Capacitive Biosensors and Molecularly Imprinted Electrodes.

Capacitive biosensors belong to the group of affinity biosensors that operate by registering direct binding between the sensor surface and the target molecule. This type of biosensors measures the changes in dielectric properties and/or thickness of the dielectric layer at the electrolyte/electrode interface. Capacitive biosensors have so far been successfully used for detection of proteins, nucleotides, heavy metals, saccharides, small organic molecules and microbial cells. In recent years, the microcontact imprinting method has been used to create very sensitive and selective biorecognition cavities on surfaces of capacitive electrodes. This chapter summarizes the principle and different applications of capacitive biosensors with an emphasis on microcontact imprinting method with its recent capacitive biosensor applications.

Why Using Molecularly Imprinted Polymers in Connection to Biosensors?

The area of biosensor-oriented research has grown rapidly during recent years.

Molecular Imprinting Techniques Used for the Preparation of Biosensors.

Molecular imprinting is the technology of creating artificial recognition sites in polymeric matrices which are complementary to the template in their size, shape and spatial arrangement of the functional groups. Molecularly imprinted polymers (MIPs) and their incorporation with various transducer platforms are among the most promising approaches for detection of several analytes. There are a variety of molecular imprinting techniques used for the preparation of biomimetic sensors including bulk imprinting, surface imprinting (soft lithography, template immobilization, grafting, emulsion polymerization) and epitope imprinting. This chapter presents an overview of all of these techniques with examples from particular publications.

Molecularly imprinted cryogels for human interferon-alpha purification from human gingival fibroblast culture.

Interferons are important proteins for the immune system because of their antiviral, anti-proliferating and immunomodulatory activities. Therapeutic value of these proteins against certain types of tumors caused interest and investigations aimed to obtain highly purified interferons. Molecular imprinting is an efficient method for purification with high selectivity, specificity and good reproducibility. In this study, we utilized advantages of molecular imprinting technique for the purification of interferon from human gingival fibroblast culture. For this purpose, interferon α-2b imprinted poly(hydroxyethyl methacrylate) cryogel (hIFN-α-MIP) was prepared. Optimum adsorption conditions were determined, and maximum adsorption capacity of hIFN-α-MIP cryogel was found as 254.8 × 10(4) IU/g from aqueous solution. All interferon measurements are expressed as International Unit (IU), which is a unit measurement used to quantify biologically active substances like interferon based on their biological activity or effect. Selectivity experiments were performed using competitive proteins and repeated adsorption-desorption studies showed that the adsorption capacity maintained almost at a constant value after ten cycles. For the purification of interferon from human gingival fibroblast culture, fast protein liquid chromatography was used and the specific activity of the purified interferon α-2b on HeLa cell line was found between the values 3.45 × 10(8) IU/mg and 3.75 × 10(8) IU/mg. The results are promising, and the molecular imprinting technique is effective for the purification of interferon α-2b.

Oriented immobilized anti-hIgG via F(c) fragment-imprinted PHEMA cryogel for IgG purification.

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F(c) fragment-imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti-hIgG for IgG purification from human plasma. Non-imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti-hIgG to compare the adsorption capacities of oriented (MIP/anti-hIgG) and random (NIP/anti-hIgG) cryogel columns. The amount of immobilized anti-hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti-hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti-hIgG column (29.7 mg/g) for the MIP/anti-hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti-hIgG cryogel column. Adsorbed IgG was eluted using 1.0 M NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti-hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification.

Clinical experience: Outcomes of mesenchymal stem cell transplantation in five stroke patients.

