Biosensors and Bioelectronics 64 (2015) 161–164

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Short communication

Sensitive point-of-care monitoring of cardiac biomarker myoglobin using aptamer and ubiquitous personal glucose meter Qing Wang, Fang Liu, Xiaohai Yang, Kemin Wang n, Hui Wang, Xin Deng State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China

art ic l e i nf o

a b s t r a c t

Article history: Received 13 June 2014 Received in revised form 8 August 2014 Accepted 27 August 2014 Available online 3 September 2014

Myoglobin (Myo), which is one of the early markers to increase after acute myocardial infarction (AMI), plays a major role in urgent diagnosis of cardiovascular diseases. Hence, monitoring of Myo in point-ofcare is fundamental. Here, a novel assay for sensitive and selective detection of Myo was introduced using a personal glucose meter (PGM) as readout. In the presence of Myo, the anti-Myo antibody immobilized on the surface of polystyrene microplate could capture the target Myo. Then the selected aptamer against Myo, which was obtained using our screening process, was conjugated with invertase, and such aptamer-invertase conjugates bound to the immobilized Myo due to the Myo/aptamer interaction. Subsequently, the resulting “antibody-Myo–aptamer sandwich” complex containing invertase conjugates hydrolyzed sucrose into glucose, thus establishing direct correlation between the Myo concentration and the amount of glucose measured by PGM. By employing the enzyme amplification, as low as 50 pM Myo could be detected. This assay also showed high selectivity for Myo and was successfully used for Myo detection in serum samples. This work may provide a simple but reliable tool for early diagnosis of AMI in the world, especially in developing countries. & 2014 Elsevier B.V. All rights reserved.

Keywords: Point-of-care Myoglobin Aptamer Glucose meter Invertase

1. Introduction Since acute myocardial infarction (AMI) is the leading cause of morbidity and mortality worldwide, the accurate evaluation of patients who show symptoms suggestive of AMI is of great clinical relevance (WHO, 2011; Zhu et al., 2013). Cardiac biomarker has a prime role in the early diagnosis of AMI. Among many cardiac biomarkers, myoglobin (Myo), although not cardiac specific, has been widely suggested as one of the best candidate markers for an early diagnosis of AMI (Aldous, 2013; Pervaiz et al., 1997). Recently, various approaches have been used for Myo detection, such as mass spectrometry (Naveena et al., 2010), liquid chromatography (Giaretta et al., 2013), luminescence (Yue and Song, 2006), colorimetric (Zhang et al., 2011), electrochemical (Lee et al., 2011; Mandal et al., 2013; Moreira et al., 2013a, 2013b; Mishra et al., 2012) and surface plasmon resonance (SPR) (Gnedenko et al. 2013; Masson et al., 2007; Matveeva et al., 2004; Osman et al., 2013). Most of these methods showed high sensitivity, but relatively expensive equipment and trained operators were required, which was limited in a remote area or at home. Therefore, there is an

n

Corresponding author. Tel./fax: þ 86 731 88821566. E-mail address: [email protected] (K. Wang).

http://dx.doi.org/10.1016/j.bios.2014.08.079 0956-5663/& 2014 Elsevier B.V. All rights reserved.

unmet need for developing a simple method to detect Myo at the point of care (POC) (Moreira et al., 2013a, 2013b; Zhu et al., 2013). Personal glucose meter (PGM) is a successful device for POC testing, due to its compact size, low cost, reliable quantitative results and simple operation. Generally, PGM is only used for the detection of glucose (Carroll et al., 2007). Recently, it has been reported that some targets, including metal ions (Su et al., 2013; Xiang and Lu, 2013), small molecules (Xiang and Lu, 2011), nucleic acids (Xiang and Lu, 2012), proteins (Ma et al., 2014; Mohapatra and Phillips, 2013; Su et al., 2012; Xiang and Lu, 2011) and pathogenic bacteria (Joo et al., 2013), could be detected by this successful portable device. In our previous work, even multiple targets detection has been achieved using PGM in combination with microfluidic chip technology (Wang et al., 2014a, 2014b). Aptamers against Myo, which exhibited dissociation constants in the nanomolar range, were successfully screened using positive and negative selection units integrated microfluidic chip in our previous work (Wang et al., 2014a, 2014b). More interestingly, it was found that the aptamer and anti-Myo antibody can bind to Myo synchronously here. On the basis of this finding, a novel method for Myo detection was introduced by using the PGM as readout. The principle of this novel assay for Myo detection was shown in Fig. 1. Anti-Myo antibody, which was first noncovalently immobilized on microplate, was used to capture target Myo. Since aptamer can easily bear labels at each end of the strand (Wang

