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Homogeneous Immunosensor Based on Luminescence Resonance Energy Transfer for Glycated Hemoglobin (HbA1c) Detection Using Upconversion Nanoparticles Eun-Jung Jo, Hyoyoung Mun, and Min-Gon Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04255 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 12, 2016

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Homogeneous Immunosensor Based on Luminescence Resonance Energy Transfer for Glycated Hemoglobin (HbA1c) Detection Using Upconversion Nanoparticles Eun-Jung Jo,† Hyoyoung Mun,† and Min-Gon Kim *,†

†Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea

*

Corresponding author. Min-Gon Kim: Tel: +82-62-715-3330; Fax: +82-62-715-3419; E-mail address: [email protected]

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ABSTRACT: We report an immunosensor based on luminescence resonance energy transfer (LRET) to detect homogeneous glycated hemoglobin (HbA1c). This system uses near-infrared (NIR)-tovisible rare-earth upconversion nanoparticles (UCNPs), such as NaYF4:Yb3+, Er3+, as the donor and HbA1c as the acceptor. The HbA1c used as target molecules showed absorption at 541 nm, which corresponded with the emission of the UCNPs. When HbA1c was added, LRET occurred between the donor and acceptor under laser irradiation of 980 nm because of the specific recognition between the anti-HbA1c monocolonal antibody-functionalized UCNPs and HbA1c. In the absence of HbA1c, there was strong upconversion luminescence intensity; however, in its presence, the distance between the donor and acceptor decreased to enable energy transfer, consequently quenching the luminescence of the UCNPs. The proposed method was successfully applied to HbA1c detection in blood samples. Our results indicate that the LRET-based immunosensor allows for specific and sensitive detection of HbA1c in a homogeneous manner.

Keywords Luminescence resonance energy transfer, Upconversion nanoparticles, Glycated hemoglobin, Quenching, Immunosensor

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■ INTRODUCTION

Glycated hemoglobin (HbA1c) is a stable glucose adduct containing the N-terminal valine of the βchain of hemoglobin. The level of HbA1c, which is measured as the percentage of HbA1c in the total hemoglobin content, indicates the average blood glucose level over the previous 2 to 3 months.1, 2 The HbA1c level is unaffected by the daily fluctuation in the blood glucose level, and is currently a very useful diagnostic index and allows long-term monitoring for diabetes.3-7 HbA1c levels are usually determined via high-performance liquid chromatography (HPLC) coupled with either mass spectrometry or capillary electrophoresis, which is a standard reference method according to the International Federation of Clinical Chemistry (IFCC) Working Group. This method is generally precise, sensitive, reproducible, and applicable to the analysis of various samples; however, it can provide false results because of the lack in specificity by the coexistence of genetic substance and other chemically modified derivatives of hemoglobin. Additionally, it is timeconsuming, expensive, and requires trained personnel as it involves complicated processes. Therefore, it hardly meets the requirements for rapid, easy, and portable HbA1c detection.4, 7-10 Accordingly, a number of sensors based on colorimetric, fluorometric, and electrochemical methods have been developed. Although the colorimetric method is easy, sensitive, and inexpensive, it requires the addition of electrolytes, such as sodium chloride, for aggregation of gold nanoparticles in the presence or absence of HbA1c.11 The fluorometric method uses boronic acid reporters to monitor boronate formation: as they interact with the cis-diol moieties of glucose and form boronate esters, the fluorescence intensity increases. Although this method is sensitive, it measures the total glycated hemoglobin, including HbA1c and hemoglobin glycated at other sites; therefore, it is not selective for HbA1c.4, 12-14 The electrochemical biosensors can be applied to portable HbA1c detection devices because of their low cost, high sensitivity, and small size.9 However, they require electrode modification,15 separation of total hemoglobin content,4 and fabrication of the sensing interface involving step-wise attachment of a molecular wire, redox species, and an HbA1c analog for competitive inhibition.9 Hence, there is a need to develop a novel homogeneous biosensor for HbA1c 3

