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Selective Artificial Receptors Based on Micropatterned Surface‐Imprinted Polymers for Label‐Free Detection of Proteins by SPR Imaging
Advanced Functional Materials
Abstract
A novel concept to generate micropatterned surface-imprinted polymers (SIPs) for protein recognition by using standard photolithographic technology is introduced. Avidin-imprinted poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) conducting polymer microbands are prepared directly on surface plasmon resonance (SPR) chips, which enable convenient label-free monitoring of the binding events. The novel surface-imprinted microstructures bind avidin, the template protein, with dissociation constants in the submicromolar range (125 nM). The SIPs have an avidin binding capacity approximately one order of magnitude higher than the corresponding nonimprinted polymers and are able to discriminate among functional homologues of avidin, i.e., neutravidin, extravidin, and streptavidin.
... The precise control over electrosynthesis enables the fine tuning of the polymer layer thickness, which is particularly important for surface imprinting with protein templates. Electrosynthesis can be used straightforwardly to create several nanometer thick polymer films [52,53] and with the aid of sacrificial materials, to prepare micro-and nanostructures with surface confined binding sites [5,[54][55][56]. It must be noted that beside direct electrooxidation of suitable monomers the versatility of electrochemical synthesis enables surface confined polymer deposition also by electrochemically generating the "active" initiator [57] or electrochemically changing the local pH in the close vicinity of the electrode [58]. ...
... These properties make PEDOT-PSS an excellent candidate for protein imprinting. However, electrosynthesized PEDOT MIPs have only been prepared to date for a single model template, avidin [55,56,65]. ...
... This concept works best with conducting polymers alleviating the demand for ultrathin films while providing a higher binding capacity owing to the increased specific surface of the polymer. Most importantly, a significantly better discrimination could be achieved between avidin (used as target) and its analogues when the template was immobilized through its biotin-binding site [54], than with the same type of polymer imprinted with randomly immobilized avidin [56]. The sphere lithography concept could be extended to generate surface imprinted microporous MIPs by self-assembling several sacrificial bead layers [85] (Fig. 4F) as opposed to just one [54]. ...
Molecularly imprinted polymers (MIPs) for the recognition of proteins are expected to possess high affinity through the establishment of multiple interactions between the polymer matrix and the large number of functional groups of the target. However, while highly affine recognition sites need building blocks rich in complementary functionalities to their target, such units are likely to generate high levels of non-specific binding. This paradox, that nature solved by evolution for biological receptors, needs to be addressed by the implementation of new concepts in molecular imprinting of proteins. Additionally, the structural variability, large size and incompatibility with a range of monomers made the development of protein MIPs to take a slow start. While the majority of MIP preparation methods are variants of chemical polymerization, the polymerization of electroactive functional monomers emerged as a particularly advantageous approach for chemical sensing application. Electropolymerization can be performed from aqueous solutions to preserve the natural conformation of the protein templates, with high spatial resolution and electrochemical control of the polymerization process. This review compiles the latest results, identifying major trends and providing an outlook on the perspectives of electrosynthesised protein-imprinted MIPs for chemical sensing.
... Electroactive functional monomers were used in Lautner et al. for the preparation of surface-imprinted microbands on SPR chips performed by potentiostatic electropolymerization [23] . Electropolymerized MIPs have attracted considerable interest in the development of chemical sensors and biosensor thanks to their electronic properties (magnetic, conducting, and optical), similar to those of metals [24] . ...
... From a different point of view, MIPs were used as a platform in SPR imaging for protein recognition [23] . The surfaceimprinted polymers (SIPs) were prepared on SPR chips using photolithographic technology. ...
Medical diagnostics aims at specific localization of molecular targets as well as detection of abnormalities associated with numerous diseases. Molecularly imprinted polymers (MIPs) represent an approach of creating a synthetic material exhibiting selective recognition properties toward the desired template. The fabricated target-specific MIPs are usually well reproducible, economically efficient, and stable under critical conditions as compared to routinely used biorecognition elements such as fluorescent proteins, antibodies, enzymes, or aptamers and can even be created to those targets for which no antibodies are available. In this review, we summarize the methods of polymer fabrication. Further, we provide key for selection of the core material with imaging function depending on the imaging modality used. Finally, MIP-based imaging applications are highlighted and presented in a comprehensive form from different aspects.
