A quartz crystal microbalance-based biosensor for enzymatic detection of hemoglobin A1c in whole blood

Sensors and Actuators B 258 (2018) 836–840

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

A quartz crystal microbalance-based biosensor for enzymatic detection of hemoglobin A1c in whole blood Hyeoun Ji Park, Soo Suk Lee ∗ Department of Pharmaceutical Engineering, Soonchunhyang University, 22 Soonchunhyang-ro, Shinchang-myeon, Asan-si, Chungcheongnam-do, 31538, Republic of Korea

a r t i c l e

i n f o

Article history: Received 26 September 2017 Received in revised form 27 November 2017 Accepted 27 November 2017 Available online 28 November 2017 Keywords: Hemoglobin A1c (HbA1c) Enzymatic assay Quartz crystal microbalance (QCM) Resonance frequency Gold nanoparticle (AuNP) Signal amplification

a b s t r a c t Here we present a quartz crystal microbalance (QCM) biosensor for enzymatic detection of hemoglobin A1c (HbA1c). It shows average plasma glucose concentration readings over the prior three months. HbA1c can be quantitatively measured based on changes of resonance frequency of QCM following mass changes on QCM sensor surface. These mass changes were caused by size enlargement of conjugated gold nanoparticle with thiol-terminated SAMs on the sensor surface due to gold staining by hydrogen peroxide (H2 O2 ) generated from enzymatic HbA1c assay. Finally, we investigated sensor responses due to mass changes on various concentrations of applied H2 O2 . We also demonstrated its capability for analyzing HbA1c in whole blood sample with enzymatic assay. Our results showed that the proposed QCM biosensor could quantitatively analyze HbA1c with a detection limit of 0.147% HbA1c with respect to hemoglobin and a coefficient variation of less than 10%. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Hemoglobin A1c (HbA1c) is generated by the binding of glucose to N-terminal valine residue of one or both hemoglobin ␤-chains [1]. HbA1c is primarily measured as average plasma glucose concentration in three-month because the lifespan of red blood cells in circulation is four months [2]. Thus, measurement of HbA1c level has become an important indicator for the diagnosis and treatment of diabetes [3–8]. Normal HbA1c levels fall within the range of 4–6% [9]. Various methods for measuring HbA1c have been reported, including ion-exchange high-performance liquid chromatography (HPLC), immunoassay, electrophoresis, boronate affinity chromatography, and enzymatic assays. These techniques have been recently reviewed [10–13]. In clinical laboratories, these techniques can provide accurate determination of HbA1c. Among these methods, enzymatic HbA1c assay using fructosyl amino acid oxidase (FAOD) has received increasing attention because this assay is rapid and reproducible [14–16]. Active research is being conducted to improve properties of this enzyme and develop novel FAOD-based detection techniques. FAOD is expected to become a major component of HbA1c detection. Usually, fructosyl amino acid reacts with H2 O and O2 in the presence of FAOD to generate H2 O2 as

∗ Corresponding author. E-mail address: [email protected] (S.S. Lee). https://doi.org/10.1016/j.snb.2017.11.170 0925-4005/© 2017 Elsevier B.V. All rights reserved.

shown in Eq. (1). The concentration of H2 O2 is proportional to that of HbA1c in the blood [17]. Enzyme peroxidase then catalyzes the reaction between H2 O2 and chromogen to form an oxidized colored chromogen as shown in Eq. (2). The concentration of chromogen is proportional to the concentration of HbA1c in the sample [18]. Fructosyl amino acid + H2 O + O2 → Amino acid + glucosone + H2 O2

(1)

H2 O2 + Chromogen (colorless) + Peroxidase → H2 O + Oxidized chromogen (color) + Peroxidase

(2)

