprising a non-porous substrate having one or more microchannels formed on a surface thereof, each microchannel comprising a porous, substantially homogenous microband adapted for fluid flow driven by capillary action.

[0012] In another aspect, a method of making a thin layer chromatography device includes: (a) forming one or more microchannels in a non-porous substrate; (b) filling one or more microchannels with a flowable porous microband precursor composition; and (c) subjecting the non-porous substrate having one or more microchannels filled with a flowable porous microband precursor composition to conditions that convert the composition into porous microbands to obtain a thin layer chromatography device. Includes.

[0013] In a further aspect, the present invention provides a method for thin layer chromatography of a single cell, a group of cells, or the contents of a microsample using the chromatography device described herein.

[0014] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated and better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 illustrates the concept of separating compounds from small-scale samples, such as single cells or other small-scale samples, by picoscale thin-layer chromatography (pTLC). Panel A shows a representative pTLC plate, including a porous microband (single microband) formed on a non-porous substrate (e.g., a glass slide). Panel B shows compounds within a cell stained with a marker (e.g., a fluorophore), and a single cell or other sample is dispensed to the left of the microband. Panel C shows solvent application at the left edge of the microband and migration of the solvent to the right edge. Compounds stained in the cell or other deposited sample are extracted by the solvent. Compounds are separated due to differences in the compound's migration rates and interactions with the matrix. [Figure 2A]Fabrication of pTLC plates. Figure 2A is a schematic of a representative fabrication process (PDMS: polydimethylsiloxane, TMOS: tetramethylorthosilicate, PEG: polyethylene glycol). Figure 2B shows bright-field microscopy images of a pTLC plate (top: low-magnification top view, middle: high-magnification top view, bottom: high-magnification side view). Figure 2C shows the porous texture of the silica microbands. The two bands on the left are dry and dark. The two bands on the right have been immersed in water and are transparent. [Figure 2B] Fabrication of pTLC plates. Figure 2A is a schematic of a representative fabrication process (PDMS: polydimethylsiloxane, TMOS: tetramethylorthosilicate, PEG: polyethylene glycol). Figure 2B shows bright-field microscopy images of a pTLC plate (top: low-magnification top view, middle: high-magnification top view, bottom: high-magnification side view). Figure 2C shows the porous texture of the silica microbands. The two bands on the left are dry and dark. The two bands on the right have been immersed in water and are transparent. [Figure 2C] Fabrication of pTLC plates. Figure 2A is a schematic of a representative fabrication process (PDMS: polydimethylsiloxane, TMOS: tetramethylorthosilicate, PEG: polyethylene glycol). Figure 2B shows bright-field microscopy images of a pTLC plate (top: low-magnification top view, middle: high-magnification top view, bottom: high-magnification side view). Figure 2C shows the porous texture of the silica microbands. The two bands on the left are dry and dark. The two bands on the right have been immersed in water and are transparent. [Figure 3A] Figure 3 shows the separation of spotted lipid mixtures by pTLC. Figure 3A is a schematic of the setup for development and image processing. Figure 3B shows the separation of 18:1 PE CF and Texas Red DHPE by 1-butanol:water:acetic acid (18:4:4, vol:vol:vol). Figure 3C shows the chemical structures and the separation of sphingosine fluorescein (SpF) and sphingosine-1-phosphate fluorescein (S1PF) by 1-butanol:water:acetic acid (18:1:1, vol:vol:vol). [Figure 3B]Figure 3 shows the separation of spotted lipid mixtures by pTLC. Figure 3A is a schematic of the setup for development and image processing. Figure 3B shows the separation of 18:1 PE CF and Texas Red DHPE by 1-butanol:water:acetic acid (18:4:4, vol:vol:vol). Figure 3C shows the chemical structures and the separation of sphingosine fluorescein (SpF) and sphingosine-1-phosphate fluorescein (S1PF) by 1-butanol:water:acetic acid (18:1:1, vol:vol:vol). [Figure 3C] Figure 3 shows the separation of spotted lipid mixtures by pTLC. Figure 3A is a schematic of the setup for development and image processing. Figure 3B shows the separation of 18:1 PE CF and Texas Red DHPE by 1-butanol:water:acetic acid (18:4:4, vol:vol:vol). Figure 3C shows the chemical structures and the separation of sphingosine fluorescein (SpF) and sphingosine-1-phosphate fluorescein (S1PF) by 1-butanol:water:acetic acid (18:1:1, vol:vol:vol). [Figure 4A] Figure 4 shows the separation of lipophilic red / green lipids from single cells by pTLC. Figure 4A shows the chemical structures of DiO and DiD. Figure 4B shows U937 cells stained with DiO and DiD. Figure 4C shows time-lapse images showing the separation of DiO and DiD by 1-pentanol from single U937 cells. [Figure 4B] Figure 4 shows the separation of lipophilic red / green lipids from single cells by pTLC. Figure 4A shows the chemical structures of DiO and DiD. Figure 4B shows U937 cells stained with DiO and DiD. Figure 4C shows time-lapse images showing the separation of DiO and DiD by 1-pentanol from single U937 cells. [Figure 4C] Figure 4 shows the separation of lipophilic red / green lipids from single cells by pTLC. Figure 4A shows the chemical structures of DiO and DiD. Figure 4B shows U937 cells stained with DiO and DiD. Figure 4C shows time-lapse images showing the separation of DiO and DiD by 1-pentanol from single U937 cells. [Figure 5A] Figure 5A shows the separation of sphingosine metabolites from single cells by pTLC. Figure 5A shows a schematic of the process. Sphingosine (Sp) alkyne is loaded into U937 cells. Inside the cells, the sphingosine alkyne is converted to sphingosine-1-phosphate (S1P) alkyne by sphingosine kinase. The cells are then fixed and the alkyne moiety is reacted with an azide-containing fluorophore (e.g., CY5-azide) through click chemistry (chemical structures of Sp alkyne, S1P alkyne and CY5-azide are shown). Figure 5B shows U937 cells loaded with Sp alkyne and clicked with CY5-azide. Figure 5C shows time-lapse images (0, 2, 4, 6, and 11 min) showing the separation of Sp-CY5 and S1P-CY5 from single U937 cells. The developing solution was 1-butanol:1-propanol:water:triethylamine (18:2:4:1, vol:vol:vol:vol). Figure 5D is a TLC chromatogram at 11 minutes showing that Sp-CY5 was completely separated from S1P-CY5. [Figure 5B] Figure 5A shows the separation of sphingosine metabolites from single cells by pTLC. Figure 5A shows a schematic of the process. Sphingosine (Sp) alkyne is loaded into U937 cells. Inside the cells, the sphingosine alkyne is converted to sphingosine-1-phosphate (S1P) alkyne by sphingosine kinase. The cells are then fixed and the alkyne moiety is reacted with an azide-containing fluorophore (e.g., CY5-azide) through click chemistry (chemical structures of Sp alkyne, S1P alkyne and CY5-azide are shown). Figure 5B shows U937 cells loaded with Sp alkyne and clicked with CY5-azide. Figure 5C shows time-lapse images (0, 2, 4, 6, and 11 min) showing the separation of Sp-CY5 and S1P-CY5 from single U937 cells. The developing solution was 1-butanol:1-propanol:water:triethylamine (18:2:4:1, vol:vol:vol:vol). Figure 5D is a TLC chromatogram at 11 minutes showing that Sp-CY5 was completely separated from S1P-CY5. [Figure 5C]Figure 5A shows the separation of sphingosine metabolites from single cells by pTLC. Figure 5A shows a schematic of the process. Sphingosine (Sp) alkyne is loaded into U937 cells. Inside the cells, the sphingosine alkyne is converted to sphingosine-1-phosphate (S1P) alkyne by sphingosine kinase. The cells are then fixed and the alkyne moiety is reacted with an azide-containing fluorophore (e.g., CY5-azide) through click chemistry (chemical structures of Sp alkyne, S1P alkyne and CY5-azide are shown). Figure 5B shows U937 cells loaded with Sp alkyne and clicked with CY5-azide. Figure 5C shows time-lapse images (0, 2, 4, 6, and 11 min) showing the separation of Sp-CY5 and S1P-CY5 from single U937 cells. The developing solution was 1-butanol:1-propanol:water:triethylamine (18:2:4:1, vol:vol:vol:vol). Figure 5D is a TLC chromatogram at 11 minutes showing that Sp-CY5 was completely separated from S1P-CY5. [Figure 5D] Figure 5A shows the separation of sphingosine metabolites from single cells by pTLC. Figure 5A shows a schematic of the process. Sphingosine (Sp) alkyne is loaded into U937 cells. Inside the cells, the sphingosine alkyne is converted to sphingosine-1-phosphate (S1P) alkyne by sphingosine kinase. The cells are then fixed and the alkyne moiety is reacted with an azide-containing fluorophore (e.g., CY5-azide) through click chemistry (chemical structures of Sp alkyne, S1P alkyne and CY5-azide are shown). Figure 5B shows U937 cells loaded with Sp alkyne and clicked with CY5-azide. Figure 5C shows time-lapse images (0, 2, 4, 6, and 11 min) showing the separation of Sp-CY5 and S1P-CY5 from single U937 cells. The developing solution was 1-butanol:1-propanol:water:triethylamine (18:2:4:1, vol:vol:vol:vol). Figure 5D is a TLC chromatogram at 11 minutes showing that Sp-CY5 was completely separated from S1P-CY5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description of the Invention The present invention provides chromatography devices (thin layer chromatography (TLC) devices), methods of making the devices, and methods of using the devices.