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2.2. Synthesis of invertase–aptamer conjugate The synthesis of invertase–aptamer conjugate was similar to the previous works (Wang et al., 2013a, 2013b, 2014a, 2014b). The aptamer was first modified with sulfhydryl at the 5′ end. Then sulfo-SMCC was used as a linker to conjugate invertase and aptamer. 2.3. The immobilization of antibody on the microplate The polystyrene microplate was first noncovalently coated with anti-Myo antibodies as follows. Coating solution of anti-Myo antibodies (0.5 mg/ml dissolved 50 mM carbonic acid buffer, pH 9.6) was added to each well, and the microplate was incubated for 12 h at 4 °C in a humid chamber. After the microplate was rinsed thoroughly, it was incubated in 1% BSA for 1 h to decrease nonspecific binding. Next, the microplate was washed repeatedly, and it was ready for Myo detection. 2.4. Characterization using surface plasmon resonance (SPR) Fig. 1. Schematic illustration of Myo detection using aptamer and personal glucose meter.

et al., 2013a, 2013b), the invertase–aptamer conjugate was synthesized and served as the signal amplification element. When the invertase–aptamer conjugate was introduced, it could be captured on the microplate due to the aptamer/Myo interaction. Upon the addition of sucrose, the bound invertase–aptamer conjugate could catalyze the hydrolysis of sucrose into glucose, resulting in an obvious signal detected by the PGM. According to the relationship between PGM signal and the Myo concentration, Myo could be detected using this simple and sensitive method. Theoretically, this format of “antibody–Myo–aptamer sandwich” may have the potential to be used to design other biosensors for Myo detection or other relevant research about Myo, such as electrochemical biosensor, fluorescence biosensor and colorimetric biosensor, as long as the appropriate label groups can be found and replaced. More importantly, this work may provide the new tool for early diagnosis of AMI in the world, especially in developing countries.

2. Experimental 2.1. Materials and reagents Myoglobin protein (from human heart tissue) and monoclonal anti-myoglobin (anti-Myo) antibody were purchased from Abcam (USA). C reactive protein (CRP) and hemoglobin was purchased from Biovision (USA). Bovine serum albumin (BSA), human serum albumin (HSA) and human immunoglobulin G (IgG) were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (China). Invertase (300 U/mg), tris (2-carboxyethyl) phosphine hydrochloride (TCEP), sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) were purchased from Sigma-Aldrich (USA). All of the chemical reagents were of analytical grade or higher. Amicon ultra centrifugal filters (3 K, 30 K) were purchased from Millipore Corporation (Billerica, MA, USA). The aptamer (5′-(T12) CCC TCC TTT CCT TCG ACG TAG ATC TGC TGC GTT GTT CCG A-3′), which was screened by our group (Wang et al., 2014a, 2014b), and control probe (5′-TGG GAG GAG TTG GGG GAG GAG ATT AGG TTA AAG GT-3′) were synthesized by Sangon Biotech. (Shanghai) Co., Ltd. Ultrapure water (18.2 MΩ cm) was used throughout.

The feasibility of the principle was investigated using our home-made SPR instrument (Yang et al., 2007). Au surface was first incubated with 0.5 mg/ml anti-Myo antibodies for 12 h at 4 °C in a humid chamber, followed by a thorough rinsing with 10 mM PBS buffer (pH 7.4). After 60 min of incubation with 1% BSA, the anti-Myo modified Au surface was thoroughly rinsed with 10 mM PBS and then incubated with 500 nM Myo for 40 min. Next, Au surface was rinsed thoroughly, and 500 nM random DNA solution was added and reacted for 1 h. Then after Au surface was washed repeatedly, 1 mg/ml invertase was injected for 1 h. Finally, Au surface was washed and then invertase–aptamer conjugate was introduced for 1 h. SPR spectra were recorded after each step described above. 2.5. Procedures of Myo detection Different concentrations of Myo solution was incubated in antiMyo modified microplate for 60 min at room temperature, followed by a thorough rinsing with 10 mM PBS (pH 7.4). After 60 min of incubation with invertase–aptamer conjugate, the microplate was thoroughly rinsed with 10 mM PBS buffer and then reacted with 0.5 M sucrose for 30 min. Next, the above reaction solution was measured using PGM. PGM with a dynamic range of 2.2–27.8 mM was a product from Sinocare Inc. (China). A signal of PGM larger than 2.2 mM was regarded as signal. Since the PGM can only detect glucose in a solution of neutral pH, this assay was achieved under neutral pH condition. For identifying the target-specificity of this assay, four proteins, i.e. IgG, hemoglobin, HAS and CRP, were respectively reacted with anti-Myo modified on microplate for 60 min at room temperature, followed by a thorough washing with 10 mM PBS. Next, the invertase–aptamer conjugate was added and incubated for 60 min, and then the microplate was rinsed repeatedly with 10 mM PBS. Finally, 0.5 M sucrose was reacted for 30 min and the reaction solution was recoded using PGM.

