polymers

Article -Conjugated Gold Nanoclusters for Qualitative Analysis of Haptoglobin Phenotypes

Shih-Hua Tan 1, Sibidou Yougbaré 2,3 , Hsueh-Liang Chu 4, Tsung-Rong Kuo 1,2 and Tsai-Mu Cheng 4,5,*

1 Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan; [email protected] (S.-H.T.); [email protected] (T.-R.K.) 2 International PhD Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan; [email protected] 3 Institut de Recherche en Sciences de la Santé (IRSS-DRCO)/Nanoro, 03 B.P 7192 Ouagadougou 03, Burkina Faso 4 Graduate Institute of Translational Medicine, College of Medicine and Technology, Taipei Medical University, Taipei 11031, Taiwan; [email protected] 5 TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei 11031, Taiwan * Correspondence: [email protected]

 Received: 29 August 2020; Accepted: 26 September 2020; Published: 29 September 2020 

Abstract: Designing a facile and rapid detection method for haptoglobin (Hp) phenotypes in human blood plasma is urgently needed to meet clinic requirements in theranostics. In this work, a novel approach to qualitatively analyze Hp phenotypes was developed using a fluorescent probe of gold nanoclusters (AuNCs). Hemoglobin-conjugated (Hb)-AuNCs were successfully synthesized with blue-green fluorescence and high biocompatibility via one-pot synthesis. The fluorescence of Hb-AuNCs comes from the ligand-metal charge transfer between surface ligands of Hb and the gold cores with high oxidation states. The biocompatibility assays including viability and fluorescence imaging, demonstrated high biocompatibility of Hb-AuNCs. For the qualitative analysis, three Hp phenotypes in plasma, Hp 1-1, Hp 2-1, and Hp 2-2, were successfully discriminated according to changes in the fluorescence intensity and peak position of the maximum intensity of Hb-AuNCs. Our work provides a practical method with facile and rapid properties for the qualitative analysis of three Hp phenotypes in human blood plasma.

Keywords: haptoglobin; gold nanoclusters; hemoglobin; phenotypes; qualitative analysis

1. Introduction Fluorescent nanoclusters (NCs) have been extensively utilized for biomedical applications in fields such as bioimaging, detection, and medicine [1–6]. With many kinds of metal NCs, recent advancements have focused on the development of gold NCs (AuNCs) with various surface ligand modifications for detection applications [7–11]. For example, glucose-conjugated AuNCs were applied as a target-specific fluorescent probe to quantitatively detect glucose metabolism rates by glucose transporters overexpressed by U-87 MG brain cancer cells [12]. Glutathione-protected AuNCs were employed to detect the biomarker of dipicolinic acid for sensing potential infections by strongly hazardous anthrax spores with a much lower limit of detection (600-fold) than the infectious dosage [13]. Hyperbranched polyethyleneimine-assisted dual-emissive AuNCs were designed to detect highly toxic cyanide anions based on changes in the fluorescence color caused by their surface Au(I) etched by cyanide anions [14]. Fluorescent AuNC-modified metal-organic frameworks have served as a highly sensitive fluorescent nanoprobe for rapid detection of nitro explosives of 2,4,6-trinitrotoluene [15].

Polymers 2020, 12, 2242; doi:10.3390/polym12102242 www.mdpi.com/journal/polymers PolymersPolymers 20202020,, 1122,, x 2242 FOR PEER REVIEW 22 of of 11 11 trinitrotoluene [15]. With the conjugation of surface ligands of small molecules, polymers, and proteins,With the conjugationAuNCs have of been surface intensively ligands of used small for molecules, biomedical polymers, applications and proteins, due to their AuNCs easy have surface been modification,intensively used tunable for biomedical fluorescence, applications high biocompatibility, due to their easy surface and superior modification, specificit tunabley [16] fluorescence,. Several studieshigh biocompatibility, have demonstrated andsuperior the syntheses specificity and [applications16]. Several studiesof Hb conjugated have demonstrated AuNCs (Hb the syntheses-AuNCs) [17and–22 applications]. However, of as Hb far conjugated as we know, AuNCs there is (Hb-AuNCs) still no design [17 – with22]. the However, use of Hb as- farAuNCs as we for know, the qualitativethere is still detection no design of with haptoglobin the use of (Hp)Hb-AuNCs phenotypes for the. The qualitative acute-phase detection Hp of protein haptoglobin has three (Hp) phenotypesphenotypes. in Thehuman acute-phase blood plasma: Hp protein Hp 1-1, has Hp three2-1, and phenotypes Hp 2-2 [23 in–27 human]. In the blood clinic, plasma: concentrations Hp 1-1, ofHp these 2-1, and three Hp Hp 2-2 [phenotypes23–27]. In the are clinic, related concentrations to kidney of failure, these three diabetes, Hp phenotypes autoimmune are disorders, related to cardiovascularkidney failure, diabetes, diseases, autoimmune and hemolytic disorders, anemia cardiovascular [28–34] diseases,. Three and ordinary hemolytic approaches anemia [28 –34 of]. spectrophotometry,Three ordinary approaches immunoreactivity, of spectrophotometry, and gel electrophoresis immunoreactivity, are and used gel to electrophoresis measure Hp. are For used the spectrophotometricto measure Hp. For analysis, the spectrophotometric the concentration analysis, of Hp the is determined concentration from of Hp the is change determined in absorption from the duechange to thine absorption formation due of to Hp the–Hb formation complexes of Hp–Hb [35]. Moreover,complexes [ with35]. Moreover, analysis ofwith different analysis absorption of different spectraabsorption of Hpspectra–Hb of complexes, Hp–Hb complexes, Hp–Hb Hp–Hb complexes, complexes, free Hb, free and Hb, and free free Hp Hp can can be be quantitatively quantitatively measuredmeasured with with varying varying degrees degrees of ofhemolysis. hemolysis. For immunoreactive For immunoreactive assays, assays, Hp and Hp antis andera antisera are mixed are tomixed form to precipitates, form precipitates, and then and then the precipitatesthe precipitates are are analyzed analyzed by by radial radial immunodiffusion immunodiffusion and and nephelometrynephelometry [36] [36].. To To detect detect the the precipitates, precipitates, nephelometry nephelometry is is a a commonly commonly utilized utilized method method by by light light irradiationirradiation with with Hp Hp–Hb-antibody–Hb-antibody complexes complexes in in solutio solutionn to to obtain obtain reflected reflected light light for for further further analysis. analysis. WithWith the the use use of of gel electrophoresis, free free Hb Hb and and Hp Hp–Hb–Hb complexes complexes can can respectively respectively be be isolated isolated via via alkalinealkaline electrophoresis electrophoresis [37 ].[37] Although. Although spectrophotometry, spectrophotometry, immunoreactivity, immunoreactivity, and gel electrophoresis and gel electrophoresishave been developed have betoen detect developed Hp, the qualitative to detect Hp, analysis the of qualitative the three Hpanalysis phenotypes of the still three lacks Hp a phenotypessuitable approach. still lacks Recently, a suitable an immunoturbidimetric approach. Recently, assayan immunoturbidimetric and enzyme-linked immunosorbent and enzyme assay- linkedwere conducted immunosorbent to discriminate assay were Hp phenotypes. conducted to Designing discriminate a rapid Hp assay phenotypes. for Hp phenotypes Designing is a still rapid an assurgentay for task Hp to phenotypes meet clinical is still requirements. an urgent task to meet clinical requirements. InIn this this work,work, forfor the the qualitative qualitative analysis analysis of threeof three Hp Hp phenotypes, phenotypes, Hb wasHb chosenwas chosen as a surface as a surface ligand ligandto prepare to prepare Hb-AuNCs Hb-AuNCs due to its due high-a to itsffinity high binding-affinity with binding Hp and with cysteine Hp and residues. cysteine Hb-AuNCs residues. wereHb- AuNCsprepared were by prepared a facile one-pot by a facile synthesis one-pot strategy synthesis as strategy shown as in shown Figure 1in. Figure The structural 1. The structural and optical and opticalproperties properties of Hb-AuNCs of Hb- wereAuNCs characterized were characterized by ultraviolet–visible by ultraviolet (UV–Vis)–visible absorption (UV–Vis) spectroscopy, absorption spectroscopy,fluorescence spectroscopy, fluorescencex-ray spectroscopy, photoelectron x-ray spectroscopy photoelectron (XPS), spectroscopy transmission (XPS), electron transmission microscopy electron(TEM), and microscopy Fourier transform (TEM), infraredand Fourier (FTIR) spectroscopy. transform infrared The biocompatibility (FTIR) spectroscopy of Hb-AuNCs. The biocompatibilitywas assessed by of a Hb resazurin-AuNCs dye was reduction assessed by assay a resazurin and fluorescence dye reduction imaging. assay Mostand fluorescence importantly, imaging.fluorescent Most Hb-AuNCs importantly, were used fluorescent to qualitatively Hb-AuNCs analyze were Hp phenotypes used to qualitatively according to changes analyze in Hp the phenotypesfluorescence according spectra of to Hb-AuNCs. changes in the fluorescence spectra of Hb-AuNCs.

