A Near-infrared Turn-on Fluorescent Sensor for Sensitive and Specific Detection of Albumin from Urine Samples

A readily synthesizable fluorescent probe DMAT-π-CAP was evaluated for sensitive and selective detection of human serum albumin (HSA). DMAT-π-CAP showed selective turn-on fluorescence at 730 nm in the presence of HSA with more than 720-fold enhancement in emission intensity ([DMAT-π-CAP] = 10 μM), and rapid detection of HSA was accomplished in 3 s. The fluorescence intensity of DMAT-π-CAP was shown to increase in HSA concentration-dependent manner (Kd = 15.4 ± 3.3 μM), and the limit of detection of DMAT-π-CAP was determined to be 10.9 nM (0.72 mg/L). The 1:1 stoichiometry between DMAT-π-CAP and HSA was determined, and the displacement assay revealed that DMAT-π-CAP competes with hemin for the unique binding site, which rarely accommodates drugs and endogenous compounds. Based on the HSA-selective turn-on NIR fluorescence property as well as the unique binding site, DMAT-π-CAP was anticipated to serve as a fluorescence sensor for quantitative detection of the HSA level in biological samples with minimized background interference. Thus, urine samples were directly analyzed by DMAT-π-CAP to assess albumin levels, and the results were comparable to those obtained from immunoassay. The similar sensitivity and specificity to the immunoassay along with the simple, cost-effective, and fast detection of HSA warrants practical application of the NIR fluorescent albumin sensor, DMAT-π-CAP, in the analysis of albumin levels in various biological environments.


Introduction
Human serum albumin (HSA, 67 kDa) is the liver-produced most abundant protein (0.6 mM) in blood plasma [1]. As HSA is synthesized in the liver, hypoalbuminemia is related with liver failure which includes cirrhosis, liver cancer, hepatitis, alcohol-related liver disease, and fatty liver disease [2]. Some people with acute heart failure [3], kidney damage [4], protein-losing nephropathy and enteropathy [5] and malnutrition [6] are also susceptible to low albumin levels. On the other hand, hyperalbuminemia, an increased concentration of albumin in blood, is caused by dehydration and high protein diets [7]. HSA also serves as "taxis" for various endogenous compounds by using several ligand binding sites located on the net negatively charged globular surface: HSA binds and transports thyroid hormones, lipid-soluble hormones, unconjugated bilirubin, free fatty acids, and divalent cations (Ca 2+ and Mg 2+ ) [8]. By the same token, many drugs bind to HSA [9], and this event has great pharmaceutical interest because, upon binding to HSA, both the pharmacokinetic and pharmacodynamic properties of the drugs are affected.
Taken together, concentration of HSA can serve as prognostic marker of morbidity and mortality [7] and a predictive factor of the pharmacokinetic properties of the drugs [10]. Therefore, development of sensitive and selective detection method for quantification of HSA in biological samples is of clinical significance.
Various analytical methods have thus been developed for detection of HSA in blood, urine, and cell extracts, which include spectrophotometric (absorbance and fluorescence spectroscopy, and LCMS-proteomics), immunochemical (antibody), and radiochemical techniques [11][12][13]. Among those, due to high sensitivity and non-destructive nature, fluorescent measurement of HSA has attracted special addition. For this purpose, a series of molecular sensors that emit fluorescence upon binding to HSA have been discovered (Table 1). Table 1. Fluorescent HSA sensors: structures, fluorescence properties, HSA sensitivity (fluorescence fold increase), binding sites and sensing properties (limit of detection, detection range).
Sensors 2020, 20, x FOR PEER REVIEW 2 of 13 has great pharmaceutical interest because, upon binding to HSA, both the pharmacokinetic and pharmacodynamic properties of the drugs are affected. Taken together, concentration of HSA can serve as prognostic marker of morbidity and mortality [7] and a predictive factor of the pharmacokinetic properties of the drugs [10]. Therefore, development of sensitive and selective detection method for quantification of HSA in biological samples is of clinical significance. These sensors are featured with HSA sensitivity and significant increases in the fluorescence intensities (9~400 fold) were observed upon binding to HSA (Table 1) [14][15][16][17][18][19][20][21][22][23][24]. In addition to the turn-on properties, sensors emitting fluorescent light in the near-infrared (NIR) range (650 nm-900 nm) has additional advantage of minimized interference of background fluorescence as well as deep tissue penetration. A dicyanomethylene-4H-chromene derivative (TG-SA) [14] and an indolium salt [16] represent the turn-on NIR fluorescent HSA sensors ( Table 1). The fluorescent HSA sensors also exhibit favorable sensing properties including low detection limit (0.022~1.68 mg/L, Table 1) and wide detection range (Table 1). On the other hand, almost all HSA fluorescence sensors bind either to drug binding site 1 or to 2 and, due to competition with other drugs or endogenous compounds binding to the same binding site, they suffer from inaccurate determination of HSA (Table 1) [25][26][27]. In this context, sensors with the unique binding site have been expected to show enhanced HSA specificity but, to the best of our knowledge, ACDM is the sole example which targets neither drug site 1 nor 2 [15]. The accurate binding site of ACDM is still obscure, but the molecular dynamics study revealed the subdomain IB as the most probable binding site [15]. However, as ACDM emits fluorescent light at wavelength outside the NIR range (λ em 612 nm), an HSA sensor with a unique binding site as well as NIR fluorescence emission has been highly anticipated to demonstrate HSA specificity with diagnostic applicability: NIR fluorescent sensors characterized by minimal background interference as well as high sample penetration depth are more appropriate to detect trace HSA in clinical samples.
Taken together, HSA sensors that have turn-on fluorescence in the NIR region with the characteristic binding site have not been disclosed, which prompted us to investigate novel scaffolds. Previously, we came to realize that a chlorinated α-cyanoacetophenone can serve as a good turn-on fluorescence sensor [28]. In this study, for fine control of HSA-selectivity as well as fluorescence emission wavelength, various aromatic moieties were incorporated into the α-cyanoacetophenone scaffold. Thus, we prepared a series of α-cyanoacetophenone derivatives conjugated with aromatic rings and, among those, (2E,4E,6E)-2-(3-chlorobenzoyl)-7-(5-(dimethylamino)thiophen-2-yl)hepta-2,4,6-trienenitrile (DMAT-π-CAP) showed HSA-selective turn-on NIR fluorescence by binding to hereto unexplored hemin-binding site.