[0017] Thin-layer chromatography (TLC) is a widely used analytical technique for various applications, but its use on very small specimens such as single cells with volumes as small as 1 picoliter has not been realized so far. The main challenge is that the dimensions of the adsorbent layer in conventional TLC significantly exceed those of a single cell. The large volume of the adsorbent does not confine the spread of diffusion of trace amounts, leading to dilution of the sample during the chromatographic separation step. Detection of such microsamples requires a very small number of sample molecules (10 -21 ~10 -15 This is a major limitation due to the low off-rate (mol) and background noise generated by the adsorbent (e.g., light scattering and light absorption by the matrix). Nonspecific binding of samples to the adsorbent with zero or very slow off-rates (or a range of off-rates) creates further difficulties that must be overcome for assaying these constrained samples. Other challenges include the methods used to deposit small amounts of sample at precise locations, but for very small samples, conventional TLC does not have sufficient volumetric or spatial resolution. To be successful in assaying very small volumes, dispersion due to large sample volumes (necessary for conventional TLC), or sample spreading due to surface tension, must be overcome, along with the ability to position the sample at the entrance to the sample area with high spatial resolution (≦50 μm). Conventional TLC cannot meet these stringent specifications.

[0018] To address this challenge, the present invention provides a TLC platform (termed picoscale thin layer chromatography (pTLC)) that combines sol-gel chemistry with microfabrication techniques. pTLC consists of single or arrays of microscale bands made from highly porous monolithic silica (or other materials) designed to accept picoliter-scale volumes of samples. In certain aspects, the present invention combines highly sensitive detection methods with separation optimization to overcome further limitations of TLC methodologies to date. In certain embodiments, the present invention provides a TLC device in which the width and depth of each band are comparable to the size of a single cell (≦100 μm). As described below, to demonstrate proof-of-principle for ultrasmall sample analysis, model fluorescent compounds (18:1 PE CF and Texas Red DHPE, sphingosine fluorescein and sphingosine 1-phosphate fluorescein) were microspotted onto the microbands and successfully separated by pTLC. As described further below, to demonstrate the ability to separate picoliter volumes, mammalian single cells loaded with fluorescent compounds (DiO and DiD, CY5-labeled sphingosine metabolites) were spotted onto a microplate and the compounds were extracted from the single cells and separated by pTLC. Thus, in certain aspects, the present invention provides devices and methods for single cell analysis and femto- to nanoliter-scale samples.

[0019] The devices and methods described herein are useful in applications requiring small specimens and high-throughput parallel analysis, such as disease diagnosis, cell heterogeneity analysis, droplet-based reaction monitoring, detection of microenvironmental, food, forensic and pharmaceutical samples. A strong advantage of the devices and methods described herein is their simplicity and high-speed separation capabilities. For example, the devices described herein are easily operated without active fluid control (pumps, valves, flow meters), current flow, or fluid level balancing. Separation can be terminated for post-chromatographic detection by simple evaporation of the solvent. These features make the devices robust yet simple to operate, allowing for fully sealed cassettes (e.g., highly scalable for automated high-throughput operation, such as home assays and drug discovery or combinatorial chemistry).

