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Dendrimer modified 8-channel screen-printed electrochemical array system for impedimetric detection of activated protein C
Sensors and Actuators B 196 (2014) 168–174
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Dendrimer modified 8-channel screen-printed electrochemical array system for impedimetric detection of activated protein C Arzum Erdem a,b,∗ , Gulsah Congur a,b a b
Ege University, Faculty of Pharmacy, Analytical Chemistry Department, 35100 Bornova, Izmir, Turkey Ege University, The Institute of Natural and Applied Sciences, Biotechnology Department, 35100 Bornova, Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 8 November 2013 Received in revised form 17 January 2014 Accepted 28 January 2014 Available online 6 February 2014 Keywords: PAMAM dendrimer 8-channel screen-printed electrochemical array system Electrochemical impedance spectroscopy (EIS) Activated protein C (APC) DNA aptamer
a b s t r a c t The 8-channel screen-printed electrochemical array system (MULTI-SPE8) was developed as an impedimetric aptasensor, and applied for monitoring the interaction between DNA aptamer (DNA-APT) and its cognate protein, human activated protein C (APC), which is the key enzyme of the protein C pathway. Poly(amidoamine) (PAMAM) dendrimer having 16 succinamic acid surface groups (generation 2, G2-PS) was utilized in order to modify the surface of each carbon-based working electrode in MULTI-SPE8, and accordingly, an enhanced sensor response was recorded. Amino linked DNA-APT was then immobilized onto the surface of G2-PS/MULTI-SPE8, and its interaction with APC was explored. After the optimization of the experimental conditions; such as G2-PS, DNA-APT and APC concentration, the selectivity of the electrochemical aptasensor array system was tested in the presence of numerous biomolecules: protein C (PC), thrombin (THR), bovine serum albumin (BSA), factor Va (FVa) and chromogenic substrate (KS) in buffer media, or in the artificial serum: fetal bovine serum (FBS). The dendrimer-modified aptasensor technology based on MULTI-SPE8 has several advantages, such as disposable, fast screening of analyte at eight channels in one batch with low cost per measurement and resulting in a sensitive and selective indirect method for the analysis of APC, with the detection limits of 1.81 g/mL (0.64 pmol in 20 L sample) in buffer solution and 0.02 g/mL (8.22 fmol in 20 L sample) in diluted FBS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Biosensors are analytical tools developed for sensitive and selective detection of analytes: nucleic acids, drugs or proteins that have a key importance in diagnostic area. Although there have been many techniques such as HPLC, GC, mass spectroscopy, QCM and SPR, the electrochemical techniques offer more sensitive, selective, practical, time-saving and fast analyzing data as well as they are appropriate techniques to design miniaturized portable point-ofcare tools. Thus, the area of electrochemical biosensor technology has expanded day by day [1–5]. Screen-printed electrodes have been fabricated as the miniaturized forms of the electrochemical analysis systems. These disposable sensors can be easily modified with different nanomaterials such as carbon nanotubes [6–10], nanoparticles [11,12] and dendrimers [13]. They are appropriate candidates for on-line measurement of numerous biological samples as they require low sample volumes. Since dendrimers were introduced in the literature by two different groups [14,15], the number of studies presenting the development of dendrimer-based biosensors has also considerably increased [13,16–18]. Dendrimers comprise well-defined cavities
∗ Corresponding author. Tel.: +90 232 311 51 31; fax: +90 232 388 52 58. E-mail addresses: [email protected], [email protected] (A. Erdem). 0925-4005/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2014.01.103
[19] and terminal groups [20] in contrast to the linear polymers [14,15]. Moreover, they have a wide range of applications in the bioimaging and drug delivery area due to their biocompatibilities [21–24]. Aptamers comprise a new class of nucleic acids, which are designed for the purpose of specific recognition of biomolecules including nucleic acids, proteins, drugs, and toxins. Since they were identified by in vitro selections [25,26], they have a great potential for biosensor progress due to their excellent properties such as being stable at different physical conditions, and sensitive and selective recognition of the target molecule. Thus, there have been many aptamer-based biosensors (aptasensors) developed in combination with different analytical techniques [1,9,27–29]. Protein C (PC) is a zymogen protein, which is involved in anticoagulant mechanism for the inhibition of overcoagulation. Severe deficiencies of PC are associated with venous thrombosis or neonatal purpura fulminans [30]. Activated protein C (APC) is the key enzyme of the PC pathway. It is a serine protease generated from zymogen protein C [30,31] and has three surface loops named as exosites (active sites); the 37-loop [30,32], the 60-loop, and the 70–80 loop that play an important role for inactivation of factors Va and VIIIa [32]. Another exosite determines the specificity of APC in the interaction with protease activated receptor-1 (PAR-1), which is formed by acidic residues of the 162 helix and located on the left side of the active site towards the back of the molecule [33].
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APC is generated by two steps: first, PC binds to the endothelial cell PC receptor (EPCR); second, the thrombin/thrombomodulin complexes are formed by proteolytic activation of PC. Cytoprotective, anti-inflammatory and antiapoptotic properties, which are related to protection of endothelial barrier function, are known [30]. APC detection has been receiving great attention due to its importance in the PC pathway. It has been reported that resistance to APC can cause diseases that affect quality of life, such as microvascular thrombosis in septicemia. In the recent years, recombinant human APC has been used as prospective therapeutic intervention for the treatment of sepsis [34,35]. Thus, the development of sensitive and selective detection platforms has become an urgent need for the recognition of APC and its monitoring by a fast, cost-effective and reliable approach. To our best knowledge, no report has been published yet on impedimetric detection of human activated protein C (APC), which is the key enzyme of the protein C pathway. In the present study, an impedimetric aptasensor based on poly-amidoamine (PAMAM) dendrimer (generation 2, G2-PS) modified 8-channel screen-printed electrochemical array system (MULTI-SPE8) was developed for the indirect analysis of human APC. The surface confined interaction between amino-linked DNA aptamer (DNA-APT) and its target protein, APC was explored at the surface of G2-PS modified MULTI-SPE8. The impedimetric responses were recorded before and after each immobilization and interaction step, and consequently, the changes in the charge transfer resistance (Rct ) value were evaluated. The experimental parameters, such as G2PS, APT and APC concentration, were optimized. The selectivity of the aptamer-based impedimetric array system was explored by means of APT interaction with different biomolecules: protein C (PC), thrombin (THR), bovine serum albumin (BSA), factor Va (FVa) and chromogenic substrate (KS) in the buffer, or in diluted fetal bovine serum (FBS). 2. Experimental 2.1. Apparatus Electrochemical measurements were performed by using electrochemical impedance spectroscopy (EIS) by dropping 20 L of the required solution in order to cover each of the electrode surfaces. All experimental measurements were carried out using AUTOLAB–PGSTAT 302 electrochemical analysis system supplied with a FRA 2.0 module for impedance measurements, and GPES 4.9 software package (Eco Chemie, The Netherlands). All measurements were carried out in the Faraday cage (Eco Chemie, The Netherlands). The 8-channel screen-printed electrochemical array (MULTI-SPE8) measured 33 mm × 78 mm × 1 mm system (length × width × height). Each electrode at the MULTI-SPE8 comprised three main parts: a carbon-based working electrode, a carbon counter electrode and a silver reference electrode. The carbon working electrode surface was 2.56 mm in diameter. The disposable MULTI-SPE8 was commercially purchased from DropSens (Oviedo-Asturias, Spain), and further details about them can be found at the following web site: www.dropsens.com. A connector (ref. DRP-CAST8X) was used to connect the electrodes and MUX.MULTI4 module (Eco Chemie, The Netherlands) in the electrochemical analyzer AUTOLAB–PGSTAT 302-FRA 2.0.