Stem cell therapy, which has promising results in acute disorders such as stroke, supports treatment by providing rehabilitation in the chronic stage patients. In acute stroke, thrombolytic medical treatment protocols are clearly defined in neurologic emergencies, but in neurologic patients who miss the "thrombolytic treatment intervention window," or in cases of hypoxic-ischemic encephalopathy, our hands are tied, and we are still unfortunately faced with hopeless clinical implementations. We consider mesenchymal stem cell therapy a viable option in these cases. In recent years, novel research has focused on neuro-stimulants and supportive and combined therapies for stroke. Currently, available treatment options are limited, and only certain patients are eligible for acute treatment. In the scope of our experience, five stroke patients were evaluated in this study, who was treated with a single dose of 1-2 × 106 cells/kg allogenic umbilical cord-mesenchymal stem cells (UC-MSCs) with the official confirmation of the Turkish Ministry of Health Stem Cell Commission. The patients were followed up for 12 months, and clinical outcomes are recorded. NIH Stroke Scale/Scores (NIHSS) decreased significantly (p = 0.0310), and the Rivermead Assessment Scale (RMA) increased significantly (p = 0.0234) for all patients at the end of the follow-up. All the patients were followed up for 1 year within a rehabilitation program. Major clinical outcome improvements were observed in the overall clinical conditions of the UC-MSC treatment patients. We observed improvement in the patients' upper extremity and muscle strength, spasticity, and fine motor functions. Considering recent studies in the literature together with our results, allogenic stem cell therapies are introduced as promising novel therapies in terms of their encouraging effects on physiological motor outcomes.

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor.

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Highly sensitive detection and quantification of the secreted bacterial benevolence factor RoxP using a capacitive biosensor: A possible early detection system for oxidative skin diseases.

The impact of the microbiota on our health is rapidly gaining interest. While several bacteria have been associated with disease, and others being indicated as having a probiotic effect, the individual biomolecules behind these alterations are often not known. A major problem in the study of these factors in vivo is their low abundance in complex environments. We recently identified the first secreted bacterial antioxidant protein, RoxP, from the skin commensal Propionibacterium acnes, suggesting its relevance for maintaining the redox homeostasis on the skin. In order to study the effect, and prevalence, of RoxP in vivo, a capacitive biosensor with a recognition surface based on molecular imprinting was used to detect RoxP on skin in vivo. In vitro analyses demonstrated the ability to detect and quantify RoxP in a concentration range of 1 x 10-13 M to 1 x 10-8 M from human skin swabs; with a limit of detection of 2.5 x 10-19 M in buffer systems. Further, the biosensor was highly selective, not responding to any other secreted protein from P. acnes. Thus, it was possible to demonstrate the presence, and quantity, of RoxP on human skin. Therefore, the developed biosensor is a very promising tool for the detection of RoxP from clinical samples, offering a rapid, cost-effective and sensitive means of detecting low-abundant bacterial proteins in vivo in complex milieus.

Revisiting Antibiotic Resistance Spreading in Wastewater Treatment Plants - Bacteriophages as a Much Neglected Potential Transmission Vehicle.

The spread of antibiotic resistance is currently a major threat to health that humanity is facing today. Novel multidrug and pandrug resistant bacteria are reported on a yearly basis, while the development of novel antibiotics is lacking. Focus to limit the spread of antibiotic resistance by reducing the usage of antibiotics in health care, veterinary applications, and meat production, have been implemented, limiting the exposure of pathogens to antibiotics, thus lowering the selection of resistant strains. Despite these attempts, the global resistance has increased significantly. A recent area of focus has been to limit the spread of resistance through wastewater treatment plants (WWTPs), serving as huge reservoirs of microbes and resistance genes. While being able to quite efficiently reduce the presence of resistant bacteria entering any of the final products of WWTPs (e.g., effluent water and sludge), the presence of resistance genes in other formats (mobile genetic elements, bacteriophages) has mainly been ignored. Recent data stress the importance of transduction in WWTPs as a mediator of resistance spread. Here we examine the current literature in the role of WWTPs as reservoirs and hotspots of antibiotic resistance with a specific focus on bacteriophages as mediators of genetic exchange.

From imprinting to microcontact imprinting-A new tool to increase selectivity in analytical devices.

