Bioprocess Biosyst Eng (2015) 38:815–822 DOI 10.1007/s00449-014-1323-1
ORIGINAL PAPER
Voltammetric detection of anti-HIV replication drug based on novel nanocomposite gold-nanoparticle–CaCO3 hybrid material Jagriti Narang • Nitesh Malhotra • Gajendra Singh C. S. Pundir
•
Received: 2 July 2014 / Accepted: 4 November 2014 / Published online: 22 November 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract A novel bionanocomposite, horse radish peroxidase- gold-nanoparticle–Calcium carbonate (HRPAuNPs-CaCO3), hybrid material was encapsulated by silica sol on a glassy carbon electrode (GCE). The fabricated modified electrode was used as a novel voltammetric sensor for electrochemical sensing of anti-HIV replication drug i.e. deferiprone. The surface morphology of the modified electrode was characterized by scanning electron microscopy (SEM). Results obtained from the voltammetric measurements show that HRP-AuNPs-CaCO3 modified GCE offers a selective and sensitive electrochemical sensor for the determination of deferiprone. Under experimental conditions, the proposed voltammetric sensor has a linear response range from 0.01 to 10,000 lM with a detection limit of 0.01 lM. Furthermore, the fabricated sensor was successfully applied to determine deferiprone level in spiked urine and serum samples. Keywords Deferiprone Gold-nanoparticle–Calcium carbonate Horse radish peroxidase GCE electrode
J. Narang (&) Amity Institute of Nanotechnology, AMITY University, Noida, UP, India e-mail: [email protected] N. Malhotra Amity Institute of Physiotherapy, AMITY University, Noida, UP, India G. Singh Faculty of Pharmaceutical Science PGIMS, Rohtak, India C. S. Pundir Department of Biochemistry, M. D. University, Rohtak 124001, Haryana, India
Introduction HIV/AIDS is a global pandemic disease [1]. As of 2012, approximately 35.3 million people are living with HIV globally [2]. There were about 1.8 million deaths from AIDS in 2010, down from 2.2 million in 2005 [2]. Researchers are locking on drug i.e. deferiprone which kills HIV on infected host cells by inducing apoptosis. Deferoxamine (DFO) in some combination appears to be the emerging ‘‘treatment of choice’’ for significant cardiac dysfunction from iron overload, as in evidence supports the biochemical rationale [3]. Deferiprone (DEF) causes apoptosis or programmed cell death or triggered suicide in HIV infected cells. DEF has been shown to inhibit HIV-1 replication in latently-infected cells after phorbol ester induction [4], and in peripheral blood lymphocytes. Overdoses can cause serious damage which may result in various diseases. Drug monitoring has a widespread impact on public health. Monitoring the drug is most important to minimize the risk of side effects [5]. Extensive efforts should be made to detect the methods for the accurate measurement of the drug if necessary. Till date, a variety of methods are available for determination of drug like chromatography [6], HPLC [7] and liquid chromatography coupled with mass spectrometry (LC/MS) [8]. Despite exceptional advancement in the field of biomedicine, major challenges still stay behind in translating biomedical knowledge of disease into clinically relevant devices that could be used as diagnostic or monitoring tools for disease management [9]. Electrochemical methods/enzyme electrodes are considered as one of the most convenient methods, because of their high sensitivity, simplicity and rapidity and no sample preparation [10–16].
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Nowadays there is rapid growth in the field of nanomaterials, materials in this size range show attractive physical properties, distinct from both the molecular and bulk scales, presenting new opportunities for biomedical research and applications [9]. Gold nanoparticles (AuNPs) are ideal material for nanobiosensors fabrication as it is suitable for the binding of biomolecules, and the metal show fast electron transfer kinetics [17]. In the present work, gold nanoparticles were coated on the surface of porous carbonate microsphere to fabricate AuNP-CaCO3 hybrid material. A coating method is generally used to fabricate nanocomposite for improving its properties. Calcium carbonate has immense potential of biocompatibility and also provides favorable environment for enzyme loading [18]. Therefore, the negatively charged AuNPs could assemble on positively charged vaterite surface via electrostatic interaction and other supramolecular interactions [9]. In this we describe novel results of immobilization of enzyme on the surface of AuNPs–CaCO3 composite to construct an amperometric biosensor. We describe herein, the therapeutic drug monitoring of deferiprone using biosensor based on AuNPs–CaCO3/silica sol–gel-modified GCE.