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factor Va (FVa), chromogenic substrate (KS), bovine serum albumin (BSA), and thrombin (THR) were purchased from Sigma. APC was purchased from Haemtech. The 5 -amino-linked single-stranded DNA aptamer (DNA-APT), which was specific for APC [37,38], and also random DNA aptamer (Random DNA-APT) and the short synthetic oligonucleotide (random DNA) were purchased from TIBMOLBIOL (Berlin, Germany). DNA-APT 5 -NH2 -C6 -GCC TCC TAA CTG AGC TGT ACT CGA CTT ATC CCG GAT GGG GCT CTT AGG AGG C-3 . Random DNA-APT 5 -NH2 -C6 -GGT TGG TGT GGT TGG AAA AAA AAA AAA AAA AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 . Random DNA 5 -NH2 -C6 -CAAA GAA GTG GCA GGA AGA GTC GAA GGT CTT GTT GTC ATT GCT GCA CAC CTT-3 . 2.2.1. The preparation of G2-PS The diluted G2-PS solutions were prepared by using frozen stock solution of G2-PS (106 g/mL) in phosphate buffer solution containing 20 mM NaCl (PBS, pH 7.4). 2.2.2. The preparation of DNA-APT, random DNA-APT and random DNA The DNA-APTs and random DNA stock solutions (500 g/mL) were prepared in ultrapure water and kept frozen. More diluted DNA solutions were prepared in Tris–buffer solution (TBS, 10 mM Tris–HCl, 150 mM NaCl, 1 mM CaCl2 , 1 mM MgCl2 , pH 7.40) containing 1 mg/mL BSA [37]. 2.2.3. The preparation of solutions of APC and other proteins The stock solutions of proteins were prepared with ultrapure water and kept frozen at −20 ◦ C. Their diluted solutions were then prepared in TBS (pH 7.40). Other chemicals were of analytical reagent grade and were supplied by Sigma and Merck. Ultrapure and deionized water was used in all solutions. 2.3. G2-PS modification onto the electrode surface at MULTI-SPE8 Each carbon electrode at MULTI-SPE8 was pretreated by applying a potential of +0.90 V for 60 s in 0.50 M acetate buffer solution containing 20 mM NaCl (ABS, pH 4.80). Each disposable pretreated electrode was then covered with 20 L droplet containing the required amount of G2-PS in PBS (pH 7.4) for dendrimer modification during 1 h. Each of the electrodes was then rinsed with PBS (pH 7.40) for 5 s to remove the non-specific binding of G2-PS. 2.4. Preparation of DNA-APT immobilized G2-PS/MULTI-SPE8 and interaction with APC Each G2-PS modified electrode of the multiscreen-printed array system was covered with 20 L droplet containing the required amount of APT in TBS (pH 7.40) containing 1 mg/mL BSA for 1 h. Each of the electrode was then rinsed with TBS (pH 7.40) for 5 s to remove the unbound DNA-APT. Then, the electrodes were covered with a 20 L droplet containing the required amount of APC in TBS (pH 7.40) for performing surface interaction between APT and APC for 1 h. Each of the electrode was then rinsed with TBS (pH 7.40) for 5 s to remove the unbound APC.
2.2. Chemicals 2.5. Impedance measurements Second-generation poly(amidoamine) (PAMAM) dendrimer with a 1,4-diaminobutane core (G2-PS), which has 16 succinamic acid surface groups [36], fetal bovine serum (FBS), protein C (PC),
The impedimetric measurements were performed before/after each modification/immobilization step. The interaction between
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Fig. 1. Representative Nyquist diagrams obtained by using EIS that was performed in 2.5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) containing 0.1 M KCl. Unmodified (bare) MULTI-SPE8 (a), 1 g/mL (b), 2 g/mL (c), 5 g/mL (d) G2-PS modified MULTI-SPE8. Inset was the equivalent electrical model used to fit the impedance data, the parameters of which are listed in the text; RS is the solution resistance. The constant phase element Q is then related to the double layer capacitance at the electrode–electrolyte interface. Rct is related to the charge transfer resistance at the electrode–electrolyte interface. The constant phase element W is the Warburg impedance due to mass transfer to the electrode surface.
the aptamer and its cognate protein APC was evaluated impedimetrically by means of the changes in Rct. values. The EIS measurements were done by using 2.5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture as a redox probe prepared in 0.1 M KCl. The impedance was measured in the frequency range of 100 mHz to 100 kHz at the potential of +0.23 V with a sinusoidal signal of 10 mV. The frequency interval was divided into 98 logarithmically equidistant measure points. The respective semicircle diameter corresponds to the charge-transfer resistance, Rct , the values of which were calculated using the fitting programme AUTOLAB 302 (FRA, version 4.9 Eco Chemie, The Netherlands). 3. Results and discussion The modification of each surface of the MULTI-SPE8 with G2-PS, and the interaction of DNA-APT with APC at the surface of G2-PS modified MULTI-SPE8 surface are presented in Scheme 1. Each electrode surface of the array system was first modified using G2-PS and the changes in the charge transfer resistance (Rct ) values obtained by EIS were evaluated. As shown in Fig. 1, the highest Rct value was obtained after surface modification with 1 g/mL G2-PS (Fig. 1b) in contrast to that of the unmodified (bare) electrodes (Fig. 1a, recorded as 315 ± 76 , n = 8). This increase claimed the coverage of MULTI-SPE8 with G2-PS and it may be explained by the repulsive electrostatic interaction between the negatively charged G2-PS [30] and ferricyanide ions [39–41]. After eight repetitive measurements, the average Rct value was estimated as 1496.5 ± 193.4 with the relative standard deviation % (RSD%) of 12.9%. Thus, the concentration of G2-PS of 1 g/mL was chosen for further studies in this work. The apparent fractional coverage ( IS R ) of G2-PS modified MULTI-SPE8 was also calculated [42]. The information is given in Table S1 related to the calculated IS R for the surface coverage of MULTI-SPE8 with G2-PS in different dendrimer concentrations from 1 to 5 g/mL. Fig. 2 shows the effect of DNA-APT concentration on the response of dendrimer modified array system. The Rct value increased with the increase in the concentration of DNA-APT from 50 to 100 g/mL (Fig. 2d), and then it began to decrease. The average Rct value was calculated as 5367 ± 904.9 with the RSD % of 16.7% (n = 16; i.e., the total number of electrodes in two different
Fig. 2. The impedimetric investigation presenting the effect of DNA-APT concentration upon to the sensor response. The representative Nyquist diagrams obtained by using unmodified MULTI-SPE8 (a), G2-PS modified MULTI-SPE8 (b), after immobilization of 50 (c), 100 (d), 150 (e), 200 g/mL (f) DNA-APT immobilization onto the surface of G2-PS/MULTI-SPE8. Other conditions were same as in Fig. 1. Inset was the equivalent electrical model used to fit the impedance data.