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detection with high selectivity and simplicity. Upconversion nanoparticles (UCNPs) are lanthanide-doped nanomaterials that convert near-infrared (NIR) radiation into visible radiation (anti-Stokes optical properties) via a nonlinear optical process. These nanoparticles exhibit a sharp emission bandwidth, low toxicity, high chemical stability, deep NIR light penetration into tissue, and a high signal-to-noise ratio as compared to traditional fluorescent labels, such as organic dyes and quantum dots.16-18 Therefore, UCNPs are promising luminescent materials for bioanalysis and cancer imaging.19-21 In particular, the use of NIR light for the excitation of UCNPs can prevent background autofluorescence, photobleaching, and photodamage in biological samples.22, 23 Taking advantage of these properties, a number of luminescence resonance energy transfer (LRET)-based biosensors using UCNPs as the donor have been developed.24-32 In this study, we designed a homogeneous LRET-based immunosensor to detect HbA1c (acceptor) levels by quenching the luminescence signals of the UCNPs (donor). This biosensor may be used as a diagnostic tool for rapid, homogeneous, and easy detection of HbA1c in blood.

■ EXPERIMENTAL SECTION Materials and sampling. Yttrium(III) chloride hexahydrate (YCl3·6H2O), ytterbium(III) chloride hexahydrate (YbCl3·6H2O), erbium(III) chloride hexahydrate (ErCl3·6H2O), oleic acid, 1octadecene, ammonium fluoride (NH4F), sodium hydroxide (NaOH), methanol (CH3OH), ethanol (CH3CH2OH),

cyclohexane,

aminopropyl)trimethoxysilane

IGEPAL®

CO-520,

(APTMS),

tetraethyl

dimethyl

orthosilicate

sulfoxide

(TEOS),

(DMSO),

(32-(N-

morpholino)ethanesulfonic acid (MES) hydrate, Tris, sodium bicarbonate (NaHCO3), succinic anhydride, N-hydroxysuccinimide (NHS), glycerol, dithiothreitol, bromophenol blue, brilliant blue G, glycated human albumin, glucose, ethylenediaminetetraacetic acid (EDTA), and Triton™ X-100 were purchased from Sigma-Aldrich Chemical (St Louis, MO, USA). Bovine serum albumin (BSA) was obtained from Fitzgerald (Acton, MA, USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The antihemoglobin A1c antibody (anti-HbA1c) and anti-E. coli antibody were purchased from Abcam 4

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(Cambridge, England). Phosphate-buffered saline (PBS, pH 7.4) and Tris-HCl (pH 6.8) were purchased from Biosesang (Seongnam, South Korea). All other chemicals and organic solvents used were of reagent grade or better. The Lyphochek® Hemoglobin A1C Linearity Set (standard samples), a human whole blood product designed to verify linearity throughout the reportable range of HbA1c, was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Real blood samples (IFCC HbA1c Monitoring Programme, HBAIFCC-mon) were obtained from the ERL/MCA Laboratory (Winterswijk, Netherlands), which is part of the International Federation of Clinical Chemistry (IFCC) Network. The HbA1c value (mmol/mol) of the each specimen was assigned by the ERL/MCA Laboratory according to the IFCC reference measurement procedure. IFCC HbA1c values (mmol/mol) were converted to NGSP (National Glycohaemoglobin Standardisation Programme) HbA1c values (%). The blood specimens were stored at −80 °C until subsequent analysis.

Instrumentation. Upconversion luminescence spectra were recorded with a fluorescence spectrometer (FluoroMate FS-2; SCINCO, Seoul, Korea) that was modified with a 980 nm cw laser (SSL-LM-980-600-D; Shanghai Sanctity Laser Technology, Shanghai, China) for upconversion excitation. The absorbance was recorded using an Infinite 200 PRO and a microplate reader from TECAN (Mannedorf, Switzerland). The shape, size, and uniformity of synthesized UCNPs were measured with a transmission electron microscope (JEM-2100F; JEOL, Tokyo, Japan). The surface charges and hydrodynamic sizes were determined using a zeta potential and particle size analyzer (ELSZ-1000; Otsuka Electronics, Japan).

Synthesis of NaYF4:Yb3+, Er3+ upconversion nanoparticles (UCNPs). Synthesis of NaYF4:Yb3+, Er3+ was conducted according to a previously reported procedure.33 YCl3·6H2O (0.78 mmol), YbCl3·6H2O (0.20 mmol), and ErCl3·6H2O (0.02 mmol) were mixed with 8 mL oleic acid and 15 mL 1-octadecene in a 50-mL flask under an atmosphere of nitrogen and heated to 160 °C to form a clear solution. The solution was stirred for 30 min at 160 °C under vacuum. The reaction mixture was then cooled down to room temperature and 10 mL methanol containing NaOH (0.25 M) and NH4F (0.4 M) was added into the flask and stirred for 30 min. In order to remove methanol, the solution was 5

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heated to 100 °C and stirred for 10 min. Subsequently, the solution was heated to 300 °C for 1 h. After cooling to room temperature, the UCNPs were precipitated and washed by addition of ethanol for three times via centrifugation. The pellet was redispersed in cyclohexane and this was used for silica coating.