Figure 1. Schematic illustration of hemoglobin-conjugated gold nanoclusters (Hb-AuNCs) synthesis Figure 1. Schematic illustration of hemoglobin-conjugated gold nanoclusters (Hb-AuNCs) synthesis by a facile one-pot synthesis strategy. by a facile one-pot synthesis strategy. 2. Materials and Methods

2.1. Synthesis of Hemoglobin-Conjugated Gold Nanoclusters (Hb-AuNCs)

An HAuCl4 aqueous solution (2.8 mM, 5 mL) (Alfa Aesar, Ward Hill, MA, USA) was added to 5 mL of an Hb aqueous solution (2 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA). The reaction solution

Polymers 2020, 12, 2242 3 of 11

was stirred at 400 rpm in a water bath (37 ◦C) for 10 min. Afterward, 1 mL of an NaOH aqueous solution (1 M) (Fluka, St. Gallen, Swiss) was added to the reaction. The reaction solution containing HAuCl4, Hb, and NaOH was stirred at 400 rpm in a water bath (37 ◦C) and a dark environment for 24 h. After reacting for 24 h, the solution containing Hb-AuNCs was centrifuged at 12,000 rpm for 10 min. After centrifugation, the upper greenish-black solution containing Hb-AuNCs was collected and stored at 4 ◦C in the dark for subsequent experiments.

2.2. Characterization Techniques UV–Vis absorption spectrometer (JASCO V-770 Spectrophotometer, Easton, MD, USA) was applied to measure the absorption of Hb-AuNCs. XPS (VG Microtech MT-500, London, United Kingdom) with Al Kα x-ray source was used to analyze the oxidation state and binding properties of Hb-AuNCs. A fluorescence spectrometer (JASCO spectrofluorometer FP-8500, Easton, MD, USA) was utilized to detect the fluorescence of Hb-AuNCs. The Systat PeakFit 4.12 (Systat Software, San Jose, CA, USA) software was employed to simulate fluorescence spectra. TEM (Hitachi HT-7700, Tokyo, Japan) was applied to characterize the morphology of the Hb-AuNCs. FTIR spectroscopy (Thermo Scientific Nicolet iS10, Waltham, MA, USA) was utilized to measure the amide groups of Hb-AuNCs.

2.3. Assay of Hb-AuNCs by Vero Cells Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, MA, USA) with 10% (v/v) fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) was applied to culture Vero cells. The cytotoxicity of Hb-AuNCs was measured before and after incubation with Vero cells by a resazurin dye reduction assay. Vero cells were cultured in a 96-well plate (104 cells/well) for 24 h. After 24 h, the media in the 96-well plate were removed, and then a phosphate-buffered saline (PBS) solution (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to wash each well cultured with Vero cells. Sequentially, Vero cells in the 96-well plates were incubated with Hb-AuNCs dispersed in DMEM. Concentrations of Hb-AuNCs of 2.7, 5.5, 11.0, 21.9, 43.8, 87.5, 175, 350, and 700 µg/mL were used to test the cytotoxicity. After 24 h of incubation, resazurin (0.02 mg/mL) (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well and further incubated for an additional 4 h. The absorbances at 570 and 600 nm were measured by a plate reader (Thermo Varioskan Flash, Waltham, MA, USA). For the cell viability assay, each concentration of Hb-AuNCs was repeated independently at least eight times.

2.4. Measurement of Vero Cell Death by Fluorescence Imaging To measure cell death, the SYTOX green fluorescent dye (Thermo Fisher Scientific, Waltham, MA, USA) was applied to stain dead cells for fluorescence imaging. Thus, 5 µM of SYTOX green was added to culture media of Vero cells in each well and incubated at room temperature for 15 min without light irradiation. After washing with PBS, nucleic acids of total cells were stained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence images of total/dead cells were obtained with fluorescence microscopy.

2.5. Detection of Hp Phenotypes The method for detecting Hp phenotypes was modified from a previous study [38]. Briefly, 7 µL of a serum sample was mixed with 5 µL Hb (8 mg/mL) and then equilibrated with 3 µL of sample buffer (0.625 M Tris-base at pH 6.8, 0.125 mg/L bromophenol blue (Sigma-Aldrich, St. Louis, MO, USA) and 50% glycerol (v/v) (BioShop Canada Inc., Burlington, ON, Canada)). The mixture was separated by non-reducing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (6% acrylamide (Sigma-Aldrich, St. Louis, MO, USA) for the separating gel (pH 8.8) and 4% acrylamide for the stacking gel (pH 6.8)). After electrophoresis, the gel was stained by freshly prepared peroxidase substrate (0.05% 3,30-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA) and 0.07% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) in PBS), and the Hp–Hb complex was visualized. Polymers 2020, 12, 2242 4 of 11