batches of MULTI-SPE8), which was 2.6 times higher than the one obtained by G2-PS modified SPE. The negatively charged phosphate groups of DNA-APT prevented the anionic [Fe(CN)6 ]3−/4− ions from reaching the electrode surface. The charge repulsion between negatively charged DNA-APT and redox couple occurred due to limited electron transfer at the G2-PS/MULTI-SPE8 surface, similar to the previous reports in the literature [43–46]. In addition to the impedimetric studies, the electrochemical behaviour of MULTI-SPE8 was explored by using cyclic voltammetry before and after the modification of 1 g/mL of G2-PS at MULTI-SPE8, and also after the immobilization of 100 g/mL DNA-APT at G2PS modified MULTI-SPE8 (Fig. S1). After G2-PS modification onto the MULTI-SPE8 surface, decrease % in anodic (Ia ) and cathodic (Ic ) currents of Fe(CN)6 3−/4− was found to be 20% and 18% respectively (shown in Table S2). However a decrease in Ia was obtained after DNA-APT immobilization and a significant decrease in Ic was recorded (Fig. S1B-b to c, i.e. about 47%). Moreover, the peak-topeak separation (Ep ) of unmodified MULTI-SPE8 indicated that the electrochemical behaviour of unmodified MULTI-SPE8 was quasi-reversible, whereas it was irreversible after modification of G2-PS at MULTI-SPE8 and immobilization of DNA-APT onto the surface of G2-PS/MULTI-SPE8 due to increase in Ep value obtained after each immobilization/modification step (Table S2), similar to earlier work presented by Qian et al. [46]. In next step, the effect of APC concentration on the aptasensor response was examined and the surface confined interaction between DNA-APT and APC was consequently performed at G2-PS modified MULTI-SPE8 (Fig. 3). The Rct value increased while APC concentration increased due to the fact that aptamers interacted with more APC molecules, which had negative structure in the redox probe (pH 5.93) [47]. A linear graph based on Rct values was obtained in the APC concentration range of 0–7.5 g/mL (shown in Fig. 3B). The detection limit (DL) was calculated using the method described by Miller and Miller [48] with coefficient of determination (R2 ) = 0.9663, and it was found to be 1.81 g/mL (0.64 pmol in 20 L sample). After surface-confined interaction of DNA-APT with 7.5 g/mL APC, the Rct was recorded as 6615.3 ± 411.2 (RSD % = 6.2%, n = 4). Since the most reproducible and the highest Rct was obtained at 7.5 g/mL APC, it was chosen as the optimum APC concentration for further experiments in our study. The selectivity of the APC selective DNA-APT modified MULTISPE8 was then examined against other biomolecules, which may present any possible interferences. The impedimetric results are shown in Fig. 4. The interaction was carried out in the presence of
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Scheme 1. Schematic representation of impedimetric detection of APC at DNA-APT immobilized G2-PS modified MULTI-SPE8 surface.
100 g/mL DNA-APT and 7.5 g/mL APC, or other biomolecules: PC, BSA, THR, FVa and KS in TBS (pH 7.4) at the surface of G2-PS/MULTISPE8. The Rct value which was recorded after aptamer interaction with APC, PC or BSA increased respectively about 28%, 9% and 16%,
Fig. 3. (A) Nyquist diagrams obtained after surface confined interaction between 100 g/mL DNA-APT and its cognate protein APC in the different concentrations of APC by using MULTI-SPE8. DNA-APT immobilized G2-PS/MULTI-SPE8 (a), after interaction of 2.5 (b), 5 (c), 7.5 (d), 10 (e), 12.5 (f), and 15 g/mL APC (g) with DNAAPT at the surface of G2-PS/MULTI-SPE8 surface. Other conditions were same as in Figs. 1 and 2. (B) Calibration graph representing the Rct values obtained after interaction between 100 g/mL DNA-APT and APC in the concentration range of 0–7.5 g/mL. Inset was the equivalent electrical model used to fit the impedance data.
whereas 21%, 6% and 54% decrease in Rct value were obtained after aptamer interaction with THR, FVa or KS, respectively. Compared to the average Rct value recorded by DNA-APT immobilized G2PS/MULTI-SPE8 surface, the highest increase ratio was found for specific interaction between DNA-APT and its cognate protein, APC.
Fig. 4. Nyquist diagrams (A), histograms (B) representing the Rct values recorded at 100 g/mL DNA-APT immobilized G2-PS/MULTI-SPE8 (a), after interaction with 7.5 g/mL APC (b), PC (c), BSA (d), THR (e), FVa (f) and KS (g) in TBS (pH 7.4) solution. Inset was the equivalent electrical model used to fit the impedance data.
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Fig. 5. (A) Nyquist diagrams, (B) histograms obtained by bare MULTI-SPE8 (a), 1 g/mL G2-PS modified MULTI-SPE8 (b), 100 g/mL DNA-APT (c), random APT (d), random DNA (e) immobilized G2-PS/MULTI-SPE8, after interaction between 7.5 g/mL APC and 100 g/mL DNA-APT (c ), random APT (d ) or random DNA (e ) in TBS (pH 7.4). Inset was the equivalent electrical model used to fit the impedance data.
Thus, it could be concluded that this impedimetric aptasensor based on 8-channel screen-printed electrochemical array system (MULTISPE8) presented a selective behaviour to the target analyte APC in comparison to many other biomolecules. In another study, the selectivity of the aptasensor was also investigated by measuring the changes in the Rct value after the interaction of APC with a control DNA aptamer (random APT) or a control DNA sequence (random DNA) (Fig. 5) in comparison to APC selective DNA-APT. After the immobilization of 100 g/mL DNA-APT (Fig. 5A and B-c), or random APT (Fig. 5A and B-d), or random DNA (Fig. 5A and B-e) onto the electrode surface, the average Rct value was recorded respectively as 5858 ± 566 (RSD % = 9.6%, n = 3), 2548 ± 119 (RSD% = 4.7%, n = 3), and 2695 ± 275 (RSD% = 10.2%, n = 3). In the case of APC interaction with DNA-APT, or random APT, or random DNA, an increase was observed in the average Rct value as 29.7% (Fig. 5A and B-c ), 10% (Fig. 5A and Bd ) and a decrease in the average Rct value as 1% (Fig. 5A and B-e ) respectively. These results claimed that there was a specific binding between the DNA-APT and its cognate protein APC in comparison to the results obtained using the random APT, or the random DNA sequence. Although biosensors have mostly presented promising properties in an aqueous buffer for monitoring numerous biomolecular interactions, the detection may be more difficult in a serum media than that in an aqueous buffer due to the fact that serum contains several interference factors, such as a large number of proteins and other biomolecules [49]. Thus, impedimetric APC analysis was
Fig. 6. Effect of APC concentration upon the sensor response before and after surface confined interaction process between 100 g/mL APT and APC in different concentrations of APC from 1 to 2.5 g/mL in 1:5 FBS: TBS diluted solution. Nyquist diagrams (A). Inset was the equivalent electrical model used to fit the impedance data. Histograms (B) of Rct values obtained from before (a) and after interaction between APT and 1 (b), 1.5 (c), 2 (d), 2.5 (e) g/mL APC by using EIS, that was performed in 2.5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) containing 0.1 M KCl. Inset was calibration graph representing the Rct values obtained after interaction between 100 g/mL DNA-APT and APC at 1–2.5 g/mL concentration.