3. Results and discussion 3.1. SPR characterization of the “antibody-Myo-aptamer sandwich” complex formation As shown in Fig. 1, it is the key that the selected aptamer and anti-Myo antibody could bind to target Myo simultaneously to form a “sandwich” complex. Thus, the feasibility of forming this

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3.2. Optimization of the experimental conditions The capture ability of anti-Myo antibody modified on the microplate may depend on the incubation time between antiMyo antibody and target Myo, so the effect of incubation time was studied. As shown in Fig. S1A, the signal of PGM shifted obviously as the incubation time with Myo increased, and then became saturated at 60 min. Hence, 60 min was selected as the incubation time of target Myo. Since both protein activity and aptamer construct were influenced obviously by the reaction temperature, the temperature was also investigated. As shown in Fig. S1B, when the temperature was either lower or higher than 37 °C, the PGM signal obviously decreased. Thus, 37 °C was chosen as the reaction temperature. 3.3. Detection of Myo Different concentrations of Myo was detected at the following experimental conditions: incubation time between anti-Myo antibody and Myo, reaction time between Myo and invertase–aptamer conjugate, concentration of sucrose, reaction temperature were 60 min, 60 min, 0.5 M and 37 °C, respectively. As shown in Fig. 3A, the PGM signal gradually elevated as the Myo concentration increased from 50 pM to 200 nM. When the concentration of Myo reached 50 nM, the PGM signal reached a saturation plateau. According to the 3s rule, the signal could be identified even at a Myo concentration as low as 50 pM, which was lower than clinical cutoff for Myo in healthy patients. Moreover, this sensitive was comparable to or better than that of those previous work (Lee et al., 2011; Mandal et al., 2013; Masson et al., 2007; Matveeva

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“sandwich” complex was first investigated using SPR technology. As shown in Fig. 2, as target Myo was added and incubated with anti-Myo antibody coated Au surface, an obvious resonance wavelength red shift (ca. 3.7 nm) was observed, suggesting that target Myo was captured on the Au surface through the interaction between antigen and antibody. When either random DNA or invertase was introduced, no significant change in the resonance wavelength was observed. While the invertase–aptamer conjugate was added, an obvious resonance wavelength red shift (ca. 7.6 nm) was observed again, implying that invertase–aptamer conjugate can bind to Myo which was captured on the Au surface. The results indicated that the selected aptamer and anti-Myo antibody could bind to target Myo simultaneously, resulting in the formation of the “antibody–Myo–aptamer sandwich” complex.

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Fig. 3. Detection of Myo (A) and other proteins (B) using PGM. Incubation time between anti-Myo antibody and Myo: 60 min; concentration of sucrose: 0.5 M; reaction time between Myo and invertase–aptamer conjugate: 60 min; reaction temperature: 37 °C. The error bars represent the standard deviation of three measurements. A signal of PGM larger than 2.2 mM was regarded as signal.

et al., 2004; Mishra et al., 2012; Moreira et al., 2013a, 2013b; Osman et al., 2013; Zhang et al., 2011). Besides sensitivity, the selectivity of this approach was also investigated. Four proteins, i.e. IgG, Hemoglobin, HSA and CRP, were used as contrasts. As shown in Fig. 3B, the presence of Myo led to remarkable increase of the PGM signal, while very low signal could be detected in the presence of other four proteins. It implied that this assay showed high selectivity for Myo detection. Notably, since the PGM is influenced by some interferences, such as ascorbic acid and uric acid, these interferences may influence this assay. This method was also used to detect Myo in human serum. Since the normal Myo concentration ranges in human serum are from 6 ng/mL (i.e. 0.34 nM) to 85 ng/mL (i.e. 4.8 nM) (De Winter et al., 1995), the human serum samples were first diluted 10 times using 10 mM PBS. The Myo-spiked serum samples were prepared by adding Myo in diluted serum samples. These samples were filtered using centrifugal filtration devices (30 K) to remove macromolecules, e.g. HSA and other abundant proteins. Next, these Myo-spiked serum samples were detected. As shown in Table S1, the recovery of Myo from human serum samples was 98.2–107.2%, which demonstrated that the other components of the serum did not interfere significantly in this method. The results demonstrated its potentials for practical applications in disease diagnostics.

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4. Conclusion In conclusion, a novel and sensitive method was developed for Myo detection at the POC. This method did not require any expensive instruments (only using commercially available PGM). Moreover, this assay showed excellent sensitive due to the enzyme amplification. Surely, since AMI requires immediate medical attention, further attempts to shorten the detection time is worthwhile. For example, the introduction of microfluidic technologies may reduce the time necessary for the Myo-antibody/aptamer reaction because of size effects of the liquid microspace. The simple and sensitive platform has great potential application in clinical biological analysis, especially in developing countries.

Acknowledgments This work was supported by the National Natural Science Foundation of China (21190040, 21375034, 21175035), National Basic Research Program (2011CB911002), International Science & Technology Cooperation Program of China (2010DFB30300), the Fundamental Research Funds for the Central Universities and the China Scholarship council (201308430175).

Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.079.

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