Colorimetric and electrochemical HbA1c sensing techniques are very popular in clinical laboratory. Although these detection techniques have proven their efficacy in a wide range of applications, they require the use of expensive equipment for measurement. They also require complex and extensive optimization. For these reasons, piezoelectric detection methods, especially quartz crystal microbalance (QCM) based detection, are attractive alternatives for detecting HbA1c. They have become versatile techniques, leading to the successful HbA1c biosensor [19,20]. QCM sensor is a typical piezoelectric sensor. It measures mass change per unit area by measuring changes in frequency of a quartz crystal resonator. QCM

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Fig. 1. The custom-made fluidic modules: schematic description (left) and practicality photograph (right).

biosensor has superb sensitivity, speed, and reliability [21–25]. QCM biosensors allow sensitive detection of target analytes in samples. In addition, their simple construction provides experimental simplicity and cost efficiency. Changes of resonance frequency are directly proportional to mass changes, thus enabling real-time detection of biochemical molecules on the sensor surface without labeling requirement. Moreover, QCM biosensors can be used to determine kinetic parameters of affinity interactions between target analytes and biochemical recognition molecules when such interactions are implemented on the sensor surface with selective sensing layer. In this contribution, we present an innovative quantitative enzymatic assay to detect HbA1c using gold nanoparticle-based QCM biosensor. In this assay, H2 O2 was generated from proteolytic digestion of glycated hemoglobin with subsequent addition of fructosyl amino acid oxidase (FAOD). It used the same process used for Direct Enzymatic HbA1c AssayTM developed by Diazyme Laboratory. To detect H2 O2 , Direct Enzymatic HbA1c AssayTM uses enzymatic process (horseradish peroxidase catalyzed reaction of a suitable chromogen to an oxidized colored chromogen). However, we used colloidal gold staining process in which H2 O2 catalyzed the reduction of HAuCl4 to Au (0) which was stacked on the surface of colloidal gold added in advance. (Eq. (3)) 2HAuCl4 + 3 H2 O2 + AuNP → 8HCl + 3O2 + Size-enhanced AuNP (by produced 2Au)

(3)

For H2 O2 detection, our process might be no less simple than Direct Enzymatic HbA1c AssayTM because we don’t use enzyme or unique long wavelength chromogen (>700 nm) to avoid absorption interferences from hemoglobin. We only use HAuCl4 and homemade gold nanoparticles. In the aspect of detection novelty and sensitivity, the size enhancement approach described in this study is novel method for the detection of HbA1c. In addition, the high sensitivity of our QCM biosensor for detecting HbA1c comes from the signal amplification strategy through size enhancement of gold nanoparticles by colloidal gold staining chemistry. Meanwhile, we measured changes of frequency by mass loading effect from size enlargement of gold nanoparticles. This reaction was run on QCM sensor surface. In this study, gold nanoparticles were captured on the sensor surface through direct binding with gold-thiol self-assembled monolayers (SAMs) formation. Formation of thiol SAMs and conjugation of gold nanoparticle on the sensor surface was discussed. We demonstrated the dependence of sensor response on H2 O2 concentration and quantitative detection of HbA1c in whole blood sample. 2. Materials and methods 2.1. Reagents and materials All chemicals used in the synthesis of gold nanoparticles, including gold (III) chloride trihydrate (chloroauric acid,