Experimental Materials Deferiprone was procured from vendors of pharmaceutical company. Calcium chloride, sodium carbonate and Gold chloride tetrahydrate, Horse radish peroxidase (HRP) and tetraethyl orthosilicate (TEOS) were procured from Sisco Research Laboratory, Mumbai, India. All other chemicals were of analytic reagent grade. Double distilled water (DW) was used throughout the experiments. Preparation of hybrid nanocomposite Gold nanoparticles (GNPs) were prepared as described by Xu et al. [19] with minor modification. The morphological characterization of the gold nanoparticles was carried out at Department of Anatomy, AIIMS, New Delhi by a transmission electron microscope (TEM) CaCO3 microspheres were prepared by adding Na2CO3 (0.33 M) solution into an equal volume of a solution of CaCl2 (0.33 M) with continuous stirring [20]. Precipitate formed was centrifuged at 6000 rpm for 15 min. Then these microspheres obtained were washed and dried. Then these prepared microspheres (0.5 gm) were poured in of gold colloid (50 ml) solution. Then sonication was done for 20 min. Then light purple hybrid nanocomposite (AuNP-CaCO3) composites were prepared. CaCO3 microspheres were positively charged at
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pH 7.0, whereas AuNPs were negatively charged at the same pH. Due to this electrostatic interaction caused the assembly of AuNPs on the surface of the oppositely charged CaCO3 microspheres; it facilitated the assembly process [21]. Preparation of bioconjugates Enzyme solution (HRP 20 ll) was added into hybrid nanocomposite (20 mg). Then this solution was continuously stirred for 1 h. These bioconjugates were centrifuged and dried at room temperature. Preparation of encapsulating material Silica sol. was prepared by mixing ethanol (600 ll), TEOS (50 ll), 5 mM NaOH (10 ll) and water (60 ll). This solution was sonicated for further use [22] (Scheme 1). Construction of enzyme electrode (HRP/AuNP-CaCO3/ GCE) for electrochemical sensing of anti-HIV replication drug. A glassy carbon electrode was polished with alumina powder (diameter 0.05 lm) followed by ultrasonic cleaning with DW. The prepared HRP-AuNP-CaCO3 bioconjugates (20 ll) were absorbed on the surface of GCE. Silica sol (10 ll) was added for encapsulation of the bioconjugates. The electrode was dried and stored at 4 °C for further electrochemical measurements. Electrochemical characterization of anti-HIV replication drug biosensor Cyclic voltammetry studies were carried out using an electrochemical cell composed of HRP/AuNP-CaCO3/ GCE as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. To detect the role of individual components, cyclic voltammograms of bare GC electrode, AuNP-CaCO3/GCE and HRP/AuNP-CaCO3/ GCE were recorded in sodium phosphate buffer (0.1 M, pH 7.0) containing 0.1 mM deferiprone at a scan rate of 0.0 to ?1.5 V s-1 at an interval of 50 mV s-1. Preparation of anti-HIV replication drug solution The standard solution of authentic deferiprone was prepared in phosphate buffer solution (pH 7.0). Solutions of different concentrations of deferiprone ranging from 0.01 to 10,000 lM were prepared in 0.1 M sodium phosphate buffer (pH 7.0) and stored at 4 °C until use. Optimization and evaluation of anti-HIV replication drug biosensor Various experimental conditions like pH, incubation temperature, time were optimized for effectual functioning of
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Scheme 1 Graphical illustration of the stepwise anti-HIV replication drug biosensor fabrication process and its application for electrochemical detection of deferiprone 1.05 1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0
2000
4000
6000
8000
10000
12000
measurements were performed after successive additions of spiked samples. After each addition, cyclic voltammograms were recorded by cycling the potential between 0.0 and ?1.5 V s-1 at a scan rate of 100 mV s-1. Deferiprone content was determined by the present biosensor by replacing deferiprone with spiked serum/urine samples and recording the current (mA) under its optimal working conditions. The amount of pharmaceutical product was extrapolated from standard curve between deferiprone concentrations and current in mA (Fig. 1).
Fig. 1 Linear response of concentrations of deferiprone (Substrate concentration/lM) vs. current (I/mA)
Storage stability of anti-HIV replication drug biosensor
biosensor. For assessment of functioning of biosensor, parameters like analytical recovery and accuracy were also studied.
Storage stability of the bioconjugated electrode and its electrochemical sensing toward deferiprone was examined over a period of 100 days at 4 °C.
