Advanced Healthcare Materials (Tiwari/Advanced) || Novel Biomaterials for Human Health: Hemocompatible Polymeric Micro-and Nanoparticles and Their Application in Biosensor

Part 2

POINT-OF-CARE DIAGNOSTICS

Ashutosh Tiwari (ed.) Advanced Healthcare Materials, (181–181) 2014 © Scrivener Publishing LLC

183

5

Novel Biomaterials for Human Health: Hemocompatible Polymeric Micro-

and Nanoparticles and Th eir Application in Biosensor

Chong Sun, Xiaobo Wang, Chun Mao* and Jian Shen

Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional

Materials Collaborative Innovation Center, College of Chemistry and Materials

Science, Nanjing Normal University, Nanjing, P. R. China

AbstractIn the past two decades, the development of nanomaterials for the ultra-sensitive detection of biological species has received great attention because of their unique optical, electronic, chemical, and mechanical properties. Diff erent nanomaterials were investigated to determine their properties and possible applications in bio-sensors. Novel nanomaterials for use in bioassay applications represent a rapidly advancing fi eld. Th e strategy for decorating electrode of glucose biosensor with the hemocompatible polymeric micro- and nanoparticles that exhibit excellent antibiofouling property was suggested by our research group. Th is smart strat-egy demonstrates a methodology for the incorporation of actively antibiofouling moieties onto a passively antibiofouling electrode, and thus expands the range of applications of electrochemical biosensors, especially in whole blood.

Keywords: Nanomaterials, hemocompatible materials, biosensors

5.1 Introduction

Blood is a bodily fl uid in animals that delivers necessary substances such as oxygen and nutrients to the cells and transports metabolic waste products

*Corresponding author: [email protected]

Ashutosh Tiwari (ed.) Advanced Healthcare Materials, (183–202) 2014 © Scrivener Publishing LLC

184 Advanced Healthcare Materials

away from them. It plays a very important role in the body. Because improved detection of blood disease can save lives, blood tests have been used for approximately 50 years to detect substances that are present in the blood that indicate either disease or a future risk of the development of a disease. Blood tests detect substances that normally are not present or measure substances that, when elevated above normal levels, indicate disease [1].

At present, the clinical conditions of diabetes mellitus are well known and well understood, yet remain a growing concern as the prevalence of the disease increases worldwide at an alarming rate [2]. Accurate blood glucose values especially play an important role in the diagnosis of dia-betes. Th e primary methods of detecting blood glucose concentration are performed by biochemical analyzer and glucose meter [3–5]. For biochem-ical analyzer, the quantifi cation of the concentration of glucose is mainly involved in serum samples, which are isolated from whole blood separation by centrifugation process, but not untreated whole blood. Th e test results are infl uenced by the diff erent model numbers of test instruments and detection reagents, treatment processes of blood samples, factitious opera-tions, especially additional centrifuge and too long a measure of time from collecting blood specimens to examination. Th is method for the diagnosis of diabetes is not recommended. Further, the red blood cells have a higher concentration of protein (e.g., hemoglobin) than serum, and serum has higher water content and consequently more dissolved glucose than whole blood. To convert from whole-blood glucose, multiplication by 1.15 has been shown to generally give the serum/plasma level. In principle, blood glucose values should be given in terms of whole blood, but most hospitals and laboratories now measure and report the serum glucose levels.

As for commercial glucose meters, there are some defects that cannot be ignored during its operation. For example, the blood samples are obtained from fi ngertip peripheral but not vein, and doped easily with tissue fl uid. So the accurate results of glucose concentration cannot be provided by commercial glucose meters.

However, it is very diffi cult to design and prepare an electrochemical biosensor that can be used in whole blood just because the biofouling of electrode surface can be developed by platelet, fi brin and blood cell adhe-sion in the complex environment of whole blood media. And the biofoul-ing of electrode surface will bring catastrophic damage to the electron transfer between enzyme and electrode redox center. So the development of novel glucose biosensors for antifouling, rapid, highly sensitive, and selective detection is of paramount importance for blood glucose concen-tration monitoring in whole blood samples.