3. Results

3.1. Optical and Structural Characterizations of Hb-AuNCs In Figure2a, the UV–Vis absorption spectrum of Hb exhibited a strong Soret band at a wavelength of 405 nm. After the formation of Hb-AuNCs, the signal intensity of the Soret band decreased, and its peak position also revealed a blue-shift from 405 to 367 nm, as shown in the UV–Vis absorption spectrum of Hb-AuNCs in Figure2a. The decrease in the signal intensity and blue-shift of the peak position can be attributed to a change in the hydrophobicity of heme groups due to the formation of Hb-AuNCs [39]. Furthermore, there was no obvious surface plasmon absorption peak at a wavelength of ~520 nm due to the absence of gold nanoparticles [40]. The disappearance of the surface plasmon absorption of gold nanoparticles can be ascribed to Hb-AuNCs exhibiting high oxidation states of gold cores, which produced a shortage of free electrons to generate coherent oscillations [41–43]. To further examine the oxidation state and binding properties, XPS spectrum of Hb-AuNCs were analyzed. In the XPS spectrum of Hb-AuNCs in Figure2b, the peaks of Au 4f 5/2 and Au 4f7/2 were separately located at 88.1 and 84.5 eV. For bulk gold, peaks of Au 4f5/2 and Au 4f7/2 were located at 87.4 and 84.0 eV, respectively. Compared to bulk gold, the positive shifts of Au 4f5/2 and Au 4f7/2 indicated that Hb-AuNCs contained a lot of gold with high oxidation states in their cores, corresponding to the results of the UV–Vis absorption spectrum of Hb-AuNCs [44]. Moreover, the fluorescence spectrum of Hb-AuNCs was measured. Under an excitation wavelength of 314 nm, Hb-AuNCs exhibited a maximum intensity of fluorescence at a wavelength of 430 nm as shown in Figure2c. A small shoulder was observed at a wavelength of 408 nm due to the Raman signal of water. Raman signal of water was measured with the excitation wavelength of 314 nm by fluorescence spectrometer, as shown in Figure2d. Based on the peak simulations, the fluorescence spectrum of Hb-AuNCs was separated into two peaks including a fluorescence spectrum of Hb-AuNCs and the Raman signal of water, as shown in Figure2c. The fluorescence of AuNCs was proven to be due to the ligand-metal charge transfer between thiolate ligands and the core of AuNCs [45]. For Hb-AuNCs, based on the ligand-metal charge transfer, electrons in the ligand of Hb were delivered to the gold cores with high oxidation states to generate fluorescence. Polymers 2020, 12, x FOR PEER REVIEW 5 of 11

Figure 2. (a) Ultraviolet–visibleFigure 2. (a) Ultraviolet (UV–Vis)–visible (UV absorption–Vis) absorption spectra spectra of hemoglobin of hemoglobin and hemoglobin hemoglobin-conjugated- conjugated gold nanocomposites (Hb-AuNCs); (b) X-ray photoelectron (XPS) spectra of Hb-AuNCs gold nanocomposites(black line), (Hb-AuNCs); the simulated peak (b of) Au X-ray 4f5/2 (blue photoelectron line) and the simulated (XPS) peak spectra of Au 4f of7/2 (red Hb-AuNCs line); (c) (black line), the simulated peakfluorescence of Au spectrum 4f5/2 (blue of Hb line)-AuNCs and composed the simulated of the simulated peak fluorescence of Au 4f7 / spectrum2 (red line); of Hb- (c) fluorescence AuNCs and the simulated Raman signal of water. The excitation wavelength was 314 nm; (d) Raman spectrum of Hb-AuNCssignal of watercomposed was measured with of thethe excitation simulated wavelength fluorescence of 314 nm by fluorescence spectrum spectrometer. of Hb-AuNCs and the simulated Raman signal of water. The excitation wavelength was 314 nm; (d) Raman signal of water was measured withIn the theTEMexcitation image of Figure wavelength 3a, Hb-AuNCs of revealed 314 nm an by approximately fluorescence globular spectrometer. shape. As shown in the histogram of Figure 3b, the size distribution of Hb-AuNCs was systematically counted according to 100 nanoclusters in the TEM image. The average size of Hb-AuNCs was calculated to be 3.7 nm. Overall, from a combination of optical and structural characterizations, we determined that fluorescent Hb-AuNCs were successfully prepared by a facile hydrothermal approach. FTIR spectra of Hb and Hb-AuNCs were applied to investigate their characteristic amide I and amide II bands. The amide I band (1600–1700 cm−1) can be ascribed to the stretching vibrations of CO from peptide linkages in the protein backbone. The amide II band (1500–1600 cm−1) was associated with NH bending and CN stretching vibrations. As shown in the FTIR spectra of Figure 3c, Hb exhibited characteristic IR bands of amide I and amide II located at 1642 and 1534 cm−1, respectively. After formation of Hb-AuNCs, the amide I band showed no significant change. However, the amide II band revealed the increase of intensity and the peak position was shifted from 1534 cm−1 to 1553 cm−1. Previous studies have demonstrated that the increase in peak intensity and the shift of peak position can be attributed to the formation of Hb-AuNCs. Based on the results of the FTIR spectra, the conjugation between Hb and AuNCs was successfully obtained [46,47].

Polymers 2020, 12, 2242 5 of 11

In the TEM image of Figure3a, Hb-AuNCs revealed an approximately globular shape. As shown in the histogram of Figure3b, the size distribution of Hb-AuNCs was systematically counted according to 100 nanoclusters in the TEM image. The average size of Hb-AuNCs was calculated to be 3.7 nm. Overall, from a combination of optical and structural characterizations, we determined that fluorescent Hb-AuNCs were successfully prepared by a facile hydrothermal approach. FTIR spectra of Hb and Hb-AuNCs were applied to investigate their characteristic amide I and amide II bands. The amide I 1 band (1600–1700 cm− ) can be ascribed to the stretching vibrations of CO from peptide linkages in 1 the protein backbone. The amide II band (1500–1600 cm− ) was associated with NH bending and CN stretching vibrations. As shown in the FTIR spectra of Figure3c, Hb exhibited characteristic IR bands 1 of amide I and amide II located at 1642 and 1534 cm− , respectively. After formation of Hb-AuNCs, the amide I band showed no significant change. However, the amide II band revealed the increase 1 1 of intensity and the peak position was shifted from 1534 cm− to 1553 cm− . Previous studies have demonstrated that the increase in peak intensity and the shift of peak position can be attributed to the formation of Hb-AuNCs. Based on the results of the FTIR spectra, the conjugation between Hb and AuNCs was successfully obtained [46,47]. Polymers 2020, 12, x FOR PEER REVIEW 6 of 11