Electrochemical sensing of anti-HIV drug in spiked serum and urine samples
Results and discussions
Serum samples were procured from University of Health Sciences (UHS), Rohtak. Stock solutions of deferiprone were spiked into 1.0 mL of the diluted serum sample. Urine samples were taken from a healthy volunteer persons and diluted with phosphate buffer (supporting electrolyte, PB), for subsequent electrochemical sensing. The
Characterization of AuNP-CaCO3 Figure 2 shows TEM images were done to confirm the formation of hybrid nanocomposites. Microscopic image was revealing the formation of hybrid nanocomposites. The AuNPs covers the surface of the CaCO3 microspheres, with
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i / mA
Fig. 2 Transmission electron microscope (TEM) image of AuNPCaCO3
0.225 0.200 0.175 0.150 0.125 0.100 0.075 0.050 0.025
c b a
-0.025 -0.050 -0.075
0
0.250
0.500
0.750
1.000
1.250
1.500
1.750
E/V Fig. 3 Cyclic voltammogram of a bare GCE b AuNPs–CaCO3/silica sol–gel-modified GCE (c) and HRP–AuNPs–CaCO3/silica sol–gelmodified GCE in a sodium phosphate buffer 0.05 M (pH 7.0) at a scan rate of 50 mV s-1
a diameter of less than 100 nm. These observations confirm formation of AuNP-CaCO3. Preparation and characterization of bioconjugated electrode (HRP/AuNP-CaCO3/GCE) The bioconjugated electrode was prepared as follow: CV pattern was done using the bioconjugated electrode HRP/ AuNP-CaCO3/GCE as the working electrode in the solution containing deferiprone in scanning potential range of 0.0 to ?1.5 V s-1 for five consecutive cycles at the scan rate 50 mV s-1 in 0.1 M phosphate buffer pH 7.0 (Fig. 3). A bare GC electrode, AuNPs–CaCO3/silica sol–gel-
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modified GCE and HRP–AuNPs–CaCO3/silica sol–gelmodified GCE were studied for the comparative study. Low sensing signal was observed in case of unmodified electrode, while more sensing signal was obtained in the case of the AuNPs–CaCO3/silica sol–gel-modified GCE (Fig. 3b) due to oxidation of deferiprone by AuNPs– CaCO3/silica sol–gel. So, hybrid nanocomposite accelerates the electron transfer kinetics. When enzyme gets conjugated with hybrid nanocomposite modified electrode, HRP–AuNPs–CaCO3/silica sol–gel-modified GCE showed amplification in the sensing signal because of catalyzed oxidation reactions (Fig. 3c) [23]. The surface morphologies of the (a) bare GCE (b) AuNPs–CaCO3/silica sol–gel-modified GCE (c) and HRP–AuNPs–CaCO3/silica sol–gel-modified GCE were also examined with a scanning electronic microscope (SEM). Figure 4 shows SEM image of the AuNPs–CaCO3/ silica sol–gel-modified GCE, which was different from that of the bare GCE (Fig. 4a). A generally porous structure was observed in the SEM image of AuNPs–CaCO3/silica sol–gel-modified GCE (Fig. 4b). The spherical structures other than porous structure can be apparent from the image which confirms that enzyme get immobilize (Fig. 4c). An electrochemical impedance spectrum (EIS) is a useful technique for probing the features of a surface modified electrode. In the EIS, the semicircle portion at higher frequencies corresponds to the electron transfer limited process and the linear portion at lower frequencies may ascribe diffusion. The semicircle diameter equals the Rct, which depends on the dielectric and insulating features at the electrode/electrolyte interface [24]. Figure 5 shows electrochemical impedance spectra (EIS) of bare GCE (a), AuNPs–CaCO3/silica sol–gel-modified GCE (b) and HRP/ AuNPs–CaCO3/silica sol–gel-modified GCE (c) in a solution containing 1 mM. Fe(CN)6 3-/4- with 0.1 M KCl at 0.20 mV s-1 (frequency range of 0.01 Hz–10 kHz). The Rct values for the bare GCE electrode, AuNPs–CaCO3/ silica sol–gel-modified GCE and HRP/AuNPs–CaCO3/silica sol–gel-modified GCE were obtained as 1,200, 600 and 525 X, respectively. After addition of nanocomposite Rct value gets decreased as nanocomposite provides conductive path for faster electron kinetics. While after immobilization of enzyme, Rct value of HRP/AuNPs–CaCO3/ silica sol–gel-modified GCE gets further decreased. It is due to the fact that catalysed reactions of drug were catalyzed by enzyme. Principle for detection of anti-HIV replication drug using modified electrode A mechanism for the oxidation of deferiprone is as follows: It is likely that an electrocatalytic mechanism initiated by
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Fig. 4 SEM of a bare GCE, b AuNPs–CaCO3/silica sol–gel-modified GCE (c) and HRP–AuNPs–CaCO3/silica sol–gel-modified GCE
because of the participation of proton(s) in the oxidation reaction of deferiprone within a quasi-reversible twoelectron process. It can be observed from the cyclic voltammogram that two peaks are associated with diffusioncontrolled processes while other peaks are corresponded to surface-confined processes and can be related to the adsorption of produced reaction products. It is well known that the adsorption of reaction product on the electrode surface represents a pre-peak [26]. So, two peaks are due to diffusion-controlled processes and another peaks are their corresponding pre-peaks. Voltammetric determination of anti-HIV replication drug
Fig. 5 EIS of (a) bare GCE (b) AuNPs–CaCO3/silica sol–gelmodified GCE (c) and HRP–AuNPs–CaCO3/silica sol–gel-modified GCE in a solution containing 1 mM Fe(CN)6 3-/4- with 0.1 M KCl at 0.20 mV s-1 (frequency range of 0.01 Hz–10 kHz)
HRP catalyzes the oxidation of deferiprone to corresponding dione. The dione formed undergoes a anodic hydroxylation of the methyl side chain [25]. It is expected
The detection limit and the linear working range of the biosensor were evaluated for determination of deferiprone. There was a linear relationship between current vs. concentration of deferiprone in the range of 0.01 to 10,000 lM (Fig. 1). The limit of detection of the present biosensor was calculated as the lowest quantity of deferiprone required to give a signal to a background (blank) ?3 times SD of blank and found to be 0.01 lM (Fig. 6).