Novel Biomaterials for Human Health 185

5.2 Design and Preparation of Hemocompatible Polymeric Micro- and Nanoparticles

Polymeric micro- or nanoparticles are successively employed for delivery of conventional drugs, recombinant proteins, vaccines and nucleotides [6–19]. Th e polymeric matrixes should be compatible with the body in the terms of adaptability (non-toxicity) and (non-antigenicity) and should be biodegradable and biocompatible [20]. Moreover, one of the impor-tant issues in developing biomedical materials with small scale that con-tact blood is improving their blood compatibility [21]. How can design of hemocompatible polymeric micro- and nanoparticles? Th ree methods are suggested as follows.

(1) Polymeric micro- and nanoparticles loaded anticoagulant drug

As we know, to improve the blood compatibility of the polymeric fi lms, heparin has been used to modify their surface [22–25]. It is one of the most intensively studied glycosaminoglycans (GAGs) as a result of its antico-agulant properties. It is a potent anticoagulant agent that interacts strongly with antithrombin III to prevent the formation of fi brin clot. So heparin immobilized to a surface enhances various surface properties, improving blood compatibility and biocompatibility. Th e immobilized forms include soluble heparin and heparin immobilized to supporting matrices by physi-cal adsorption, by covalent chemical methods, and by photochemical attachment [22]. Th e effi ciency, stability, and activity of heparin are deter-mined by the diff erent immobilized methods and support materials [26]. Besides its anticoagulant property, heparin also has great signifi cance in regulating many biological pathways including cell-cell recognition, signal transduction, growth processes, coagulation cascade and the cellular inter-action of growth factors [27]. So the development of novel heparin-loaded microspheres for anticoagulant drug-release, hemocompatible coating, and selective cellular interaction is of paramount importance for the biomedi-cal application of biomaterials or bio-devices in whole blood environment. Our research group reported a novel kind of heparin-loaded polyurethane microsphere (Hep-PU MS), which was synthesized by a single-step phase separation method. Th e morphology of the Hep-PU MS was depicted in Fig. 5.1a. As shown in Fig. 5.1a, the Hep-PU MS were formed. Th e aver-age particle size of Hep-PU MS was about 540 nm. With the simplicity of the loaded method, the excellent hemocompatibility and the slow-release of heparin, the Hep-PU MS with desirable bioproperties can be readily tailored to cater to various biomedical applications.


(2) Polymeric micro- and nanoparticles hybrided with hydrophilic molecules

Surface modifi cation with increasing hydrophilicity is believed to be a useful method for improving blood compatibility, and various polymer materials have been modifi ed by water-soluble polymer for biomedical use such as poly(ethylene glycol) (PEG) or poly(ethylene oxide) (PEO) that can prevent plasma protein adsorption, platelet adhesion, and thrombus formation by the steric repulsion mechanism. Steric repulsion by surface-bound water-soluble polymer chains occurs as a result of overlapping polymer layers that could lead to loss in confi gurational entropy because of volume restriction and/or osmotic repulsion between interdigitated polymer chains. Th e accepted mechanism for preventing protein adsorption by the graft ed PEO chains is that such a technique decreased interfacial free energy and the steric repul-sion force between PEO chains and the proteins [28]. Similarly, hydrophilic polysulfone membranes, polyvinylpyrrolidone-polysulfone (PVP-PSf), were prepared from PSf membranes covalently conjugated with PVP on the sur-face. It was found that PVP-PSf membranes gave lower protein adsorption from a plasma solution than PSf membranes. Th is is also attributed to the hydrophilic surface of the PVP-PSf membranes [29].

How can prepare hemocompatible polymeric micro- and nanoparticles by surface modifi cation with increasing hydrophilicity? It became a target for our research group. Pluronic F127 (triblock copolymer PEO106PPO70PEO106) has good blood compatibility [30, 31]. Th e preparation and hemocompat-ibility of PU-F127 nanospheres by a spontaneous emulsion solvent diff usion method were investigated by our research group. As displayed in Fig. 5.1b, typical TEM photograph of the PU-F127 hybrid nanospheres showed the average diameter was about 200 nm. It is very interesting that every PU-F127 hybrid nanosphere has a porous structure. Th e results of blood test indicate the PU-F127 hybrid nanospheres have good blood compatibility.