investigated in an artificial serum medium, e.g., foetal bovine serum (FBS), by using an aptamer modified G2-PS/MULTI-SPE8 (Fig. S2). The interaction between 100 g/mL DNA-APT and 7.5 g/mL APC was performed in the non-diluted FBS solution, and also in the diluted FBS solutions prepared using TBS in different ratios of FBS:TBS, such as 1:1, 1:5 and 1:10. The Rct value was obtained as 6610 ± 594 (RSD % = 9%, n = 3) in the presence of APT interaction with APC in 1:5 FBS:TBS diluted solution (Fig. S2-d), which was found to be similar to the Rct value obtained in TBS (Fig. S2-b). It was concluded that the surface confined interaction process between aptamer and APC was successfully achieved in 1:5 FBS:TBS diluted solution. In addition, the effect of APC concentration on the aptasensor response was investigated in 1:5 FBS:TBS diluted solution (Fig. 6). A linear graph was obtained in the APC concentration range between 1 and 2.5 g/mL. Consequently, the DL was calculated according to the method described by Miller and Miller [48] with the R2 = 0.9999, and it was found to be 0.02 g/mL (8.22 fmol in 20 L sample). 4. Conclusion Screen-printed biosensors (SPBs) are the one of the appropriate candidates for on-line measurement of biological samples due to the fact that sensitive and selective results in a low sample volume could be obtained in a short time using SPBs. Different types
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of screen-printed electrochemical array system are commercially available and they have the proportional properties to design the chip technologies that could detect various types of bioanalytes fastly, selectively and sensitively in the future. No report has been introduced yet on the impedimetric detection of human activated protein C (APC) in the literature. Herein, an impedimetric aptasensor based on poly-amidoamine (PAMAM) dendrimer (generation 2, G2-PS) modified 8-channel screenprinted electrochemical array system (MULTI-SPE8) was developed for the first time and applied for the detection of human activated protein C (APC), which is the key enzyme of the protein C pathway. Under optimum experimental conditions, it was presented in our study that APC could be detected in both buffer and a diluted serum medium (FBS) with the low detection limits respectively as 1.81 g/mL (0.64 pmol in 20 L sample) and 0.02 g/mL (8.22 fmol in 20 L sample). In comparison to earlier reports related to aptasensor technology developed using different materials such as dendrimers, carbon nanotubes, magnetic particles [9,29,45,50], our impedimetric aptasensor array system presented an effective protocol requiring a lower sample volume for sensitive and selective detection of the analyte in buffer, and also in a serum medium, such as FBS. Moreover, this aptasensor array system was not only a single-use, time-saving system that required less chemicals compared to other ELISA-based conventional methods reported in the literature [38,51,52], but it could also determine APC selectively compared to the other biomolecules such as PC, BSA, THR, FVa and chromogenic substrate. As a conclusion, APT immobilized dendrimer modified 8channel screen-printed electrochemical array system (MULTISPE8) presented a sensitive and selective protocol for APC sensing in a short time even when the interaction process occurred in a diluted serum medium (i.e., FBS). The aptamer-based dendrimer modified array system will also bring a new perspective to design novel chip technologies for the detection of other (bio)molecules, such as nucleic acids, drugs or proteins. Acknowledgements AE acknowledges the financial support from Turkish Scientific and Technological Research Council (TUBITAK; Project no. 111T073), and she also expresses her gratitude to the Turkish Academy of Sciences (TUBA) as an associate member for its partial support. GC acknowledges a master project scholarship through project (TUBITAK Project no. 111T073). The authors also thank Prof. Günter Mayer for his valuable scientific comments during this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2014.01.103. References [1] E. Palecek, M. Bartosik, Electrochemistry of nucleic acids, Chemical Reviews 112 (2012) 3427–3481. [2] J. Wang, Stripping-based electrochemical metal sensors for environmental monitoring, in: S. Alegret, A. Merkoc¸i (Eds.), Electrochemical sensor analysis, Elsevier, Oxford, UK, 2007, p. 131. [3] S. Qu, J. Wang, J. Kong, P. Yang, G. Chen, Magnetic loading of carbon nanotube/nano-Fe3 O4 composite for electrochemical sensing, Talanta 71 (2007) 1096–1102. [4] I. Willner, M. Rosen, Y. Eichen, Characterization of the hydrogenation process of allyl alcohol at a Pt electrode using a double galvanostatic pulse technique, Journal of Electrochemical Society 138 (1991) 434–439. [5] A. Erdem, Nanomaterial based electrochemical DNA sensing strategies, Talanta 74 (2007) 318–325. [6] J. Wang, Carbon-nanotube based electrochemical biosensors, Electroanalysis 17 (2005) 7–14.