[0020] The present invention provides a TLC platform for the analysis of very small samples and specimens, such as single cells. The platform is suitable for assaying a wide range of ultra-small sample volumes (1 zeptoL (zL), 1 attoL (aL), 1 femtoL (fL), 10 fL, 100 fL, 1 pL, 10 pL, 100 pL, 1 nL, 10 nL, 100 nL). Molar sample size ranges include 10 -21 , 10 -20 , 10 -19 , 10 -18 , 10 -17 , 10 -16 , and 10 -15The concept of using pTLC to separate compounds from small sample volumes such as single cells is illustrated in Figure 1. In addition to cells, the concept can be applied to other microsamples in the form of droplets, aerosols, crystals, fibers, microcapsules, microbeads, microgels, liquids, solids, gels, oils, pico- to nanoliter containers, microplastics, microparticles, prions, aggregates, liposomes and other membrane-bound vesicles / structures, plaques, membranes, precipitates, and particles. A pTLC plate consists of an array of microscale bands made of a porous matrix on a non-porous substrate (Figure 1A, only one band shown). The dimensions of the bands are comparable to the size of a single cell (typical diameter: 10-20 μm, but also smaller and larger cells and organisms such as viruses, bacteria, fungi, yeasts, microorganisms, plankton, algae, mammalian and plant cells, oocytes, sperm, nematodes, insects, scales, hairs, etc.). Small sample sizes can include populations of cells such as organoids, cell colonies, biopsies, aspirates, and other biological samples such as plasma, blood, urine, membranes, lymph, cerebral or spinal fluid, tears, saliva, bile, feces, sweat, and other excretions / secretions. Compounds that are intracellular, extracellular, or secreted by cells can be samples. Subcellular organelles are suitable samples such as nuclei, Golgi bodies, mitochondria, endoplasmic reticulum, secretory or proteolytic vesicles or synaptic vesicles, lysosomes, vacuoles, extracellular vesicles, phagosomes, nucleoli, ribosomes, cytosol, or centrosomes. Microscale bands have widths ranging from 1 to 1000 μm, depths ranging from 0.5 to 100 μm, and lengths ranging from 0.1 to 100 mm. Thus, sample dimensions or spot sizes can be in the range of 50 nm, 100 nm, 1 micron, 10 microns, 1000 microns. Compounds found in small samples may or may not be stained with markers (e.g., fluorophores, chemical dyes, antibodies, rare metal isotopes, radioisotopes, click reagents, crosslinkers, nanoparticles, quantum dots, p-dots, lanthanides) before or after the separation step, and single cells or other small samples are dispensed to the left of the microband (Figure 1B).Solvent is applied to the left edge of the microband and its migration is drawn along the microband in the direction of capillary forces. Stained compounds are eluted from the cells or sample spots, their migration is confined along the band, and they are separated downstream due to differences in their migration speeds (Figure 1C).

[0021] TLC equipment In one aspect, the invention provides a chromatography device. In one embodiment, the invention provides a thin layer chromatography (TLC) device comprising a non-porous substrate having one or more microchannels formed thereon, each microchannel comprising a porous, substantially homogenous microband adapted for capillary action-driven fluid flow.

[0022] In some embodiments, the fluid flow is capillary fluid flow combined with electrophoresis. In other embodiments, the fluid flow is capillary fluid flow driven by intermolecular forces. In this device, the fluid flow is not driven by hydrodynamic centrifugal forces or pressure-driven fluid flow. Solvent can be drawn into the porous microband and then moved along the band by capillary forces, pressure, vacuum, centrifugal forces, electrophoretic forces, osmotic forces, or magnetic forces, among others. Combinations of solvents can be used and applied to the microband discontinuously or in gradients.

[0023] As used herein, the term "microchannel" refers to a channel formed on the surface of a non-porous substrate. During the fabrication of the TLC device described herein, each microchannel receives a flowable composition (e.g., a silica sol-gel slurry), which is then processed in situ to provide microbands. The microchannels can have a variety of shapes. For example, the microchannels can be U-shaped, V-shaped, can include three rectangular or square sides, or can include a triangular / pentagonal / hexagonal bottom. In some embodiments, the microchannel has a flat U-shape. In other embodiments, the microchannel includes a first surface (bottom) that is substantially parallel to the surface of the substrate, and second and third opposing surfaces (sides) that are substantially perpendicular to the first surface (i.e., a three-sided channel).

[0024] The term "porous microband" refers to a porous matrix suitable for chromatography. Each porous microband is formed in situ in a microchannel. The microband is a substantially homogeneous (e.g., crack-free, split-free, defect-free) porous matrix through which the sample travels. In one embodiment, the microband is a porous, crack-free, silica monolithic matrix having a width of about 1 to about 1000 μm, a depth of 1 to 100 μm, and a length of about 0.1 to about 100 mm.

[0025] A microband is a porous matrix with pore sizes from nanometers to micrometers (e.g., from about 1 nm to about 999 μm). The term "porous" refers to the microband that fills the microchannel and through which the sample travels in the TLC device. Suitable porous microbands include inorganic (e.g., silicon, silicon oxide, silica gel, aluminum oxide, titanium oxide, carbon nanostructures), metallic (e.g., gold, silver, titanium), organic (e.g., polymeric such as polymer nanofibers, polymer hydrogels, polyamides, monolithic porous polymers), natural (e.g., chitosan, diatomaceous earth, diatom biosilica, cellulose), plastic, and hybrid materials. Exemplary materials for the porous microbands include diatomaceous earth, cellulose (and modified cellulose), polyamide, modified / coated silica (amino-bonded silica, cyano-bonded silica, thiol-bonded silica, diol-bonded silica, C-18, C-8 and C-2 bonded silica, phenylsilica, etc.), hydroxylapatite, zinc carbonate, polyethylene, calcium sulfate, calcium hydroxide, dextran, polyacrylonitrile, polycaprolactone, calcium hydroxide, magnesium silicate, magnesium oxide, starch, agar, alumina, titania, indium tin oxide, and combinations of the above (such as silica-titania). Exemplary porous microbands include silica-based microbands.

[0026] In some embodiments, the porous microband is monolithic porous silica. In one embodiment, a thin layer chromatography (TLC) apparatus includes a non-porous substrate having one or more microchannels formed on a surface thereof, each microchannel comprising a monolithic porous silica microband adapted for fluid flow driven by capillary action.

[0027] Although the TLC devices described herein have been demonstrated to be advantageous for monolithic porous silica microbands, it will be appreciated that other porous matrices made from materials other than silica are useful for the microbands of the TLC devices described herein and are within the scope of the present invention.

[0028] In certain embodiments, the porous microbands have a width of about 1 to about 1000 μm (e.g., ≦5, ≦15, ≦30, ≦50, ≦100, ≦200, ≦500, or ≦1000 μm). In some of these embodiments, the porous microbands have a width of about 50 to about 150 μm. In other embodiments, the porous microbands have a width of about 50 to about 100 μm. In further embodiments, the porous microbands have a width of about 50 to about 75 μm. In certain embodiments, the porous microbands have a width of less than 200 μm (e.g., less than 190 μm). In representative embodiments, the porous microbands have a width of about 1 μm, about 5 μm, about 10 μm, about 50 μm, about 100 μm, about 200 μm, about 500 μm, or about 1000 μm. The porous microbands of the TLC devices described herein have the widths described and are free of discontinuities (eg, free of cracks, clefts, defects).