Figure 5.1 Representative TEM images of (a) PU-Hep MS, (b) PU-F127 hybrid nanospheres, (c) HBPE-SO3 nanospheres.


(3) Polymeric micro- and nanoparticles surface modifi ed by special functional groups

Hyperbranched polymers have attracted signifi cant interests because of their unique architecture and novel properties that include good solubil-ity, special viscosity behavior, and high density of their functional groups [32, 33]. Owing to the multifunctionality in hyperbranched polymers, the physical properties can be adjusted to a large extent by the chemical modi-fi cation of the end-groups [34, 45]. Th e use of hyperbranched polymers by the chemical modifi cation has attracted increasing attention in recent years [36–39]. Th ese features of hyperbranched polymers have been used extensively in diverse fi elds, such as coatings, additives, blends, nonlinear optics, composites, and copolymers [40–43]. Especially, hyperbranched polymers hold great potential as drug delivery agents because of their three-dimensional shapes and availability of a large number of surface functional groups amenable to various modifi cation chemistries for drug conjugation and targeting purposes [44–46].

Herein, water-soluble nanoparticles were synthesized by the chemical modifi cation of aliphatic hyperbranched polyester (HBPE) with sulfonic acid functional groups (HBPE-SO3). TEM photographs of the nanopar-ticles showed an average diameter of 210 nm (Fig. 5.1c). Th e blood com-patibility and cytotoxicity of HBPE-SO3 nanospheres were investigated by a series of specialized blood experiments. Th e results showed these hemocompatible polymeric micro- and nanoparticles provide a promis-ing platform of blood circulation system for diagnosis and therapy with the help of the drug-loaded capacity of hyperbranched polyester micelles. Similarly, hemocompatible and water-soluble nanoparticles were also syn-thesized by the chemical modifi cation of aliphatic HBPE with carboxylic acid functional groups.

As to the actual operation for preparation of polymeric micro- and nanoparticles, many methods in the past ten years were used to prepare polymeric micro- and nanoparticles, such as micro-emulsifi cation [47–49], phase conversion [50–54], template method [55–57], surface modifi cation by special functional groups [58, 59], and ATRP technique [60–62].

5.3 Th e Biosafety and Hemocompatibility Evaluation System for Polymeric Micro- and Nanoparticles

When polymeric micro- and nanoparticles enter biosystems, they interact with various biomolecules, especially proteins, forming a protein corona on the surface. Understanding how polymeric micro- and nanoparticles


interact with biomolecules is crucial for bioapplications and for the bio-safety of polymeric micro- and nanoparticles.

In this paper, Hep-PU MS we prepared were chosen as an example for investigating their hemocompatibility and cytotoxicity in the blood and cell experiments mentioned below [50].

5.3.1 In vitro Coagulation Time Tests

Th e activated partial thromboplastin time (APTT), prothrombin time (PT), and thromboplastin time (TT) were widely used for the clinical detection of the abnormality of blood plasma. In recent times, they were applied in the evaluation of in vitro antithrombogenicity of biomateri-als [63–65]. All the samples were dispersed in phosphate buff er solution (PBS) at the pre-determined concentrations. Th e eff ect on coagulation in the presence of the Hep-PU MS was studied aft er mixing anticoagulated rabbit blood plasma with the sample solution in the cuvette strips at 37 °C for 5 min before adding the coagulation reagents. PBS acted as the con-trol. All the assays were performed and measured by using a Semi auto-mated Coagulometer (RT-2204C, Rayto, USA). All the coagulation tests were performed in triplicate. Th e APTT, PT, and TT values of control for a healthy blood plasma were 21.3±0.2, 6.7±0.1 and 16.5±0.1 s, respectively. Th e eff ect of Hep-PU MS can be observed by comparing the APTT, PT, and TT with those of control. Th e results showed that the data of APTT/PT/TT were statistically longer in tests than in controls aft er the injection of Hep-PU MS, indicating that Hep-PU MS have excellent anticoagulant eff ects (Fig. 5.2a).