Synthetic Procedure of DMAT-π-CAP (5)
Materials and reagents. Unless otherwise stated, chemicals were obtained from either Sigma-Aldrich (St.Louis, MO, USA), TCI (Tokyo, Japan), or Thermo Fisher Scientific (Waltham, MA, USA). A 400 AMX spectrometer (Bruker, Karlsruhe, Germany) (operating at 400 MHz and 100 MHz for 1 H-NMR and 13 C-NMR, respectively) was used to produce nuclear magnetic resonance spectra. Mass spectrometry data (m/z) were obtained by a FAB mass spectrometer (FAB-MS) at Korea Basic Science Institute (Daegu, Korea). Absorbance and fluorescence were recorded using a Cytation™ 5 system (BioTek, Winooski, VT, USA).

Synthesis of (E)-3-(5-(dimethylamino) thiophen-2-yl) acrylaldehyde (3)
Under N 2 , a solution of 2 (2.40 mmol) and (1,3-dioxolan-2-ylmethyl)triphenylphosphonium bromide (3.36 mmol) in dry THF was treated with sodium methoxide (6.01 mmol). After stirring under reflux for 18 h, the reaction mixture was cooled to 0 • C. The reaction mixture was treated with 2 N HCl (30 mL), stirred at room temperature for 20 min, and neutralized with 2 N NaOH. After dilution with water, the mixture was extracted with EtOAc three times. The organic layers were combined and washed with brine. After drying over MgSO 4 , the organic phase was concentrated under reduced pressure, and purification of the residue by column chromatography (silica gel, hexane:ethyl acetate:acetone = 6:1:1) gave 3 in 34% yield as a yellow solid: 1  Under N 2 , a solution of 3 (0.81 mmol) and (1,3-dioxolan-2-ylmethyl)triphenylphosphonium bromide (1.14 mmol) in dry THF was treated with sodium methoxide (2.03 mmol). After stirring under reflux for 18 h, the reaction mixture was cooled to 0 • C. The reaction mixture was treated with 2 N HCl (30 mL), stirred at room temperature for 20 min, and neutralized with 2 N NaOH. After dilution with water, the mixture was extracted with EtOAc three times. The organic layers were combined and washed with brine. After drying over MgSO 4 ., the organic phase was concentrated under reduced pressure, and purification of the residue by column chromatography (silica gel, hexane:ethyl acetate:acetone = 6:1:1) gave 4 in 42% yield as a red solid: 1