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Biographies
Arzum Erdem is a professor at the Analytical Chemistry Department in the Faculty of Pharmacy of Ege University in Turkey. She received her PhD in analytical chemistry from Ege University in 2000. Dr. Erdem was awarded by the Turkish Academy of Sciences (TUBA) as the one of highly skilled young scientists selected in 2001, and she also received Junior Science Award 2006 given by The Scientific and Technological Research Council of Turkey (TUBITAK). She has initiated many international collaborative research on development and applications of electrochemical (bio)sensors based on drug, enzyme and nucleic acids. Her recent research is centred on the development of novel transducers and chemical and biological recognition systems by using different nanomaterials (e.g., magnetic nanoparticles, carbon nanotubes, gold and silver nanoparticles, nanowires, etc.) designed for electrochemical sensing of nucleic acid (DNA, RNA) hybridization, and also the specific interactions between drug and DNA, or protein and DNA, aptamer–protein and also the development of integrated analytical systems for environmental, industry and biomedical monitoring. Gulsah Congur has a B.Sc. in bioengineering from Faculty of Engineering, Ege University (Izmir, Turkey), and an M.Sc. in biotechnology from the Institute of Natural and Applied Sciences at Ege University. She is still continuing her Ph.D. in biotechnology from Natural and Applied Sciences, Ege University. Her current research is on the development of electrochemical biosensors for the purpose of monitoring of (bio)molecule–DNA interaction, detection of genetic disease by nucleic acid hybridization, investigation of protein–aptamer interaction.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Dendrimer modified 8-channel screen-printed electrochemical array system for impedimetric detection of activated protein C Arzum Erdem a,b,∗ , Gulsah Congur a,b a b
Ege University, Faculty of Pharmacy, Analytical Chemistry Department, 35100 Bornova, Izmir, Turkey Ege University, The Institute of Natural and Applied Sciences, Biotechnology Department, 35100 Bornova, Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 8 November 2013 Received in revised form 17 January 2014 Accepted 28 January 2014 Available online 6 February 2014 Keywords: PAMAM dendrimer 8-channel screen-printed electrochemical array system Electrochemical impedance spectroscopy (EIS) Activated protein C (APC) DNA aptamer
a b s t r a c t The 8-channel screen-printed electrochemical array system (MULTI-SPE8) was developed as an impedimetric aptasensor, and applied for monitoring the interaction between DNA aptamer (DNA-APT) and its cognate protein, human activated protein C (APC), which is the key enzyme of the protein C pathway. Poly(amidoamine) (PAMAM) dendrimer having 16 succinamic acid surface groups (generation 2, G2-PS) was utilized in order to modify the surface of each carbon-based working electrode in MULTI-SPE8, and accordingly, an enhanced sensor response was recorded. Amino linked DNA-APT was then immobilized onto the surface of G2-PS/MULTI-SPE8, and its interaction with APC was explored. After the optimization of the experimental conditions; such as G2-PS, DNA-APT and APC concentration, the selectivity of the electrochemical aptasensor array system was tested in the presence of numerous biomolecules: protein C (PC), thrombin (THR), bovine serum albumin (BSA), factor Va (FVa) and chromogenic substrate (KS) in buffer media, or in the artificial serum: fetal bovine serum (FBS). The dendrimer-modified aptasensor technology based on MULTI-SPE8 has several advantages, such as disposable, fast screening of analyte at eight channels in one batch with low cost per measurement and resulting in a sensitive and selective indirect method for the analysis of APC, with the detection limits of 1.81 g/mL (0.64 pmol in 20 L sample) in buffer solution and 0.02 g/mL (8.22 fmol in 20 L sample) in diluted FBS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Biosensors are analytical tools developed for sensitive and selective detection of analytes: nucleic acids, drugs or proteins that have a key importance in diagnostic area. Although there have been many techniques such as HPLC, GC, mass spectroscopy, QCM and SPR, the electrochemical techniques offer more sensitive, selective, practical, time-saving and fast analyzing data as well as they are appropriate techniques to design miniaturized portable point-ofcare tools. Thus, the area of electrochemical biosensor technology has expanded day by day [1–5]. Screen-printed electrodes have been fabricated as the miniaturized forms of the electrochemical analysis systems. These disposable sensors can be easily modified with different nanomaterials such as carbon nanotubes [6–10], nanoparticles [11,12] and dendrimers [13]. They are appropriate candidates for on-line measurement of numerous biological samples as they require low sample volumes. Since dendrimers were introduced in the literature by two different groups [14,15], the number of studies presenting the development of dendrimer-based biosensors has also considerably increased [13,16–18]. Dendrimers comprise well-defined cavities
∗ Corresponding author. Tel.: +90 232 311 51 31; fax: +90 232 388 52 58. E-mail addresses: [email protected], [email protected] (A. Erdem). 0925-4005/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2014.01.103
[19] and terminal groups [20] in contrast to the linear polymers [14,15]. Moreover, they have a wide range of applications in the bioimaging and drug delivery area due to their biocompatibilities [21–24]. Aptamers comprise a new class of nucleic acids, which are designed for the purpose of specific recognition of biomolecules including nucleic acids, proteins, drugs, and toxins. Since they were identified by in vitro selections [25,26], they have a great potential for biosensor progress due to their excellent properties such as being stable at different physical conditions, and sensitive and selective recognition of the target molecule. Thus, there have been many aptamer-based biosensors (aptasensors) developed in combination with different analytical techniques [1,9,27–29]. Protein C (PC) is a zymogen protein, which is involved in anticoagulant mechanism for the inhibition of overcoagulation. Severe deficiencies of PC are associated with venous thrombosis or neonatal purpura fulminans [30]. Activated protein C (APC) is the key enzyme of the PC pathway. It is a serine protease generated from zymogen protein C [30,31] and has three surface loops named as exosites (active sites); the 37-loop [30,32], the 60-loop, and the 70–80 loop that play an important role for inactivation of factors Va and VIIIa [32]. Another exosite determines the specificity of APC in the interaction with protease activated receptor-1 (PAR-1), which is formed by acidic residues of the 162 helix and located on the left side of the active site towards the back of the molecule [33].
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APC is generated by two steps: first, PC binds to the endothelial cell PC receptor (EPCR); second, the thrombin/thrombomodulin complexes are formed by proteolytic activation of PC. Cytoprotective, anti-inflammatory and antiapoptotic properties, which are related to protection of endothelial barrier function, are known [30]. APC detection has been receiving great attention due to its importance in the PC pathway. It has been reported that resistance to APC can cause diseases that affect quality of life, such as microvascular thrombosis in septicemia. In the recent years, recombinant human APC has been used as prospective therapeutic intervention for the treatment of sepsis [34,35]. Thus, the development of sensitive and selective detection platforms has become an urgent need for the recognition of APC and its monitoring by a fast, cost-effective and reliable approach. To our best knowledge, no report has been published yet on impedimetric detection of human activated protein C (APC), which is the key enzyme of the protein C pathway. In the present study, an impedimetric aptasensor based on poly-amidoamine (PAMAM) dendrimer (generation 2, G2-PS) modified 8-channel screen-printed electrochemical array system (MULTI-SPE8) was developed for the indirect analysis of human APC. The surface confined interaction between amino-linked DNA aptamer (DNA-APT) and its target protein, APC was explored at the surface of G2-PS modified MULTI-SPE8. The impedimetric responses were recorded before and after each immobilization and interaction step, and consequently, the changes in the charge transfer resistance (Rct ) value were evaluated. The experimental parameters, such as G2PS, APT and APC concentration, were optimized. The selectivity of the aptamer-based impedimetric array system was explored by means of APT interaction with different biomolecules: protein C (PC), thrombin (THR), bovine serum albumin (BSA), factor Va (FVa) and chromogenic substrate (KS) in the buffer, or in diluted fetal bovine serum (FBS). 2. Experimental 2.1. Apparatus Electrochemical measurements were performed by using electrochemical impedance spectroscopy (EIS) by dropping 20 L of the required solution in order to cover each of the electrode surfaces. All experimental measurements were carried out using AUTOLAB–PGSTAT 302 electrochemical analysis system supplied with a FRA 2.0 module for impedance measurements, and GPES 4.9 software package (Eco Chemie, The Netherlands). All measurements were carried out in the Faraday cage (Eco Chemie, The Netherlands). The 8-channel screen-printed electrochemical array (MULTI-SPE8) measured 33 mm × 78 mm × 1 mm system (length × width × height). Each electrode at the MULTI-SPE8 comprised three main parts: a carbon-based working electrode, a carbon counter electrode and a silver reference electrode. The carbon working electrode surface was 2.56 mm in diameter. The disposable MULTI-SPE8 was commercially purchased from DropSens (Oviedo-Asturias, Spain), and further details about them can be found at the following web site: www.dropsens.com. A connector (ref. DRP-CAST8X) was used to connect the electrodes and MUX.MULTI4 module (Eco Chemie, The Netherlands) in the electrochemical analyzer AUTOLAB–PGSTAT 302-FRA 2.0.