5.3.2 Complement and Platelet Activation Detection

Th e hemocompatibility of the Hep-PU MS was also evaluated by mea-suring complement and platelet activation under in vitro conditions. Complement activation is a key indicator of both adaptive and innate immunity, thus hemocompatibility when foreign material is introduced into blood [66–68]. We have used an enzyme immunoassay kit (C3a kit) for measuring the complement activation. C3a is an anaphylatoxin pro-duced during the complement cascade and its concentration in the plasma is a measure of the extent of complement activation. Hep-PU MS was incu-bated with platelet poor plasma (PPP) at 37 ºC for 1 h and the amount of C3a produced was measured. Hep-PU MS were found to be neutral to the complement system in that the amount of C3a (27.1 ng/mL) produced


by the Hep-PU MS. From the data, it is obvious that the Hep-PU MS do not activate the complement system (Fig. 5.2b). It is known that nega-tively charged sulfate and sulfonate groups of heparin play a major role in the biocompatibility acticity of heparin, which reduce the infl ammatory potential of an external additive [69, 70].

Platelet activation upon interaction with samples is another major barrier in blood incompatibility and can lead to thrombotic complica-tions under in vivo conditions [67]. We have measured the platelet acti-vation aft er incubating the Hep-PU MS in platelet rich plasma (PRP) for 30 min at 37 ºC using fl ow cytometry. Platelet activation is expressed as the percentage of platelets positive for both of the bound antibod-ies (CD 62P and CD42). Th ere was no signifi cant diff erence between the Hep-PU MS and the control plasma sample (Fig. 5.2c). No eff ect in platelet activation that occurred with the Hep-PU MS was also attributed to heparin.

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Figure 5.2 Hemocompatibility of Hep-PU MS. (a) APTT/PT/TT of PU MS and Hep-PU MS; (b) Complement activation u pon interaction of Hep-PU MS with PPP for 1 h at 37 ºC; and (c) Platelet activation upon interaction of Hep-PU MS with PRP for 30 min at 37 ºC. Plasma incubated with saline as a negative control. Data are presented as means ± standard deviation (n = 3).


5.3.3 Percent Hemolysis of RBCs

Hemolysis occurs when cells swell to the critical bulk to break up the cell membranes. Meanwhile, released adenosine diphosphate from the broken red blood cells intensifi es the assembly of blood platelets, which accelerates the formation of clotting and thrombus. So hemolysis of the blood is a very important problem associated with the bio-incompatibility of material [71]. Less than 5% hemolysis was regarded as a nontoxic eff ect level [64].

Th e experimental process was as follows. First, 5 mL of rabbit blood sample was added to 10 mL of PBS, and then red blood cells (RBCs) were isolated from whole rabbit blood by centrifugation at 1500 rpm for 10 min. Th e RBCs were further washed fi ve times with 10 mL of PBS solution. Th e RBCs were diluted to 50 mL of PBS. Herein, RBCs incubation with doubly distilled deionized water (DI water) and PBS were used as the positive and negative controls, respectively. Th en 1 mL of diluted 2% RBCs suspension was added to 1 mL Hep-PU MS solutions at required concentration. All the sample tubes were kept in static condition at 37 ºC for 3 h. Finally, the mix-tures were centrifuged at 1500 r/min for 10 min, and 200 μL of supernatants of all samples were transferred to a 96-well plate. Th e absorbance values of the supernatants at 570 nm were determined by using a microplate reader. Th e percent hemolysis of RBCs was calculated using the following formula.