Statement of significance
In this review, we summarize the methods of polymer fabrication. Key applications of Molecularly imprinted polymers (MIPs) in imaging are highlighted and discussed with regard to the selection of the core material for imaging as well as commonly used imaging targets. MIPs represent an approach of creating a synthetic material exhibiting selective recognition properties toward the desired template. The fabricated target-specific MIPs are usually well reproducible, economically efficient, and stable under critical conditions as compared to routinely used biorecognition elements, e.g., antibodies, fluorescent proteins, enzymes, or aptamers, and can even be created to those targets for which no antibodies are available.
... Performance of surface imprinted polymers was further increased by template immobilization on a core support before polymerization (Bognár et al., 2013;Dechtrirat et al., 2012;Gao et al., 2013;Lautner et al., 2011;Menaker et al., 2009). That way, the number of Scheme 1. ...
... Other than that, fluorometric detection is particularly attractive because luminescent signals are very conveniently measured with high sensitivity. Majority of fluorescence chemosensors involves imprinting of fluoro-tagged analytes (Dechtrirat et al., 2012;Lautner et al., 2011;Menaker et al., 2009). Recently, an interesting procedure described post-imprinting modification of MIP cavities with fluorescence active compounds (Suga et al., 2013;. ...
The present review article focuses on gathering, summarizing, and critical evaluating of the last decade results on separating and sensing of macromolecular compounds and microorganisms with the use of molecularly imprinted polymer (MIP) synthetic receptors. Macromolecules play an important role in biology and are termed that way to contrast micromolecules. The former are large and complex molecules with relatively high molecular weights. The article mainly considers chemical sensing of deoxyribonucleic acids (DNAs), proteins and protein fragments as well as sugars and oligosaccharides. Moreover, it briefly discusses fabrication of chemosensors for determination of bacteria and viruses that can ultimately be considered as extremely large macromolecules.
... In these reviews, the development of functional monomers with complementary functional moieties to form donor-acceptor interactions with the template molecules [30][31][32] and the effects of crosslinkers and solvents on controlling the recognition site as well as polymer morphology are discussed in detail [33,34]. With the highly selective binding sites, MIPs have found a wide range of applications such as solid-phase extraction (SPE), sensors, membranes, catalysis, synthesis, and drug delivery [35,36]. Moreover, the high thermal stability and structural rigidity of MIPs enable their use under harsh conditions. ...
Molecularly imprinted polymer (MIP)-based luminescent chemosensors combine the advantages of the highly specific molecular recognition of the imprinting sites and the high sensitivity with the luminescence detection. These advantages have drawn great attention during the past two decades. Luminescent molecularly imprinted polymers (luminescent MIPs) towards different targeted analytes are constructed with different strategies, such as the incorporation of luminescent functional monomers, physical entrapment, covalent attachment of luminescent signaling elements on the MIPs, and surface-imprinting polymerization on the luminescent nanomaterials. In this review, we will discuss the design strategies and sensing approaches of luminescent MIP-based chemosensors, as well as their selected applications in biosensing, bioimaging, food safety, and clinical diagnosis. The limitations and prospects for the future development of MIP-based luminescent chemosensors will also be discussed.
... Lautner et al. presented micropatterned surface-imprinted polymers for protein recognition obtained by photolithographic technique [108]. Avidin-imprinted poly(3,4ethylenedioxythiophene)/poly(styrenesulfonate) conducting polymer microbands were prepared directly on the SPR chip surface. ...
In recent years, plasmonic sensors have been used in various fields ranging from environmental monitoring, pharmaceutical analysis, medical diagnosis, and food quality assessment to forensics. A significant amount of information on plasmonic sensors and their applications already exists and there is a continuing development of reliable, selective, sensitive, and low-cost sensors. Combining molecularly imprinting technology with plasmonic sensors is an increasingly timely and important challenge to obtain portable, easy-to-use, particularly selective devices helpful in detecting analytes at the trace level. This review proposes an overview of the applications of molecularly imprinted plasmonic chemosensors and biosensors, critically discussing the performances, pros, and cons of the more recently developed devices.
... In the present work, we have developed surface-imprinted polymer (SIP) receptors for Campylobacter. SIPs are biomimetic receptors that have been used successfully to detect bacteria [22][23][24][25], yeast [26], mammal cells [27,28], viruses [29,30], and proteins [31]. SIPs, a subtype of molecularly imprinted polymers, are imprinted only at the surface to allow for facile template extraction after imprinting [32,33]. ...