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factor Va (FVa), chromogenic substrate (KS), bovine serum albumin (BSA), and thrombin (THR) were purchased from Sigma. APC was purchased from Haemtech. The 5 -amino-linked single-stranded DNA aptamer (DNA-APT), which was specific for APC [37,38], and also random DNA aptamer (Random DNA-APT) and the short synthetic oligonucleotide (random DNA) were purchased from TIBMOLBIOL (Berlin, Germany). DNA-APT 5 -NH2 -C6 -GCC TCC TAA CTG AGC TGT ACT CGA CTT ATC CCG GAT GGG GCT CTT AGG AGG C-3 . Random DNA-APT 5 -NH2 -C6 -GGT TGG TGT GGT TGG AAA AAA AAA AAA AAA AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 . Random DNA 5 -NH2 -C6 -CAAA GAA GTG GCA GGA AGA GTC GAA GGT CTT GTT GTC ATT GCT GCA CAC CTT-3 . 2.2.1. The preparation of G2-PS The diluted G2-PS solutions were prepared by using frozen stock solution of G2-PS (106 g/mL) in phosphate buffer solution containing 20 mM NaCl (PBS, pH 7.4). 2.2.2. The preparation of DNA-APT, random DNA-APT and random DNA The DNA-APTs and random DNA stock solutions (500 g/mL) were prepared in ultrapure water and kept frozen. More diluted DNA solutions were prepared in Tris–buffer solution (TBS, 10 mM Tris–HCl, 150 mM NaCl, 1 mM CaCl2 , 1 mM MgCl2 , pH 7.40) containing 1 mg/mL BSA [37]. 2.2.3. The preparation of solutions of APC and other proteins The stock solutions of proteins were prepared with ultrapure water and kept frozen at −20 ◦ C. Their diluted solutions were then prepared in TBS (pH 7.40). Other chemicals were of analytical reagent grade and were supplied by Sigma and Merck. Ultrapure and deionized water was used in all solutions. 2.3. G2-PS modification onto the electrode surface at MULTI-SPE8 Each carbon electrode at MULTI-SPE8 was pretreated by applying a potential of +0.90 V for 60 s in 0.50 M acetate buffer solution containing 20 mM NaCl (ABS, pH 4.80). Each disposable pretreated electrode was then covered with 20 L droplet containing the required amount of G2-PS in PBS (pH 7.4) for dendrimer modification during 1 h. Each of the electrodes was then rinsed with PBS (pH 7.40) for 5 s to remove the non-specific binding of G2-PS. 2.4. Preparation of DNA-APT immobilized G2-PS/MULTI-SPE8 and interaction with APC Each G2-PS modified electrode of the multiscreen-printed array system was covered with 20 L droplet containing the required amount of APT in TBS (pH 7.40) containing 1 mg/mL BSA for 1 h. Each of the electrode was then rinsed with TBS (pH 7.40) for 5 s to remove the unbound DNA-APT. Then, the electrodes were covered with a 20 L droplet containing the required amount of APC in TBS (pH 7.40) for performing surface interaction between APT and APC for 1 h. Each of the electrode was then rinsed with TBS (pH 7.40) for 5 s to remove the unbound APC.
2.2. Chemicals 2.5. Impedance measurements Second-generation poly(amidoamine) (PAMAM) dendrimer with a 1,4-diaminobutane core (G2-PS), which has 16 succinamic acid surface groups [36], fetal bovine serum (FBS), protein C (PC),
The impedimetric measurements were performed before/after each modification/immobilization step. The interaction between
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Fig. 1. Representative Nyquist diagrams obtained by using EIS that was performed in 2.5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) containing 0.1 M KCl. Unmodified (bare) MULTI-SPE8 (a), 1 g/mL (b), 2 g/mL (c), 5 g/mL (d) G2-PS modified MULTI-SPE8. Inset was the equivalent electrical model used to fit the impedance data, the parameters of which are listed in the text; RS is the solution resistance. The constant phase element Q is then related to the double layer capacitance at the electrode–electrolyte interface. Rct is related to the charge transfer resistance at the electrode–electrolyte interface. The constant phase element W is the Warburg impedance due to mass transfer to the electrode surface.
the aptamer and its cognate protein APC was evaluated impedimetrically by means of the changes in Rct. values. The EIS measurements were done by using 2.5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture as a redox probe prepared in 0.1 M KCl. The impedance was measured in the frequency range of 100 mHz to 100 kHz at the potential of +0.23 V with a sinusoidal signal of 10 mV. The frequency interval was divided into 98 logarithmically equidistant measure points. The respective semicircle diameter corresponds to the charge-transfer resistance, Rct , the values of which were calculated using the fitting programme AUTOLAB 302 (FRA, version 4.9 Eco Chemie, The Netherlands). 3. Results and discussion The modification of each surface of the MULTI-SPE8 with G2-PS, and the interaction of DNA-APT with APC at the surface of G2-PS modified MULTI-SPE8 surface are presented in Scheme 1. Each electrode surface of the array system was first modified using G2-PS and the changes in the charge transfer resistance (Rct ) values obtained by EIS were evaluated. As shown in Fig. 1, the highest Rct value was obtained after surface modification with 1 g/mL G2-PS (Fig. 1b) in contrast to that of the unmodified (bare) electrodes (Fig. 1a, recorded as 315 ± 76 , n = 8). This increase claimed the coverage of MULTI-SPE8 with G2-PS and it may be explained by the repulsive electrostatic interaction between the negatively charged G2-PS [30] and ferricyanide ions [39–41]. After eight repetitive measurements, the average Rct value was estimated as 1496.5 ± 193.4 with the relative standard deviation % (RSD%) of 12.9%. Thus, the concentration of G2-PS of 1 g/mL was chosen for further studies in this work. The apparent fractional coverage ( IS R ) of G2-PS modified MULTI-SPE8 was also calculated [42]. The information is given in Table S1 related to the calculated IS R for the surface coverage of MULTI-SPE8 with G2-PS in different dendrimer concentrations from 1 to 5 g/mL. Fig. 2 shows the effect of DNA-APT concentration on the response of dendrimer modified array system. The Rct value increased with the increase in the concentration of DNA-APT from 50 to 100 g/mL (Fig. 2d), and then it began to decrease. The average Rct value was calculated as 5367 ± 904.9 with the RSD % of 16.7% (n = 16; i.e., the total number of electrodes in two different
Fig. 2. The impedimetric investigation presenting the effect of DNA-APT concentration upon to the sensor response. The representative Nyquist diagrams obtained by using unmodified MULTI-SPE8 (a), G2-PS modified MULTI-SPE8 (b), after immobilization of 50 (c), 100 (d), 150 (e), 200 g/mL (f) DNA-APT immobilization onto the surface of G2-PS/MULTI-SPE8. Other conditions were same as in Fig. 1. Inset was the equivalent electrical model used to fit the impedance data.