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i / mA
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0.600 0.500 0.450 0.400 0.350 0.300 0.250
10000 µM
0.200 0.150 0.100 0.050 0 -0.050 -0.100 -0.150
1000 µM
0.250
0.500
0.750
1.000
1.250
1.500
1.750
E/V
Table 1 Determination of deferiprone by anti-HIV drug biosensor and pharmacopeia method in human spiked urine and serum samples S. no. Spiked samples (mM)
Pharmacopoeia method (mM)
Present method (mM)
Urine
Serum
Urine
Serum
Urine
Serum
1
1.50
1.50
1.52
1.50
1.50
1.51
2
2.50
2.50
2.48
2.51
2.48
2.50
3
3.50
3.50
3.50
3.48
3.47
3.48
4
4.50
4.50
4.20
4.50
4.51
4.50
5
5.50
5.50
5.00
5.48
5.51
5.45
6
6.50
6.50
6.25
6.40
6.50
6.50
7
7.50
7.50
7.40
7.45
7.51
7.48
8
8.50
8.50
8.50
8.48
8.51
8.50
9
9.50
9.50
9.40
9.50
9.50
9.50
Fig. 6 Cyclic voltammograms of anti-HIV replication drug biosensor with various concentrations of deferiprone
10
10.0
10.0
9.58
9.99
10.1
10.0
Optimization of the anti-HIV replication drug biosensor
98.1 ± 1.3, respectively. To test the reproducibility and reliability of the present biosensor deferiprone content in ten spiked serum, urine sample was estimated on single day (within batch) and five times again after storage at -20 °C. The accuracy of the proposed method was determined by spiking serum and urine samples with different concentrations of deferiprone (Table 1). Accuracy of proposed method was 99 %. The effect of some oxidizable substances that might interfere with the response of the biosensor was studied. However, insignificant current due to oxidation of the compounds in either the serum or the urine samples appeared.
The effect of the determination conditions such as the working potential, pH value, response time and temperature on the response of the HRP–AuNPs–CaCO3/silica sol– gel-modified GCE was investigated in detail. The effect of the applied potential on the response current of the HRP–AuNPs–CaCO3/silica sol–gel-modified GCE was studied. The response of the HRP–AuNPs– CaCO3/silica sol–gel-modified GCE as a function of the applied potential was between 0.0 and ?1.5 V s-1. An applied potential of ?0.5 V was selected to give a high detection sensitivity and good signal/noise ratio. The effect of the pH value on the response current of the HRP–AuNPs–CaCO3/silica sol–gel-modified GCE was studied between 5.0 and 8.0 in 0.05 M PBS. The suitable pH with the maximum activity of the immobilized HRP was at pH 7.0. The response time was less than 2 s, which shows a quick response. The faster response was mainly ascribed to the fact that HRP–AuNPs–CaCO3 is providing favorable orientation and conductive pathway to transfer electrons. The electrocatalytic mechanism initiated by HRP makes the process fast i.e. oxidation of drug. Effect of temperature on biosensor was also studied to ensure the optimization. The current response reaches a maximum at approximately 50 °C, and then goes down as the temperature turn higher. To maintain steady with the temperature of human body, 35 °C is selected for this work.
Stability of the anti-HIV replication drug biosensor The stability of the biosensor is investigated and the current response of biosensor is retained about 80 % of its original response after 40 times uninterrupted detection. In addition, the long-term stability is also tested after a month. It is revealed that the current response of the sensor maintains 84 % of the initial current response. This means that AuNPs–CaCO3/silica sol–gel-modified GCE ensures well stability of the biosensor. A comparison of analytic parameters of various nanoparticles based biosensors for detection of deferiprone with the present biosensor is summarized in Table 2.
Conclusions Analytical performance and accuracy The analytic recovery of known amount of added deferiprone was determined by the present biosensor. The mean analytic recoveries of added 5 and 10 mmol L-1 deferiprone (final conc. in reaction mixture) were 98.5 ± 1.7 and
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In summary, we report electrochemical sensing protocol using porous gold-nanoparticle–CaCO3 hybrid material for the detection of anti-HIV replication drug i.e. deferiprone. The proposed sensor matrix is evaluated, which demonstrates a very low detection limit of 0.01 lM. The present
Bioprocess Biosyst Eng (2015) 38:815–822 Table 2 Comparison of the present method with other biosensing methods
Matrix/method
821
Enzyme
Response time
Detection limit (lM)
Linearity (lM)
Stability
References
GC/cyclic voltammetry
–
5s
0.542
51–7,600
–
[25]
Platinum disk/differentialpulse voltammetry
–
–
14.3
70–4,210
–
[27]
Luminescent complex with Tb3 ? ions/ fluorometric
–
–
0.0063
0.0072–14
–
[28]
GC/differential-pulse voltammetry
–
–
2.30
50.7–2,530
–
[29]
Nickel oxyhydroxide/cyclic voltammetry
–
–
19
99–534
4 weeks
[30]
AuNPs–CaCO3/silica sol–gelmodified GCE/cyclic voltammetry
HRP
2s
0.01
0.01–10,000
1 month
Present
investigations revealed that the performance of modified sensor exhibited its suitability for enhanced as well as sensitive determination of deferiprone. Thus, the promising voltammetric approach platform makes an avenue for fast, simple, sensitive and selective detection of deferiprone. Acknowledgments In the present work, one of the author (Jagriti Narang) was supported by SERB, Department of Science and Technology (DST), India. Thanks to all scientists referenced throughout the paper whose valuable work has guided the way through to this research work.
9.
