Applications of antibiofouling PEG-coating in electrochemical biosensors for determination of glucose in whole blood

Electrochimica Acta 89 (2013) 549–554

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Applications of antibiofouling PEG-coating in electrochemical biosensors for determination of glucose in whole blood Chong Sun a,b , Jingjing Miao a , Jie Yan a , Kai Yang a , Chun Mao a,∗ , Jing Ju a , Jian Shen a,c,∗∗ a b c

Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, PR China School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, PR China School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China

a r t i c l e

i n f o

Article history: Received 1 October 2012 Received in revised form 1 November 2012 Accepted 1 November 2012 Available online 10 November 2012 Keywords: Whole blood Antibiofouling Carboxymethyl-PEG-carboxymethyl Biosensor

a b s t r a c t Polyethyleneglycol-modified surfaces have become popular for anti-biofouling applications. In this paper, a novel glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) onto the carboxymethylPEG-carboxymethyl (CM-PEG-CM) biomaterial film on the surface of glassy carbon electrode (GCE). The biosensor was applied in whole blood directly, which was based on the low values of polymer–water interfacial energy, resistance to protein adsorption and cell adhesion. The entrapped GOx could preserve its bioactivity and exhibited an excellent electrochemical behavior with a formal potential of −290 mV in phosphate buffer solution (PBS) (pH = 7.4). Response studies to glucose were carried out using differential pulsed voltammetry (DPV). The results indicated that the modified electrode can be used to determine glucose without interference from l-ascorbic acid (AA) and uric acid (UA) with the low detection limit of 1.24 × 10−5 M. The data obtained from the biosensor showed good agreement with those from a biochemical analyzer in hospital. The GOx biosensor modified with CM-PEG-CM will have essential meaning and practical application in future that attributed to the effect of anti-biofouling and good performance. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Glucose biosensors constitute a very important and widespread class of enzymatic biosensors, due to the relevance of glucose determinations in biomedical diagnosis [1–4]. Accurate blood glucose values especially play an important role in the diagnosis of diabetes. In principle, blood glucose values should be given in terms of whole blood, but most laboratories now measure and report the serum glucose levels. Because red blood cells (erythrocytes) have a higher concentration of protein (e.g., hemoglobin) than serum, serum has a higher water content and consequently more dissolved glucose than does whole blood. To convert from whole-blood glucose, multiplication by 1.15 has been shown to generally give the serum/plasma level. However, the test results are influenced by the different model numbers of test instruments and detection reagents, treatment processes of blood samples, factitious operations, especially additional centrifuge and too long measure time from collecting specimen of blood to examination. The accurate

∗ Corresponding author. Tel.: +86 25 85891635; fax: +86 25 83598280. ∗∗ Corresponding author at: Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, PR China. Tel.: +86 25 85891635; fax: +86 25 83598280. E-mail addresses: [email protected] (C. Mao), [email protected] (J. Shen). 0013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2012.11.005

results of glucose concentration were not provided by commercial glucose meter, due to the fact that some defects cannot be ignored during its operation [5]. For example, the blood samples are obtained from fingertip peripheral rather than vein, and are doped easily with tissue fluid. What is the key point of preparation for an electrochemical glucose biosensor that can be directly used in whole blood? While an electrochemical biosensor directly used in whole blood, the biofouling of electrode surface can be developed by platelet, fibrin and blood cell adhesion in the complex environment of whole blood media. And the biofouling of electrode surface will bring catastrophic damage to the electron transfer between enzyme and electrode redox center. How to solve the problem? As we know, anti-biofouling surfaces lie at the heart of several contemporary and advanced technologies, ranging from coatings for biomedical implants, ship hulls, to carriers for targeted drug delivery [6–13]. Hydrophilic surfaces of polymers like polyethyleneglycol (PEG), with low values of polymer–water interfacial energy, show resistance to protein adsorption and cell adhesion [14]. In this case, the hydrophilic polymer coating, carboxymethyl-PEG-carboxymethyl (CM-PEG-CM), was designed and explored as antibiofouling surface for preparing for electrochemical glucose biosensor that can be used directly for whole blood samples. The development of novel glucose biosensors for antifouling, rapid, highly sensitive, and selective detection

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of blood glucose concentration in whole blood samples was presented.

