Dendrimer modified 8-channel screen-printed electrochemical array system for impedimetric detection of activated protein C

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Highlights

  • An 8-channel screen-printed electrochemical array system (MULTI-SPE8) was used as sensor platform.

  • MULTI-SPE8 was modified with PAMAM dendrimer having 16 succinamic acid surface groups (generation 2, G2-PS).

  • DNA aptamer was modified onto the surface of G2-PS/MULTI-SPE8 for the development of aptasensor.

  • The impedimetric detection of human activated protein C (APC) was done at aptamer modified G2-PS/MULTI-SPE8.

  • The selectivity of the aptasensor array system was tested in the presence of numerous biomolecules both in the buffer and in the fetal bovine serum (FBS).

Abstract

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 μg/mL (0.64 pmol in 20 μL sample) in buffer solution and 0.02 μg/mL (8.22 fmol in 20 μL sample) in diluted FBS.

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-of-care tools. Thus, the area of electrochemical biosensor technology has expanded day by day [1], [2], [3], [4], [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], [7], [8], [9], [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], [17], [18]. Dendrimers comprise well-defined cavities [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], [22], [23], [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], [28], [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]. 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 G2-PS, 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).

Section snippets

Apparatus

Electrochemical measurements were performed by using electrochemical impedance spectroscopy (EIS) by dropping 20 μL 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).

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 μg/mL G2-PS (Fig. 1b) in contrast to that of the unmodified

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 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

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.

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

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  • Cited by (0)

    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.

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