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ORIGINAL PAPER
Voltammetric detection of anti-HIV replication drug based on novel nanocomposite gold-nanoparticle–CaCO3 hybrid material Jagriti Narang • Nitesh Malhotra • Gajendra Singh C. S. Pundir
•
Received: 2 July 2014 / Accepted: 4 November 2014 / Published online: 22 November 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract A novel bionanocomposite, horse radish peroxidase- gold-nanoparticle–Calcium carbonate (HRPAuNPs-CaCO3), hybrid material was encapsulated by silica sol on a glassy carbon electrode (GCE). The fabricated modified electrode was used as a novel voltammetric sensor for electrochemical sensing of anti-HIV replication drug i.e. deferiprone. The surface morphology of the modified electrode was characterized by scanning electron microscopy (SEM). Results obtained from the voltammetric measurements show that HRP-AuNPs-CaCO3 modified GCE offers a selective and sensitive electrochemical sensor for the determination of deferiprone. Under experimental conditions, the proposed voltammetric sensor has a linear response range from 0.01 to 10,000 lM with a detection limit of 0.01 lM. Furthermore, the fabricated sensor was successfully applied to determine deferiprone level in spiked urine and serum samples. Keywords Deferiprone Gold-nanoparticle–Calcium carbonate Horse radish peroxidase GCE electrode
J. Narang (&) Amity Institute of Nanotechnology, AMITY University, Noida, UP, India e-mail: [email protected] N. Malhotra Amity Institute of Physiotherapy, AMITY University, Noida, UP, India G. Singh Faculty of Pharmaceutical Science PGIMS, Rohtak, India C. S. Pundir Department of Biochemistry, M. D. University, Rohtak 124001, Haryana, India
Introduction HIV/AIDS is a global pandemic disease [1]. As of 2012, approximately 35.3 million people are living with HIV globally [2]. There were about 1.8 million deaths from AIDS in 2010, down from 2.2 million in 2005 [2]. Researchers are locking on drug i.e. deferiprone which kills HIV on infected host cells by inducing apoptosis. Deferoxamine (DFO) in some combination appears to be the emerging ‘‘treatment of choice’’ for significant cardiac dysfunction from iron overload, as in evidence supports the biochemical rationale [3]. Deferiprone (DEF) causes apoptosis or programmed cell death or triggered suicide in HIV infected cells. DEF has been shown to inhibit HIV-1 replication in latently-infected cells after phorbol ester induction [4], and in peripheral blood lymphocytes. Overdoses can cause serious damage which may result in various diseases. Drug monitoring has a widespread impact on public health. Monitoring the drug is most important to minimize the risk of side effects [5]. Extensive efforts should be made to detect the methods for the accurate measurement of the drug if necessary. Till date, a variety of methods are available for determination of drug like chromatography [6], HPLC [7] and liquid chromatography coupled with mass spectrometry (LC/MS) [8]. Despite exceptional advancement in the field of biomedicine, major challenges still stay behind in translating biomedical knowledge of disease into clinically relevant devices that could be used as diagnostic or monitoring tools for disease management [9]. Electrochemical methods/enzyme electrodes are considered as one of the most convenient methods, because of their high sensitivity, simplicity and rapidity and no sample preparation [10–16].
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Nowadays there is rapid growth in the field of nanomaterials, materials in this size range show attractive physical properties, distinct from both the molecular and bulk scales, presenting new opportunities for biomedical research and applications [9]. Gold nanoparticles (AuNPs) are ideal material for nanobiosensors fabrication as it is suitable for the binding of biomolecules, and the metal show fast electron transfer kinetics [17]. In the present work, gold nanoparticles were coated on the surface of porous carbonate microsphere to fabricate AuNP-CaCO3 hybrid material. A coating method is generally used to fabricate nanocomposite for improving its properties. Calcium carbonate has immense potential of biocompatibility and also provides favorable environment for enzyme loading [18]. Therefore, the negatively charged AuNPs could assemble on positively charged vaterite surface via electrostatic interaction and other supramolecular interactions [9]. In this we describe novel results of immobilization of enzyme on the surface of AuNPs–CaCO3 composite to construct an amperometric biosensor. We describe herein, the therapeutic drug monitoring of deferiprone using biosensor based on AuNPs–CaCO3/silica sol–gel-modified GCE.