2. Experiments 2.1. Reagents Carboxymethyl-PEG-carboxymethyl (CM-PEG-CM, MW 2000) was obtained from Seebio Co. Ltd. (China). Glucose oxidase (GOx), N-ethyl-N -(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimidobiotin (NHS) were purchased from Sigma–Aldrich (USA), ␤-d-(+)-glucose (99%) was received from J&K Chemical Co. Inc. (China), and (3-aminopropyl)-triethoxysilane (APTES) (98%) was purchased from Aladdin Chemistry Co. Ltd. (China). All the above reagents were used without further purification. Phosphate buffer solution (PBS) was prepared by mixing stock standard solution of Na2 HPO4 and NaH2 PO4 . All other chemicals were of analytical grade and were used as received. All the solutions prepared with double-distilled water have high purity and were subjected to N2 for deaeration. 2.2. Construction of the GOx/CM-PEG-CM/APTES modified electrode Prior to the modification, the glass carbon electrode (GCE) was successively polished to a mirror-like surface with 0.3 and 0.05 ␮m alumina slurry followed by rinsing thoroughly with double-distilled water. After sonicated in ethanol and doubledistilled water for 5 min, respectively, the GCE was allowed to dry at room temperature. 10 ␮L APTES was dispersed into 10 mL anhydrous alcohol to form 0.1% APTES ethanol solution. Then 8 ␮L of 0.1% APTES was dropped onto the surface of the GCE and dried under the infrared lamp. 1 mg of CM-PEG-CM was dissolved in 1 mL double-distilled water to obtain 1 mg mL−1 CM-PEG-CM solution. 8.0 ␮L of the resulting CM-PEG-CM solution was dropped onto the electrode surface and dried in air. APTES was linked to the GCE surface through the silicon oxygen bonds, the carboxylic group of CM-PEG-CM was combined with the amino at the end of APTES molecule. After that, the surface of modified GCE was activated using EDC and NHS. Then, 8.0 ␮L of 6.0 mg mL−1 GOx solution was dropped onto the pretreated GCE surface successively and dried in room temperature. Another aldehyde group of CM-PEG-CM was reacted with the amino group of GOx. In this way, the GOx/CM-PEG-CM/APTES/GCE was obtained. The other electrodes used as contrast samples were prepared by the same modified method. When not in use, the electrodes were stored at 4 ◦ C in a refrigerator. 2.3. Apparatus and measurements CD spectra in the far-UV (with the range from 190 to 260 nm) were measured on a JASCO J-715 spectropolarimeter using a 1 cm quartz cuvette. The contents of ␣-helix, ␤-sheet, ␤-turn and the random coil conformation were calculated using the JASCO710 program. All electrochemical experiments were performed on a CHI 760D electrochemical analyzer (CH Instruments, US), using a conventional three-electrode system with GCE (3 mm in diameter, Shanghai Chenhua, China) as the working electrode, a platinum wire as the auxiliary electrode and a saturated calomel electrode (SCE) as the reference electrode. Cyclic voltammogram (CV) experiments were carried out in quiescent solution at 100 mV s−1 in 5 mL of 0.1 M PBS, and the solution was purged with high purity nitrogen prior to and blanked with nitrogen during the electrochemical experiments. Differential pulse voltammetry (DPV) measurements