Determination of Fluorescence Properties of DMAT-π-CAP in PBS Buffer (pH 7.4)
Stock solutions of DMAT-π-CAP (500 µM in DMSO) and HSA (1 mM in PBS, pH 7.4) were prepared. At room temperature, 1 µL of HSA (1 mM in PBS, pH 7.4) and 1 µL of DMAT-π-CAP (500 µM in DMSO) stock solutions were mixed with 98 µL of PBS (pH 7.4). After incubation for 3 min, the mixture was transferred into 96-well plate and the fluorescence was measured (λ ex /λ em = 600/730 nm) by a Cytation™ 5 system. All measurements were carried out in triplicates.

Determination of the Quantum Efficiency of Fluorescence of DMAT-π-CAP
At various concentrations (absorbances) of fluorescein (1-10 µM), integrated fluorescence intensity was measured (λ ex /λ em = 430/515 nm) by a Cytation™ 5 system. The same experiment was performed by using various concentrations (absorbances) of DMAT-π-CAP (1-10 µM) in the absence and presence of HSA (10 µM). Absorbance of fluorescein (or DMAT-π-CAP) was then plotted against the integrated fluorescence intensity, and the resulting linear regression slope was determined to give Grad s (or Grad x ). By using fluorescein as a reference (Φ s = 0.65 in water), quantum efficiency of DMAT-π-CAP . As all experiments were performed in the same solvent, water, solvent refractive index of DMAT-π-CAP (η x ) is equal to that of fluorescein (η s ).

Time-Dependent
Fluorescence of DMAT-π-CAP after Incubation with HSA.
At room temperature, 1 µL of HSA (1 mM in PBS, pH 7.4) and 1 µL of DMAT-π-CAP (500 µM in DMSO) stock solutions were mixed with 98 µL of PBS (pH 7.4) in 96-well plate. Immediately after mixing, the fluorescence was measured (λ ex /λ em = 600/730 nm) by a Cytation™ 5 system at every second for 3 min. All measurements were carried out in triplicates.

Limit of Detection (LOD) of DMAT-π-CAP
In the absence of HSA, fluorescence of DMAT-π-CAP (5 µM, PBS pH 7.4) was measured five times, and the standard deviation of blank measurement (σ) was determined. On the other hand, in the presence of varying concentrations of HSA, fluorescence intensity of DMAT-π-CAP (5 µM, PBS pH 7.4) at 730 nm was measured and, from the linear plot of the fluorescence intensity against HSA concentrations, the slope was determined. Limit of detection (LOD) of DMAT-π-CAP was then determined by the following equation: LOD = 3σ/slope.

Job's Plot Analysis of DMAT-π-CAP
Mixtures of DMAT-π-CAP and HSA (total concentrations = 10 µM) were generated in PBS buffer (pH = 7.4) by varying molar ratios (0.1~0.9). Fluorescence measurements were performed as described above.  [23], and the dissociation constant (K d ) as well as the maximum number of binding sites (B max ) were then calculated.

Assignment of the Binding Site of DMAT-π-CAP on HSA
Stock solutions of DMAT-π-CAP (500 µM) and site-specific drugs (500 µM) in DMSO were prepared. Displacement of DMAT-π-CAP from the HSA-DMAT-π-CAP complex was performed by addition of one of the site-specific drugs (500 µM) including warfarin (drug binding site I, subdomain IIA), ibuprofen (drug binding site II, subdomain IIIA), and hemin (subdomain IB). At room temperature, 178 µL of albumin (10 µM) solution was mixed with DMAT-π-CAP (2 µL) and 20 µL of the site-specific drugs, and the resulting mixture was incubated at room temperature for 3 min. After incubation, 150 µL of the supernatant was transferred into 96-well plate and the fluorescence emission was measured (λ ex /λ em = 600/730 nm) by s Cytation™ 5 system. All measurements were carried out in triplicates.

