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

## INTRODUCTION

According to the Centers for Disease Control and Prevention diabetes statistics report in 2017, around 30.3 million people (~9.4% of the U.S. population) have diabetes, and this disease is responsible for 4.6 million deaths each year (1). Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both (2). Long-term type 2 diabetes induces severe complications such as retinopathy with potential loss of vision (3), nephropathy leading to renal failure (4), and peripheral neuropathy with risk of foot ulcers or even limb amputations (5). Being cost-effective and convenient, blood glucose measurement has been widely used as the golden standard to monitor type 2 diabetes (6). However, glucose level fluctuates (7), and blood glucose measurement often produces false-positive results (8).

As a marker of long-term glucose level, glycated hemoglobin (HbA1c) presents a valuable target for long-term type 2 diabetes monitoring (9, 10). HbA1c is formed by an irreversible nonenzymatic glycosylation of hemoglobin (Hb) exposed to glucose in the bloodstream (11). Specifically, through the Maillard reaction, glucose attaches to the NH2-terminal valine of the β-chain of Hb (12). HbA1c concentration in the blood depends on two parameters: the life span of red blood cells (RBCs) (~120 days) and the glucose concentration in the blood (13). Therefore, a strong correlation exists between HbA1c concentration and the average glucose concentration in the bloodstream over the past 3 months. Thus, HbA1c concentration could be used to monitor long-term glucose levels (14). HbA1c fraction among total Hb has been approved for screening for diabetes (HbA1c ≥ 6.5%) and prediabetes (5.7% ≤ HbA1c ≤ 6.4%) according to the American Diabetes Association (15).

Multiple methods have been developed to detect HbA1c fraction (16). Boronate affinity chromatography, for example, was used to differentiate HbA1c from Hb (17). On the basis of the polarity difference between HbA1c and Hb, capillary electrophoresis was also used to separate these two molecules (18). Through conjugating upconversion nanoparticles such as NaYF4: Yb3+, Er3+ with anti-HbA1c monoclonal antibody, HbA1c concentration is negatively proportional to the luminescence signal from the conjugated nanoparticle (19). Anti-HbA1c antibody-functionalized gold nanoprobe provides another clue for the determination of HbA1c based on a one-step colorimetric immunoassay (20). Surface-enhanced Raman spectroscopy was reported to achieve selective detection of HbA1c, as a characteristic Raman band around 770 to 830 cm−1 was found in the case of HbA1c (21). Drop coating Raman technique was used to locally enrich and quantify HbA1c concentration (22). Mass spectrometry in combination with high-performance liquid chromatography (HPLC) was also used to detect HbA1c from other Hb species (23). One major limitation of the above methods lies in the poor detection specificity (18). Moreover, none of those methods can provide HbA1c fraction at the single-RBC level, thus unable to separate the contributions by glucose and by lifetime of RBCs.

Here, through transient absorption microscopy, we found that HbA1c and Hb have different excited-state dynamics. This spectroscopic signature triggered us to apply transient absorption microscopy, an advanced label-free imaging technique for nonfluorescent chromophores (2433), to study this heme modification process and to quantify the HbA1c percent in single RBCs. At a pixel dwell time of 10 μs, we obtained a time-resolved whole blood image stack within 2 min. Through quantitative phasor analysis, we harvested the standard calibration curve for HbA1c fraction quantitation. Subsequently, combined with the time-resolved whole blood image stacks, we obtained the HbA1c fraction in single RBCs. We show that HbA1c fraction distribution behaves differently between diabetic whole blood and healthy whole blood. Single-cell HbA1c fraction distribution could offer long-term monitoring of type 2 diabetes void of the interference from the life span of RBCs. Moreover, we used a mathematical model to interpret this distribution and derived the averaged glucose concentration, which monitors the bloodstream glucose concentration over the past 3 months.