[0029] In certain embodiments, the porous microbands have a depth of about 1 to about 100 μm (e.g., ≦5, ≦15, ≦30, ≦50, or ≦100 μm). In some of these embodiments, the porous microbands have a depth of about 10 to about 50 μm. In other embodiments, the porous microbands have a depth of about 15 to about 25 μm. In further embodiments, the porous microbands have a depth of about 15 μm.

[0030] In one embodiment, the porous microband has a length that is substantially in the same range as the length of the device, with typical lengths being from about 0.1 to about 100 mm.

[0031] In one embodiment, the porous microbands have an aspect ratio (width / depth) of about 1 to about 100. Exemplary useful aspect ratios are 0.1, 0.5, 1, 5, 10, 5, 10, 50, and 100.

[0032] In one embodiment, the porous microband is about 10 μm 2 ~about 50,000μm 2 A typical useful cross-sectional area is 10 μm 2 , 100μm 2 , 500μm 2 , 1,000μm 2 , 5,000μm 2 , 10,000μm 2 , and 50,000 μm 2 It is.

[0033] In the TLC device, each microchannel contains a porous, substantially homogeneous microband. This is achieved by a fabrication process in which a flowable precursor of the porous material fills each microchannel, taking the shape of the microchannel in the process, thereby filling the channel. A solid porous microband occupying each microchannel is formed from the precursor by further processing as described herein. The solid porous microband so formed is also homogeneous by virtue of the use of a flowable sol-gel precursor to fill each microchannel. As used herein, the term "homogeneous" refers to the composition, mechanical properties (e.g., strength, stability, and continuous, crack-free, split-free, defect-free, discontinuous) of the porous microband, as well as the nature of the pores (e.g., pore size (1 nm to <10 μm), pore size distribution, and distribution of pores throughout the microband).

[0034] In certain embodiments, the microbands are patterned microbands having zones or regions with different properties, in some of these embodiments, the microbands include cross-linking regions effective to capture the analyte bands.

[0035] In certain embodiments, the microchannels and ultimately the microbands are straight or curvilinear (eg, zigzag, spiral, circular).

[0036] In some embodiments, the microchannels and ultimately the microbands are in a non-porous substrate (e.g., via photolithography or microfabrication processes). In other embodiments, the microchannels are formed on top of a non-porous substrate (e.g., via micromolding in a capillary) and then laterally clad with non-porous walls on either side of the microbands.

[0037] The term "non-porous" refers to a substrate material that is non-porous to the solvent and solvent system used to elute the sample in the TLC device. Suitable non-porous substrates include inorganic (e.g., glass, silicon, quartz), metal, plastic, or organic (e.g., polymeric) substrates. Non-porous substrates can be inorganic (e.g., glass, silicon, quartz), organic (e.g., polymer plate), or metal. In some embodiments, the substrate is glass, quartz, plastic, polymer, ceramic, metal, or silicon. In some embodiments, the substrate is glass.

[0038] In some embodiments, the device comprises a single microchannel that contains a porous, substantially homogeneous microband.

[0039] In other embodiments, the device contains between 2 and about 1,000,000 microchannels, each microchannel containing a porous, substantially homogeneous microband. A typical TLC plate is 20 cm by 20 cm. In one embodiment, for such a plate, the device contains 10,000 channels (10 μm width, 10 μm gap). In another embodiment, the device contains 2,000 or fewer channels (50 μm width, 50 μm gap). In a further embodiment, using high precision fabrication techniques (e.g., laser machining, e-beam), the device contains up to 100,000 channels (10 μm width, 5 μm gap).

[0040] The exemplary TLC apparatus of the present invention is different from other conventional TLC apparatus. Conventional TLC, HPTLC, and UTLC are continuous and do not rely on or use microbands. The narrow bands of conventional TLC apparatus are not the microbands of the TLC apparatus described herein. Although narrow bands have been described in conventional apparatus, these narrow bands have widths of more than 200 μm and none of them have the dimensions of the microbands of the exemplary TLC apparatus of the present invention as described herein.

[0041] Conventional TLC are defined as "continuous" because their porous stationary phase is not covered laterally by non-porous walls. The microbands of the devices described herein are not "continuous" because they are covered laterally by non-porous walls. As a result of this covering, the movement of analytes is restricted along the microbands, without lateral diffusion.

[0042] The TLC apparatus described herein is chromatograph the contents of single cells; chromatographing the contents of a single organism; chromatograph the contents of virus particles; chromatographing analytes in a sample having a volume of about 1 zL to about 100 nL; Chromatograph samples in forensic drug analysis, toxicology, explosives analysis, and analysis of dyes and inks for the identification and comparison of drugs, explosives, inks and dyes, toxins, and pesticides; and To assay for drug candidates and drug metabolites, It can be used.

[0043] How to make a TLC device In another aspect, a method of making a chromatography device is provided. In one embodiment, the invention provides a method of making a thin layer chromatography device, comprising: (a) forming one or more microchannels in a non-porous substrate; (b) filling one or more microchannels with a flowable porous microband precursor composition; and (c) subjecting the non-porous substrate having one or more microchannels filled with a flowable porous microband precursor composition to conditions that convert the composition into porous microbands to obtain a thin layer chromatography device. The present invention provides a method comprising:

[0044] In one embodiment, the preparation comprises the steps of: (1) forming microchannels in glass; (2) temporarily sealing the microchannel with an elastomer slab (e.g., PDMS); (3) filling the microchannel with a flowable silica sol-gel slurry; (4) converting the flowable sol-gel composition into a crack-free, monolithic silica porous structure (gelling and aging); and (5) Release of the elastomer slab from the glass to reveal the porous microbands. Includes.

[0045] The fabrication method advantageously includes step (2): sealing the microchannel with an elastomer slab, which provides the enclosed microchannel necessary to carry out steps (3) and (4).

[0046] In some embodiments of the TLC device described herein, the porous microband is a porous matrix made of monolithic porous silica. Monolithic porous silica is difficult to microfabricate into microbands due to crack formation. Traditionally, monolithic porous silica is formed inside a column or capillary tube by sol-gel chemistry. To date, the fabrication of monolithic porous silica microbands as described herein is unknown. The fabrication methods described herein, for example, the combination of steps (1)-(5) above, result in novel crack-free (split-free, defect-free) (covered with non-porous walls) monolithic porous silica microbands.

[0047] Although this preparation method has been demonstrated to be advantageous for monolithic porous silica, it will be appreciated that other porous matrices, including materials other than silica, are also useful for the microbands of the TLC devices described herein and are within the scope of the present invention.

