To demonstrate the effect of small-molecule fluorophore conjugates on cells, we first synthesized an ADPfluorescein conjugate and treated Jurkat T cells. Like fluo-rescein treatment, ADP-fluorescein conjugate was internalized by T cells (Figure ). This led to the design of a cargo molecule similar to a polymer/macromolecule carrier. We designed and synthesized a cargo backbone with only a side-chain cationic group (namely non-covalent cargo) while another cargo backbone with both side-chain cationic and phosphate groups (namely covalent cargo, Figure , Figure ). To test our hypothesis, we also synthesized phospholipid linked non-covalent and covalent cargos (Figure For phospholipid fatty acid, P=O stretching band appeared at 1170 cm -1 , and the P=O stretch was at 1156 cm -1 for non-covalent cargo (Figure ). The decrease in P=O stretching band indicated its presence as anion and/or conjugated form such as a phospho-ammonium group. Similarly, for covalent phospholipid cargo, a larger decrease in P=O stretching band suggests a covalent bond in extended conjugated form as a tri-phosphoester bond. The decrease in P=O stretching band was observed at 972 cm -1 for covalent phospholipid, compared to 1103 cm -1 in ADP and 1075 cm -1 in the covalent cargo (Figure ). After establishing bond formation between our synthetic cargos and the phospholipids, we hypothesized that our noncovalent and covalent cargo molecules will interact with the native cell-surface phospholipids, as outlined in Figure . We conjugated a fluorophore molecule (fluorescein) with both non-covalent and covalent cargo molecules to locate the presence of the cargo molecules on cells (Figure ). Next, we were interested to see whether these cargo molecules will conjugate on the membrane of Jurkat T cells. We identified both optimal molecular weight and concentration for these cargos (Figure ) and used it with an optimal concentration of 0.1 mg/mL to treat the cells at RT for 30 minutes on an orbital shaker followed by stained with Hoechst 33342. Fluorescein was internalized by the cells as observed by the perfect overlap of blue fluorescence of Hoechst 33342 and green fluorescence of fluorescein in the nucleus of the Jurkat cells. When we used the fluorescein-conjugated cargo, the blue fluorescence of Hoechst was observed in the nucleus and green fluorescence of fluorescein was observed at the periphery of the cells (Figure ). The live-cell surface conjugation efficiency of the covalent cargo was better than the non-covalent cargo; suggesting that our hypothesis to develop a dualfunctional covalent cargo was rational for cell-surface conjugation. Next, using various length D-lysine linker backbone, we synthesized three covalent cargos 1 to 3 with molecular weights of 75, 150, and 300 kD respectively (Figure ). We treated the cells with 0.1 mg/mL of the cargo 1-3 at RT for 30 minutes on an orbital shaker. Then, cells were washed, stained with Hoechst 33342, and imaged via fluorescence confocal microscopy (Figure ). The live-cell surface conjugation efficiency of the cargo 2 (150 kD) was better than the other cargos. Since keeping cells alive after cell surface conjugation is a requirement for future applications. We performed the viability test of the surface-modified Jurkat T cells for 3 days by cell-titer blue viability assay, which resulted in more than 80% viability of the surface-modified Jurkat T cells (Figure ) for all these cargos. To further validate the usability of our reagent for membrane imaging of fixed cells we also treated our dual covalent cargo with fixed Jurkat T cells and observed membrane imaging of cells (Figure ). These results suggest that our stable and non-toxic conjugate reagent can be used to image both fixed and live cells. This was a useful application of our cargo 2 -since there are very limited and very expensive non-toxic staining reagents for live cell membrane imaging that do not show long-term stability. Furthermore, to verify the need for dual-cell conjugation, we synthesized a cargo by protecting its cationic ammonium side chains with BOC-groups. We observed no surface conjugation of the Jurkat T cells with the BOC-cargo indicating the plausible role of the cargo decoration chemistry for cell-surface bond formations (Figure ).