## RESULTS

### Structure difference between HbA1c and Hb revealed by PyMOL simulation

Similar to Hb, HbA1c is a tetramer in which glucose is covalently linked to the N terminus of the β-chain of Hb (34). This modification not only confers HbA1c with different polarity from Hb (18, 35) but also alters the secondary structure of Hb (36). To have a direct visualization of the protein structure of HbA1c and Hb, we used PyMOL to analyze the protein structure of Hb and HbA1c. As shown in Fig. 1, the heme ring in Hb is hidden inside the hydrophobic pocket, whereas the porphyrin ring becomes more exposed to the outside environment in the case of HbA1c (Fig. 1, A and B), and there is some difference in the secondary structure between HbA1c (Fig. 1, A and C) and Hb (Fig. 1, B and D). The glucose at the N terminus has a polar interaction with the heme ring in the case of HbA1c, shown by blue dashed lines in Fig. 1C. This interaction indicates that the excited dynamics of heme might be altered after glycation, which can be measured by transient absorption spectroscopy.

### Characterization of HbA1c and Hb by fluorescence, time-resolved photoluminescence, and linear absorption spectroscopy

To identify the spectral difference between HbA1c and Hb, we first measured their fluorescence emission spectra. Because of the poor quantum yield of Hb (37), the fluorescence emission spectra for Hb (Fig. 2A) and HbA1c (Fig. 2B) were recorded with an integration time of 1000 s. Both HbA1c and Hb exhibit a fluorescence emission peak at 492 nm (Fig. 2, A and B), whereas HbA1c has a weaker shoulder peak (Fig. 2B). This finding confirms the structural difference between Hb and HbA1c. Then, we used time-resolved photoluminescence to understand this emission difference. It turned out that Hb demonstrates a longer fluorescence lifetime (Fig. 2C) compared with HbA1c (Fig. 2D). We further measured their absorption spectra. Consistent with previously documented literature (21, 38), we observed the absorption difference between Hb and HbA1c as well. In the Soret band (~400 nm) region, Hb shows a strong absorption at 405 nm (Fig. 2E), whereas HbA1c shows a characteristic peak at 411 nm, around a 6-nm red shift (Fig. 2F). In the Q-band region, Hb shows peaks at 498, 538, 630, and 677 nm (Fig. 2E), while HbA1c has only two peaks at 540 and 575 nm (Fig. 2F). These data altogether consolidate the fact that HbA1c and Hb are spectrally distinct from each other. However, Fourier transform Raman spectroscopy (fig. S1) and linear spectroscopy, by either fluorescence emission or electronic absorption, do not have the sensitivity to determine the fraction of HbA1c inside single RBCs. Transient absorption microscopy circumvents such difficulty, as shown in the following sections.

### Time domain signature of HbA1c and Hb unveiled by transient absorption microscopy

Given the weak fluorescence quantum yield of HbA1c and Hb, we turned to a pump-probe approach to detect the transient absorption of HbA1c in the time domain. By pumping at 520 nm and probing at 780 nm (refer to Materials and Methods and fig. S2), we obtained a time-resolved curve for Hb (Fig. 3A) and HbA1c (Fig. 3B), respectively. Then, we fitted these curves with an equation accounting for the convolution between a Gaussian function and multiexponential decay function (see Materials and Methods) (39). Through fitting, Hb exhibits two decay constants, with τ1 = 439 fs and τ2 = 3.14 ps. In the case of HbA1c, we obtained τ1 = 577 fs and τ2 = 3.61 ps. Thus, HbA1c has a slower excited-state decay compared with Hb (Fig. 3C). It is known that Hb in whole blood has two main forms, deoxy-form Hb (deoxyHb) and oxy-form Hb (oxyHb) (40). Moreover, oxyHb and deoxyHb show different time-resolved decays (32). To alleviate the interference from oxygen, we applied a protocol (41) to transfer all deoxyHb or deoxyHbA1c to oxy-form by saturating HbA1c or Hb solution with an oxygen balloon (see Materials and Methods). OxyHb decays faster compared to Hb (Fig. 3D), and oxyHbA1c decays faster compared to HbA1c as well (Fig. 3E). Nevertheless, the decay constants of oxyHbA1c stay larger compared to oxyHb (Fig. 3F). In the following experiments, we treated the samples in the same manner to ensure that the Hb and HbA1c molecules stay in the oxy form.