Determination of Urinary Albumin by Immunoassay
For the determination of urinary albumin levels with immunoassay, human albumin ELISA kit (STA-383-Human Albumin ELISA kit Cell Biolabs, Inc., San Diego, CA, USA) was used according to the manufacturer's protocol. The calibration curve obtained by using human HSA standard dissolved in assay buffer was used to estimate the HSA concentration in the urine sample.

Statistics
Data are expressed as means ± SD from at least three independent experiments. Tests used for nonparametric data included one-way analysis of variance (ANOVA) with Tukey's post hoc test using a GraphPad Prism 5 software (San Diego, CA, USA, USA). Unless otherwise indicated, p values < 0.05 were considered statistically significant.

Near-infrared Turn-on Fluorescence Properties of DMAT-π-CAP
The fluorescence properties of DMAT-π-CAP was investigated in PBS buffer (pH 7.4), which showed an absorption maxima (λex) at 600 nm. DMAT-π-CAP (5 μM) alone did not show any noticeable emission fluorescence (Figure 1a, inset) but, in the presence of HSA (10 μM, 665 mg/L), it showed strong fluorescence in the NIR region (λem = 730 nm) culminating in more than 720-fold enhancement in the emission intensity ( Figure 1a) and the quantum yield increase by 37-fold ( Figure  S1, Supplementary Material).

Near-infrared Turn-on Fluorescence Properties of DMAT-π-CAP
The fluorescence properties of DMAT-π-CAP was investigated in PBS buffer (pH 7.4), which showed an absorption maxima (λ ex ) at 600 nm. DMAT-π-CAP (5 µM) alone did not show any noticeable emission fluorescence (Figure 1a, inset) but, in the presence of HSA (10 µM, 665 mg/L), it showed strong fluorescence in the NIR region (λ em = 730 nm) culminating in more than 720-fold enhancement in the emission intensity ( Figure 1a) and the quantum yield increase by 37-fold ( Figure S1, Supplementary Material).

