Selective Near-Infrared Blood Detection Driven by Ionic Liquid-Dye-Albumin Nanointeractions

Abstract

Due to its abundance in blood, a great deal of research has been undertaken to develop efficient biosensors for serum albumin and provide insight into the interactions that take place between these biosensing molecules and the protein. Near-infrared (NIR, >700 nm) organic dyes have been shown to be effective biosensors of serum albumin, but their effectiveness is diminished in whole blood. Herein, it is shown that an NIR sulfonate indolizine-donor-based squaraine dye, SO₃SQ, can be strengthened as a biosensor of albumin through the addition of biocompatible ionic liquids (ILs). Specifically, the IL choline glycolate (1:1), at a concentration of 160 mM, results in the enhanced fluorescence emission ("switch-on") of the dye in the presence of blood. The origin of the fluorescence enhancement was investigated via methods, including DLS, ITC, and molecular dynamics. Further, fluorescence measurements were conducted to see the impact the dye-IL system had on the fluorescence of the tryptophan residue of human serum albumin (HSA), as well as to determine its apparent association constants in relation to albumin. Circular dichroism (CD) spectroscopy was used to provide evidence that the dye-IL system does not alter the secondary structures of albumin or DNA. Our results suggest that the enhanced fluorescence of the dye in the presence of IL and blood is due to diversification of binding sites in albumin, controlled by the interaction of the IL-dye-albumin complex.

Figure 1.

SO₃SQ dye shows switch-on fluorescence in the presence of blood that is enhanced 5-fold in the presence of 160 mM IL. Fluorescence scans of 10 μM dye in the (A) presence of blood and (B) absence of blood as the concentration of IL is varied from 0 (green solid line), 40 (gray dashed line), 80 (blue solid line), 120 (purple solid line), 160 (red solid line), 200 (navy dashed line), and 240 mM (black solid line) of IL in water. (C) The fluorescent emission of 10 μM SO₃SQ (no IL) in the absence (green line) and presence of blood (blue line) contrasts with the emission in the presence of 160 mM IL in the absence (purple) and presence (red line) of blood.

Figure 2.

CG (160 mM, 1:1) allows for a greater binding affinity between SO₃SQ and HSA. Normalized fluorescence intensity as a function of albumin concentration was used to compare the binding affinities of a solution of 10 μM SO₃SQ (black) and a solution of 160 mM CG (1:1) in 10 μM SO₃SQ to HSA (red).

Figure 3.

The addition of SO₃SQ, CG 1:1, or a combination of the two, to a solution of HSA causes a decrease in the fluorescence of the tryptophan residue. (A) The structure of HSA with location of tryptophan residue; Fluorescence spectra of tryptophan residues under 3 conditions: (B) HSA in water (blue) with the addition of 160 mM CG (1:1) (green); (C) HSA in 10 μM SO3SQ (black) with the addition of 160 mM CG (1:1) (red); (D) HSA in solutions containing 160 mM CG (1:1) (black) prior to adding SO₃SQ (purple).

Figure 4.

The components of the IL/dye system show a greater binding affinity for the heme cleft. Results of the Molecular Dynamics simulation for complexes of SO3SQ and glycolate with HSA within (a) heme cleft, (b) Sudlow’s site I, (c) Sudlow’s site II, (d) site near R485, and (e) results of MM/GBSA calculation for the ligand–albumin complex after MD simulation and trajectory clustering.

Figure 5.

Proximity of the glycolate anion to W214 results in interactions that lead to quenching of the W214 fluorescence. Superposition of ligand–protein complexes: (a) superimposed protein structures with backbone illustrated as a curved line; (b) superimposed W214 residue.

Figure 6.

The presence of 1:1 CG results in more favorable binding of SO₃SQ to the heme cleft of HSA, and slightly more improved binding in Sudlow’s site II. Results of the Molecular Dynamics simulation for complexes of SO₃SQ-G-HSA with (a) glycolate in the heme cleft and SO₃SQ in Sudlow’s site II and (b) glycolate in Sudlow’s site II and SO₃SQ in the heme cleft.

Figure 7.

Adding CG 1:1 to HSA results in a size increase and a charge that is less negative. Adding CG 1:1 to a system containing 10 μM SO3SQ and HSA causes the overall complex to increase in size and charge. Hydrodynamic diameter (intensity) of (A) HSA in water (black) and HSA in water with 160 mM CG 1:1 added (red). Zeta potential of (B) HSA in water (black), HSA in water with a 1:1 addition of 160 mM CG 1:1 added (red). Hydrodynamic diameter of (C) HSA in 10 μM SO3SQ (black), HSA in 10 μM SO3SQ with 1:1 added 160 mM CG 1:1 added (blue). Zeta potential of (D) HSA in 10 μM SO3SQ (black), HSA in 10 μM SO3SQ with a 1:1 addition of 160 mM CG 1:1 added (blue).

Figure 8.

The interaction between SO₃SQ and HSA is exothermic in the absence of 1:1 CG, but is endothermic in the presence of CG 1:1. Representative isothermal titration calorimetry of HSA titrations into SO₃SQ. (A) A conventional reverse titration, showing the baseline-corrected injections (top) and integrated heats (bottom) in the absence of IL. The red line indicates the best fit finding profile, which has an exothermic heat. (B) The same experiment was performed in the presence of 40 mM IL. The enthalpy becomes positive, and the complete binding profile is not observed because of solubility limitations. The red line shows the average of the first 10 injections, which was used to estimate the enthalpy of binding. Conditions are provided in the main text.

Figure 9.

The 1:1 SO₃SQ/CG system does not destroy albumin’s secondary structure or compromise the integrity of DNA. CD spectra of (a) human serum albumin (HSA) (black), HSA in 10 μM dye (red), and HSA in 10 μM dye with 160 mM CG 1:1. Overlaid CD spectra of (b) DNA only (blue) and DNA in 2.5% IL (red), (c) 9.14 μM dye in water (blue) and dye in 2.5% IL (orange), (d) DNA in 2.5% IL (black), DNA in 2.5% IL with 5 μL of 1 μM dye solution (red), DNA in 2.5% IL with 10 μL of 1 μM dye solution (yellow), and DNA in 2.5% IL with 15 μL of 1 μM dye solution (blue).

Scheme 1.

Dropwise Addition of a 1:1 Molar Ratio of Choline Bicarbonate to Glycolic Acid to Synthesize the IL Choline Glycolate (CG (1:1)) via a Salt Metathesis Reaction