Experimental Materials Deferiprone was procured from vendors of pharmaceutical company. Calcium chloride, sodium carbonate and Gold chloride tetrahydrate, Horse radish peroxidase (HRP) and tetraethyl orthosilicate (TEOS) were procured from Sisco Research Laboratory, Mumbai, India. All other chemicals were of analytic reagent grade. Double distilled water (DW) was used throughout the experiments. Preparation of hybrid nanocomposite Gold nanoparticles (GNPs) were prepared as described by Xu et al. [19] with minor modification. The morphological characterization of the gold nanoparticles was carried out at Department of Anatomy, AIIMS, New Delhi by a transmission electron microscope (TEM) CaCO3 microspheres were prepared by adding Na2CO3 (0.33 M) solution into an equal volume of a solution of CaCl2 (0.33 M) with continuous stirring [20]. Precipitate formed was centrifuged at 6000 rpm for 15 min. Then these microspheres obtained were washed and dried. Then these prepared microspheres (0.5 gm) were poured in of gold colloid (50 ml) solution. Then sonication was done for 20 min. Then light purple hybrid nanocomposite (AuNP-CaCO3) composites were prepared. CaCO3 microspheres were positively charged at
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pH 7.0, whereas AuNPs were negatively charged at the same pH. Due to this electrostatic interaction caused the assembly of AuNPs on the surface of the oppositely charged CaCO3 microspheres; it facilitated the assembly process [21]. Preparation of bioconjugates Enzyme solution (HRP 20 ll) was added into hybrid nanocomposite (20 mg). Then this solution was continuously stirred for 1 h. These bioconjugates were centrifuged and dried at room temperature. Preparation of encapsulating material Silica sol. was prepared by mixing ethanol (600 ll), TEOS (50 ll), 5 mM NaOH (10 ll) and water (60 ll). This solution was sonicated for further use [22] (Scheme 1). Construction of enzyme electrode (HRP/AuNP-CaCO3/ GCE) for electrochemical sensing of anti-HIV replication drug. A glassy carbon electrode was polished with alumina powder (diameter 0.05 lm) followed by ultrasonic cleaning with DW. The prepared HRP-AuNP-CaCO3 bioconjugates (20 ll) were absorbed on the surface of GCE. Silica sol (10 ll) was added for encapsulation of the bioconjugates. The electrode was dried and stored at 4 °C for further electrochemical measurements. Electrochemical characterization of anti-HIV replication drug biosensor Cyclic voltammetry studies were carried out using an electrochemical cell composed of HRP/AuNP-CaCO3/ GCE as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. To detect the role of individual components, cyclic voltammograms of bare GC electrode, AuNP-CaCO3/GCE and HRP/AuNP-CaCO3/ GCE were recorded in sodium phosphate buffer (0.1 M, pH 7.0) containing 0.1 mM deferiprone at a scan rate of 0.0 to ?1.5 V s-1 at an interval of 50 mV s-1. Preparation of anti-HIV replication drug solution The standard solution of authentic deferiprone was prepared in phosphate buffer solution (pH 7.0). Solutions of different concentrations of deferiprone ranging from 0.01 to 10,000 lM were prepared in 0.1 M sodium phosphate buffer (pH 7.0) and stored at 4 °C until use. Optimization and evaluation of anti-HIV replication drug biosensor Various experimental conditions like pH, incubation temperature, time were optimized for effectual functioning of
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817
Scheme 1 Graphical illustration of the stepwise anti-HIV replication drug biosensor fabrication process and its application for electrochemical detection of deferiprone 1.05 1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0
2000
4000
6000
8000
10000
12000
measurements were performed after successive additions of spiked samples. After each addition, cyclic voltammograms were recorded by cycling the potential between 0.0 and ?1.5 V s-1 at a scan rate of 100 mV s-1. Deferiprone content was determined by the present biosensor by replacing deferiprone with spiked serum/urine samples and recording the current (mA) under its optimal working conditions. The amount of pharmaceutical product was extrapolated from standard curve between deferiprone concentrations and current in mA (Fig. 1).
Fig. 1 Linear response of concentrations of deferiprone (Substrate concentration/lM) vs. current (I/mA)
Storage stability of anti-HIV replication drug biosensor
biosensor. For assessment of functioning of biosensor, parameters like analytical recovery and accuracy were also studied.
Storage stability of the bioconjugated electrode and its electrochemical sensing toward deferiprone was examined over a period of 100 days at 4 °C.
Electrochemical sensing of anti-HIV drug in spiked serum and urine samples
Results and discussions
Serum samples were procured from University of Health Sciences (UHS), Rohtak. Stock solutions of deferiprone were spiked into 1.0 mL of the diluted serum sample. Urine samples were taken from a healthy volunteer persons and diluted with phosphate buffer (supporting electrolyte, PB), for subsequent electrochemical sensing. The
Characterization of AuNP-CaCO3 Figure 2 shows TEM images were done to confirm the formation of hybrid nanocomposites. Microscopic image was revealing the formation of hybrid nanocomposites. The AuNPs covers the surface of the CaCO3 microspheres, with