Near-infrared Turn-on Fluorescence Properties of DMAT-π-CAP
The fluorescence properties of DMAT-π-CAP was investigated in PBS buffer (pH 7.4), which showed an absorption maxima (λex) at 600 nm. DMAT-π-CAP (5 μM) alone did not show any noticeable emission fluorescence (Figure 1a, inset) but, in the presence of HSA (10 μM, 665 mg/L), it showed strong fluorescence in the NIR region (λem = 730 nm) culminating in more than 720-fold enhancement in the emission intensity ( Figure 1a) and the quantum yield increase by 37-fold ( Figure  S1, Supplementary Material).  Compared with the previously reported fluorescent HSA sensors (Table 1), the remarkably high increase in fluorescence intensity of DMAT-π-CAP in the presence of HSA is noteworthy. Moreover, DMAT-π-CAP showed selective turn-on fluorescence in the presence of human (HSA) and bovine (BSA) albumin proteins, while no substantial fluorescence was observed upon co-incubation with various bioanalytes including intracellular ions, organic molecules, and proteins with wide range of isoelectric points and nucleic acid (Figure 1b). In addition, fluorescence signal from DMAT-π-CAP became stable in 3 s after incubation with HSA ( Figure S2). Taken together, these data suggested selective and rapid binding of DMAT-π-CAP to HSA resulting in turn-on fluorescence in the NIR region.
The fluorescence intensity of DMAT-π-CAP at 730 nm was shown to increase in HSA concentration-dependent manner (Figure 2a). The steep increase in fluorescence intensity at lower HSA concentrations was followed by a plateau at 50 µM (3.3 g/L) and, at this concentration of HSA, more than 1,200-fold of fluorescence intensity increase of DMAT-π-CAP was achieved (Figure 2b). From the linear region of the HSA concentration-dependent fluorescence data (inset, Figure 2b; [HSA] = 0-10 µM, correlation coefficient R 2 = 0.9892), the limit of detection (LOD) for HSA by DMAT-π-CAP (5 µM) was determined (LOD = 3σ/slope) to be 10.9 nM (0.72 mg/L), which is comparable to those of other HSA sensors (Table 1). Compared with the previously reported fluorescent HSA sensors (Table 1), the remarkably high increase in fluorescence intensity of DMAT-π-CAP in the presence of HSA is noteworthy. Moreover, DMAT-π-CAP showed selective turn-on fluorescence in the presence of human (HSA) and bovine (BSA) albumin proteins, while no substantial fluorescence was observed upon co-incubation with various bioanalytes including intracellular ions, organic molecules, and proteins with wide range of isoelectric points and nucleic acid (Figure 1b). In addition, fluorescence signal from DMAT-π-CAP became stable in 3 seconds after incubation with HSA ( Figure S2). Taken together, these data suggested selective and rapid binding of DMAT-π-CAP to HSA resulting in turn-on fluorescence in the NIR region.
The fluorescence intensity of DMAT-π-CAP at 730 nm was shown to increase in HSA concentration-dependent manner (Figure 2a). The steep increase in fluorescence intensity at lower HSA concentrations was followed by a plateau at 50 μM (3.3 g/L) and, at this concentration of HSA, more than 1,200-fold of fluorescence intensity increase of DMAT-π-CAP was achieved (Figure 2b). From the linear region of the HSA concentration-dependent fluorescence data (inset, Figure 2b; [HSA] = 0-10 μM, correlation coefficient R 2 = 0.9892), the limit of detection (LOD) for HSA by DMAT-π-CAP (5 μM) was determined (LOD = 3σ/slope) to be 10.9 nM (0.72 mg/L), which is comparable to those of other HSA sensors (Table 1).  Considering the reference range of albumin in urine (2.2-25 mg/L) [29], the wide linear dynamic range spanning two orders of magnitude (0.01-10 µM; 0.72-665 mg/L) supports the clinical use of DMAT-π-CAP for detecting HSA.

The Binding Properties of DMAT-π-CAP to HSA
In order to gain in-depth information of the binding properties and the binding site of DMAT-π-CAP on HSA, we performed Job's plot analysis as well as competition assay with site-specific binders. First, the stoichiometry of binding of DMAT-π-CAP on HSA was determined to be 1:1 by analysis of a Job's plot, which showed the maximum fluorescence intensity when the molar fraction of DMAT-π-CAP approached 0.5 (Figure 3a). The 1:1 stoichiometry between DMAT-π-CAP and HSA also suggests the single binding site and, when the one-site binding model is applied to the data shown in Figure 2b, a relatively high binding affinity with a dissociation constant (K d ) of 15.4 ± 3.3 µM was obtained. Based on the one-site binding model, the binding site of DMAT-π-CAP on HSA was then explored. HSA possesses two distinct drug binding sites, and most drugs and fluorescence dyes are classified into specific binders to one of these two sites. By the same token, DMAT-π-CAP was anticipated to bind to either of the two drug binding sites, which could be confirmed by a competition binding assay with site-specific binders such as warfarin (drug site 1) and ibuprofen (drug site 2). However, fluorescence intensity from the preformed complex of DMAT-π-CAP and HSA was not affected by addition of increasing concentrations of warfarin or ibuprofen (Figure 3b).