batches of MULTI-SPE8), which was 2.6 times higher than the one obtained by G2-PS modified SPE. The negatively charged phosphate groups of DNA-APT prevented the anionic [Fe(CN)6 ]3−/4− ions from reaching the electrode surface. The charge repulsion between negatively charged DNA-APT and redox couple occurred due to limited electron transfer at the G2-PS/MULTI-SPE8 surface, similar to the previous reports in the literature [43–46]. In addition to the impedimetric studies, the electrochemical behaviour of MULTI-SPE8 was explored by using cyclic voltammetry before and after the modification of 1 g/mL of G2-PS at MULTI-SPE8, and also after the immobilization of 100 g/mL DNA-APT at G2PS modified MULTI-SPE8 (Fig. S1). After G2-PS modification onto the MULTI-SPE8 surface, decrease % in anodic (Ia ) and cathodic (Ic ) currents of Fe(CN)6 3−/4− was found to be 20% and 18% respectively (shown in Table S2). However a decrease in Ia was obtained after DNA-APT immobilization and a significant decrease in Ic was recorded (Fig. S1B-b to c, i.e. about 47%). Moreover, the peak-topeak separation (Ep ) of unmodified MULTI-SPE8 indicated that the electrochemical behaviour of unmodified MULTI-SPE8 was quasi-reversible, whereas it was irreversible after modification of G2-PS at MULTI-SPE8 and immobilization of DNA-APT onto the surface of G2-PS/MULTI-SPE8 due to increase in Ep value obtained after each immobilization/modification step (Table S2), similar to earlier work presented by Qian et al. [46]. In next step, the effect of APC concentration on the aptasensor response was examined and the surface confined interaction between DNA-APT and APC was consequently performed at G2-PS modified MULTI-SPE8 (Fig. 3). The Rct value increased while APC concentration increased due to the fact that aptamers interacted with more APC molecules, which had negative structure in the redox probe (pH 5.93) [47]. A linear graph based on Rct values was obtained in the APC concentration range of 0–7.5 g/mL (shown in Fig. 3B). The detection limit (DL) was calculated using the method described by Miller and Miller [48] with coefficient of determination (R2 ) = 0.9663, and it was found to be 1.81 g/mL (0.64 pmol in 20 L sample). After surface-confined interaction of DNA-APT with 7.5 g/mL APC, the Rct was recorded as 6615.3 ± 411.2 (RSD % = 6.2%, n = 4). Since the most reproducible and the highest Rct was obtained at 7.5 g/mL APC, it was chosen as the optimum APC concentration for further experiments in our study. The selectivity of the APC selective DNA-APT modified MULTISPE8 was then examined against other biomolecules, which may present any possible interferences. The impedimetric results are shown in Fig. 4. The interaction was carried out in the presence of
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Scheme 1. Schematic representation of impedimetric detection of APC at DNA-APT immobilized G2-PS modified MULTI-SPE8 surface.
100 g/mL DNA-APT and 7.5 g/mL APC, or other biomolecules: PC, BSA, THR, FVa and KS in TBS (pH 7.4) at the surface of G2-PS/MULTISPE8. The Rct value which was recorded after aptamer interaction with APC, PC or BSA increased respectively about 28%, 9% and 16%,
Fig. 3. (A) Nyquist diagrams obtained after surface confined interaction between 100 g/mL DNA-APT and its cognate protein APC in the different concentrations of APC by using MULTI-SPE8. DNA-APT immobilized G2-PS/MULTI-SPE8 (a), after interaction of 2.5 (b), 5 (c), 7.5 (d), 10 (e), 12.5 (f), and 15 g/mL APC (g) with DNAAPT at the surface of G2-PS/MULTI-SPE8 surface. Other conditions were same as in Figs. 1 and 2. (B) Calibration graph representing the Rct values obtained after interaction between 100 g/mL DNA-APT and APC in the concentration range of 0–7.5 g/mL. Inset was the equivalent electrical model used to fit the impedance data.
whereas 21%, 6% and 54% decrease in Rct value were obtained after aptamer interaction with THR, FVa or KS, respectively. Compared to the average Rct value recorded by DNA-APT immobilized G2PS/MULTI-SPE8 surface, the highest increase ratio was found for specific interaction between DNA-APT and its cognate protein, APC.
Fig. 4. Nyquist diagrams (A), histograms (B) representing the Rct values recorded at 100 g/mL DNA-APT immobilized G2-PS/MULTI-SPE8 (a), after interaction with 7.5 g/mL APC (b), PC (c), BSA (d), THR (e), FVa (f) and KS (g) in TBS (pH 7.4) solution. Inset was the equivalent electrical model used to fit the impedance data.
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Fig. 5. (A) Nyquist diagrams, (B) histograms obtained by bare MULTI-SPE8 (a), 1 g/mL G2-PS modified MULTI-SPE8 (b), 100 g/mL DNA-APT (c), random APT (d), random DNA (e) immobilized G2-PS/MULTI-SPE8, after interaction between 7.5 g/mL APC and 100 g/mL DNA-APT (c ), random APT (d ) or random DNA (e ) in TBS (pH 7.4). Inset was the equivalent electrical model used to fit the impedance data.
Thus, it could be concluded that this impedimetric aptasensor based on 8-channel screen-printed electrochemical array system (MULTISPE8) presented a selective behaviour to the target analyte APC in comparison to many other biomolecules. In another study, the selectivity of the aptasensor was also investigated by measuring the changes in the Rct value after the interaction of APC with a control DNA aptamer (random APT) or a control DNA sequence (random DNA) (Fig. 5) in comparison to APC selective DNA-APT. After the immobilization of 100 g/mL DNA-APT (Fig. 5A and B-c), or random APT (Fig. 5A and B-d), or random DNA (Fig. 5A and B-e) onto the electrode surface, the average Rct value was recorded respectively as 5858 ± 566 (RSD % = 9.6%, n = 3), 2548 ± 119 (RSD% = 4.7%, n = 3), and 2695 ± 275 (RSD% = 10.2%, n = 3). In the case of APC interaction with DNA-APT, or random APT, or random DNA, an increase was observed in the average Rct value as 29.7% (Fig. 5A and B-c ), 10% (Fig. 5A and Bd ) and a decrease in the average Rct value as 1% (Fig. 5A and B-e ) respectively. These results claimed that there was a specific binding between the DNA-APT and its cognate protein APC in comparison to the results obtained using the random APT, or the random DNA sequence. Although biosensors have mostly presented promising properties in an aqueous buffer for monitoring numerous biomolecular interactions, the detection may be more difficult in a serum media than that in an aqueous buffer due to the fact that serum contains several interference factors, such as a large number of proteins and other biomolecules [49]. Thus, impedimetric APC analysis was
Fig. 6. Effect of APC concentration upon the sensor response before and after surface confined interaction process between 100 g/mL APT and APC in different concentrations of APC from 1 to 2.5 g/mL in 1:5 FBS: TBS diluted solution. Nyquist diagrams (A). Inset was the equivalent electrical model used to fit the impedance data. Histograms (B) of Rct values obtained from before (a) and after interaction between APT and 1 (b), 1.5 (c), 2 (d), 2.5 (e) g/mL APC by using EIS, that was performed in 2.5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) containing 0.1 M KCl. Inset was calibration graph representing the Rct values obtained after interaction between 100 g/mL DNA-APT and APC at 1–2.5 g/mL concentration.