were carried out with pulse amplitude of 0.05 V and pulse width of 0.2 s. 2.4. Blood compatibility analysis 2.4.1. Coagulation tests of CM-PEG-CM solution Activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) were measured with a coagulation analyzer using a Semi automated Coagulometer (RT-2204C, Rayto, USA). The effect of CM-PEG-CM on coagulation was studied after mixing platelet-poor plasma (PPP) with the CM-PEG-CM solution (9:1 dilution) in the cuvette-strips at 37 ◦ C for 5 min before adding the coagulation reagents. Control experiments were done by adding identical volumes of 0.15 M saline solution. Coagulation parameters, APTT, PT and TT were measured in the plasma (obtained from anticoagulated rabbit whole blood) within 2 h of sample collection from all the groups [15]. Each experiment was repeated three times. 2.4.2. Complement activation Hemocompatibility of CM-PEG-CM was also evaluated by measuring complement activation and platelet activation under in vitro conditions [16,17]. To assess complement activation, the cleavage of complement component C3 was monitored by measuring the formation of its activation peptides, C3a and C3a des-Arg, using a commercial C3a enzyme immunoassay kit with a flow cytometer (FACSCalibur; BD Biosciences, USA) according to the manufacturer’s instructions. Activation studies were performed on PPP isolated by centrifugation from human whole blood donations. Equal volumes of plasma and the CM-PEG-CM solution in saline were incubated at 37 ◦ C for 1 h. Briefly, the samples were diluted with the dilution buffer provided in the kit and added to a microtiter plate coated with a monoclonal antibody specific for human C3a and C3a des-Arg. After 1 h incubation at room temperature to allow any C3a in the sample to bind to the monoclonal antibody, the plates were washed and incubated with peroxidase-conjugated human anti-C3a for 15 min. Following a final wash step, the chromogenic substrate was added to detect bound C3a. Absorbance was measured at 450 nm. The sample C3a concentrations were calculated using a standard curve with net absorbance values plotted on the y-axis for each C3a concentration indicated on the x-axis. Sample values were accepted as valid if they fell on the standard curve; sample values above the top end of the curve were retested following further dilution. The measurements were done in duplicates. 2.4.3. Platelet activation To measure the platelet activation, the platelet rich plasma (PRP) was incubated at 37 ◦ C with an equal volume of the CM-PEG-CM solution with different concentrations of saline. The incubation mixture was removed at 30 min to assess the activation state of the platelets using fluorescence flow cytometry. Expression of the fluorescently labeled platelet activation marker anti-CD62P and the platelet pan-marker anti-CD42a were detected using a BD FACSCalibur and a double staining method according to the manufacturer’s instructions. All the platelet activation experiments were done in triplicates. 2.4.4. Blood-cell adhesion tests In order to determine the potential blood compatibility of CM-PEG-CM, blood-cell adhesion studies were conducted, since blood cell adhesion is one of the most important steps during blood coagulation on artificial surface [18–21]. The blank GCE and GOx/CM-PEG-CM/GCE were equilibrated with PBS (pH = 7.4) for 24 h. In this study, human whole blood was applied to clarify the blood-cell adhesion [22]. The blood cells were prewarmed to 37 ◦ C and added after removing PBS. After 60 min incubation at 37 ◦ C, the

C. Sun et al. / Electrochimica Acta 89 (2013) 549–554

[

q]*10-4/deg.cm2.dmol-1

40

Table 1 Anticoagulation properties (APTT, PT and TT) of citrated normal rabbit plasma samples after they were treated with CM-PEG-CM solution.

30 20 10 0

a b

-10 -20 -30

551

200

210

220

230

240

250

260

Wavelength/nm Fig. 1. CD spectra of (a) pure GOx, (b) GOx/CM-PEG-CM in 0.1 M PBS (pH = 7.4) in the wavelength region of 190–260 nm.

