Molecularly imprinted polymer-based SAW sensor for label-free detection of cerebral dopamine neurotrophic factor protein
Introduction
In clinical diagnostics, the research in the discovery and detection of biomarkers of human diseases, neurological and mental disorders has become of great demand due to the increase in the prevalence of these diseases over the last decades and the urgent need for their early stage diagnosis [1]. For example, neurotrophic factor (NTFs) proteins are a family of proteins secreted from neurons and glial cells, and supporting the survival of neurons [2]. NTFs were found to be associated with a number of neurological diseases (NDs) such as Alzheimer's, Parkinson’s and mental disorders and have been tested in clinical trials of several neurodegenerative diseases [[3], [4], [5], [6]].
Cerebral dopamine neurotrophic factor (CDNF), and mesencephalic astrocyte derived neurotrophic factor (MANF) form a new family of unconventional NTFs that have been shown to be promising candidates for the treatment of Alzheimer's and Parkinson’s [[6], [7], [8], [9]]. In animal models of Parkinson’s disease these NTFs can support the survival of neurons and regenerated neuronal axons opening a possibility for the development of disease modifying treatments. CDNF is currently tested in phase I-II clinical study on Parkinson’s disease patients in three Scandinavian medical centres [10]. The abnormal levels of NTFs in the blood may be associated with a number of NDs [11], mental disorders [3] and diabetes [12]. Serum and/or cerebrospinal fluid (CSF) concentration of a specific NTF could therefore be a potential biomarker for early-stage diagnosis and/or the follow-up of neuroprotective therapies.
Today, ELISA is one of the most commonly used immunological assays, applicable in both research and diagnostics, providing a quantitative detection of specific proteins, including NTFs, in serum samples [13]. In spite of its high specificity and low limit of detection (LOD) [14], ELISA suffers from several disadvantages such as laborious, lengthy procedure, the use expensive bioassay kits, low reliability and reproducibility in serum samples because of the cross-reaction with other antibodies.
To address these issues valuable alternatives to the traditional detection methods e.g. based on label-free sensor platform have been intensively studied [15]. The sensors based on the acoustic wave transduction mechanism seem to be a prospective for diagnostics purposes since they combine direct detection, simplicity in handling, real-time monitoring, and good sensitivity with a more reduced cost. Thus, Surface Acoustic Wave sensors (SAW) being fully compatible with large-scale fabrication and multiplexing technologies may provide substantial advantages in biosensing where electron transfer processes are hindered [16]. However, the reported label-free biosensing systems mostly utilize labile biological recognition elements (e.g. enzymes, DNA, antibodies) [17,18]. Moreover, applications for label-free sensing of NTF-proteins are very scarce [19,20].
Coupling label-free sensor platforms with synthetic recognition elements that avoid the disadvantages associated with the use of biological receptors in order to provide a reproducible and fast analysis of a biological sample, is of great importance. Molecular imprinting is one of the state-of-the-art techniques to generate robust synthetic molecular recognition materials with antibody-like ability to bind and discriminate between molecules [21]. The technique can be defined as the process of template-induced formation of specific molecular recognition sites in a polymer matrix material. The main benefits of these polymers, so-called Molecularly Imprinted Polymers (MIPs), are related to their synthetic nature, i.e., excellent chemical and thermal stability associated with reproducible, cost-effective fabrication. MIP receptors have been shown to be a promising alternative to natural biological receptors in biosensors providing more stable and low-cost recognition elements [22,23].
It should be noted that robust interfacing of a MIP with a sensor platform capable of responding upon interaction between the MIP and a binding analyte is a key aspect in the design of a MIP-based sensor. Recently, the use of an electrosynthesis approach for the facile integration of MIPs with label-free sensor platforms was reported [[24], [25], [26], [27]].
The application of MIP-based sensors for diagnostics has been extensively studied. Thus, the detection of cancer biomarkers - prostate specific antigen [28], epithelial ovarian cancer antigen-125 [29], carcinoembryonic antigen [30], cardiovascular disease biomarkers - myoglobin [31] and cardiac troponin T [32] by MIP-modified sensors have been reported. In addition, MIP receptors for selective extraction of Alzheimer's disease biomarker, β-amyloid peptides, has been studied by Sellergren’s group [33]. As concerns the imprinting of NTFs, so far only the selective recognition of another growth factor family protein - vascular endothelial growth factor (VEGF-A) by hybrid MIP nanoparticles as well as by MIP thin layer on SPR and screen-printed electrodes (SPE) has been reported [[34], [35], [36]]. Very recently, our group demonstrated the preparation of a photopolymerized MIP film integrated to a SPE and capable of selective recognition of brain-derived neurotrophic factor [37].