HAuCl4 ) and sodium citrate, were of analytical grade. They were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). (3-Mercaptopropyl)-trimethoxysilane (3-MPTMS), protease from Aspergillus oryzae, and fructosyl-amino acid oxidase (FAOD) from Corynebacterium sp. were also purchased from Sigma-Aldrich. An HbA1c linearity set was purchased from Bio-Rad Co. (Hercules, CA, USA). PBS buffer was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ethyl alcohol (99.5%) and other organic solvents were purchased from Samchun Pure Chemical Co. Ltd. (Seoul, South Korea). All aqueous solutions were prepared in deionized (DI) water which was obtained from a Milli-Q water-purifying system (18 M cm). 2.2. Apparatus and measurement system A QCM sensing system was comprised of fluidic and detection modules to obtain real-time sensor information during assay. Mass loading on silicon dioxide coated QCM resonators (5 mm in diameter; 9 MHz resonance frequency) was monitored by tracking resonance change of bulk acoustic waves at central frequency of 9 MHz with a Princeton Applied Research QCM922A (SEIKO EG&G, Tokyo, Japan). Flow cell was constructed with a peristaltic pump (ISM597; ISMATEC, Glattbrugg, Switzerland), a custom-made fluidic block, and a silicone rubber gasket. Accurate fluidic control is one of important factors for reproducible and user-friendly detection of biomolecules in liquid media. Therefore, we developed fluidic modules for our QCM sensor as dipicted in Fig. 1. The top piece contained recessed regions for reaction chambers and silicone rubber gaskets to prevent liquid leakage due to hydrodynamic pressure. It had fluidic connectors to permit flow across QCM devices. Sample flow and buffer solution flow to reaction chambers in the fluidic block were actuated by peristaltic pump. The flow rate was kept at 1.0 ml/min. The volume of each reaction chamber was 20 ␮L. After each run, reaction chambers and silicone rubber gaskets were thoroughly rinsed with DI water and 0.05% Tween 20 (Sigma® Aldrich, MO, USA) in PBS solution. Teflon tubing (0.032 in. I.D., The Lee Company, CT, USA) was used to connect fluidic and detection modules together. 2.3. Sensor surface preparation Silicon dioxide (SiO2 )-coated QCM sensor chip (SEIKO EG&G, Tokyo, Japan) was sequentially rinsed with deionized water and absolute ethanol followed by drying under nitrogen gas. In this QCM sensor chip, silicon dioxide layer was coated on the top of gold electrodes to prevent non-specific gold staining that might occur at the surface of gold electrodes. The QCM sensor chip was then placed in a UV/ozone chamber (144AX-220; Jelight Company, Inc., Irvine, CA) for 10 min. It was then incubated in a solution of 5% (vol./vol.) freshly prepared 3-MPTMS in methanol for 1 h followed by rinsing with methanol for 2 min and drying under nitrogen gas. The silanized sensor was then baked in an oven at 110 ◦ C for 1 h followed by rinsing with methanol for 2 min and drying under nitrogen gas.

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Gold nanoparticles (AuNPs) obtained by reduction of aqueous solution of HAuCl4 with sodium citrate were conjugated onto the surface of silanized QCM sensor chip via thiol-gold affinity interaction as described previously [26]. Briefly, thiol-terminated QCM sensor chip was exposed to a AuNP solution for 10 min at room temperature. Information related to desorption of AuNP on the thio-terminated sensor surface was then obtained by QCM experiments. 2.4. Detection of hydrogen peroxide (H2 O2 ) by QCM We performed all experiments in triplicates at room temperature (25 ◦ C). For QCM measurement, we conducted H2 O2 detection assays in an AuNP-conjugated QCM sensor surface. Resulting changes of resonance frequency were then recorded. We thoroughly mixed 10 ␮L of HAuCl4 (20 mM) and 10 ␮L of various concentrations of H2 O2 starting from 0.05 mM by diluting 30% H2 O2 solution in water (Sigma-Aldrich Chemical Co.) to make a total reaction volume of 20 ␮L. After incubating the 20 ␮L of mixture at room temperature for 10 min in the chamber formed by gasket on QCM sensor surface, changes in resonance frequency of a quartz crystal resonator were detected in real time.

Fig. 2. Decreases in resonance frequency observed for the conjugation of AuNPs onto thiol terminated SAM surface by QCM measurement.

system. After the AuNPs solution was pumped in, the frequency was decreased. Frequency change was measured for 10 min to be approximately 157 Hz. The coverage of AuNPs on the electrode surface was estimated to be 1.71 × 1011 AuNPs particles/cm2 based on the decrease in resonance frequency. The diameter of our AuNPs was 12.5 nm and their density was 19.32 g/cm3 .