GCEs were washed three times with PBS by mild shaking (fresh PBS each time) to remove non-adherent blood cells. The GCEs were then soaked in 2.5% glutaraldehyde for 30 min to fix the adhered blood cells. After being washed with PBS, the blood cells adhering to the films were dehydrated in an ethanol grade series (50, 60, 70, 80, 90, 95 and 100%) for 30 min each and allowed to dry at room temperature. The surfaces attached with blood cells were gold deposited in vacuum and examined by SEM (JSM Model 6300 scanning electron microscope, JEOL, Japan). 3. Results and discussion 3.1. Characterization of GOx/CM-PEG-CM film Circular dichroism is the spectroscopic probe that has most sensitive to polypeptide backbone conformations, especially secondary and tertiary structure of the protein [23]. Fig. 1 shows the CD spectrum of GOx in the PBS with and without CM-PEG-CM solution in the far-UV region. Two negative bands at around 209 and 220 nm were observed from curve (a) and curve (b) are in accordance to the ␲–␲* and n–␲* transition of the amide groups of the GOx polypeptide chain, respectively [24]. Compared with native GOx, band of GOx/CM-PEG-CM solution presented a similar CD spectra profile, only the intensity of the dual bands at 209 and 220 nm had a slight decrease, indicating that the secondary structure of GOx was not obviously changed. Compared with the data calculated from CD spectrum, GOx secondary structure had no significant change with and without CM-PEG-CM solution. For analyzed concretely with the GOx secondary structure with CM-PEG-CM, the content of ␣-helix conformation decreased by 4.4%; while the contents of anti-parallel, ␤-turn and random coil increased by 0.4%, 0.8% and 1.8%, respectively. These results further proved that CM-PEG-CM solution did not destroy the natural conformation of GOx. Thus, the secondary structure of GOx was not further lost in the prepared biosensor and the CM-PEG-CM solution possess good biocompatibility. 3.2. Blood compatibility of the CM-PEG-CM solution Hemocompatibility of the CM-PEG-CM solution was evaluated by measuring coagulation (APTT, PT and TT), complement activation, platelet activation, and blood-cell adhesion under in vitro conditions. A change in plasma coagulation properties upon incubation with the polymers is an indication of its interaction with blood components which might lead to thrombosis [25–29]. Data of APTT, PT,

Sample

APTT(s)

PT(s)

TT(s)

Control PEG solution

17.5 ± 0.1 18 ± 0.4

7.7 ± 0.1 7.8 ± 0.1

12.1 ± 0.1 11.9 ± 0.2

and TT for CM-PEG-CM solution are shown in Table 1. The APTT/PT were statistically longer in test than in controls after the injection of CM-PEG-CM solution. It means the CM-PEG-CM has anticoagulation property that attributed to PEG [14]. And, more importantly, an antibiofouling surface of GCE can be obtained by help of the anticoagulation property of CM-PEG-CM that immobilized onto the surface of GCE. When foreign material is introduced into blood, activation of components of the complement system could produce anaphylatoxins which will activate the immune system [26]. So the complement system is an important regulator of both adaptive and innate immunity, thus hemocompatibility [16,17]. We have used an enzyme immunoassay kit (C3a kit) for measuring the complement activation. C3a is an anaphylatoxin produced during the complement cascade and its concentration in the plasma is a measure of the extent of complement activation [26]. The CM-PEG-CM solution was incubated with citrate anticoagulated plasma at 37 ◦ C for 1 h and the amount of C3a produced was measured. Our samples were found to be neutral to the complement system in that the amount of C3a (3.76 ng/mL) produced by the CM-PEG-CM solution was not significantly different from that in the control plasma sample. Platelet activation upon interaction with polymers is another indication of blood incompatibility and could lead to thrombotic complications under in vivo conditions [26]. It is known that polycations induce aggregation and activation of platelets which could impair the platelet function [30,31]. We have measured the platelet activation after incubating CM-PEG-CM solution in PRP for 30 min at 37 ◦ C using flow cytometry. Platelet activation is expressed as the percentage of platelets positive for both of the bound antibodies (CD 62P and CD 42). The CM-PEG-CM solution did not activate platelets and the values (0.68%) were similar to that of saline control (0.50%). These results are consistent with the previous research work from Bradley et al. that PEG may reduce the inflammatory potential of an external additive and the platelet activation [32]. The antibiofouling property of the CM-PEG-CM was evaluated by whole blood adhesion tests. And the results were observed by SEM (Fig. 2). Many adherent blood cells and fibrins were observed on the surface of blank GCE after contact with whole blood (Fig. 2a). On the other hand, whole blood adhesion was suppressed on the surface of GCE modified with GOx/CM-PEG-CM (Fig. 2b). The hydrated PEG chains attached on a surface probably prevented adhesion of blood cells and fibrins. Thus, CM-PEG-CM might provide a desirable microenvironment for GOx to undergo facile electron-transfer reactions. 3.3. Direct electrochemistry of GOx/CM-PEG-CM/APTES/GCE Cyclic voltammetric method is used to evaluate the electrochemical performance of the electrodes. Fig. 3 shows the CVs of different electrodes in 0.1 M PBS (pH = 7.4) at a scan rate of 100 mV s−1 . No obvious redox peak could be observed at CMPEG-CM film electrode (curve a). When GOx was embedded in the CM-PEG-CM film (curve b), the observed oxidation–reduction peaks on GOx/CM-PEG-CM/APTES/GCE electrode were more clearly. This result demonstrated that the good biocompatibility of CM-PEG-CM could provide the favorable microenvironment for GOx. From the CVs of CM-PEG-CM/APTES/GCE and GOx/CM-PEGCM/APTES/GCE, we can get the results that the observed oxidation