Polymers 2020, 12, x FOR PEER REVIEW 6 of 11

Figure 3. Figure(a) Transmission 3. (a) Transmission electron electron microscopy microscopy ( (TEM)TEM) image image of hemoglobin of hemoglobin-conjugated-conjugated gold gold nanoclustersnanoclusters (Hb-AuNCs); (Hb-AuNCs) (b) histogram; (b) histogram of of the the sizesize distribution distribution of Hb of-AuNCs Hb-AuNCs;; (c) Fourier (c) transform Fourier transform infrared (FTIR) spectra of Hb and Hb-AuNCs. infrared (FTIR)Figure spectra 3. (a) Transmission of Hb and electron Hb-AuNCs. microscopy (TEM) image of hemoglobin-conjugated gold nanoclusters (Hb-AuNCs); (b) histogram of the size distribution of Hb-AuNCs; (c) Fourier transform 3.2. Biocompatibility Assays of Hb-AuNCs 3.2. Biocompatibilityinfrared Assays(FTIR) spectra of Hb-AuNCs of Hb and Hb-AuNCs. For further application as a biomedical sensor, the cell viability of Hb-AuNCs was evaluated by For further3.2.a resazurin Biocompatibility application dye reduction Assays as ofassay. a Hb biomedical-AuNCs Vero cells cultured sensor, in the DMEM cell without viability Hb-AuNCs of Hb-AuNCs were used wasas the evaluated by a resazurincontrol.For dye Waterfurther reduction-soluble application Hb assay.-AuNCs as a biomedical Vero at different cells sensor, culturedconcentrations the cell in viability DMEM of 2.7, of5.5, Hb without 11.0,-AuNCs 21.9, Hb-AuNCs43.8, was 87.5,evaluated 175, 350, were by used as and 700 μg/mL were separately incubated with Vero cells to examine cell viability. As shown in the control.a resazurin Water-soluble dye reduction Hb-AuNCs assay. Vero at cells diff culturederent concentrations in DMEM without of Hb 2.7,-AuNCs 5.5, 11.0,were used 21.9, as 43.8, the 87.5, 175, control.Figure 4,Water all cell-soluble viabilities Hb-AuNCs of Hb- AuNCsat different at con concentrationscentrations of of 2.7~7002.7, 5.5, μ11.0,g/mL 21.9, were 43.8, >80%. 87.5, High175, 350, cell 350, and 700 µg/mL were separately incubated with Vero cells to examine cell viability. As shown in andviabilities 700 μ g/mLindicated were that separately Hb-AuNCs incubated exhibited with no Verosignificant cells tocytotoxicity. examine cell viability. As shown in Figure4, allFigure cell 4, viabilities all cell viabilities of Hb-AuNCs of Hb-AuNCs at at concentrations concentrations of of 2.7~700 2.7~700 μg/mLµ gwere/mL >80%. were High>80%. cell High cell viabilitiesviabilities indicated indicated that Hb-AuNCs that Hb-AuNCs exhibited exhibited nono significant significant cytotoxicity. cytotoxicity.

Figure 4. Cell viabilities of hemoglobin-conjugated gold nanoclusters (Hb-AuNCs) at concentrations of 2.7~700 μg/mL via a resazurin dye reduction assay.

Figure 4. FigureCell viabilities 4. Cell viabilities of hemoglobin-conjugated of hemoglobin-conjugated goldgold nanoclusters nanoclusters (Hb (Hb-AuNCs)-AuNCs) at concentrations at concentrations of To further investigate the cytotoxicity of Hb-AuNCs, fluorescence images of Vero cells incubated 2.7~700 µgof/ mL2.7~700 via μg/mL a resazurin via a resazur dyein reduction dye reduction assay. assay. with Hb-AuNCs were examined. In Figure 5, blue and green pseudocolors indicate fluorescence signalsTo furtherof total investigateVero cells includingthe cytotoxicity both live of Hb and-AuNCs, dead cells fluorescence (stained withimages Hoechst of Vero 33342) cells incubated and dead withVero Hbcells-AuNCs (stained were with examined.SYTOX green), In Figure respectively. 5, blue For and the green control, pseudocolors not incubated indicate with fluorescence Hb-AuNCs, signalsVero cells of total homogeneously Vero cells including grew as indicatedboth live byand the dead blue cells pseudocolor (stained with as shown Hoechst in Figure 33342) 5 and (top dead left). VeroThere cells was (stained no detectable with SYTOX green pseudocolor, green), respectively. indicating For no the dead control, Vero not cells incubated in the control with ,Hb as -shownAuNCs, in VeroFigure cells 5 (tophomogeneously middle). After grew incubation as indicated with by Hbthe- AuNCs,blue pseudocolor Vero cells as alsoshown revealed in Figure homogeneous 5 (top left). Theregrowth was as noindicated detectable by greenthe blue pseudocolor, pseudocolor indicating, as shown no indead Figur Veroe 5 cells (bottom in the left). control There, as wereshown only in Figureslight signals 5 (top middle). of green After pseudocolor incubation in the with Vero Hb cells-AuNCs, incubated Vero cells with also Hb- AuNCs revealed, indicating homogeneous high growthbiocompatibility as indicated of Hb by- AuNCsthe blue, aspseudocolor shown in Figure, as shown 5 (bottom in Figur middle).e 5 (bottom Overall, left). the Thereresults were of the only cell slightviability signals assays of suggest green pseudocolorthat Hb-AuNCs in thecould Vero be a cells potential incubated fluorescent with Hbprobe-AuNCs for biomedical, indicating sensors high biocompatibilitybased on their biocompatibility. of Hb-AuNCs, as shown in Figure 5 (bottom middle). Overall, the results of the cell viability assays suggest that Hb-AuNCs could be a potential fluorescent probe for biomedical sensors

based on their biocompatibility.

Polymers 2020, 12, 2242 6 of 11

To further investigate the cytotoxicity of Hb-AuNCs, fluorescence images of Vero cells incubated with Hb-AuNCs were examined. In Figure5, blue and green pseudocolors indicate fluorescence signals of total Vero cells including both live and dead cells (stained with Hoechst 33342) and dead Vero cells (stained with SYTOX green), respectively. For the control, not incubated with Hb-AuNCs, Vero cells homogeneously grew as indicated by the blue pseudocolor as shown in Figure5 (top left). There was no detectable green pseudocolor, indicating no dead Vero cells in the control, as shown in Figure5 (top middle). After incubation with Hb-AuNCs, Vero cells also revealed homogeneous growth as indicated by the blue pseudocolor, as shown in Figure5 (bottom left). There were only slight signals of green pseudocolor in the Vero cells incubated with Hb-AuNCs, indicating high biocompatibility of Hb-AuNCs, as shown in Figure5 (bottom middle). Overall, the results of the cell viability assays suggest that Hb-AuNCs could be a potential fluorescent probe for biomedical sensors Polymersbased 2020 on, 12 their, x FOR biocompatibility. PEER REVIEW 7 of 11