Molecular imprinting technology has been successfully applied to small molecular templates but a slow progress has been made in macromolecular imprinting owing to the challenges in natural properties of macromolecules, especially proteins. In this review, the macromolecular imprinting approaches are discussed with examples from recent publications. A new molecular imprinting strategy, microcontact imprinting is highlighted with its recent applications.

A sensitive and real-time assay of trypsin by using molecular imprinting-based capacitive biosensor.

Use of a highly sensitive, selective capacitive biosensor is reported for label-free, real-time, easy and rapid detection of trypsin by using the microcontact imprinting method. Real-time trypsin detection was performed with trypsin-imprinted (trypsin-MIP) capacitive electrodes using standard trypsin solutions in the concentration range of 1.0×10(-13)-1.0×10(-7)M with a detection limit of 3.0×10(-13)M. Selectivity and cross-reactivity of the system were tested by using competing proteins including chymotrypsin (chy), bovine serum albumin (BSA), lysozyme (lyz) and cytochrome c (cyt c) in singular and competitive manner and the selectivity of the system was determined with the selectivity coefficients of approximately 705.1, 6.5, 6.4 and 5.1 for chy, BSA, lyz and cyt c, respectively. The trypsin-MIP capacitive electrode was used for ~80 assays during 2 months and retained its binding property during all that time with a decrease of approximately 2.3% in the signal amplitude. In the last step, trypsin activity was measured by using Nα-Benzoyl-D, l-arginine 4-nitroanilide hydrochloride (BAPNA) as the substrate with spectrophotometer at 410nm. The trypsin activity was measured as 9mU/mL by spectrophotometer while the amount of captured enzyme calculated from the capacitive system was 7.9mU/mL which shows the correlation between two methods. From the comparison it is obvious that the new method is an attractive alternative for assaying trypsin and the developed capacitive system might be used successfully to monitor label-free, real-time enzymatic activity of different proteases in a sensitive, rapid, cost-effective manner for different applications.

Real-time prostate-specific antigen detection with prostate-specific antigen imprinted capacitive biosensors.

Prostate specific antigen (PSA) is a valuable biomarker for early detection of prostate cancer, the third most common cancer in men. Ultrasensitive detection of PSA is crucial to screen the prostate cancer in an early stage and to detect the recurrence of the disease after treatment. In this report, microcontact-PSA imprinted (PSA-MIP) capacitive biosensor chip was developed for real-time, highly sensitive and selective detection of PSA. PSA-MIP electrodes were prepared in the presence of methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker via UV polymerization. Immobilized Anti-PSA antibodies on electrodes (Anti-PSA) for capacitance measurements were also prepared to compare the detection performances of both methods. The electrodes were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV) and real-time PSA detection was performed with standard PSA solutions in the concentration range of 10 fg mL(-1)-100 ng mL(-1). The detection limits were found as 8.0 × 10(-5) ng mL(-1) (16 × 10(-17) M) and 6.0 × 10(-4) ng mL(-1) (12 × 10(-16) M) for PSA-MIP and Anti-PSA electrodes, respectively. Selectivity studies were performed against HSA and IgG and selectivity coefficients were calculated. PSA detection was also carried out from diluted human serum samples and finally, reproducibility of the electrodes was tested. The results are promising and show that when the sensitivity of the capacitive system is combined with the selectivity and reproducibility of the microcontact-imprinting procedure, the resulting system might be used successfully for real-time detection of various analytes even in very low concentrations.

Cryogels-versatile tools in bioseparation.

Cryogels are polymeric materials formed from monomeric or polymeric precursors in moderately frozen state by polymerization or crosslinking. The process applied for cryogel formation is called cryogelation. These macroporous gel matrices can be produced with different shapes and the gels are of interest in the bioseparation area since they can meet needs that conventional chromatographic media are less suitable to fulfill. High porosity, high mechanical and chemical stability make them appropriate carriers for immobilization of biomolecules and cells thereby making them attractive gel matrices for separation and purification of various molecules. This review highlights the preparation and properties of cryogels, and applications of these materials especially in bioseparation science.