The transient absorption signal comes from three major processes, that is, excited-state absorption, ground-state depletion, and stimulated emission (30). To identify the dominant mechanism in the case of Hb and HbA1c under our transient absorption settings, we adopted a method developed by Jung et al. (42) and retrieved the intensity and phase information of both the X channel and the Y channel from a lock-in amplifier (fig. S3; see Materials and Methods). The lock-in amplifier detects the in-phase channel

$(Xchannel,ΔIprobeIprobecos(ϕprobe−ϕpump))$

$(Ychannel,ΔIprobeIprobesin(ϕprobe−ϕpump))$

, where phase is given by ϕprobe − ϕpump. We found that at a phase of 180°, the intensity from the X channel has a maximal positive signal, whereas that of the Y channel is the same as background (fig. S3), indicating that the sign of

$ΔIprobeIprobe$

is negative. Together, this means that under our transient absorption settings, we observed a gain in absorption. Thus, both Hb and HbA1c abide by excited-state absorption. The difference in decay constants between oxyHb and oxyHbA1c led us to explore intramolecular dynamics. A laser at 520 nm in this study pumps Hb or HbA1c from the ground state to the excited 1Q state (Fig. 3G). Because of ring-to-metal charge transfer (43), the excited 1Q state would relax to the Hb*I state (~50 fs). Then, the Hb*I state would jump to the Hb*II state because of ring-to-iron charge transfer (~300 fs) (43), and last, Hb*II relaxes back to the ground state [~3 to 5 ps (43)]. On the basis of the difference in linear absorption in the Q-band region (Fig. 2, E and F), it is reasonable to elucidate that HbA1c and Hb exhibit different excited-state dynamics. This difference provides a foundation for mapping the fraction of HbA1c in single RBCs.

### Transient absorption mapping of HbA1c fraction in diabetic versus healthy whole blood

For mapping HbA1c fraction in single RBCs, we first coated the glass substrate with poly-l-lysine so that a single layer of blood cells could adhere to the substrate (see Materials and Methods). Then, we obtained time-resolved image stacks from whole blood under the transient absorption microscope. The RBCs exhibit strong intensity when pump and probe beams are spatially and temporally overlapped with each other (Fig. 5A). Different RBCs exhibit distinct decay time constants, as observed in regions of interest ROI 1 (Fig. 5B) and ROI 2 (Fig. 5C), suggesting that HbA1c fraction varies from cell to cell.

By combining the time-resolved decay curves from RBCs along with the standard calibration curve obtained from whole blood samples with known HbA1c percent, we calculated HbA1c fraction for each RBC. Then, we compiled the HbA1c fractions from seven diabetic whole blood samples (at least 150 RBCs for each sample) and seven healthy whole blood samples (at least 150 RBCs for each sample). The distributions of HbA1c fraction in the diabetic whole blood samples (Fig. 5, D to F, and fig. S5) are notably different from those of healthy whole blood samples (Fig. 5, G to I, and fig. S6).

## DISCUSSION

Type 2 diabetes mellitus has become an expanding global health problem, which is closely linked to the epidemic of obesity (48). Moreover, patients with type 2 diabetes are at high risk of microvascular and macrovascular complications due to hyperglycemia and insulin resistance (49). A conventional method to diagnose type 2 diabetes mellitus is to monitor the bloodstream glucose concentration (6). However, blood glucose measurement often causes false positives due to fluctuation (8). As a stable biomarker, HbA1c has drawn increasing attention to be a new target to diagnose type 2 diabetes mellitus (50). Multiple methods such as boronate affinity chromatography and surface-enhanced Raman spectroscopy have been developed to detect HbA1c (18, 21). Nevertheless, none of the above methods could provide HbA1c fraction from single RBCs. Moreover, they could not separate the HbA1c fraction contributed by other factors such as diseases related to lifetime of RBCs. Therefore, other innovative methods are highly needed to provide accurate HbA1c fraction in single RBCs.