[0048] As noted above, in some embodiments, the method includes surrounding one or more microchannels (e.g., with a PDMS slab) prior to filling with the precursor composition. In some embodiments, the precursor composition includes tetramethyl orthosilicate, polyethylene glycol, and urea.

[0049] In certain embodiments, the step of subjecting the non-porous substrate to conditions to convert the precursor composition to porous microbands includes polymerizing the precursor composition to obtain a gel, subjecting the gel to heat (120° C.) and pressure (15 PSI) to obtain a porous gel (Ostwald ripening), exposing the porous gel by removing the enclosure, and then subjecting the non-porous substrate with the exposed porous gel to heat (330° C.) to calcinate the porous gel, thereby obtaining a non-porous substrate comprising one or more monolithic porous silica microbands.

[0050] The shape, width, depth, length, aspect ratio, cross-sectional area, and composition of the microbands are as described above for the TLC apparatus.

[0051] The non-porous substrate is as described above for the TLC instrument. In some embodiments, the microchannels are on the surface of a non-porous substrate (e.g., via photolithography or microfabrication processes) or are formed on top of a non-porous substrate (e.g., via micromolding in a capillary) and then laterally covered with a non-porous wall.

[0052] A schematic of a representative fabrication process is shown in FIG. 2A. The process illustrates sol-gel micromolding in microcapillaries (i.e., microchannels). With reference to FIG. 2A, soda-lime glass slides (3 inches by 1 inch) carrying linear microfluidic channels with an array of openings were fabricated by photolithography and wet etching (see, for example, Castano-Alvarez, M.; Ayuso, DFP; Granda, MG; Fernandez-Abedul, MT; Garcia, JR; Costa-Garcia, A., Critical points in the fabrication of microfluidic devices on glass substrates, Sens. Actuator B-Chem. 2008, 130(1), 436-448). The channels in this example have a width of 60-80 μm, a depth of 13 μm, and a length of 60 mm. To enhance the bonding between the glass and the silica monolith, the channel surface was roughened by etching with 7.5% aqueous sodium bicarbonate solution at 70 °C for 24 h. The channel was enclosed by attaching a polydimethylsiloxane (PDMS) slab. A monolithic silica sol-gel precursor was prepared by mixing 5.6 mL of tetramethyl orthosilicate (TMOS), 1.2–1.5 g of polyethylene glycol (PEG, MW = 10,000 g / mol), 0.9 g of urea, and 10 mL of aqueous acetic acid (10 mM) for 45 min on ice. The channel was then filled with the precursor and incubated in a humidified chamber at 40 °C for 24 h to polymerize the silica gel. PEG acted as a phase separation inducer to form a macroporous silica structure. After gelation, the glass slide (with the PDMS slab attached) was immersed in 45 mL of 10 mM acetic acid in water containing urea (resulting in a urea concentration of 0.09 g / mL) in a polypropylene Coplin jar and autoclaved at 120° C. and 15 pounds per square inch (PSI) for 4 hours to form mesopores via the Ostwald ripening process. The plate was then rinsed with water and dried in air.The PDMS slab was then cut away from the glass slide to expose the silica microbands. The slide was then incubated in water for 2 hours to leach the PEG and urea from the silica monolith. To further remove the organic polymer PEG from the silica monolith, the slide was immersed in sulfuric acid for 24 hours. The slide was then incubated in water for 24 hours to remove the sulfuric acid. Finally, the monolith / slide was calcined by baking on a hotplate at 330 °C for 24 hours to provide a mechanically stable silica monolith.

[0053] To confirm the formation of porous silica monolithic microbands, the glass slides were examined under a bright-field microscope (Figures 2B and 2C). The glass channel was filled with silica monoliths, with the top surface open to air (Figure 2B). The silica bands are dark and opaque due to their porous structure and the difference in refractive index between silica (1.475) and air (1). As silica monoliths are hydrophilic and water easily enters the porous structure, the bands become clear and transparent upon immersion in water due to the similar refractive index between silica (1.475) and water (1.33) (Figure 3C).

[0054] The above description shows an example of a fabrication method (micromolding in microcapillaries or microchannels) for generating pTLC bands. However, pTLCs can be fabricated from alternative strategies such as 3D printing, microtransfer molding, solvent-assisted micromolding, replica micromolding, microcontact molding, photolithography, soft lithography, stereolithography, micro / nano-imprint lithography, electrospinning, and inkjet printing.

[0055] How to Use a TLC Instrument In a further aspect, the present invention provides methods of using the chromatography devices described herein.

[0056] In certain embodiments, the present invention provides methods for thin layer chromatography of the contents of single cells, cell populations, or microsamples using the chromatography devices (i.e., TLC devices) described herein. In some of these embodiments, the contents are biological analytes. In certain embodiments, the biological analytes are lipids. In other embodiments, the biological analytes are fatty acids, amino acids, peptides, lipopeptides, vitamins, hormones, proteins, carbohydrates, enzyme substrates, and metabolites.

[0057] In other embodiments, the invention provides methods for thin layer chromatography of an analyte in a sample having a volume of about 1 zL to about 100 nL. In some of these embodiments, the analyte is a fatty acid, an amino acid, a peptide, a lipopeptide, a protein, a carbohydrate, a metabolite, a hormone, an antigen, pollen, a spore, a vitamin, a cytokine, an indole, an antibiotic, a pigment, a steroid, a phenol, a bile acid, a coumarin, a pharmaceutical, a reactive analyte, a toxin, a pesticide, an insecticide, a poison, or a pollution contaminant.

[0058] In the methods described herein, samples for chromatography may be deposited by inkjet printing, contact printing, microfluidic delivery, micropipette deposition, nebulization (electrospray, electrodeposition, etc.), electrowetting, optoelectrowetting, dielectrophoresis, surface acoustic wave deposition, magnetic or electrostatic droplet deposition, immiscible fluid flow, microtraps, microweirs, microholes, docking sites, microvalving, optical tweezers, or pressure- and magnetic-based manipulation.

[0059] In the methods described herein, the chromatographic sample may be moved by capillary forces, pressure, vacuum, centrifugal forces, electrophoretic forces, osmotic forces or magnetic forces.

[0060] In the methods described herein, the analytes may be detected by detection techniques known in the art for detecting analytes separated on a chromatographic support. Exemplary detection techniques include fluorescence absorption (UV, visible, and infrared light), chemical dyes, luminescence, infrared spectroscopy, surface-enhanced Raman spectroscopy, mass spectrometry, radioactivity, colorimetry, precipitate formation, electrochemistry, nuclear magnetic resonance, or electrochemiluminescence.