Microcontact-BSA imprinted capacitive biosensor for real-time, sensitive and selective detection of BSA.

An analytical method is presented, combining novel microcontact imprinting technique and capacitive biosensor technology for the detection of BSA. Glass cover slips were used for preparation of protein stamps. The microcontact-BSA imprinted gold electrodes were prepared in the presence of methacrylic acid (MAA) and poly-ethylene glycol dimethacrylate (PEGDMA) as the cross-linker by bringing the protein stamp and the gold electrode into contact under UV-polymerization. Real-time BSA detection studies were performed in the concentration range of 1.0 × 10-20-1.0 × 10-8 M with a limit of detection (LOD) of 1.0 × 10-19 M. Cross-reactivity towards HSA and IgG were 5 and 3%, respectively. The electrodes were used for >70 assays during 2 months and retained their binding properties during all that time. The NIP (non-imprinted) electrode was used as a reference. The microcontact imprinting technology combined with the biosensor applications is a promising technology for future applications.

Molecularly imprinted supermacroporous cryogels for myoglobin recognition.

Myoglobin is a primary iron, and oxygen-binding protein of muscle tissues and levels can be an important diagnostic biomarker for acute myocardial infarction, myocardial necrosis, or other cardiac diseases. The establishment of myoglobin recognition systems is important because of its protein's structural and functional values in physiology, biochemistry, and diagnostic value in some damaged muscle tissue and cardiac diseases. For this purpose, we used molecular imprinting technique for myoglobin recognition from aqueous solutions and human plasma. In the first step, myoglobin-imprinted poly(hydroxyethyl methacrylate) (PHEMA) cryogels (MGb-MIP) were prepared, and optimum myoglobin adsorption conditions were determined. Selectivity experiments have been done with the competitive proteins, and myoglobin adsorption from IgG and albumin-free human plasma was studied. The purity of the desorbed samples was determined with SDS-PAGE. The desorption efficiency and reusability of the MGb-MIP cryogels were tested, and it was shown that without any significant loss in the adsorption capacity, MGb-MIP cryogels can be used a number of times for myoglobin recognition and separation.

Fab fragments imprinted SPR biosensor for real-time human immunoglobulin G detection.

F(ab) fragments imprinted surface plasmon resonance (SPR) chip was prepared for the real-time detection of human immunoglobulin G (IgG). In order to attach polymerization precursor on SPR chip, the SPR chip surface was modified with allyl mercaptan. F(ab) fragments of the IgG molecules were prepared by papain digestion procedure and collected by fast protein liquid chromatography (FPLC) system using Hi-Trap_r Protein A FF column. The collected F(ab) fragments were complexed with histidine containing specific monomer, N-methacryloyl-l-histidine methyl ester (MAH). Molecular imprinted polymeric nanofilm was prepared on SPR chip in the presence of ethylene glycol dimethacrylate and 2-hydroxyethylmethacrylate. The template molecules, F(ab) fragments, were removed from the polymeric nanofilm using 1M NaCl solution (pH: 7.4, phosphate buffer system). The molecular imprinted SPR chip was characterized by contact angle, atomic force microscopy and Fourier transform infrared spectroscopy. By the real-time IgG detection studies carried out using aqueous IgG solutions in different concentrations, the kinetics and isotherm parameters of the molecular imprinted SPR chip-IgG system were calculated. To show selectivity and specificity of the molecular imprinted SPR chip, competitive kinetic analyses were performed using bovine serum albumin (BSA), IgG, F(ab) and F(c) fragments in singular and competitive manner. As last step, IgG detection studies from human plasma were performed and the measured IgG concentrations were well matched with the results determined by enzyme-linked immunosorbent assay (ELISA). The results obtained with the molecular imprinted SPR chip were well fitted to Langmuir isotherm and the detection limit was found as 56 ng/mL. In the light of the results, we can conclude that the proposed molecular imprinted SPR chip can detect IgG molecules from both aqueous solutions and complex natural samples.