FigureFigure 5. Fluorescence 5. Fluorescence images images of Vero of Vero cells cells incubated incubated with with hemoglobin-conjugatedhemoglobin-conjugated gold gold nanoclusters nanoclusters (Hb-AuNCs).(Hb-AuNCs). In the In thecontrol control experiments, experiments, Vero Vero c cellsells were culturedcultured with with Dulbecco’s Dulbecco’s modified modified Eagle’s Eagle’s mediummedium (DMEM (DMEM).). 3.3. Qualitative Analysis of Hp Phenotypes by Hb-AuNCs 3.3. Qualitative Analysis of Hp Phenotypes by Hb-AuNCs There are three different phenotypes of haptoglobin of Hp 1-1, Hp 2-1, and Hp 2-2, as shown in ThereFigure 6area. Hp three 1-1 different is composed phenotypes of two units of of haptoglobinα1β. The β ofchain Hp is 1 formed-1, Hp 2 of-1, 245 and amino-acid Hp 2-2, as residues shown in Figure(40 6a. kDa), Hp and1-1 is the composedα1 chain isof formedtwo un ofits 83of amino-acidα1β. The β residueschain is (9formed kDa) [of38 ].245 In amino Hp 2-1,-acid the residuesα1β (40 kDa),chain and is conjugated the α1 chain with is di formedfferent units of 83 of aminoα2β and-acid finally residues conjugated (9 kDa) with [38]α1.β Into Hp form 2- (1,α1 theβ)2 (αα12β )chainn is conjugated(n = 0, 1, 2with... ). different Hp 2-2 is units composed of α2β of and various finally numbers conjugated of (α2 βwith)n (n α=13,β to 4, 5form... ) ( toα1 formβ)2(α a2 linearβ)n (n = 0, 1, 2…).structure, Hp 2-2 and is composed the linear structure of various sequentially numbers forms of (α the2β cyclic)n (n structure= 3,4,5…) of to Hp form 2-2. Fora linear the native-PAGE structure, and the linearof Figure structure6b, plasma sequentially revealed phenotypesforms the cyclic including structure the monomeric of Hp 2-2. form For ofthe Hp native 1-1 and-PAGE polymeric of Figure α β 6b, plasmapatterns revealed of Hp 2-1 phenotypes and Hp 2-2. including The monomeric the monomeric form of Hp form 1-1 was of composedHp 1-1 and of (polymeric1 )2 as indicated patterns of in Figure6a. For Hp 2-1, the monomeric form of ( α1β)2 and polymeric form of (α1β)2(α2β)1 were Hp 2-1 and Hp 2-2. The monomeric form of Hp 1-1 was composed of (α1β)2 as indicated in Figure 6a. observably revealed in the native-PAGE results in Figure6b. Moreover, Hp 2-2 also showed polymeric For Hp 2-1, the monomeric form of (α1β)2 and polymeric form of (α1β)2(α2β)1 were observably forms of (α2β)n. For subsequent phenotype detection, three different phenotypes of Hp 1-1, Hp 2-1, revealedand Hpin the 2-2 native in plasma-PAGE were results respectively in Figure measured 6b. Moreover, by Hb-AuNCs. Hp 2-2 also showed polymeric forms of (α2β)n. For subsequent phenotype detection, three different phenotypes of Hp 1-1, Hp 2-1, and Hp 2- 2 in plasma were respectively measured by Hb-AuNCs.

Figure 6. (a) Schematic illustration of haptoglobin (Hp) phenotypes of Hp 1-1, Hp 2-1, and Hp 2-2; (b) native-PAGE of plasma phenotypes of Hp 1-1, Hp 2-1, and Hp 2-2.

To detect Hp phenotypes, human blood plasma with three different Hp phenotypes of Hp 1-1, Hp 2-1, and Hp 2-2 was prepared. In this work, plasma (5 μL) with three different Hp phenotypes

Polymers 2020, 12, x FOR PEER REVIEW 7 of 11

Figure 5. Fluorescence images of Vero cells incubated with hemoglobin-conjugated gold nanoclusters (Hb-AuNCs). In the control experiments, Vero cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM).

3.3. Qualitative Analysis of Hp Phenotypes by Hb-AuNCs There are three different phenotypes of haptoglobin of Hp 1-1, Hp 2-1, and Hp 2-2, as shown in Figure 6a. Hp 1-1 is composed of two units of α1β. The β chain is formed of 245 amino-acid residues (40 kDa), and the α1 chain is formed of 83 amino-acid residues (9 kDa) [38]. In Hp 2-1, the α1β chain is conjugated with different units of α2β and finally conjugated with α1β to form (α1β)2(α2β)n (n = 0, 1, 2…). Hp 2-2 is composed of various numbers of (α2β)n (n = 3,4,5…) to form a linear structure, and the linear structure sequentially forms the cyclic structure of Hp 2-2. For the native-PAGE of Figure 6b, plasma revealed phenotypes including the monomeric form of Hp 1-1 and polymeric patterns of Hp 2-1 and Hp 2-2. The monomeric form of Hp 1-1 was composed of (α1β)2 as indicated in Figure 6a. For Hp 2-1, the monomeric form of (α1β)2 and polymeric form of (α1β)2(α2β)1 were observably revealed in the native-PAGE results in Figure 6b. Moreover, Hp 2-2 also showed polymeric forms of (α2Polymersβ)n. For2020 subsequent, 12, 2242 phenotype detection, three different phenotypes of Hp 1-1, Hp 2-1, and7 ofHp 11 2- 2 in plasma were respectively measured by Hb-AuNCs.

FigureFigure 6. ( 6.a) (Schematica) Schematic illustration illustration of ofhaptoglobin haptoglobin (Hp) (Hp) phenotypes phenotypes of of Hp Hp 1- 1-1,1, Hp Hp 2- 2-1,1, and and Hp Hp 2- 2-2;2; (b) native(b) native-PAGE-PAGE of plasma of plasma phenotypes phenotypes of Hp of Hp1-1, 1-1,Hp Hp2-1, 2-1, and and Hp Hp 2-2. 2-2.