Percent hemolysis % = ((sample absorbance – negative control absor-bance)/(positive control absorbance – negative control absorbance)) × 100

In this case, Hep-PU MS did not cause any hemolysis (0.89±0.10%) on rabbit erythrocyte comparing with the negative control (normal saline, hae-molysis rate is 0%) and the positive control (water, haemolysis rate is 100%)

5.3.4 Morphological Changes of RBCs

Morphologically aberrant forms of RBCs can give insights into the diagno-sis of various medical conditions such as hemolytic anemia. Th e RBC pel-let was resuspended in 0.9% saline solution. Th e Hep-PU MS were diluted to a required concentration in cell suspension. Observation of morpholog-ical changes by light microscopy was captured aft er 1.5 h of the Hep-PU MS exposure to analyze the morphological variation at the early stages of haemolysis. Th e pellet obtained aft er centrifugation was diluted in 0.9% saline solution and mounted on clean glass slides covered with cover slips and observed under Olympus BX41 microscope and photographed with an Olympus E-620 camera (Olympus Ltd., Japan). As shown in Fig. 5.3a, the untreated RBCs in saline appeared in a normal biconcave shape. It is known to all, exposure to materials with the bad blood compatibility will


induce appearance of morphological aberrant forms for RBCs such as echi-nocyte-like forms with numerous surface spikes, the RBCs appear swollen, and the phenomena of ghost cells (lysed RBCs) [72]. Th e RBCs treated by Hep-PU MS was imaged as shown in Fig. 5.3b. Th e result showed that there were no obvious changes of cell morphology. Such a result was in agreement with the above hemolysis analysis.

5.3.5 Cytotoxic Assessment

Cytotoxicity of Hep-PU MS was evaluated by MTT (3-[4,5-dimethylthia-zolyl-2]-2,5-diphenyltetrazolium bromide) assays. Briefl y, human embry-onic kidney 293 (HEK 293) cells were seeded to a 96-well culture plate and the cells would come to about 50% confluence aft er 24 h of culture. Th e media were changed by fresh ones, and the mixtures containing required concentration of Hep-PU MS samples were added to the wells. Th e cells of positive control were only incubated with equal Dulbecco’s modifi ed eagle’s medium (DMEM) (10% fetal bovine serum) and the cell viability was set as 100%. All of the cells were allowed to grow for 24 h before 10 mL MTT (5 mg/mL) was added to each well. Th en, the cells were incubated at 37 ºC for an additional 4 h until the purple precipitates were visible. Th e medium was replaced by 100 mL dimethyl sulfoxide (DMSO) and the cell plate was vibrated for 15 min at room temperature to dissolve the crystals formed by the living cells. Finally, the absorption at 490 nm of each well was mea-sured by an ultramicroplate reader [73]. Th e eff ect of the Hep-PU MS on cell viability (90±1.5%) was compared to nontreated cells (control sample), which are considered as 100%. According to the relationship between cell proliferation rate and cytotoxicity grade of United States pharmacopeia (USP), we got the conclusion that Hep-PU MS have no cytotoxicity, which is very important for their potential use in vivo [74–76].

Figure 5.3 Optical images of (a) saline solution-treated RBCs, and (b) Hep-PU MS treated RBCs.


5.4 Construction of Biosensor for Direct Detection in Whole Blood

As we know, when in direct contact with blood, the foreign materials are prone to initiate the formation of clots, as platelets and other compo-nents of the blood coagulation system are activated. So it is very diffi cult to design and prepare an electrochemical biosensor that can be used in whole blood directly just because the biofouling of electrode surface can be developed by platelet, fi brin and blood cell adhesion in the complex environment of whole blood media. As for the glucose biosensor that used in whole blood directly, the biofouling of electrode surface will bring cata-strophic damage to the electron transfer between enzyme and electrode redox center. In Reichert’s review paper, he suggested that biofouling is one of several causes for the failure of in vivo biosensors due to the accu-mulation of proteins, cells and other biological materials on the surface of biosensors [77]. Anti-biofouling is the process of removing or preventing these accumulations include platelets and other components of the blood from forming. So improving the hemocompatibility and anti-biofouling property of biomaterials or biodevices has become a very important task for biomedical material scientists. In the past years, diff erent approaches were taken by various research groups to combat the challenge of forming anti-biofouling and fouling release surfaces. Particular attention was given to the chemical structures produced and the various techniques utilized to demonstrate these surfaces inherent anti-biofouling character.

Nowadays, nanomaterials of polymers are found to have superior per-formance compared with conventional polymeric materials due to their much larger exposed surface area and better biocompatibility. In this case, Hep-PU MS were prepared and chosen as an example to modify the glassy carbon electrode (GCE) for its antibiofouling eff ect that due to the coher-ence between blood compatibility and antifouling property of Hep. Herein, using hemocompatible polymeric nanospheres to do the study of antibiofouling of the glucose biosensor was attempted by our research group.