[0061] In certain embodiments, the devices described herein are used to chromatograph analytes in (i) the contents of a single cell, (ii) the contents of a single organism, (iii) the contents of a viral particle, or (iv) a sample having a volume of about 1 zL to about 100 nL.

[0062] The devices described herein may be used to chromatograph samples in forensic drug analysis, toxicology, explosives analysis, and analysis of dyes and inks for identification and comparison of drugs, explosives, inks and dyes, toxins, and pesticides; and to chromatograph and assay drug candidates and drug metabolites.

[0063] The following description demonstrates the separation efficiency of a representative TLC apparatus of the present invention prepared as described herein.

[0064] Separation of Spotted Lipid Mixtures by pTLC. The separation efficiency of a representative pTLC device prepared as described above was evaluated using model lipids. Two model fluorescent lipids (18:1 PE CF and Texas Red DHPE) were selected to determine whether pTLC can separate organic compounds. Parafilm was cut into a sharp triangular shape and its tip was used to microspot 300-1,000 picoliters of sample onto the microband of the pTLC device. To spot the sample, the parafilm tip was immersed in the fluorescent lipid solution (1 μg / mL in ethanol), removed, and immediately placed against the sample zone of the pTLC plate. The parafilm was soft enough not to damage the silica monolith. After spotting the sample, the pTLC device was placed face down and developed in a Petri dish in a horizontal position, as shown in Figure 3A. Solvent was drawn from a wiper at one end into the silica microband. The separation was monitored with an Olympus FluoView FV3000 confocal laser scanning microscope. 18:1 PE CF and Texas Red DHPE were successfully separated using 1-butanol:water:acetic acid (18:4:4, vol:vol:vol) in only 3 minutes with a short migration distance of 360 μm (Figure 3B). The average migration speed of 264 μm / min over 9 minutes for 18:1 PE CF is faster than that of 101 μm / min for Texas Red DHPE. The migration speed is related to the interaction between the compound and the silica. Texas Red DHPE has more charged groups (two tertiary amines, one sulfonate) than 18:1 PE CF (one carbonate). Therefore, Texas Red DHPE has more interactions with the silica (whose surface is charged) and moves slower than 18:1 PE CF.

[0065] To further demonstrate the utility of pTLC, we isolated two biologically important compounds, sphingosine and sphingosine 1-phosphate (Figure 3C). Sphingosine 1-phosphate (S1P) is a metabolic product of sphingosine (Sp), formed by the action of sphingosine kinase (SK) in cells. Analysis of S1P and Sp in biological samples is useful to elucidate lipid signaling in pathology and therapy, and it has been recognized as a key mediator in infections by lipid-enveloped viruses, such as SARS, MERS and coronavirus (COVID 19). Due to the cell proliferation properties of S1P, the majority of tumors exploit this pathway to accelerate their malignant behavior via gene mutations, overexpression and other strategies to upregulate this pathway. The SK pathway is also involved in immunopathologies such as autoimmune diseases. As an example, fingolimod is a sphingosine-1-phosphate receptor modulator used to treat multiple sclerosis. Other morpholino analogs as well as ozanimod and siponipod are envisioned as new drug candidates targeting the sphingosine pathway in autoimmune diseases, as well as in encephalitis, arthritis, traumatic brain injury, cancer, ulcerative colitis, viral and bacterial infections, fibrosis, renal disease, allograft rejection and other pathologies.

[0066] Fluorescein-labeled Sp and S1P solutions (10 μM in ethanol) were spotted onto a pTLC plate using a parafilm tip. The only difference between sphingosine fluorescein (SpF) and sphingosine 1-phosphate fluorescein (S1PF) is the negative phosphate group in S1PF. SpF was successfully separated from S1PF by developing with 1-butanol:water:acetic acid (18:1:1, vol:vol:vol). The average migration speed of SpF over 16 min, 170 μm / min, is faster than that of S1PF, 36 μm / min. The negative phosphate group of S1PF hinders the migration of S1PF due to its interaction with silica. The above results demonstrate that pTLC can separate microspotted organic compounds. One of the unique advantages of pTLC is that migration is confined along the microband without lateral diffusion. Other derivatized lipids (bearing fluorescent or clickable moieties) are similarly suitable for separation and assay by pTLC.

[0067] Representative lipids that can be analyzed using pTLC include saturated fatty acids, unsaturated fatty acids, phospholipids, sphingolipids, glycolipids, glycerophospholipids, cholesterol, steroids, triacylglycerols, triacylglycerides, fatty acids, bile salts, eicosanoids, ketones, fatty acyls, glycerolipids, glycolipids, polyketides, glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoserine, glycerophosphoglycerol, glycerophosphate, glycerophosphoinolates, and glycerophosphoinolates. These include glycerophosphoinositol, glycerophosphoinositol monophosphate, glycerophosphoinositol bis-phosphate, glycerophosphoinositol tris-phosphate, glycerophosphoglycerophosphoglycerol, cardiolipin, glycerophosphoglycerophosphate, glyceropyrophosphate, CDP-glycerol CDP-DG, glycosylglycerophospholipids, glycerophosphoinositol glycan, glycerophosphonocholine, and glycerophosphonoethanolamine.

[0068] Other sample deposition strategies with sufficient spatial and volumetric resolution include inkjet printing, contact printing, microfluidic delivery, micropipette deposition, nebulization (electrospray, electrodeposition, etc.), electrowetting, optoelectrowetting, dielectrophoresis, surface acoustic wave deposition, magnetic or electrostatic droplet deposition, immiscible fluid flow, and others. Droplets can be generated by instruments (such as flow cytometry) and deposited directly onto pTLC bands. Additionally, microtraps, microwells, microholes, docking sites, microvalving, optical tweezers, or pressure- and magnetic-based manipulations can be used for precise sample positioning at the sample deposition area.

[0069] The above description provides two examples of lipids that can be separated by pTLC. In addition to lipids, pTLC can separate other biological analytes, including fatty acids, amino acids, peptides, lipopeptides, proteins, carbohydrates, metabolites, hormones, antigens, pollen, spores, vitamins, cytokines, indoles, antibiotics, pigments, steroids, phenols, bile acids, coumarins, medicines, reaction analytes, toxins, pesticides, insecticides, poisons, and pollution contaminants. In addition to biological analytes, pTLC can be used for forensic drug analysis, toxicology, explosives analysis, and the analysis of dyes and inks for the identification and comparison of drugs, explosives, inks and dyes, toxins, and pesticides. Particularly valuable are the assays of drug candidates, drug metabolites, and synthetic reaction contaminants or by-products.