To detect Hp phenotypes, human blood plasma with three different Hp phenotypes of Hp 1-1, To detect Hp phenotypes, human blood plasma with three different Hp phenotypes of Hp 1-1, Hp 2-1, and Hp 2-2 was prepared. In this work, plasma (5 µL) with three different Hp phenotypes was Hp 2-1, and Hp 2-2 was prepared. In this work, plasma (5 μL) with three different Hp phenotypes respectively added to 100 µL of an Hb-AuNC solution (700 µg/mL), and then the mixtures were mixed by vortexing for 5 min. After vortexing for 5 min, the fluorescence spectra of the mixtures containing Hb-AuNCs and plasma were measured, as shown in Figure7. Compared to the fluorescence spectrum of Hb-AuNCs, the fluorescence spectrum of Hb-AuNCs with Hp 1-1 revealed an increase in the maximum intensity from 2892 to 6082, and the peak position of the maximum intensity was red shifted from 430 to 448 nm. For Hp 2-1 with Hb-AuNCs, the fluorescence spectrum showed an increase in the maximum intensity from 2892 to 3953, and the peak position of the maximum intensity was red shifted from 430 to 439 nm. After Hb-AuNCs were mixed with Hp 2-2, the fluorescence spectrum exhibited a slight increase in the maximum intensity from 2892 to 3054, and the peak position of the maximum intensity was red shifted from 430 to 431 nm. A previous study demonstrated that the domain of Hp serine protease exhibited extensive conjugation with the α- and β-subunits of Hb to form tightly bound Hp–Hb complexes [48]. Furthermore, cysteine residues of proteins are an important ligand to encapsulate and stabilize AuNCs due to the strong Au–S bonds between the surface of AuNCs and thiol groups of cysteine. The number of cysteines can cause a shift in the fluorescence spectrum of AuNCs [49]. Moreover, concentrations of Hp 1-1, Hp 2-1, and Hp 2-2 in plasma were demonstrated to be in the order of Hp 1-1 (184 42 mg/dL) > Hp 2-1 (153 55 mg/dL) > Hp 2-2 (93 54 mg/dL) [38]. ± ± ± Therefore, after Hb-AuNCs were mixed with Hp, Hp-Hb-AuNCs were formed. With the increase in cysteines, Hp-Hb-AuNCs revealed an increase in the maximum fluorescence intensity and a red shift of the peak position of the maximum intensity compared to that of Hb-AuNCs. With the highest concentration in plasma, Hp 1-1 conjugated with Hb-AuNCs exhibited the strongest fluorescence intensity and greatest red shift of the peak position of the maximum intensity compared to those of Hp 2-1 and Hp 2-2 conjugated with Hb-AuNCs. Overall, based on changes in the fluorescence intensity and the peak position of the maximum intensity of Hp-Hb-AuNCs, the three different Hp phenotypes in plasma of Hp 1-1, Hp 2-1, and Hp 2-2 were successfully qualitatively analyzed by fluorescent Hb-AuNCs. Polymers 2020, 12, x FOR PEER REVIEW 8 of 11 was respectively added to 100 μL of an Hb-AuNC solution (700 μg/mL), and then the mixtures were mixed by vortexing for 5 min. After vortexing for 5 min, the fluorescence spectra of the mixtures containing Hb-AuNCs and plasma were measured, as shown in Figure 7. Compared to the fluorescence spectrum of Hb-AuNCs, the fluorescence spectrum of Hb-AuNCs with Hp 1-1 revealed an increase in the maximum intensity from 2892 to 6082, and the peak position of the maximum intensity was red shifted from 430 to 448 nm. For Hp 2-1 with Hb-AuNCs, the fluorescence spectrum showed an increase in the maximum intensity from 2892 to 3953, and the peak position of the maximum intensity was red shifted from 430 to 439 nm. After Hb-AuNCs were mixed with Hp 2-2, the fluorescence spectrum exhibited a slight increase in the maximum intensity from 2892 to 3054, and the peak position of the maximum intensity was red shifted from 430 to 431 nm. A previous study demonstrated that the domain of Hp serine protease exhibited extensive conjugation with the α- and β-subunits of Hb to form tightly bound Hp–Hb complexes [48]. Furthermore, cysteine residues of proteins are an important ligand to encapsulate and stabilize AuNCs due to the strong Au–S bonds between the surface of AuNCs and thiol groups of cysteine. The number of cysteines can cause a shift in the fluorescence spectrum of AuNCs [49]. Moreover, concentrations of Hp 1-1, Hp 2-1, and Hp 2- 2 in plasma were demonstrated to be in the order of Hp 1-1 (184 ± 42 mg/dL) > Hp 2-1 (153 ± 55 mg/dL) > Hp 2-2 (93 ± 54 mg/dL) [38]. Therefore, after Hb-AuNCs were mixed with Hp, Hp-Hb- AuNCs were formed. With the increase in cysteines, Hp-Hb-AuNCs revealed an increase in the maximum fluorescence intensity and a red shift of the peak position of the maximum intensity compared to that of Hb-AuNCs. With the highest concentration in plasma, Hp 1-1 conjugated with Hb-AuNCs exhibited the strongest fluorescence intensity and greatest red shift of the peak position of the maximum intensity compared to those of Hp 2-1 and Hp 2-2 conjugated with Hb-AuNCs. Overall, based on changes in the fluorescence intensity and the peak position of the maximum intensityPolymers 2020 of, Hp12, 2242-Hb-AuNCs, the three different Hp phenotypes in plasma of Hp 1-1, Hp 2-1, and8 ofHp 11 2-2 were successfully qualitatively analyzed by fluorescent Hb-AuNCs.

FigureFigure 7. FluorescenceFluorescence spectra spectra of of hemoglobin hemoglobin-conjugated-conjugated gold gold nanoclusters nanoclusters (Hb (Hb-AuNCs)-AuNCs) before before and afterafter adding adding Hp Hp 1 1-1,-1, Hp 2 2-2,-2, and Hp 2 2-2.-2.

4. Conclusions 4. Conclusions In summary, fluorescent Hb-AuNCs were successfully synthesized via a facile hydrothermal In summary, fluorescent Hb-AuNCs were successfully synthesized via a facile hydrothermal synthesis method. With the formation of Hb-AuNCs, the absorption spectrum of Hb-AuNCs revealed a synthesis method. With the formation of Hb-AuNCs, the absorption spectrum of Hb-AuNCs decrease in the signal intensity and a blue-shift from 405 to 367 nm of the Hb Soret band due to a change revealed a decrease in the signal intensity and a blue-shift from 405 to 367 nm of the Hb Soret band in the hydrophobicity of the heme groups. Based on results of the XPS analysis, Hb-AuNCs exhibited due to a change in the hydrophobicity of the heme groups. Based on results of the XPS analysis, Hb- peaks of Au 4f5/2 and Au 4f7/2 separately located at 88.1 and 84.5 eV because of a lot of gold with AuNCs exhibited peaks of Au 4f5/2 and Au 4f7/2 separately located at 88.1 and 84.5 eV because of a lot high oxidation states in their cores. The fluorescence spectrum of Hb-AuNCs demonstrated the maximum fluorescence intensity at a wavelength of 430 nm under an excitation wavelength of 314 nm. For structural characterization, Hb-AuNCs showed an approximately globular shape, and their average size was calculated to be 3.7 nm. Moreover, Hb-AuNCs were verified to have high biocompatibility according to a cell viability assay and fluorescence images of Vero cells. To detect the three different Hp phenotypes, fluorescent Hb-AuNCs were successfully qualitatively analyzed in plasma because of the formation of Hp-Hb-AuNCs. Overall, our work demonstrated that fluorescent Hb-AuNCs can be applied for the clinical diagnosis of three different Hp phenotypes.

Author Contributions: Conceptualization, S.-H.T., S.Y., H.-L.C., T.-R.K., and T.-M.C.; Methodology, S.-H.T., S.Y., and H.-L.C.; Writing—original draft preparation, T.-M.C.; Writing—review and editing, T.-R.K. and T.-M.C. All authors read and agreed to the published version of the manuscript. Funding: This work was financially supported by grants from the Ministry of Science and Technology (MOST 109-2113-M-038-005-MY2), Taipei Medical University, and the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. Acknowledgments: We would like to acknowledge Yuan-Chin Hsiung, Chun-Chih Liu, and Chi-Ming Lee for their excellent technical support at the TMU Core Facility Center. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Zhang, H.; Liu, H.; Tian, Z.; Lu, D.; Yu, Y.; Cestellos-Blanco, S.; Sakimoto, K.K.; Yang, P. Bacteria photosensitized by intracellular gold nanoclusters for solar fuel production. Nat. Nanotechnol. 2018, 13, 900–905. [CrossRef] 2. Sakimoto, K.K.; Zhang, S.J.; Yang, P.Cysteine–cystine photoregeneration for oxygenic photosynthesis of acetic acid

from CO2 by a tandem inorganic–biological hybrid system. Nano Lett. 2016, 16, 5883–5887. [CrossRef] Polymers 2020, 12, 2242 9 of 11

