Research ArticleCHEMICAL PHYSICS

Label-free quantitation of glycated hemoglobin in single red blood cells by transient absorption microscopy and phasor analysis

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Science Advances  10 May 2019:
Vol. 5, no. 5, eaav0561
DOI: 10.1126/sciadv.aav0561
  • Fig. 1 Protein structure of N terminus of the β chain of HbA1c and Hb through PyMOL simulation.

    (A and B) Protein structure of the glycation site of HbA1c, and normal Hb, respectively. The crystal structure of HbA1c (3B75) and Hb (1LFZ) are from Protein Data Bank. Cyan sphere, water molecule; red sphere, oxygen atom; green sphere, carbon atom; blue sphere, nitrogen atom. In (A), the polar force between glucose and heme and surrounding water is highlighted by blue dashed lines. Regions of interest (ROIs) are highlighted by red dashed circles. (C and D) Zoom-in view of the interaction between glucose and porphyrin ring from glycated Hb (C) and normal Hb (D).

  • Fig. 2 Comparative characterization of Hb and HbA1c by fluorescence, time-resolved photoluminescence, and absorption spectroscopy.

    (A and B) Fluorescence spectra of Hb (0.025 mg/ml) (A) along with HbA1c (0.025 mg/ml) (B), respectively. Excitation wavelength, 447 nm; integration time, 1000 s; band-pass filter, 488 ± 10 nm; power on the sample, 150 μW. 20× air objective. (C and D) Time-resolved photoluminescence (PL) measurements of Hb (0.025 mg/ml) (C) and HbA1c (0.025 mg/ml) (D), respectively. a.u., arbitrary units. (E and F) Absorption spectra (normalized) of Hb (E) and HbA1c (F), respectively.

  • Fig. 3 Comparison of transient absorption decay signatures between Hb and HbA1c.

    (A and B) Time-resolved decay curves (normalized) of Hb (A) and HbA1c (B), respectively. int., intensity. (C) Merged time-resolved curves (normalized) of Hb and HbA1c. (D and E) Time-resolved decay curves (normalized) of oxyHb (D) and oxyHbA1c (E), respectively. (F) Merged time-resolved curves (normalized) of oxyHb and oxyHbA1c. (G) Proposed excited-state dynamic pathway of Hb when pumped at 520 nm and probed at 780 nm.

  • Fig. 4 Quantitation of HbA1c in a series of solutions by phasor analysis of transient absorption traces.

    (A) Time-resolved decay curves (normalized) of standard HbA1c solutions (human whole blood based) at different concentrations. (B) Zoom-in view of (A) from delay time of 1 to 4 ps. (C) Component s versus different ω from 0 to 2π THz for pure HbA1c and Hb. (D) Phasor plot of standard HbA1c solutions of different concentrations when ω = 0.8π THz. (E) Calibration curve of standard HbA1c solution at different concentrations (component s versus HbA1c%).

  • Fig. 5 Transient absorption imaging of diabetic whole blood and healthy whole blood.

    (A) Pseudocolor transient absorption images (delay time, 0 ps) of single RBCs with ROIs are highlighted by blue dashed circles. Scale bar, 10 μm. Pump: 520 nm, 2 mW on the sample; probe: 780 nm, 10 mW on the sample. int., intensity. (B and C) Time-resolved decay curves (normalized) of ROIs shown in (A). (D to F) HbA1c fraction (in single RBCs) distribution along with the fitted glucose concentration from three diabetic whole blood samples. (G to I) HbA1c fraction (in single RBCs) distribution along with the derived glucose concentration from three healthy whole blood samples. Curve fitted by Eq. 8.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaav0561/DC1

    Fig. S1. Fourier transform Raman spectra of HbA1c (0.025 mg/ml) and Hb (0.025 mg/ml).

    Fig. S2. Schematic of a visible-pump (520 nm), near-infrared probe (780 nm) transient absorption microscope.

    Fig. S3. Transient absorption signal of Hb solution from in-phase channel (cosine channel) and quadrature channel (sine channel) at a phase of 180°.

    Fig. S4. Phasor plots of two standard HbA1c solutions.

    Fig. S5. HbA1c fraction (in single RBC) distribution along with the derived glucose concentration from four new type 2 diabetic whole blood samples.

    Fig. S6. HbA1c fraction (in single RBC) distribution along with the derived glucose concentration from four new healthy whole blood samples.

    Table S1. Key parameter comparison between type 2 diabetic whole blood and healthy whole blood.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Fourier transform Raman spectra of HbA1c (0.025 mg/ml) and Hb (0.025 mg/ml).
    • Fig. S2. Schematic of a visible-pump (520 nm), near-infrared probe (780 nm) transient absorption microscope.
    • Fig. S3. Transient absorption signal of Hb solution from in-phase channel (cosine channel) and quadrature channel (sine channel) at a phase of 180°.
    • Fig. S4. Phasor plots of two standard HbA1c solutions.
    • Fig. S5. HbA1c fraction (in single RBC) distribution along with the derived glucose concentration from four new type 2 diabetic whole blood samples.
    • Fig. S6. HbA1c fraction (in single RBC) distribution along with the derived glucose concentration from four new healthy whole blood samples.
    • Table S1. Key parameter comparison between type 2 diabetic whole blood and healthy whole blood.

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