5.4.1 Evaluation of GOx/(Hep-PU) Hybrids

Th e maintenance of enzyme activity on the supporting materials is crucial in the biosensor designs, because the secondary conformational variations of the enzyme can aff ect its activity markedly. Herein, circular dicroism (CD) were utilized to check the secondary conformation variations of the polypeptide chain of GOx on the Hep-PU MS [78]. Th e CD spectra in the far-UV (with the range from 185 to 260 nm) were measured on a JASCO


J-715 spectropolarimeter using a 1 cm quartz cuvette. Th e contents of α-helix, β-sheet, β-turn, and the random coil conformation were calcu-lated using the JASCO710 program.

Fig. 5.4 showed the CD spectra of GOx in PBS without (curve a) and with Hep-PU MS (curve b) in the far-UV region (185–260 nm). Th e posi-tive bands at 191 nm in both curves correspond to the π–π* transition of the amide groups in the GOx peptide chain. Two negative bands at 209 and 219 nm are in accordance to the π–π* and n–π* transition of the amide groups of the GOx polypeptide chain, respectively [79]. In the presence of Hep-PU MS, the intensity of the positive band at 191 nm signifi cantly decreases compared with that of pure GOx, indicating the interactions between the amino acid residues of GOx and Hep-PU nanocomposites. Additionally, the intensity of the dual bands at 209 and 219 nm had a slight decrease for the GOx/(Hep-PU), indicating that the secondary structure of GOx was not obviously changed.

In addition, the contents of α-helix and β-turn conformation of GOx/(Hep-PU) changed 4.3% and 0.6% compared with pure GOx. All results indicate that Hep-PU MS could essentially maintain the native con-formation of GOx [80]. Th us, the secondary structure of GOx was well maintained in the prepared biosensor and Hep-PU MS indeed have good biocompatibility.

5.4.2 Evaluation of Whole Blood Adhesion Tests

Th e whole blood adhesion test has already become a recognized tech-nique to estimate the blood compatibility or anticoagulation of a pre-pared material. Th us, our work also employed this test to evaluate the

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blood compatibility of the surface of electrode that modifi ed by Hep-PU MS. Th e blood was obtained from a healthy adult volunteer anticoagu-lated with sodium citrate solution. Th e blank substrate without Hep-PU MS and GOx/(Hep-PU) modifi ed substrate were immersed in PBS for 24 h before they were placed in the 24-well microplates. Each well was added with 1.0 mL of whole blood. Aft er being incubated for 60 min at 37 ºC in humidifi ed air, the samples were taken out, and rinsed by PBS for three times to remove the physically attached blood cells. Aft er that, the adhered blood cells were fi xed with 2.5% glutaraldehyde in PBS for 30 min. Finally, the sample was washed with PBS and dehydrated with a series of ethanol/water mixtures of increasing ethanol concentration (50, 60, 70, 80, 90, 95 and 100% of ethanol) for 10 min in each mixture respectively. Th e surfaces membranes were air-dried, coated with gold and blood cells visualized by scanning electron microscope (SEM, JEOL JSM Model 6300, Japan).

Fig. 5.5 showed SEM photographs of blood cells that adhered to the surfaces of the substrates with or without Hep-PU MS. Numerous adher-ent blood cells and some aggregates were observed on the blank GCE (Fig. 5.5a), while the blood cells and platelets adhering were remarkably suppressed on the surface of electrode substrate modifi ed with GOx/(Hep-PU) (Fig. 5.5b and c). Based on the above observations, it was believed that thrombus was diffi cult to form onto the surface of Hep-PU MS without fused blood cells and platelets, which was caused by the anti-biofouling eff ect. Th e results strongly display that the anticoagulation of Hep-PU MS could effi ciently suppress blood-cell adhesion and increase the microenvironment for GOx to undergo facile electron-transfer reactions.