3. Weng, B.; Lu, K.-Q.; Tang, Z.; Chen, H.M.; Xu, Y.-J. Stabilizing ultrasmall Au clusters for enhanced photoredox catalysis. Nat. Commun. 2018, 9, 1543. [CrossRef][PubMed] 4. Wen, F.; Dong, Y.; Feng, L.; Wang, S.; Zhang, S.; Zhang, X. Horseradish peroxidase functionalized fluorescent gold nanoclusters for hydrogen peroxide sensing. Anal. Chem. 2011, 83, 1193–1196. [CrossRef] 5. Liu, C.-L.; Wu, H.-T.; Hsiao, Y.-H.; Lai, C.-W.; Shih, C.-W.; Peng, Y.-K.; Tang, K.-C.; Chang, H.-W.; Chien, Y.-C.; Hsiao, J.-K.; et al. Insulin-directed synthesis of fluorescent gold nanoclusters: Preservation of insulin bioactivity and versatility in cell imaging. Angew. Chem. Int. Ed. 2011, 50, 7056–7060. [CrossRef] 6. Wu, X.; He, X.; Wang, K.; Xie, C.; Zhou, B.; Qing, Z. Ultrasmall near-infrared gold nanoclusters for tumor fluorescence imaging in vivo. Nanoscale 2010, 2, 2244–2249. [CrossRef] 7. Li, C.-H.; Kuo, T.-R.; Su, H.-J.; Lai, W.-Y.; Yang, P.-C.; Chen, J.-S.; Wang, D.-Y.; Wu, Y.-C.; Chen, C.-C. Fluorescence-guided probes of aptamer-targeted gold nanoparticles with computed tomography imaging accesses for in vivo tumor resection. Sci. Rep. 2015, 5, 15675. [CrossRef] 8. Chen, L.Y.; Wang, C.W.; Yuan, Z.; Chang, H.T. Fluorescent gold nanoclusters: Recent advances in sensing and imaging. Anal. Chem. 2015, 87, 216–229. [CrossRef] 9. Lakkakula, J.R.; Divakaran, D.; Thakur, M.; Kumawat, M.K.; Srivastava, R. Cyclodextrin-stabilized gold nanoclusters for bioimaging and selective label-free intracellular sensing of Co2+ ions. Sens. Actuators B Chem. 2018, 262, 270–281. [CrossRef] 10. Xie, Y.; Xianyu, Y.; Wang, N.; Yan, Z.; Liu, Y.; Zhu, K.; Hatzakis, N.S.; Jiang, X. Functionalized gold nanoclusters identify highly reactive oxygen species in living organisms. Adv. Funct. Mater. 2018, 28, 1702026. [CrossRef] 11. Zang, J.; Li, C.; Zhou, K.; Dong, H.; Chen, B.; Wang, F.; Zhao, G. Nanomolar Hg2+ detection using β-lactoglobulin-stabilized fluorescent gold nanoclusters in beverage and biological media. Anal. Chem. 2016, 88, 10275–10283. [CrossRef][PubMed] 12. Cheng, T.M.; Chu, H.L.; Lee, Y.C.; Wang, D.Y.; Chang, C.C.; Chung, K.L.; Yen, H.C.; Hsiao, C.W.; Pan, X.Y.; Kuo, T.R.; et al. Quantitative analysis of glucose metabolic cleavage in glucose transporters overexpressed cancer cells by target-specific fluorescent gold nanoclusters. Anal. Chem. 2018, 90, 3974–3980. [CrossRef] 13. Halawa, M.I.; Li, B.S.; Xu, G.B. Novel synthesis of thiolated gold nanoclusters induced by lanthanides for ultrasensitive and luminescent detection of the potential anthrax spores’ biomarker. ACS Appl. Mater. Interfaces 2020, 12, 32888–32897. [CrossRef] 14. Yang, H.W.; Yang, Y.; Liu, S.L.; Zhan, X.X.; Zhou, H.; Li, X.S.; Yuan, Z.Q. Ratiometric and sensitive cyanide sensing using dual-emissive gold nanoclusters. Anal. Bioanal. Chem. 2020, 412, 5819–5826. [CrossRef] 15. Zhao, Y.; Pan, M.; Liu, F.; Liu, Y.H.; Dong, P.; Feng, J.; Shi, T.H.; Liu, X.Q. Highly selective and sensitive detection of trinitrotoluene by framework-enhanced fluorescence of gold nanoclusters. Anal. Chim. Acta 2020, 1106, 133–138. [CrossRef] 16. Ungor, D.; Dékány, I.; Csapó, E. Reduction of tetrachloroaurate (iii) ions with bioligands: Role of the thiol and amine functional groups on the structure and optical features of gold nanohybrid systems. Nanomaterials 2019, 9, 1229. [CrossRef] 17. Sarparast, M.; Molaabasi, F.; Ghazfar, R.; Ashtiani, M.M.; Qarai, M.B.; Taherpour, A.A.; Amyab, S.P.; Shamsipur, M. Efficient ethanol oxidation by hemoglobin-capped gold nanoclusters: The critical role of fe in the heme group as an oxophilic metal active site. Electrochem. Commun. 2019, 103, 42–47. [CrossRef] 18. Shamsipur, M.; Samandari, L.; Farzin, L.; Sarparast, M.; Molaabasi, F.; Mousazadeh, M.H. Dual-modal label-free genosensor based on hemoglobin@ gold nanocluster stabilized graphene nanosheets for the electrochemical detection of BCR/ABL fusion gene. Talanta 2020, 217, 121093. [CrossRef] 19. Li, Y.; Peng, W.; You, X. Determination of dopamine by exploiting the catalytic effect of hemoglobin–stabilized gold nanoclusters on the luminol–naio 4 chemiluminescence system. Microchim. Acta 2017, 184, 3539–3545. [CrossRef] 20. Shamsipur, M.; Molaabasi, F.; Shanehsaz, M.; Moosavi-Movahedi, A.A. Novel blue-emitting gold nanoclusters confined in human hemoglobin, and their use as fluorescent probes for copper (ii) and histidine. Microchim. Acta 2015, 182, 1131–1141. [CrossRef] 21. Molaabasi, F.; Hosseinkhani, S.; Moosavi-Movahedi, A.A.; Shamsipur, M. Hydrogen peroxide sensitive hemoglobin-capped gold nanoclusters as a fluorescence enhancing sensor for the label-free detection of glucose. RSC Adv. 2015, 5, 33123–33135. [CrossRef] Polymers 2020, 12, 2242 10 of 11