The Binding Properties of DMAT-π-CAP to HSA
In order to gain in-depth information of the binding properties and the binding site of DMAT-π-CAP on HSA, we performed Job's plot analysis as well as competition assay with site-specific binders. First, the stoichiometry of binding of DMAT-π-CAP on HSA was determined to be 1:1 by analysis of a Job's plot, which showed the maximum fluorescence intensity when the molar fraction of DMAT-π-CAP approached 0.5 (Figure 3a). The 1:1 stoichiometry between DMAT-π-CAP and HSA also suggests the single binding site and, when the one-site binding model is applied to the data shown in Figure 2b, a relatively high binding affinity with a dissociation constant (Kd) of 15.4 ± 3.3 μM was obtained. Based on the one-site binding model, the binding site of DMAT-π-CAP on HSA was then explored. HSA possesses two distinct drug binding sites, and most drugs and fluorescence dyes are classified into specific binders to one of these two sites. By the same token, DMAT-π-CAP was anticipated to bind to either of the two drug binding sites, which could be confirmed by a competition binding assay with site-specific binders such as warfarin (drug site 1) and ibuprofen (drug site 2). However, fluorescence intensity from the preformed complex of DMAT-π-CAP and HSA was not affected by addition of increasing concentrations of warfarin or ibuprofen (Figure 3b).  Instead, hemin effectively replaced DMAT-π-CAP from HSA to result in sharp decrease in fluorescence intensity (>95% displacement by 25 µM of hemin), which suggests that DMAT-π-CAP and hemin share the same binding site in HSA. Collectively, these results demonstrate that the specific binding to the hereto unexplored hemin-binding site of HSA is responsible for the turn-on fluorescence of DMAT-π-CAP. Hemin, a porphyrin, is a rare example of the HSA-binders targeting its subdomain IB [30]. Bilirubin [31] and lidocaine [32] are also known to bind to subdomain IB but, while bilirubin and lidocaine did not compete with DMAT-π-CAP for binding to HSA (Figure 1b), hemin effectively displaced DMAT-π-CAP from its complex with HSA (Figure 3b). This result is not surprising in light of the distinct differences in the binding sites of these subdomain IB-binding molecules ( Figure S3). Thus, it might be proposed that, among the various binding sites located in the subdomain IB of HSA, only the hemin-binding site specifically accommodates DMAT-π-CAP.

Determination of Urinary HSA Levels by Using DMAT-π-CAP
The unique binding mode of DMAT-π-CAP is reminiscent of ACDM, which demonstrated selective and sensitive detection of HSA through binding to a non-drug binding site [14]. In addition, NIR fluorescence property of DMAT-π-CAP locates its emission wavelength at 730 nm, which is far away from the interfering background fluorescence of biological samples such as plasma (~520 nm) [33] and urine (400~450 nm) [34]. Taken together, the HSA-selective turn-on NIR fluorescence property of DMAT-π-CAP along with its unique HSA-binding site was anticipated to allow detection and monitoring of the HSA levels in biological samples with minimized background interference. Thus, clinical applicability of DMAT-π-CAP as an HSA sensor was evaluated by measurement of urinary HSA levels. Urine samples were obtained from three healthy donors, and urinary HSA levels were determined by using direct fluorometric method as well as fluorometric standard addition method. The direct fluorometric analysis is based on the proportional relationship between fluorescence emission of DMAT-π-CAP and concentration of HSA (Figure 2b). Therefore, fluorescence intensities of DMAT-π-CAP (5 µM) were measured (λ ex /λ em = 600/730 nm) in the presence of undiluted urine samples and, by using the linear relationship between fluorescence intensity and HSA concentration (Figure 2b), the urinary HSA concentrations were determined (4.9 mg/L, 7.5 mg/L, and 24.2 mg/L) (Figure 4).
Sensors 2020, 20, x FOR PEER REVIEW 10 of 13 Instead, hemin effectively replaced DMAT-π-CAP from HSA to result in sharp decrease in fluorescence intensity (>95% displacement by 25 μM of hemin), which suggests that DMAT-π-CAP and hemin share the same binding site in HSA. Collectively, these results demonstrate that the specific binding to the hereto unexplored hemin-binding site of HSA is responsible for the turn-on fluorescence of DMAT-π-CAP. Hemin, a porphyrin, is a rare example of the HSA-binders targeting its subdomain IB [30]. Bilirubin [31] and lidocaine [32] are also known to bind to subdomain IB but, while bilirubin and lidocaine did not compete with DMAT-π-CAP for binding to HSA (Figure 1b), hemin effectively displaced DMAT-π-CAP from its complex with HSA (Figure 3b). This result is not surprising in light of the distinct differences in the binding sites of these subdomain IB-binding molecules ( Figure S3). Thus, it might be proposed that, among the various binding sites located in the subdomain IB of HSA, only the hemin-binding site specifically accommodates DMAT-π-CAP.