In this study, we report for the first time on the fabrication of MIP-based SAW sensor for label-free detection of CDNF. In this sensor, CDNF-MIP prepared by a surface imprinting approach, was utilized as a synthetic recognition element firmly interfaced with SAW sensing surface in the course of a simple electrochemical synthesis. As compared to electrochemical detection, we reported previously, the use of a mass-sensitive transducer such as SAW benefits from the absence of necessity to employ a redox pair as an electrochemical indicator as well as for ensuring the sufficient electrical conductivity at the electrode/solution interface for reliable and fast sensing of CDNF.
Section snippets
Chemical and materials
Sodium chloride (NaCl), 4-aminothiophenol (4-ATP), 2-mercaptoethanol, m-phenylenediamine (mPD), dimethyl sulfoxide (DMSO), human serum albumin (HSA), and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich. Human recombinant Cerebral Dopamine Neurotrophic Factor (CDNF, 18.5 kDa, calculated pI 7.68), human recombinant mesencephalic astrocyte-derived neurotrophic factor (MANF, 18.1 kDa, calculated pI 8.55), and mouse recombinant mCD48 (cluster of differentiation 48, 22.2 kDa, pI 9.36)
Optimization of CDNF-MIP sensor preparation
The molecular imprinting strategy used in this study employs the bottom-up approach [26] in order to generate the macromolecular imprints resided on at/close to the surface of the polymeric film. Thus, for such MIP, the deposition of a polymer with an appropriate thickness is one of the most crucial tasks in order to avoid irreversible entrapment of a macromolecular template and infeasibility of its removal during the subsequent washing out procedures. The effect of polymer thickness on the
Conclusions
We have demonstrated the possibility of using a MIP-based synthetic receptor to build a SAW sensor capable of detecting a neurotrophic factor protein, CDNF, in label-free manner. The electrochemical surface imprinting approach enabled a simple and rapid preparation of the polymeric matrix possessing the selectivity to CDNF, i.e. CDNF-MIP, directly on the sensor surface. We found that the thickness of the polymer matrix is critical and should be precisely adjusted in order to achieve the notable
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Estonian Research Council grant (PRG307) and by the Jane and Aatos Erkko Foundation. The authors thank Dr. Andreas Furchner from Leibniz-Institut für Analytische Wissenschaften – ISAS – e. V. for the VIS ellipsometry measurements. The authors thank Icosagen AS and prof. Mart Ustav personally, for kindly providing the neurotrophic factor proteins.
Anna Kidakova graduated from Astrakhan State Technical University in 2007. She received her MSc (2014) in material sciences from TalTech. She is currently a PhD student at the Laboratory of Biofunctional Materials of TalTech. Her research interest includes the development of molecularly imprinted polymer-based sensors for point-of care application.
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Anna Kidakova graduated from Astrakhan State Technical University in 2007. She received her MSc (2014) in material sciences from TalTech. She is currently a PhD student at the Laboratory of Biofunctional Materials of TalTech. Her research interest includes the development of molecularly imprinted polymer-based sensors for point-of care application.
Roman Boroznjak received his MSc (2007) in organic chemistry and PhD (2017) in natural and exact sciences from TalTech. His research interests include computational modelling and rational design of molecularly imprinted polymers.
Jekaterina Reut is currently a research scientist at the Department of Material and Environmental Technology in TalTech. She received her PhD (2004) in the field of electrically conducting polymers from TalTech. Her research interest is in the area of the design and synthesis of molecularly imprinted polymers for biosensing applications.
Andres Öpik received his PhD in chemistry from the University of Tartu in 1980. He is currently Professor of physical chemistry at the Department of Material and Environmental Technology in TalTech. His main research field is material science and technology: investigation of the physical and chemical properties and possibilities of practical applications of different electronic materials such as electrically conductive polymers and inorganic semiconductive compounds. Currently his research interests include the development of novel functional materials based on molecularly imprinted polymers for biomedical diagnostics.
Mart Saarma received his PhD in 1975 in Molecular Biology at the University of Tartu. Currently he is the head of the Laboratory of Molecular Neuroscience at the Institute of Biotechnology, HiLIFE, University of Helsinki. He is investigating the structure, biology and therapeutic potential of neurotrophic factors. His group has characterized several new GDNF family receptors and novel neurotrophic factor CDNF that is in Phase III clinical trials on Parkinson’s disease patients.
Vitali Syritski received his PhD in Chemistry at Tallinn University of Technology in 2004. Currently he is the head of the Laboratory of Biofunctional Materials in the Department of Material and Environmental Technology at TalTech. His present research interests include molecularly imprinted technology and electrochemical analysis. In particular, he has focused on development of chemical and biosensors for accurate and fast detection of disease biomarkers and environmental contaminants.