2.5. Detection of HbA1c in whole blood samples by QCM

3.2. Dependence of QCM sensor response on H2 O2 concentration

To measure HbA1c in human whole blood control, a Lyphocheck Hemoglobin A1C Linearity Set from Bio-Rad was used. We mixed level 2 and level 5 (out of 6 levels, 4.61% and 13.5% HbA1c with respect to hemoglobin, respectively) at various ratios (5:0, 4:1, 3:2, 2.5:2.5, 2:3, 1:4, and 0:5). We then mixed 5 ␮L of each of whole blood samples with 100 ␮L of hemolysis buffer followed by incubation at room temperature for 10 min. We thoroughly mixed 12.5 ␮L of the hemolysate solution and 40 ␮L of protease from Aspergillus oryzae in plates and incubated at 25 ◦ C for 5 min. To this mixture, we gently added 10 ␮L of HAuCl4 (20 mM) and 5 ␮L of FAOD from Corynebacterium sp. (106.4 U/ml) without bubble formation. Then we incubated the 20 ␮L of above mixture at room temperature for 10 min in the chamber formed by gasket on QCM sensor surface and detected changes in resonance frequency of the quartz crystal resonator in real time.

Fig. 3(a) highlights the detection of H2 O2 using a combination of formation of gold(0) oxidation state and signal amplification by size enlargement of gold nanoparticle. The growth of gold nanoparticles was carried out through chemical reduction of chlorolauric acid (HAuCl4 ) by H2 O2 . Based on this concept, we investigated QCM sensor response due to gold staining depending on applied H2 O2 concentrations in a fluidic module. As expected, addition of H2 O2 resulted in decrease in resonance frequency due to significant mass increase caused by catalytic deposition of gold on the conjugated gold nanoparticles over the sensor surface. Measurements were performed three times for each concentration of H2 O2 . Results are displayed in Fig. 3(b). Changes of resonance frequency (f) increased linearly with increasing addition of H2 O2 in the range of 0.05 mM to 0.35 mM. The correlation coefficient was

3. Results and discussion 3.1. Analysis of gold-conjugated sensor chip by QCM It is well-known that QCM provides very sensitive sensor device by measuring mass loading effect because resonance frequency will change upon the deposition of a given mass on the electrode. The resonance frequency has been found to decrease upon increase of mass on the QCM sensor surface. In this research, adsorption kinetics of AuNPs was studied. Decrease in frequency observed for the adsorption kinetics of AuNPs is shown in Fig. 2. An increase in mass corresponds to a decrease in frequency according to Sauerbrey’s Eq. (4) [27]. f = −[2f 0 2 /A(q ␮q )1/2 ]m 6

(4)

2

f = −2.3 × 10 f 0 (m/A) m = (−1.1 × 10

−9

(5) 2

g/Hz cm ) f

(6) (8.924 × 106

Where f0 is resonance frequency Hz), f is change in frequency (Hz), m is mass change (g), A is piezoelectrically active crystal area (0.196 cm2 ), q is density of quartz (q = 2.648 g/cm3 ), and ␮q is shear modulus of quartz for AT-cut crystal (q = 2.947 × 1011 g cm−1 s−2 ). From Eq. (4), we obtained Eq. (5) by substituting several constants. We obtained Eq. (6) by substituting parameters for our QCM

Fig. 3. (a) Principles of H2 O2 detection assay using the combination of gold (0) formation and signal amplification by size enlargement of AuNPs and (b) Plot of changes of resonance frequency (f) after the addition of H2 O2 versus concentrations of H2 O2 .

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Fig. 5. Plot of changes of resonance frequency (f) versus concentrations of% HbA1c with respect to hemoglobin.

Fig. 4. HbA1c detection procedure utilized in this study in combination with size enlargement of AuNPs on the QCM sensor surface.