Here, through high-speed label-free transient absorption microscopy, we showed that HbA1c and Hb have different excited-state dynamics, thus having distinct time-resolved signatures. Then, quantitative phasor analysis generated the standard calibration HbA1c fraction curve without any a priori information input. Subsequently, combined with the standard calibration curve from whole blood samples with known HbA1c percent, HbA1c fraction in single RBCs is derived, and we found that HbA1c fraction distribution in diabetic whole blood is significantly different from that in healthy whole blood. A mathematical model is further developed to convert such distribution into clinically relevant information for diagnosis of type 2 diabetes stage.

Both Hb and HbA1c have poor fluorescence quantum yield yet strong absorption due to the existence of the heme ring (37). Transient absorption microscopy is an absorption-based imaging technique, which is able to image chromophores with undetectable fluorescence (33). Here, both Hb and HbA1c present strong intensity under the transient absorption microscope. Moreover, Hb and HbA1c have distinct time-resolved decay signatures, which allows for the quantitation of HbA1c at the single-RBC level. The approach reported here can be extended to study heme modification in other conditions, such as detection of hemozoin in malaria infection.

Phasor analysis has been widely used to interpret the fluorescence signal of relevant biological fluorophores by using their phasor fingerprints (51). Compared with other methods, such as principal components analysis, phasor analysis does not need any a priori information (52). Moreover, phasor analysis is not dependent on the concentration of analyte, thus eliminating fluctuation caused by intensity (53). Through normalization, phasor analysis could also amplify the molecular contrast, especially when the concentration of analyte is low in a mixture. For example, the time-resolved curves of HbA1c at 3.3 and 5.9% look similar (Fig. 4A); however, they demonstrate significant difference in the phasor plot. Therefore, combined with mean-shift theory, phasor analysis provides an optimal method to quantify the HbA1c fraction in single RBCs.

It is worth noting that the HbA1c fraction distribution in the diabetic whole blood samples is significantly different from that of healthy whole blood samples. Most of the diabetic RBCs have high HbA1c fraction. Even for the healthy whole blood samples, there is still quite a portion of RBCs whose HbA1c fraction is higher than 6.5%. In addition, the mean value of HbA1c fraction obtained from the distribution is consistent with that from the traditional HPLC approach (table S1). Through modeling, we derived that the stable average glucose concentration from the past 3 months in the diabetic whole blood sample is higher compared with that of healthy whole blood, which coincides with the real condition in type 2 diabetes. Moreover, this method only needs around microliters of whole blood samples instead of around milliliters of the whole blood sample. In the future, a portable fiber laser–based transient absorption microscope can be developed to achieve noninvasive diagnosis of type 2 diabetes through imaging the artery whole blood. In summary, work reported here underlies the profound potential of using label-free transient absorption microscopy to study the heme modification process and to determine the accurate long-term glucose level in the clinic.

Acknowledgments: We thank P. Hipskind at Eli Lilly for motivating us to develop an in situ HbA1c quantitation technology. We appreciate the help from T. Zhu in L. Huang’s laboratory at Purdue University for the time-resolved photoluminescence experiment. Funding: This work is partially supported by a Keck Foundation grant to J.-X.C. Author contributions: J.-X.C. conceived the idea of imaging glycated Hb (HbA1c) by transient absorption microscopy. P.-T.D. conducted the experiments and data acquisition. K.-C.H. and P.-T.D. built the visible-pump and the near-infrared probe microscope. H.L. figured out the mathematical model to fit the HbA1c fraction distribution. P.-T.D., H.L., and J.-X.C. cowrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.