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818
Bioprocess Biosyst Eng (2015) 38:815–822
i / mA
Fig. 2 Transmission electron microscope (TEM) image of AuNPCaCO3
0.225 0.200 0.175 0.150 0.125 0.100 0.075 0.050 0.025
c b a
-0.025 -0.050 -0.075
0
0.250
0.500
0.750
1.000
1.250
1.500
1.750
E/V Fig. 3 Cyclic voltammogram of a bare GCE b AuNPs–CaCO3/silica sol–gel-modified GCE (c) and HRP–AuNPs–CaCO3/silica sol–gelmodified GCE in a sodium phosphate buffer 0.05 M (pH 7.0) at a scan rate of 50 mV s-1
a diameter of less than 100 nm. These observations confirm formation of AuNP-CaCO3. Preparation and characterization of bioconjugated electrode (HRP/AuNP-CaCO3/GCE) The bioconjugated electrode was prepared as follow: CV pattern was done using the bioconjugated electrode HRP/ AuNP-CaCO3/GCE as the working electrode in the solution containing deferiprone in scanning potential range of 0.0 to ?1.5 V s-1 for five consecutive cycles at the scan rate 50 mV s-1 in 0.1 M phosphate buffer pH 7.0 (Fig. 3). A bare GC electrode, AuNPs–CaCO3/silica sol–gel-
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modified GCE and HRP–AuNPs–CaCO3/silica sol–gelmodified GCE were studied for the comparative study. Low sensing signal was observed in case of unmodified electrode, while more sensing signal was obtained in the case of the AuNPs–CaCO3/silica sol–gel-modified GCE (Fig. 3b) due to oxidation of deferiprone by AuNPs– CaCO3/silica sol–gel. So, hybrid nanocomposite accelerates the electron transfer kinetics. When enzyme gets conjugated with hybrid nanocomposite modified electrode, HRP–AuNPs–CaCO3/silica sol–gel-modified GCE showed amplification in the sensing signal because of catalyzed oxidation reactions (Fig. 3c) [23]. The surface morphologies of the (a) bare GCE (b) AuNPs–CaCO3/silica sol–gel-modified GCE (c) and HRP–AuNPs–CaCO3/silica sol–gel-modified GCE were also examined with a scanning electronic microscope (SEM). Figure 4 shows SEM image of the AuNPs–CaCO3/ silica sol–gel-modified GCE, which was different from that of the bare GCE (Fig. 4a). A generally porous structure was observed in the SEM image of AuNPs–CaCO3/silica sol–gel-modified GCE (Fig. 4b). The spherical structures other than porous structure can be apparent from the image which confirms that enzyme get immobilize (Fig. 4c). An electrochemical impedance spectrum (EIS) is a useful technique for probing the features of a surface modified electrode. In the EIS, the semicircle portion at higher frequencies corresponds to the electron transfer limited process and the linear portion at lower frequencies may ascribe diffusion. The semicircle diameter equals the Rct, which depends on the dielectric and insulating features at the electrode/electrolyte interface [24]. Figure 5 shows electrochemical impedance spectra (EIS) of bare GCE (a), AuNPs–CaCO3/silica sol–gel-modified GCE (b) and HRP/ AuNPs–CaCO3/silica sol–gel-modified GCE (c) in a solution containing 1 mM. Fe(CN)6 3-/4- with 0.1 M KCl at 0.20 mV s-1 (frequency range of 0.01 Hz–10 kHz). The Rct values for the bare GCE electrode, AuNPs–CaCO3/ silica sol–gel-modified GCE and HRP/AuNPs–CaCO3/silica sol–gel-modified GCE were obtained as 1,200, 600 and 525 X, respectively. After addition of nanocomposite Rct value gets decreased as nanocomposite provides conductive path for faster electron kinetics. While after immobilization of enzyme, Rct value of HRP/AuNPs–CaCO3/ silica sol–gel-modified GCE gets further decreased. It is due to the fact that catalysed reactions of drug were catalyzed by enzyme. Principle for detection of anti-HIV replication drug using modified electrode A mechanism for the oxidation of deferiprone is as follows: It is likely that an electrocatalytic mechanism initiated by
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Fig. 4 SEM of a bare GCE, b AuNPs–CaCO3/silica sol–gel-modified GCE (c) and HRP–AuNPs–CaCO3/silica sol–gel-modified GCE
because of the participation of proton(s) in the oxidation reaction of deferiprone within a quasi-reversible twoelectron process. It can be observed from the cyclic voltammogram that two peaks are associated with diffusioncontrolled processes while other peaks are corresponded to surface-confined processes and can be related to the adsorption of produced reaction products. It is well known that the adsorption of reaction product on the electrode surface represents a pre-peak [26]. So, two peaks are due to diffusion-controlled processes and another peaks are their corresponding pre-peaks. Voltammetric determination of anti-HIV replication drug
Fig. 5 EIS of (a) bare GCE (b) AuNPs–CaCO3/silica sol–gelmodified GCE (c) and HRP–AuNPs–CaCO3/silica sol–gel-modified GCE in a solution containing 1 mM Fe(CN)6 3-/4- with 0.1 M KCl at 0.20 mV s-1 (frequency range of 0.01 Hz–10 kHz)
HRP catalyzes the oxidation of deferiprone to corresponding dione. The dione formed undergoes a anodic hydroxylation of the methyl side chain [25]. It is expected
The detection limit and the linear working range of the biosensor were evaluated for determination of deferiprone. There was a linear relationship between current vs. concentration of deferiprone in the range of 0.01 to 10,000 lM (Fig. 1). The limit of detection of the present biosensor was calculated as the lowest quantity of deferiprone required to give a signal to a background (blank) ?3 times SD of blank and found to be 0.01 lM (Fig. 6).