0.973. In addition, the detection limit was calculated to be as low as 0.038 mM [28]. These results revealed that the H2 O2 sensor herein had a detection limit and linearity range comparable to those of sensors reported recently [29]. 3.3. Detection of hemoglobin A1c (HbA1c) Fig. 4 highlights HbA1c detection procedure on the sensing surface used in this study, including formation of thiol-terminated self-assembled monolayer (SAM), conjugation of AuNPs via thiolgold bonding onto the SAM surface, generation of H2 O2 by enzymatic reaction using FAOD, and subsequent gold stainingbased signal enhancement strategy. Thiol-terminated SAMs were formed on SiO2 -coated QCM sensor chip from methanol solution of 3-MPTMS, a well-defined protocol [30]. The resulting surface thiol groups reacted with citrate stabilized AuNPs, thus forming thiol-gold conjugate. To this sensor surface, human whole blood controls treated with hemolysis buffer for lysis of red blood cells was added. Protease was added to release amino acids, including glycated valines from hemoglobin ␤-chains. FAOD enzyme was also added to cleave N-terminal valines and produce H2 O2 [31]. Finally, HAuCl4 was added, resulting in the generation of H2 O2 and catalyzed deposition of gold onto the captured AuNPs on the QCM sensor surface.

that AuNP-based QCM biosensor provided a satisfactory platform for detecting HbA1c in whole blood sample. With this QCM biosensor platform, we obtained a detection limit of 0.147% for HbA1c with respect to hemoglobin with coefficient variation of less than 10%. 4. Conclusion We developed a QCM biosensor that could quantitatively analyze HbA1c in whole blood sample controls. In this study, our QCM biosensor in combination with size enhancement of AuNPs detected H2 O2 in a sensitive, rapid, and reproducible manner. Using FAOD, H2 O2 was enzymatically generated. It reduced HAuCl4 to gold (0) which could be deposited to the surface on AuNPs that were conjugated with thiol head group of SAMs in advance. We showed that responses of such QCM sensor depended on concentrations of HbA1c. It had a detection limit of 0.147% HbA1c with respect to hemoglobin. Due to its high sensitivity and reproducibility, this platform is expected to be useful for developing valuable devices for HbA1c sensing. Our future efforts will be focused on using this strategy to increase sizes of AuNPs to allow other targets to detect mass changes. Acknowledgments This work was supported by Soonchunhyang University. It was also supported by a grant (NRF-2016R1D1A3B03934762) of the National Research Foundation (NRF) funded by the Korean Government.

3.4. Quantitative measurements of HbA1c in whole blood samples

Appendix A. Supplementary data

To prove our detection principle in an environment similar to real application, we analyzed the dependence of QCM sensor responses on AuNP size enhancement using HbA1c concentrations in human whole blood control samples. The sensor responses to HbA1c were obtained with commercial human whole blood control samples that had different concentrations of HbA1c using the QCM sensor. We used human whole blood controls with 4.61% and 13.5% HbA1c with respect to hemoglobin and mixed them in various ratios (5:0, 4:1, 3:2, 2.5:2.5, 2:3, 1:4, and 0:5). Measurements were performed three times for each concentration of HbA1c. Results are shown in Fig. 5. With increasing concentration from 4.6% to 13.5% HbA1c with respect to hemoglobin, QCM sensor responses due to size enlargement of AuNPs were also increased, indicating that the amount of enzymatically generated H2 O2 by FAOD was proportional to the concentration of HbA1c with respect to hemoglobin in human whole blood controls. In addition, increasing values of changes of resonance frequency (f) versus HbA1c levels indicated

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Biographies Hyeoun Ji Park is an undergraduate majoring in pharmaceutical engineering, Soonchunhyang University. Her major research activities are development of biosensors, synthesis of organic molecules, etc. Soo Suk Lee obtained Ph.D. in Chemistry in 1998 from the Pohang University of Science and Technology (POSTECH), Korea. He is currently an assistant professor of department of pharmaceutical engineering, Soonchunhyang University. His major research activities are development of biosensors, surface chemistry, synthesis of organic molecules and analysis of circulating tumor cell, etc.