investigated in an artificial serum medium, e.g., foetal bovine serum (FBS), by using an aptamer modified G2-PS/MULTI-SPE8 (Fig. S2). The interaction between 100 g/mL DNA-APT and 7.5 g/mL APC was performed in the non-diluted FBS solution, and also in the diluted FBS solutions prepared using TBS in different ratios of FBS:TBS, such as 1:1, 1:5 and 1:10. The Rct value was obtained as 6610 ± 594 (RSD % = 9%, n = 3) in the presence of APT interaction with APC in 1:5 FBS:TBS diluted solution (Fig. S2-d), which was found to be similar to the Rct value obtained in TBS (Fig. S2-b). It was concluded that the surface confined interaction process between aptamer and APC was successfully achieved in 1:5 FBS:TBS diluted solution. In addition, the effect of APC concentration on the aptasensor response was investigated in 1:5 FBS:TBS diluted solution (Fig. 6). A linear graph was obtained in the APC concentration range between 1 and 2.5 g/mL. Consequently, the DL was calculated according to the method described by Miller and Miller [48] with the R2 = 0.9999, and it was found to be 0.02 g/mL (8.22 fmol in 20 L sample). 4. Conclusion Screen-printed biosensors (SPBs) are the one of the appropriate candidates for on-line measurement of biological samples due to the fact that sensitive and selective results in a low sample volume could be obtained in a short time using SPBs. Different types
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of screen-printed electrochemical array system are commercially available and they have the proportional properties to design the chip technologies that could detect various types of bioanalytes fastly, selectively and sensitively in the future. No report has been introduced yet on the impedimetric detection of human activated protein C (APC) in the literature. Herein, an impedimetric aptasensor based on poly-amidoamine (PAMAM) dendrimer (generation 2, G2-PS) modified 8-channel screenprinted electrochemical array system (MULTI-SPE8) was developed for the first time and applied for the detection of human activated protein C (APC), which is the key enzyme of the protein C pathway. Under optimum experimental conditions, it was presented in our study that APC could be detected in both buffer and a diluted serum medium (FBS) with the low detection limits respectively as 1.81 g/mL (0.64 pmol in 20 L sample) and 0.02 g/mL (8.22 fmol in 20 L sample). In comparison to earlier reports related to aptasensor technology developed using different materials such as dendrimers, carbon nanotubes, magnetic particles [9,29,45,50], our impedimetric aptasensor array system presented an effective protocol requiring a lower sample volume for sensitive and selective detection of the analyte in buffer, and also in a serum medium, such as FBS. Moreover, this aptasensor array system was not only a single-use, time-saving system that required less chemicals compared to other ELISA-based conventional methods reported in the literature [38,51,52], but it could also determine APC selectively compared to the other biomolecules such as PC, BSA, THR, FVa and chromogenic substrate. As a conclusion, APT immobilized dendrimer modified 8channel screen-printed electrochemical array system (MULTISPE8) presented a sensitive and selective protocol for APC sensing in a short time even when the interaction process occurred in a diluted serum medium (i.e., FBS). The aptamer-based dendrimer modified array system will also bring a new perspective to design novel chip technologies for the detection of other (bio)molecules, such as nucleic acids, drugs or proteins. Acknowledgements AE acknowledges the financial support from Turkish Scientific and Technological Research Council (TUBITAK; Project no. 111T073), and she also expresses her gratitude to the Turkish Academy of Sciences (TUBA) as an associate member for its partial support. GC acknowledges a master project scholarship through project (TUBITAK Project no. 111T073). The authors also thank Prof. Günter Mayer for his valuable scientific comments during this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2014.01.103. References [1] E. Palecek, M. Bartosik, Electrochemistry of nucleic acids, Chemical Reviews 112 (2012) 3427–3481. [2] J. Wang, Stripping-based electrochemical metal sensors for environmental monitoring, in: S. Alegret, A. Merkoc¸i (Eds.), Electrochemical sensor analysis, Elsevier, Oxford, UK, 2007, p. 131. [3] S. Qu, J. Wang, J. Kong, P. Yang, G. Chen, Magnetic loading of carbon nanotube/nano-Fe3 O4 composite for electrochemical sensing, Talanta 71 (2007) 1096–1102. [4] I. Willner, M. Rosen, Y. Eichen, Characterization of the hydrogenation process of allyl alcohol at a Pt electrode using a double galvanostatic pulse technique, Journal of Electrochemical Society 138 (1991) 434–439. [5] A. Erdem, Nanomaterial based electrochemical DNA sensing strategies, Talanta 74 (2007) 318–325. [6] J. Wang, Carbon-nanotube based electrochemical biosensors, Electroanalysis 17 (2005) 7–14.
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Biographies
Arzum Erdem is a professor at the Analytical Chemistry Department in the Faculty of Pharmacy of Ege University in Turkey. She received her PhD in analytical chemistry from Ege University in 2000. Dr. Erdem was awarded by the Turkish Academy of Sciences (TUBA) as the one of highly skilled young scientists selected in 2001, and she also received Junior Science Award 2006 given by The Scientific and Technological Research Council of Turkey (TUBITAK). She has initiated many international collaborative research on development and applications of electrochemical (bio)sensors based on drug, enzyme and nucleic acids. Her recent research is centred on the development of novel transducers and chemical and biological recognition systems by using different nanomaterials (e.g., magnetic nanoparticles, carbon nanotubes, gold and silver nanoparticles, nanowires, etc.) designed for electrochemical sensing of nucleic acid (DNA, RNA) hybridization, and also the specific interactions between drug and DNA, or protein and DNA, aptamer–protein and also the development of integrated analytical systems for environmental, industry and biomedical monitoring. Gulsah Congur has a B.Sc. in bioengineering from Faculty of Engineering, Ege University (Izmir, Turkey), and an M.Sc. in biotechnology from the Institute of Natural and Applied Sciences at Ege University. She is still continuing her Ph.D. in biotechnology from Natural and Applied Sciences, Ege University. Her current research is on the development of electrochemical biosensors for the purpose of monitoring of (bio)molecule–DNA interaction, detection of genetic disease by nucleic acid hybridization, investigation of protein–aptamer interaction.