22. Shamsipur, M.; Pashabadi, A.; Molaabasi, F. A novel electrochemical hydrogen peroxide biosensor based on hemoglobin capped gold nanoclusters–chitosan composite. RSC Adv. 2015, 5, 61725–61734. [CrossRef] 23. Hochberg, I.; Roguin, A.; Nikolsky, E.; Chanderashekhar, P.V.; Cohen, S.; Levy, A.P. Haptoglobin phenotype and coronary artery collaterals in diabetic patients. Atherosclerosis 2002, 161, 441–446. [CrossRef] 24. Bamm, V.V.; Tsemakhovich, V.A.; Shaklai, M.; Shaklai, N. Haptoglobin phenotypes differ in their ability to inhibit heme transfer from hemoglobin to LDL. Biochemistry 2004, 43, 3899–3906. [CrossRef][PubMed] 25. Langlois, M.R.; Martin, M.E.; Boelaert, J.R.; Beaumont, C.; Taes, Y.E.; De Buyzere, M.L.; Bernard, D.R.; Neels, H.M.; Delanghe, J.R. The haptoglobin 2-2 phenotype affects serum markers of iron status in healthy males. Clin. Chem. 2000, 46, 1619–1625. [CrossRef] 26. Levy, A.P.; Hochberg, I.; Jablonski, K.; Resnick, H.E.; Lee, E.T.; Best, L.; Howard, B.V. Haptoglobin phenotype is an independent risk factor for cardiovascular disease in individuals with diabetes: The strong heart study. J. Am. Coll. Cardiol. 2002, 40, 1984–1990. [CrossRef] 27. Levy, A.P.; Roguin, A.; Hochberg, I.; Herer, P.; Marsh, S.; Nakhoul, F.M.; Skorecki, K. Haptoglobin phenotype and vascular complications in patients with diabetes. N. Engl. J. Med. 2000, 343, 969–970. [CrossRef] 28. Maeda, N.; Smithies, O. The evolution of multigene families-human haptoglobin genes. Annu. Rev. Genet. 1986, 20, 81–108. [CrossRef] 29. Gutteridge, J.M.C. The antioxidant activity of haptoglobin towards hemoglobin-stimulated lipid-peroxidation. Biochim. Biophys. Acta 1987, 917, 219–223. [CrossRef] 30. Melamed-Frank, M.; Lache, O.; Enav, B.I.; Szafranek, T.; Levy, N.S.; Ricklis, R.M.; Levy, A.P. Structure-function analysis of the antioxidant properties of haptoglobin. Blood 2001, 98, 3693–3698. [CrossRef] 31. Ebersole, J.L.; Machen, R.L.; Steffen, M.J.; Willmann, D.E. Systemic acute-phase reactants, c-reactive protein and haptoglobin, in adult periodontitis. Clin. Exp. Immunol. 1997, 107, 347–352. [CrossRef][PubMed] 32. Langlois, M.R.; Delanghe, J.R. Biological and clinical significance of haptoglobin polymorphism in humans. Clin. Chem. 1996, 42, 1589–1600. [CrossRef][PubMed] 33. Dobryszycka, W. Biological functions of haptoglobin-new pieces to an old puzzle. Eur. J. Clin. Chem.·Clin. Biochem. 1997, 35, 647–654. 34. Okuyama, N.; Ide, Y.; Nakano, M.; Nakagawa, T.; Yamanaka, K.; Moriwaki, K.; Murata, K.; Ohigashi, H.; Yokoyama, S.; Eguchi, H.; et al. Fucosylated haptoglobin is a novel marker for pancreatic cancer: A detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation. Int. J. Cancer 2006, 118, 2803–2808. [CrossRef][PubMed] 35. Bong-Sop, S.; Dae-Myung, J. Simple spectrophotometric determination of haptoglobin level in serum. Clin. Chim. Acta 1984, 136, 145–153. [CrossRef] 36. Braun, H.; Aly, F. Problems in the quantitative estimation of human serum haptoglobin by single radial immunodiffusion. Clin. Chim. Acta 1969, 26, 588–590. [CrossRef] 37. Van Lente, F.; Marchand, A.; Galen, R.S. Evaluation of a nephelometric assay for haptoglobin and its clinical usefulness. Clin. Chem. 1979, 25, 2007–2010. [CrossRef] 38. Cheng, T.-M.; Pan, J.-P.; Lai, S.-T.; Kao, L.-P.; Lin, H.-H.; Mao, S.J. Immunochemical property of human haptoglobin phenotypes: Determination of plasma haptoglobin using type-matched standards. Clin. Biochem. 2007, 40, 1045–1056. [CrossRef] 39. Dou, X.; Yuan, X.; Yu, Y.; Luo, Z.; Yao, Q.; Leong, D.T.; Xie, J. Lighting up thiolated Au@ Ag nanoclusters via aggregation-induced emission. Nanoscale 2014, 6, 157–161. [CrossRef] 40. Shamsipur, M.; Molaabasi, F.; Sarparast, M.; Roshani, E.; Vaezi, Z.; Alipour, M.; Molaei, K.; Naderi-Manesh, H.; Hosseinkhani, S. Photoluminescence mechanisms of dual-emission fluorescent silver nanoclusters fabricated by human hemoglobin template: From oxidation-and aggregation-induced emission enhancement to targeted drug delivery and cell imaging. ACS Sustain. Chem. Eng. 2018, 6, 11123–11137. [CrossRef] 41. Shang, L.; Dong, S.; Nienhaus, G.U. Ultra-small fluorescent metal nanoclusters: Synthesis and biological applications. Nano Today 2011, 6, 401–418. [CrossRef] 42. Muhammed, M.A.H.; Verma, P.K.; Pal, S.K.; Kumar, R.A.; Paul, S.; Omkumar, R.V.; Pradeep, T. Bright, NIR-emitting Au23 from Au25: Characterization and applications including biolabeling. Chem. Eur. J. 2009, 15, 10110–10120. [CrossRef][PubMed] 43. Wu, Z.; Jin, R. On the ligand’s role in the fluorescence of gold nanoclusters. Nano Lett. 2010, 10, 2568–2573. [CrossRef][PubMed] Polymers 2020, 12, 2242 11 of 11

44. Negishi, Y.; Nobusada, K.; Tsukuda, T. Glutathione-protected gold clusters revisited: Bridging the gap between gold (i) thiolate complexes and thiolate-protected gold nanocrystals. J. Am. Chem. Soc. 2005, 127, − 5261–5270. [CrossRef] 45. Luo, Z.; Yuan, X.; Yu, Y.; Zhang, Q.; Leong, D.T.; Lee, J.Y.; Xie, J. From aggregation-induced emission of Au (i)–thiolate complexes to ultrabright Au (0)@ Au (i)–thiolate core–shell nanoclusters. J. Am. Chem. Soc. 2012, 134, 16662–16670. [CrossRef][PubMed] 46. Ungor, D.; Horváth, K.; Dékány, I.; Csapó, E. Red-emitting gold nanoclusters for rapid fluorescence sensing of tryptophan metabolites. Sens. Actuators B Chem. 2019, 288, 728–733. [CrossRef] 47. Del Caño, R.; Mateus, L.; Sánchez-Obrero, G.; Sevilla, J.M.; Madueño, R.; Blázquez, M.; Pineda, T. Hemoglobin bioconjugates with surface-protected gold nanoparticles in aqueous media: The stability depends on solution pH and protein properties. J. Colloid Interface Sci. 2017, 505, 1165–1171. 48. Andersen, C.B.F.; Torvund-Jensen, M.; Nielsen, M.J.; de Oliveira, C.L.P.; Hersleth, H.-P.; Andersen, N.H.; Pedersen, J.S.; Andersen, G.R.; Moestrup, S.K. Structure of the haptoglobin–haemoglobin complex. Nature 2012, 489, 456–459. [CrossRef][PubMed] 49. Xu, Y.; Sherwood, J.; Qin, Y.; Crowley, D.; Bonizzoni, M.; Bao, Y. The role of protein characteristics in the formation and fluorescence of au nanoclusters. Nanoscale 2014, 6, 1515–1524. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).