Silica coating of UCNPs. The surface of UCNPs was coated via a procedure reported by Jalil and Zhang.34 CO-520 (0.5 mL), cyclohexane (5 mL), and 0.05 M UCNP solution (20 mL) in cyclohexane were mixed and sonicated for 10 min. Then, 2 mL of CO-520 and 0.4 mL of aqueous ammonia (wt 30%) were added and the flask was sealed and sonicated for 30 min until a clear emulsion was formed. TEOS (0.2 mL) was then added into the solution, and the solution was stirred for 2 days. The silica-coated UCNPs were precipitated by adding acetone, washed with ethanol and collected via centrifugation twice, and then stored in 10 mL of ethanol. The concentration of the asprepared silica-coated UCNPs solution was denoted as “1×”.

Functionalization of silica-coated UCNPs. Amino-functionalized silica-coated UCNPs were prepared as follows: 40 µL of APTMS was added to 0.04× silica-coated UCNPs in ethanol to yield an APTMS solution with a final concentration of 2%, and the resulting solution was incubated at room temperature for 1 h. The precipitates were separated by centrifugation, washed with ethanol several times, and then dried at 60 °C overnight. Then, 10 mg of amino-modified UCNPs were dissolved in 1 mL of ethanol, centrifuged, and finally redispersed in 1 mL succinic anhydride solution. The succinic anhydride solution was composed of 0.75 mg succinic anhydride in a 1-mL solution [DMSO/0.1 M NaHCO3 (pH 9) = 1/9 (v/v)]. The amino-modified UCNPs were then stirred with a succinic anhydride solution to convert the amine group to a carboxylate group for 1 h. The resulting carboxylate-modified UCNPs were washed with 10 mM borate buffer (pH 8) 3 times, and then redispersed in MES buffer (25 mM, pH 6).

Conjugation of antibodies to UCNPs. Antibodies (anti-HbA1c or anti-E. coli) were covalently conjugated to UCNPs using EDC/NHS coupling. A solution of the carboxylate-modified UCNPs (1 mg/mL, 0.3 mL) was washed twice with MES buffer (25 mM, pH 6) and centrifuged. EDC solution (5 mg/mL, 50 µL) and NHS solution (5 mg/mL, 50 µL) were added to the washed 6

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carboxylate-modified UCNPs. UCNPs were activated via EDC/NHS chemistry with slow shaking at room temperature for 30 min. The activated UCNPs were centrifuged and washed twice with MES buffer (25 mM, pH 6). After discarding the supernatant, antibodies (1 mg/mL, 15 µL) and 85 µL of MES buffer (25 mM, pH6) were added to the activated UCNPs to a final volume of 100 µL. Then, the solution was incubated at room temperature for 1 h. In order to quench the excess activated carboxylic acid groups, the UCNPs coated with antibodies were incubated with Tris (50 mM, pH 7.4) at room temperature for 15 min. This was followed by washing the particles twice with water, with a centrifugation step (10000 rpm for 10 min) in between the washings. The coated UCNPs were washed three times with PBS buffer (pH 7.4) containing 0.1% BSA. Finally, the UCNP-antibody conjugates were resuspended and stored in 0.15 mL of PBS. The concentration of the as-prepared UCNPantibody conjugate solution was denoted as “1×”. The UCNP-antibody conjugates were quantitatively confirmed using the Bradford assay as follows: Standard solutions of anti-HbA1c antibody at concentrations of 0, 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/mL were prepared. These standards and UCNP-antibody conjugates were each mixed with Coomassie Brilliant Blue G-250 dye at a ratio of 1:4. After 5 min of incubation at room temperature, the absorbance at 595 nm was measured for all samples. The concentration of anti-HbA1c antibody conjugated to the UCNPs was calculated based on the standard curve created from the standard antiHbA1c antibody solutions.