Figure 5.5 Representative SEM images of (a) blank electrode substrate, (b) electrode substrate modifi ed with GOx/(Hep-PU), and (c) enlarged view of (b) exposed to human whole blood for 60 min, respectively.


5.4.3 Direct Electrochemistry of GOx/(Hep-PU)/GCE and Calibration Curve

All electrochemical experiments were performed on a CHI 760D electro-chemical workstation (Chenhua Co. Ltd., China) in a three-electrode con-fi guration. A saturated calomel electrode (SCE) and a platinum electrode served as reference and counter electrode, respectively. All potentials given below were relative to the SCE. Th e working electrode was a GCE. Hep-PU MS suspension was dropped onto the electrode surface and dried in air. Aft er that, GOx solution was dropped onto the surface of (Hep-PU)/GCE and kept overnight at 4 ºC, then the GCE modifi ed with GOx/(Hep-PU) was obtained and the same modifi ed method was used to get the (Hep-PU)/GCE. When not in use, the electrodes were stored at 4 ºC in a refrigerator.

Cyclic voltammogram (CV) measurements were conducted in a 5 mL PBS cell at room temperature and the solution was purged with high purity nitrogen fi rstly and blanked with nitrogen during the electrochemical experiments.

Fig. 5.6 showed the electrochemical behaviors of (Hep-PU)/GCE, GOx/GCE and GOx/(Hep-PU)/GCE in PBS. Th ere was no apparently redox process in CVs of the (Hep-PU)/GCE (curve a) and GOx/GCE (curve b), while GOx/(Hep-PU)/GCE displayed a pair of well-defined and quasi-reversible CV peak with a formal potential value (E0’) of -0.408 V (curve b). Th e good electrochemical response of GOx/(Hep-PU)/GCE indicated

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Figure 5.6 CVs of (a) (Hep-PU)/GCE, (b) GOx/GCE, and (c) GOx/(Hep-PU)/GCE in 0.1 M PBS (pH=7.4). Scan rate: 100 mV/s. Th e insert: Amperometric responses of the biosensor upon additions of glucose (1.0 mM), AA (0.1 mM), glucose (1.0 mM) and UA (0.1 mM), respectively, in PBS. Th e biosensor was biased on the potential of -0.41 V.


Hep-PU MS played an important role in facilitating the electron exchange between the electroactive center of GOx and GCE, and provided a mild environment so that the bioactivity of GOx could be retained. In addi-tion, the anodic (Epa) and cathodic (Epc) peak potential were detected at −0.374 V and −0.442 V, respectively, and the separation of peak poten-tials (ΔEp) was 68 mV, at a scan rate of 100 mV/s. Th e ratio of anodic to cathodic peak currents was about 0.85. Th ese results indicate that GOx underwent a quasi-reversible redox process (FeIII/FeII redox couple) at the GCE modifi ed with Hep-PU MS.

An important analytical parameter for a biosensor is its ability to dis-criminate between the interfering species commonly present in similar physiological environment and the target analyte [81]. Electrochemical response of the GOx/(Hep-PU)/GCE was examined in the presence of some electroactive interfering substances like ascorbic acid (AA) and uric acid (UA) at higher levels than normal physiological [81]. Th e experiment was carried out by adding 1.0 mM glucose followed by 0.1 mM AA, 1.0 mM glucose and 0.1 mM UA. As shown in the insert of Fig. 5.6, the elec-troactive species did not cause interference signifi cantly for the determina-tion of glucose. Th e results imply that the GOx/(Hep-PU)/GCE has a good anti-interference ability.