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i / mA
820
Bioprocess Biosyst Eng (2015) 38:815–822
0.600 0.500 0.450 0.400 0.350 0.300 0.250
10000 µM
0.200 0.150 0.100 0.050 0 -0.050 -0.100 -0.150
1000 µM
0.250
0.500
0.750
1.000
1.250
1.500
1.750
E/V
Table 1 Determination of deferiprone by anti-HIV drug biosensor and pharmacopeia method in human spiked urine and serum samples S. no. Spiked samples (mM)
Pharmacopoeia method (mM)
Present method (mM)
Urine
Serum
Urine
Serum
Urine
Serum
1
1.50
1.50
1.52
1.50
1.50
1.51
2
2.50
2.50
2.48
2.51
2.48
2.50
3
3.50
3.50
3.50
3.48
3.47
3.48
4
4.50
4.50
4.20
4.50
4.51
4.50
5
5.50
5.50
5.00
5.48
5.51
5.45
6
6.50
6.50
6.25
6.40
6.50
6.50
7
7.50
7.50
7.40
7.45
7.51
7.48
8
8.50
8.50
8.50
8.48
8.51
8.50
9
9.50
9.50
9.40
9.50
9.50
9.50
Fig. 6 Cyclic voltammograms of anti-HIV replication drug biosensor with various concentrations of deferiprone
10
10.0
10.0
9.58
9.99
10.1
10.0
Optimization of the anti-HIV replication drug biosensor
98.1 ± 1.3, respectively. To test the reproducibility and reliability of the present biosensor deferiprone content in ten spiked serum, urine sample was estimated on single day (within batch) and five times again after storage at -20 °C. The accuracy of the proposed method was determined by spiking serum and urine samples with different concentrations of deferiprone (Table 1). Accuracy of proposed method was 99 %. The effect of some oxidizable substances that might interfere with the response of the biosensor was studied. However, insignificant current due to oxidation of the compounds in either the serum or the urine samples appeared.
The effect of the determination conditions such as the working potential, pH value, response time and temperature on the response of the HRP–AuNPs–CaCO3/silica sol– gel-modified GCE was investigated in detail. The effect of the applied potential on the response current of the HRP–AuNPs–CaCO3/silica sol–gel-modified GCE was studied. The response of the HRP–AuNPs– CaCO3/silica sol–gel-modified GCE as a function of the applied potential was between 0.0 and ?1.5 V s-1. An applied potential of ?0.5 V was selected to give a high detection sensitivity and good signal/noise ratio. The effect of the pH value on the response current of the HRP–AuNPs–CaCO3/silica sol–gel-modified GCE was studied between 5.0 and 8.0 in 0.05 M PBS. The suitable pH with the maximum activity of the immobilized HRP was at pH 7.0. The response time was less than 2 s, which shows a quick response. The faster response was mainly ascribed to the fact that HRP–AuNPs–CaCO3 is providing favorable orientation and conductive pathway to transfer electrons. The electrocatalytic mechanism initiated by HRP makes the process fast i.e. oxidation of drug. Effect of temperature on biosensor was also studied to ensure the optimization. The current response reaches a maximum at approximately 50 °C, and then goes down as the temperature turn higher. To maintain steady with the temperature of human body, 35 °C is selected for this work.
Stability of the anti-HIV replication drug biosensor The stability of the biosensor is investigated and the current response of biosensor is retained about 80 % of its original response after 40 times uninterrupted detection. In addition, the long-term stability is also tested after a month. It is revealed that the current response of the sensor maintains 84 % of the initial current response. This means that AuNPs–CaCO3/silica sol–gel-modified GCE ensures well stability of the biosensor. A comparison of analytic parameters of various nanoparticles based biosensors for detection of deferiprone with the present biosensor is summarized in Table 2.
Conclusions Analytical performance and accuracy The analytic recovery of known amount of added deferiprone was determined by the present biosensor. The mean analytic recoveries of added 5 and 10 mmol L-1 deferiprone (final conc. in reaction mixture) were 98.5 ± 1.7 and
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In summary, we report electrochemical sensing protocol using porous gold-nanoparticle–CaCO3 hybrid material for the detection of anti-HIV replication drug i.e. deferiprone. The proposed sensor matrix is evaluated, which demonstrates a very low detection limit of 0.01 lM. The present
Bioprocess Biosyst Eng (2015) 38:815–822 Table 2 Comparison of the present method with other biosensing methods
Matrix/method
821
Enzyme
Response time
Detection limit (lM)
Linearity (lM)
Stability
References
GC/cyclic voltammetry
–
5s
0.542
51–7,600
–
[25]
Platinum disk/differentialpulse voltammetry
–
–
14.3
70–4,210
–
[27]
Luminescent complex with Tb3 ? ions/ fluorometric
–
–
0.0063
0.0072–14
–
[28]
GC/differential-pulse voltammetry
–
–
2.30
50.7–2,530
–
[29]
Nickel oxyhydroxide/cyclic voltammetry
–
–
19
99–534
4 weeks
[30]
AuNPs–CaCO3/silica sol–gelmodified GCE/cyclic voltammetry
HRP
2s
0.01
0.01–10,000
1 month
Present
investigations revealed that the performance of modified sensor exhibited its suitability for enhanced as well as sensitive determination of deferiprone. Thus, the promising voltammetric approach platform makes an avenue for fast, simple, sensitive and selective detection of deferiprone. Acknowledgments In the present work, one of the author (Jagriti Narang) was supported by SERB, Department of Science and Technology (DST), India. Thanks to all scientists referenced throughout the paper whose valuable work has guided the way through to this research work.
9.
10.
11.
12.
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