Preparation of the LRET-based immunosensor system for HbA1c detection. The standard samples (Level 1–4; Lyphochek Hemoglobin A1C Linearity Set) and real blood samples of HbA1c were diluted with PBS buffer (pH 7.4) containing 0.07 % Triton™ X-100 and 5 mM EDTA. The optical density (OD) at 541 nm of the diluted HbA1c samples was measured using a microplate reader and adjusted to 0.01. For preparation of the LRET-based immunosensor, 20 µL of UCNPantibody conjugate solution was added to the upconversion luminescence measurement cell along with 160 µL of PBS buffer (pH 7.4). After the addition of 20 µL of HbA1c (OD 541 nm = 0.01) solution in PBS buffer (pH 7.4) containing 0.07 % Triton™ X-100 and 5 mM EDTA, the cell was incubated at room temperature prior to measurement. 7

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■ RESULTS AND DISCUSSION

Principle of the LRET-based immunosensor. We developed an LRET-based immunosensor (Figure 1) with NaYF4:Yb3+, Er3+ UCNPs and HbA1c as the donor and acceptor of the LRET pair, respectively. Strong upconversion luminescence intensity was observed in the absence of HbA1c (Figure 1A). In the presence of HbA1c, the specific recognition between the anti-HbA1c monoclonal antibody-functionalized UCNPs and HbA1c leads to positioning of the energy donor and acceptor in close proximity, ultimately quenching the upconversion luminescence of the UCNPs through LRET (Figure 1B). The UCNPs showed upconversion luminescence around 545 and 660 nm upon excitation by 980-nm lasers (Figure S1). The predominantly green emissions (emission maximum at 545 nm) of the UCNPs can easily be seen by the naked eye (Figure S1 inset). As we can see from Figure 2, the green emission spectrum of the UCNPs ranging from 500 to 570 nm suitably overlaps with the absorption spectrum of HbA1c. Therefore, the luminescence of the anti-HbA1c-functionalized UCNPs can be strongly quenched by HbA1c and very weakly by glycated albumin (Figure 3). In addition, the luminescence of anti-E.coli monoclonal antibody-functionalized UCNPs was weakly quenched by HbA1c (Figure 3), possibly due to the inner filter effect35 by HbA1c absorbance. This result suggests that the anti-HbA1c monoclonal antibody-functionalized conjugates bind to HbA1c specifically and are useful in HbA1c detection through quenching by LRET.

Characterization of the prepared UCNPs and UCNP-antibody conjugates. Figure S2 displays TEM images (Figure S2A) and size distribution (Figure S2B) of the synthesized UCNPs before surface modification and conjugation. The UCNPs were well dispersed and uniform in size. The selected area electron diffraction (SAED) pattern confirmed a perfect hexagonal closepacked (HCP) structure of the synthesized UCNPs (Figure S2A (1)). The high-resolution (HR) TEM image (Figure S2A (2)) reveals the perfect single-crystalline structure with a uniform lattice distance of 0.51 nm. The size distribution was analyzed based on the HR-TEM images; 100 UCNPs were randomly picked. The average diameter of the UCNPs was 41.8 ± 2.1 nm. Therefore, both the TEM 8

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images and SAED pattern demonstrated that the prepared hexagonal UCNPs were single crystals. After surface modification by silica, the size of the UCNPs increased owing to the formation of a silica layer on the surface of the bare UCNPs in Figure S3 (A). Functional groups, such as oligonucleotides, antibodies, enzymes, which are necessary for the biological application of the UCNPs, were not present on the surface.36 Therefore, the bare UCNPs were functionalized with a carboxyl group. The resultant carboxyl-functionalized UCNPs could be easily conjugated to the amino groups in monoclonal antibodies and to confirm this, the zeta potential of the bare and carboxyl-functionalized UCNPs was compared. Figure S3 (B) and (C) indicate that the bare UCNPs were positively charged, and changed from 5.80 mV to -17.39 mV after modifying with carboxyl groups. The results of the Bradford assay demonstrated successful conjugation between 4.6 µg/mL antibodies and the UCNPs (Figure S4).

Optimization of the LRET-based immunosensor. Quenching efficiency was determined as [(I0-I)/I0] × 100 (%); I0 is intensity in the absence of HbA1c, and I is intensity in the presence of HbA1c at 545 nm. We found that the luminescence quenching efficiency of the green emission at 545 nm reached a maximum upon the addition of 0.005× UCNP-antibody conjugate and decreased at higher concentrations (Figure S5). Therefore, a conjugate concentration of 0.005× was chosen for subsequent experiments. The time course of the relative upconversion luminescence quenching was investigated. The specific recognition stabilized at an incubation time of about 10 min. With increasing incubation time, the quenching efficiency sharply increased and tended to stabilize after 10 min (Figure S6.). To keep the detection time as short as possible, 10 min was chosen as the optimal incubation time for the LRETbased immunosensor.