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Fig. 2. SEM images of (a) blank GCE, and (b) GOx/CM-PEG-CM modified surfaces exposed to human whole blood for 60 min, respectively.

The DPV studies of GOx/CM-PEG-CM/APTES/GCE have been carried out in whole blood in the voltage range from −0.7 to −0.1 V (Fig. 4). Blood samples were supplied by volunteer, within sodium fluoride to prevent glucose metabolism by blood cells prior to glucose determination [5,33]. Moderate stirring was kept for more than 5 min when glucose solution was successively added to the blood samples prior to recording the DPV voltammogram. An anodic peak was observed at −0.31 V and the peak current gradually increases with the increasing concentration of glucose. The calibration curve is presented in the insert of Fig. 4. The biosensor exhibit super highly sensitive response to glucose and the calculated detection limit is 1.24 × 10−5 M (S/N = 3) in whole blood which is lower than those obtained for GOx immobilized on other polymers [34–37]. The linear relation has a regression equation of I (␮A) = −0.00243c (pM) − 0.1714 with a correlation coefficient (R) of 0.9958. Such results may be due to the good biocompatibility of CM-PEG-CM and the favorable electrical conductivity of the biosensor. Biomimetic surface of CM-PEG-CM provides a friendly microenvironment to retain GOx’s bioactivity.

-0.2 -0.3

a

-0.4 -0.5

g

-0.6

-0.6 -0.7 -0.8 -0.9

-0.5

Response/ μA

3.4. Ameperometric determination of glucose in whole blood

-0.1

Current/μA

and reduction peaks were come from GOx, not CM-PEG-CM. Moreover, only when GOx was embedded in the CM-PEG-CM film, a pair of well-defined oxidation and reduction peaks could be observed with a formal potential value (E0 ) of −0.290 V. In addition, the ratio of anodic to cathodic peak currents was about 0.86 and the separation of peak potentials (Ep ) was 45 mV, which indicated that GOx undergone a quasi-reversible redox process (FeIII/FeII redox couple) at the GCE modified with CM-PEG-CM.

-0.4 -0.3 -0.2 0

-0.6

90

120 150 180

-0.5

-0.4

-0.3

-0.2

-0.1

Fig. 4. DPV obtained at GOx/CM-PEG-CM/APTES/GCE in whole human blood samples at 25 ◦ C with glucose of (a) 0 pM, (b) 30 pM, (c) 60 pM, (d) 90 pM, (e) 120 pM, (f) 150 pM, and (g) 180 pM; the insert: relationship between the peak current and the concentration of glucose in whole blood samples.