Diff erential pulse voltammetry (DPV) measurements were carried out with pulse amplitude of 0.05 V and pulse width of 0.2 s. Diff erent concen-trations of glucose solution were added under intensive stirring, then CV was performed until the currents did not change any more, and DPV was immediately carried out. Th en correlation between response currents and diff erent concentrations of glucose solution was obtained. Fig. 5.7 showed typical DPVs for the detection of glucose in PBS and whole blood at the GOx/(Hep-PU)/GCE. Firstly, the GOx/(Hep-PU)/GCE was applied in PBS and the linear response range was from 0.2 mM to 20 mM. Th e linear regression equation was I (μA) = 0.074c (mM) + 0.122 (R = 0.9990), where I was current and c was the glucose concentration. Th e enhanced linear range expands the applications of the biosensor, especially in the glucose determination in whole blood samples. Th en the proposed biosensor was used to determine blood glucose in whole blood. Blood samples were sup-plied by volunteer, within sodium fl uoride to prevent glucose metabolism by blood cells prior to glucose determination [82, 83]. Th e current response was determined in 5 mL of 0.1 M, pH 7.4 PBS containing whole blood sample of 500 μL. It can be observed in Fig. 5.7B that with the increasing of glucose concentration, an anodic peak at -0.48 V emerges gradually. Th e calibration curve by plotting the current response with glucose concen-tration was presented in the insert of Fig. 5.7B. Th e linear relation had a


regression equation of I (μA) = 0.074c (mM) + 0.122 (R = 0.9990) with a correlation coeffi cient (R) of 0.9939 and the calculated detection limit is 1.4 × 10–5 M (S/N = 3) in whole blood, which is lower than those obtained for GOx immobilized on poly(pyrrole propylic acid)/Au nanocomposite fi lms (5 × 10–5 M) [84] and PtPd-MWCNTs fi lms (3.1 × 10–5 M) [85].

5.4.4 Human Blood Samples Measurement

In an attempt to explore the GOx/(Hep-PU)/GCE for practical applica-tions, the biosensor was applied to determine glucose in human blood

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0.15

0.20

0.25

Figure 5.7 (A) DPVs obtained at GOx/(Hep-PU)/GCE in 0.1 M PBS (pH=7.4) with the concentration of glucose (from a to h) 0, 0.2, 1, 2, 5, 10, 15, 20 mM; and the insert: DPVs obtained at GOx/(Hep-PU)/GCE in whole blood samples at 25 ºC with glucose of (a) 6.20 mM, (b) 6.25 mM, (c) 6.30 mM, (d) 6.35 mM, (e) 6.40 mM, (f) 6.45 mM. (B) Relationship between the peak current and the concentration of glucose in PBS; and the insert: relationship between the peak current and the concentration of glucose in whole blood samples.


samples of diabetic and healthy people. Th e glucose concentration in the serum of the diabetic is as high as 12.2 mM, while it is only 4.7–5.3 mM for the healthy ones [86]. Table 5.1 displayed the determination results of fi ve samples including three diabetic and two healthy people.

Th e samples were fi rst analyzed in the hospital biochemistry laboratory, then reanalyzed with the GOx/(Hep-PU)/GCE in our laboratory. Th e val-ues of glucose was determined by the proposed biosensor are very close with the data provided by hospital. Furthermore, the values we measured direct in whole blood by the GOx/(Hep-PU)/GCE are more closed to the real values probably compared to the values that were measured in serum samples. Deeper investigation will be performed by our group in future.

5.5 Conclusion and Prospect

Considering the signifi cance of blood in the area of biomedical and health for life, this paper has practical importance. Th is idea and technique of innovative hemocompatible polymeric micro- and nanoparticles provide a promising platform for the development of novel electrochemical biosen-sors that can be directly used in whole blood-contact system for illness diagnosis. In other words, the integration of nanotechnology, anticoagu-lant design and biosensor that will, without doubt, bring signifi cant input to practical biomedical devices relevant to diagnostics and therapy of inter-est for human health.

Table 5.1 Determination of glucose in whole blood samples using the GOx/(Hep-PU)/GCE.

NO. Referenced valuesa (mM) Determined valuesb (mM)

1 12.2±0.29 12.1±0.31

2 11.6±0.33 11.4±0.37

3 8.9±0.35 8.7±0.36

4 5.2±0.25 5.0±0.29

5 4.9±0.28 4.8±0.32a Referenced values were provided by the hospital biochemistry laboratory. b Th e values were determined by the GOx/(Hep-PU)/GCE; they were average values of fi ve measurements for each sample


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Advanced Healthcare Materials (Tiwari/Advanced) || Novel Biomaterials for Human Health: Hemocompatible Polymeric Micro-and Nanoparticles and Their Application in Biosensor