[0070] Fluorescence was used as an example of analyte detection. Other detection strategies are suitable for pTLC (with or without signal amplification), including absorption (UV, visible, and infrared light), chemical dyes, luminescence, infrared and other spectroscopy, surface-enhanced Raman spectroscopy, mass spectrometry, radioactivity, colorimetry, precipitate formation, electrochemistry, nuclear magnetic resonance, and electrochemiluminescence.

[0071] Isolation of lipophilic red / green lipids from single cells. To demonstrate the potential of pTLC for single cell analysis, two model fluorescent lipids (DiO and DiD) were first loaded into U937 cells (monocyte-derived tumor cells, ~1 pL in volume, 10-20 μm in size), and then single U937 cells were spotted onto the microband of a representative pTLC device prepared as described above for separation (Figure 4). DiO or DiD (chemical structures shown in Figure 4A) are lipophilic fluorescent stains for labeling cell membranes and other hydrophobic structures. These dyes have high extinction coefficients and exhibit distinct fluorescent colors (DiO: green fluorescence; DiD: red fluorescence). U937 cells were incubated with DiO and DiD (5 μM in Hank's balanced salt solution) for 20 min at 37 °C to allow for lipid uptake by the cells. Cells were fixed with 4% paraformaldehyde in PBS for 15 min, followed by rinsing five times with PBS. The cells showed both red and green fluorescence, with the expected cell-to-cell heterogeneity (Figure 4B). To spot single cells, a parafilm tip was dipped into the cell suspension (10,000 cells / mL), removed, and immediately contacted with the pTLC microband. By Poisson statistics, most microbands were spotted with 0 and >1 cells. The number of deposited single cells is easily optimized using microfluidics, inkjet printing, microdroplets, cell docking sites, electrophoresis, contact printing, micropipette deposition, spraying (electrospray, electrodeposition, etc.), electrowetting, optoelectrowetting, dielectrophoresis, immiscible fluid flow, and others. In addition, microtraps, microwells, microholes, microvalving, optical tweezers, or pressure- and magnetic-based manipulations can be used for precise cell positioning at the sample deposition area. Only microbands with one cell spotted were used for the assay. The presence of a single cell was confirmed by microscopy. The pTLC plate was developed with 1-pentanol in the setup shown in FIG. 3A.The elution of DiO / DiD from single cells and migration along the microband were imaged using an Olympus FluoView FV3000 confocal laser scanning microscope (Figure 4C). DiO and DiD were extracted from cells and separated in only 2 min. In 5 min, DiD was completely separated from DiO. DiO is more polar, so migration of DiD is faster than DiO. These results demonstrate that the TLC technique described herein can be applied to assay the contents of single cells. This result was achieved by generating a cytocompatible sample loading zone, a microband containing cell contents to minimize dilution, a single-cell deposition strategy, and an adsorbent with features appropriate for cell size. The ability to assay single-cell contents was further enhanced by combining a highly sensitive detection method.

[0072] In addition to the red and green fluorophores described herein, other fluorophores with emission colors in the blue, light green, dark green, yellow, orange, red, far red, and infrared ranges are suitable for pTLC detection. These fluorophores span the near UV, visible, and near IR spectra.

[0073] Although U937 cells are monocyte-derived tumor cells, other tumor and normal cell types can also be assayed, such as lymphocytes, neutrophils, eosinophils, basophils, macrophages, erythrocytes, platelets, megakaryocytes, platelets, mast cells, natural killer cells, plasma cells, epithelial cells, neurons, muscle cells, adipocytes, skin cells, nerve cells, endothelial cells, bone cells, fibroblasts, egg cells, sperm cells, embryonic stem cells, tissue-specific stem cells (e.g., hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, skin stem cells), induced pluripotent stem cells, cardiac cells, intestinal cells, muscle, fibroblasts, kidney, liver, neurons / nerves, retina, lung, spleen, bone, and any other cells or tissues found in mammals, humans, plants, insects or animals.

[0074] Isolation of sphingosine metabolites from single cells. Although DiO or DiD can be isolated and detected from single cells by pTLC as described above, many biologically important compounds inside cells, such as sphingosine and sphingosine 1-phosphate, are non-fluorescent or present at very low concentrations. To overcome these challenges, analyte amplification strategies, highly sensitive detection methods, and sensors can be developed. Examples of such strategies follow. Using click chemistry, the non-fluorescent sensor loaded inside the cells was conjugated with a highly fluorescent dye for detection purposes (Figure 5A). U937 cells were incubated with the sensor sphingosine alkyne (10 μM) at 37 °C for 30 min. The sphingosine alkyne sensor enters the cells and is metabolized by cellular enzymes in the same way as sphingosine, since the alkyne is minimally perturbative. For example, sphingosine kinase inside the cells converts sphingosine alkyne to sphingosine 1-phosphate alkyne. The ratio of sphingosine alkyne to its metabolites reveals the activity of sphingosine kinase. Cells are incubated with the loaded sphingosine alkyne and then fixed with 4% paraformaldehyde in PBS for 15 min, followed by rinsing five times with PBS. To detect non-fluorescent alkynes, CY5-azide was conjugated intracellularly with an alkyne moiety using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), commonly known as click chemistry. CY-5 exhibits a sharp absorption band, a high molar extinction coefficient (250,000 cm in ethanol), and a high NMR spectrum. -1 M -1 ), high quantum efficiency, and excellent resistance to photobleaching (Figure 5B).

[0075] To detect the ratio of Sp-CY5 to S1P-CY5 in single cells, single cells were spotted onto the pTLC microband of a representative pTLC device prepared as described above using a parafilm tip. The pTLC plate was developed with 1-butanol:1-propanol:water:triethylamine (18:2:4:1, vol:vol:vol:vol). Sp-CY5 and S1P-CY5 eluted from the single cells and migrated along the microband. CY5 fluorescence was monitored using a microscope (Figure 5C). Sp-CY5 and S1P-CY5 migrated away from the cells within 2 min. The lipids were separated as early as 4 min. After 4 min, S1P-CY5 was completely separated from Sp-CY5 (resolution=1). Migration of S1P-CY5 is slower than Sp-CY5 because S1P-CY5 has an extra phosphate group and is therefore more polar. Quantitative analysis of the pTLC chromatograms at 11 min shows that the S1P-CY5 peak area is 33.8% and the Sp-CY5 peak area is 66.2% of the total CY5 fluorescence measured. This data demonstrates that this particular cell converts 33.8% of the loaded Sp alkyne to S1P alkyne in 30 min via the action of sphingosine kinase. This result demonstrates that the pTLC technique described herein can be used to analyze biologically important compounds in single cells.