3.5. Anti-interference of GOx/CM-PEG-CM/APTES/GCE biosensor l-Ascorbic acid (AA) and uric acid (UA) are coexisting electroactive species in whole blood which might affect the biosensors response [38]. The anti-interference advantages of the biosensor are displayed in Fig. 5. While a well-defined glucose response was observed at the biosensor, relevant level of AA and UA (10 mM,

0.12 0.13

3

b

Current/1e-6A

Current/μA

60

Potential/V

4

2

a 1

0.1 mM glucose

0.14

UA 0.15

AA

0.16 0.17

0 -1 0.2

30

Concentration/pM

-1.0

0.18

0.0

-0.2

-0.4

-0.6

-0.8

-1.0

40

60

80

100

120

140

160

180

Time/sec

Potential/V Fig. 3. CV of (a) CM-PEG-CM/APTES/GCE, (b) GOx/CM-PEG-CM/APTES/GCE in 0.1 M PBS (pH = 7.4). Scan rate: 100 mV s−1 .

Fig. 5. Amperometric responses of the biosensor upon additions of glucose (0.1 mM), AA (10 mM), and UA (10 mM), in PBS. The biosensor was biased on the potential of −0.29 V.

C. Sun et al. / Electrochimica Acta 89 (2013) 549–554 Table 2 Determination of glucose in whole human blood samples using the GOx/CM-PEGCM/APTES/GCE. No.

Referenced valuesa (mM)

1 2 3 4 5

4.2 6.7 8.2 12.1 19.8

± ± ± ± ±

0.15 0.19 0.12 0.15 0.12

Determined valuesb (mM) 3.9 6.5 8.1 11.9 19.6

± ± ± ± ±

0.24 0.18 0.28 0.22 0.23

a

Values were provided by the hospital biochemistry laboratory. Values were determined by the GOx/CM-PEG-CM/APTES/GCE; they were average values of the response of five measurements for each sample. b

respectively) resulted in negligible signals, while both of the physiological normal levels of UA and AA are about 0.2 mM [36]. Hereby, we can conclude that the resulting biosensor has a strong antiinterference ability. 3.6. Real sample analysis To evaluate its applicability, the biosensor was used for determination of the concentration of glucose in whole blood. Five blood samples obtained from hospital were analyzed by using five independently prepared biosensors (one biosensor for per sample). Only an appropriate dilution of the samples with PBS (pH 7.4) was needed before the measurements were performed. The determined results were compared with those provided by the hospital (Table 2). It was shown that the values measured by the proposed biosensor were in good agreement with the data provided by hospital. These results demonstrated the great potential of the biosensor for the analysis of glucose in whole blood samples. 4. Conclusion Advances in high performance materials for structural and functional applications depend on the development of materials processing [39–43]. In this paper, Polyethyleneglycol-modified electrode surfaces were used for anti-biofouling application and a high sensitivity glucose biosensor was assembled by immobilizing enzymes on the surface of CM-PEG-CM biomaterial film on the surface of GCE. The entrapped GOx could preserve its bioactivity and exhibited an excellent electrochemical behavior with the help of anti-biofouling of PEG. The biosensor can be used for accurate quantification of the concentration of glucose in whole blood samples directly. The data obtained from the biosensor showed good agreement with those from a biochemical analyzer in hospital. Such novel assembly of the biosensor provided a promising platform for clinical detection in the future. Acknowledgments The work is supported by NSFC (21144002), NSFJS (BK2011781), RFDP (20113207120005), Major Program for the Natural Science Fundamental Research of the Higher Education Institutions of Jiangsu Province (12KJA150006), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Base of production, education & research of prospective joint research project of Jiangsu Province (BY2011109). References [1] E.H. Yoo, S.Y. Lee, Glucose biosensors: an overview of use in clinical practice, Sensors 10 (2010) 4558. [2] J. Wang, Glucose biosensors: 40 years of advances and challenges, Electroanalysis 13 (2001) 983. [3] G.S. Wilson, R. Gifford, Biosensors for real-time in vivo measurements, Biosensors and Bioelectronics 20 (2005) 2388.

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