HbA1c detection by the LRET-based immunosensor. The upconversion luminescence quenching efficiency increased gradually with increasing concentrations of HbA1c in the standard samples (Lyphochek Hemoglobin A1C Linearity Set), i.e., from level 1 (2.5–4.0%) to 4 (17.0–22.0%), suggesting that the efficiency of energy transfer from donor to the acceptor had increased (Figure 4). To estimate the clinical applicability of the LRET-based immunosensor, we used 9

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this system to detect HbA1c (5.6–11.5%) in real blood samples (Figure 5). We observed a linear relationship (r2 = 0.9631) between the quenching efficiency and HbA1c concentration in the tested samples in the range of 5.6–11.5%. In addition, the HbA1c concentrations obtained for the clinical samples by using the IFCC HbA1c Monitoring Programme closely matched those measured by our novel method (Figure S7). Thus, we found that the LRET-based immunosensor can be successfully used for the quantitative measurement of HbA1c level in real blood samples.

Specificity of the LRET-based immunosensor. The specificity of the sensor was tested by evaluating possible interference and the coexistence performance. HbA1c showed strong quenching efficiency, whereas other interference alone showed weak quenching efficiency (Figure S8). Additionally, coexistence of HbA1c with other interference did not affect the quenching efficiency (Figure S8). These results indicated that the proposed system shows specificity towards HbA1c regardless of the addition of competing interference. The high specificity of this system to HbA1c may be useful in the analysis of complex samples.

■ CONCLUSIONS We established a sensitive, simple, rapid and homogeneous LRET-based immunosensor for HbA1c detection using UCNPs. The proposed system was successfully applied to real blood samples. Most current LRET-based systems require quenchers, such as quantum dots, dyes, and gold nanoparticles, to be tagged to receptors for detecting the target of interest. However, our model is based on the intrinsic absorbance of HbA1c, and hence, it is more cost- and time-effective. Therefore, this platform can be used for quick, homogeneous, and easy detection of HbA1c in blood samples without the need for washing steps and has significant potential as a diagnostic tool.

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

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■ AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENT This work was financially

supported

by

grants

from

the

GRL

Program

(NRF-

2013K1A1A2A02050616) funded by the Ministry of Science, ICT and Future Planning; and the GSR(GIST Specialized Research) Project through a grant provided by Gwangju Institute of Science and Technology. ■ Supporting Information Additional information includes upconversion luminescence spectrum and TEM analysis of the upconversion nanoparticles (UCNPs); characterization of functionalized UCNPs; quantitative confirmation of the UCNP-antibody conjugates; optimization of this proposed method; matching of the clinical-samples; specificity of HbA1c (PDF)

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Figure Legends Figure 1. Schematic illustration of the LRET-based immunosensor: (A) in the absence of HbA1c; no quenching and strong upconversion luminescence, (B) in the presence of HbA1c; high quenching and weak upconversion luminescence. Figure 2. Spectral overlap: the absorption spectrum of HbA1c ( of NaYF4:Yb3+, Er3+ (

).

Figure 3. Luminescence emission spectra of UCNPs ( + glycated albumin (

) and the upconversion spectrum

), anti- HbA1c antibody-UCNP conjugates

), anti-E. coli antibody-UCNP conjugates + HbA1c (

antibody-UCNP conjugates+HbA1c (

), and anti-HbA1c

). Conditions: Conjugate 0.005×; glycated albumin (1

mg/mL); HbA1c real sample 11.5 %. Figure 4. Quenching of luminescence by HbA1c in standard samples. HbA1c values: Level 1 (2.5– 4.0%), Level 2 (5.0–7.0%), Level 3 (9.0–12.5%), Level 4 (17–22%). Each data point is the average of N = 3 individual measurements and error bars indicate the standard deviation. Figure 5. Quenching of luminescence by HbA1c (5.6–11.5%) in real blood samples.

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Figure 1.

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Figure 2.

Figure 3. 16

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Figure 4.

Figure 5. 17

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Homogeneous Immunosensor Based on Luminescence Resonance Energy Transfer for Glycated Hemoglobin Detection Using Upconversion Nanoparticles.

We report an immunosensor based on luminescence resonance energy transfer (LRET) to detect homogeneous glycated hemoglobin (HbA1c). This system uses n...
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