Determination of Urinary HSA Levels by Using DMAT-π-CAP
The unique binding mode of DMAT-π-CAP is reminiscent of ACDM, which demonstrated selective and sensitive detection of HSA through binding to a non-drug binding site [14]. In addition, NIR fluorescence property of DMAT-π-CAP locates its emission wavelength at 730 nm, which is far away from the interfering background fluorescence of biological samples such as plasma (~520 nm) [33] and urine (400 ~ 450 nm) [34]. Taken together, the HSA-selective turn-on NIR fluorescence property of DMAT-π-CAP along with its unique HSA-binding site was anticipated to allow detection and monitoring of the HSA levels in biological samples with minimized background interference. Thus, clinical applicability of DMAT-π-CAP as an HSA sensor was evaluated by measurement of urinary HSA levels. Urine samples were obtained from three healthy donors, and urinary HSA levels were determined by using direct fluorometric method as well as fluorometric standard addition method. The direct fluorometric analysis is based on the proportional relationship between fluorescence emission of DMAT-π-CAP and concentration of HSA (Figure 2b). Therefore, fluorescence intensities of DMAT-π-CAP (5 μM) were measured (λex/λem = 600/730 nm) in the presence of undiluted urine samples and, by using the linear relationship between fluorescence intensity and HSA concentration (Figure 2b), the urinary HSA concentrations were determined (4.9 mg/L, 7.5 mg/L, and 24.2 mg/L) (Figure 4).   The second fluorometric measurement of urinary HSA levels was accomplished by using the fluorometric standard addition method [23], and the urine samples were spiked with various concentrations of HSA (0.2-2.0 µM; 13.3-133.0 mg/L). The fluorescence of DMAT-π-CAP (5 µM) in each HSA-spiked urine sample was then measured (λ em = 730 nm), and the fluorescence intensity of DMAT-π-CAP increased linearly as the spiked HSA increased ( Figure S4). From the linear plots, the urinary albumin levels of the three donors were determined to be 4.8 mg/L, 7.6 mg/L, and 22.7 mg/L, respectively, which are in good agreement with those obtained by direct fluorometric method (Figure 4). On the other hand, immunoassay using anti-HSA antibody was also performed, and the urinary albumin levels determined by immunoassay (1.6 mg/L, 4.6 mg/L, and 21.4 mg/L, respectively) ( Figure  S5) was comparable to those obtained by the fluorometric analyses of DMAT-π-CAP (Figure 4). These results demonstrate convincingly that the sensitivity and selectivity of DMAT-π-CAP toward HSA remains unperturbed in urine and DMAT-π-CAP can serve as a suitable fluorescence probe for precise determination of the HSA levels in urine samples.

Conclusions
In summary, a readily synthesizable fluorescent probe DMAT-π-CAP was evaluated for sensitive and selective detection of HSA. DMAT-π-CAP showed selective turn-on fluorescence at 730 nm (λ em ) in the presence of HSA with more than 720-fold enhancement in emission intensity ([DMAT-π-CAP] = 10 µM), and rapid detection of HSA was accomplished in 3 s. The fluorescence intensity of DMAT-π-CAP was shown to increase in HSA concentration-dependent manner (K d = 15.4 ± 3.3 µM), and the LOD of DMAT-π-CAP was determined to be 10.9 nM (0.72 mg/L). The 1:1 stoichiometry between DMAT-π-CAP and HSA was determined, and the displacement assay revealed that DMAT-π-CAP competes with hemin for the unique binding site, which rarely accommodates drugs and endogenous compounds. Based on the HSA-selective turn-on NIR fluorescence property as well as the unique binding site, DMAT-π-CAP was anticipated to serve as a fluorescence sensor for quantitative detection of the HSA level in biological samples with minimized background interference. Thus, urine samples were directly analyzed by DMAT-π-CAP to assess albumin levels, and the results were comparable to those obtained from immunoassay. The similar sensitivity and specificity to the immunoassay along with the simple, cost-effective, and fast detection of HSA warrants practical application of the NIR fluorescent albumin sensor, DMAT-π-CAP, in the analysis of albumin levels in various biological environments.