[0076] Examples of other clickable lipids include photoclick sphingosine [(2S,3R,E)-2-amino-13-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tridec-4-ene-1,3-diol], 27-alkyne cholesterol, alkyne-cholesterol, photoclick cholesterol, click PI(4,5)P2-azide, 18:0 propargyrin, and 18:0 propargyrin. 16:0 propargyl SM (d18:1-16:0), C6(6-azido)ceramide, C6(6-azido)LacCer, C6(6-azido)GalCer, C6(6-azido)GluCer, 16:0 azido coenzyme A, N3C14SOBRAC, trifunctional sphingosine, 16:0 azidocaproyl PE, 16:0 hexinoyl PE, 16:0 DBCO PE, 18:1 DBCO PE, 18:0 azidoethyl PC, 16:0 azidoethyl SM (d18:1 / 16:0), IKS02, 18:0-16:0(16-azido)PC, DSPE PEG(2000)azide, DSPE-PEG(2000)-DBCO, trifunctional fatty acid, palmitic acid(15-yne), pacFA, pacFA ceramide, pacFA GlcCer, pacFA GalCer, 16:0-pacFA PC, pacFA-18:1 PC, arachidonic acid-alkyne, oleic acid(17-yne), 16:0(alkyne)-18:1 PC, 16:0(alkyne)-18:1 These include PE, oleic acid (18-azide), Photoclick sphingosine-1-phosphate, Photoclick C6 ceramide, Photoclick GM1 (synthetic), including sphingosine alkyne and sphingosine-1-phosphate alkyne, all of which are commercially available.A wide range of other clickable probes suitable for cell-based assays include, among others: other fluorophores, gelling agents, PEG, nucleotides, bis-dPEG 11-DBCO, azidopropyl vinylsulfonamide bifunctional click reagents, biotin, docosahexaenoic acid alkyne, 4-hydroxynonenal alkyne, farnesyl alcohol azide, 6-azidohexanoic acid, oleic acid alkyne, eicosapentaenoic acid alkyne, palmitic acid alkyne, 12(S)-HETE-19,20-alkyne, arachidonic acid alkyne, myristic acid alkyne, palmitoyl alkyne-coenzyme A ... Nal alkyne, farnesyl alcohol azide, 6-azidohexanoic acid, oleic acid alkyne, L-homopropargylglycine, 4-pentynoyl-Coenzyme A, eicosapentaenoic acid alkyne, palmitic acid alkyne, 5-azidopentanoic acid, 12(S)-HETE-19,20-alkyne, arachidonic acid alkyne, myristate acid alkyne, palmitoyl alkyne-Coenzyme A (trifluoroacetate), 15-hexadecynoyl-CoA, palmitoyl alkyne-CoA.

[0077] In certain embodiments, the analytes can be labeled after separation and before detection, hi certain embodiments, the analytes can be labeled in situ within the sample itself, before separation.

[0078] In some embodiments, compounds within a cell can have both crosslinkable and clickable moieties (e.g., azides, alkynes). In these methods, compounds can be separated by pTLC, crosslinked with microbands to immobilize the compounds, and then clicked with, for example, a fluorescent dye for visualization.

[0079] In an embodiment, the pTLC device of the present invention can be used for high-throughput screening, for example for parallel analysis in drug discovery. Because of the microscale width of each band, a high-density array of microbands can be generated. For example, if the band width is 50 μm and the band gap is 50 μm, 2,000 bands can be generated on a 20×20 cm plate. A microdispenser or printer can be used to spot samples onto the bands.

[0080] Thus, the TLC techniques described herein can be advantageously applied to integrated high-throughput and highly parallel microfluidic devices; assays for drug screening; assays for combinatorial chemistry screening; reaction screening; cell-to-cell heterogeneity analysis; subcellular organelle assays; and virus detection assays, including chickenpox, influenza, herpes, HIV / AIDS, human papillomavirus, parotitis, measles, rubella, shingles, hepatitis, meningitis, pneumonia, coronaviruses such as MERS, SARS, and COVID 19, among others. Enveloped viruses are amenable to these assays.

[0081] As noted above, in certain embodiments, the pTLC devices of the present invention can be used at very small scale sample volumes (e.g., 1 zeptoliter to 100 nL) and are therefore suitable for the analysis of precious, rare or very small samples (such as forensic specimens).

[0082] As used herein, the term "about" refers to ±5% of the stated value. While illustrative embodiments have been illustrated and described, it will be recognized that various changes can be made therein without departing from the spirit and scope of the invention.

[0083] The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

Claims

1. A thin layer chromatography (TLC) device comprising a non-porous substrate having one or more microchannels formed on a surface thereof, each microchannel comprising a porous, substantially homogeneous microband adapted for fluid flow driven by capillary action.

2. The apparatus of claim 1 , wherein the porous microband is a monolithic porous silica microband.

3. 2. The device of claim 1, wherein the fluid flow is capillary fluid flow combined with electrophoresis, or the fluid flow is capillary fluid flow driven by intermolecular forces, or the fluid flow is not driven by hydrodynamic centrifugal forces or pressure-driven fluid flow.

4. 4. The device according to claim 1, wherein the microchannels are at the surface of the non-porous substrate or are formed on top of the non-porous substrate and subsequently covered laterally with a non-porous wall.

5. The device according to claim 1 , wherein the microchannel is straight or curved.

6. The device of any one of claims 1 to 3, wherein the porous microbands have a width of about 1 to about 1000 μm.

7. The device of any one of claims 1 to 3, wherein the porous microband has a depth of about 1 to about 100 μm.

8. 4. The device of claim 1, wherein the porous microband has a length that is substantially in the same range as the length of the device.

9. The device of any one of claims 1 to 3, wherein the porous microbands have an aspect ratio (width / depth) of about 1 to about 100.

10. The porous microband is about 10 μm ２ ~Approx. 50,000μm ２ 4. The device of claim 1 having a cross-sectional area of

11. The device of claim 1 , wherein the substrate is glass, quartz, plastic, polymer, ceramic, metal, or silicon.

12. 4. The device of claim 1, wherein the device comprises a single microchannel that contains the porous, substantially homogeneous microband.

13. 1. A method of making a thin layer chromatography device, comprising: (a) forming one or more microchannels in a non-porous substrate; (b) filling said one or more microchannels with a flowable porous microband precursor composition; and (c) subjecting said non-porous substrate having one or more microchannels filled with a flowable porous microband precursor composition to conditions that convert said composition into porous microbands to obtain a thin layer chromatography device. A method comprising:

14. A method for thin layer chromatography of single cells, cell populations or the contents of a microsample using a device according to any one of claims 1 to 12.

15. About 1 zL to about 100 nL of solution using the device according to any one of claims 1 to 12. A method for thin layer chromatography of an analyte in a sample having a volume.

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