Thermotolerant Campylobacter bacteria, most notably Campylobacter jejuni and Campylo-bacter coli, are a major cause of human foodborne gastroenteritis, which is usually related to consumption of contaminated poultry. In this work, we present a sensor and the associated assay for the on-site detection of the prevalent species C. jejuni and C. coli. The sensor uses surface-imprinted polymer (SIP) layers as selective, biomimetic recognition elements in combination with a modified heat-transfer method (M-HTM) as a label-free, quantitative readout principle. The selectivity for C. coli and C. jejuni was evaluated against six other morphologically similar Campylobacterales species, confirming that the sensor is selective at species level while responding uniformly to different strains within the same species. For the relevant matrix, that is chicken cecal droppings suspended in PBS buffer, the detection limits are 1.1 × 10³ CFU/ml for C. coli and 2.7 × 10⁴ CFU/ml for C. jejuni, which is both low enough for a meaningful diagnostic test. The sensor concept requires only a minimum of sample preparation and a given concentration can be measured within less than one hour: Both are important assets for on-site detection such as on a poultry farm or in a slaughterhouse, keeping in mind that Campylobacter detection with established methods in analytical laboratories takes 2 to 4 days for obtaining the result.
... This is achieved by the semiconductor in which the absorbed energy should be equal to or larger than the band gap in order to form electron hole pair. Nanoscience research has a strong influence on the development of novel and more influential catalysts through the pattern and properties through with energy gap, composition and modification [9,10]. Since nanotechnology emerges, QDs are promising materials for photo-catalysis, ions sensing, biological imaging and heavy metal detection. ...
The industrial pollutants in water bodies tend to unsuitable for living organisms and irrigation uses. Water contamination is exaggerating at regular pace and the universe is holding carcinogenic agents. Therefore, there is a necessity of immediate action to generate a potential and efficient technology for water management. By means of this, quantum dots (QDs) have emerged as an effective probe for the removal process. This review discusses the methods for removing and degrading the coloured components, pesticides, pathogens from waste water and contaminants removal ability of QDs.
... SPR can be considered as one of the most powerful, and therefore popular, readout technologies and has been combined with MIPs for the detection of microorganisms [174,175], amino acids, peptides and proteins [176][177][178], dangerous explosives [179][180][181], antibiotics [182][183][184] and other low-molecular weight compounds [185][186][187][188][189]. Detection upon rebinding of the target is based on changes in electron density at the surface of the sensor chip. Ertürk Bergdahl et al. reported on the development of a biosensor with high diagnostic potential last year [190]. ...
Molecularly imprinted polymers (MIPs) have emerged over the past few decades as interesting synthetic alternatives due to their long-term chemical and physical stability and low-cost synthesis procedure. They have been integrated into many sensing platforms and assay formats for the detection of various targets, ranging from small molecules to macromolecular entities such as pathogens and whole cells. Despite the advantages MIPs have over natural receptors in terms of commercialization, the striking success stories of biosensor applications such as the glucose meter or the self-test for pregnancy have not been matched by MIP-based sensor or detection kits yet. In this review, we zoom in on the commercial potential of MIP technology and aim to summarize the latest developments in their commercialization and integration into sensors and assays with high commercial potential. We will also analyze which bottlenecks are inflicting with commercialization and how recent advances in commercial MIP synthesis could overcome these obstacles in order for MIPs to truly achieve their commercial potential in the near future.
... Avidin-imprinted poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) conducting polymer microbands were prepared on the SPR platform. The target analyte, avidin, was bound to the SIP with dissociation constants in the submicromolar range (125 nM), and the sensor demonstrated selectivity among functional homologues of avidin, i.e., neutravidin, extravidin and streptavidin [68]. A microcontact printing technique was proposed by the group of Denizli to produce MIP-SPR for procalcitonin with an LoD of 9.9 ng/mL [69] and for the detection of the bacterium monitoring Salmonella paratyphi in food supplies or contaminated water [70]. ...
Optical sensing, taking advantage of the variety of available optical structures, is a rapidly expanding area. Over recent years, whispering gallery mode resonators, photonic crystals, optical waveguides, optical fibers and surface plasmon resonance have been exploited to devise different optical sensing configurations. In the present review, we report on the state of the art of optical sensing devices based on the aforementioned optical structures and on synthetic receptors prepared by means of the molecular imprinting technology. Molecularly imprinted polymers (MIPs) are polymeric receptors, cheap and robust, with high affinity and selectivity, prepared by a template assisted synthesis. The state of the art of the MIP functionalized optical structures is critically discussed, highlighting the key progresses that enabled the achievement of improved sensing performances, the merits and the limits both in MIP synthetic strategies and in MIP coupling.
... [1][2][3] However, protein templates are more challenging to imprint than low-molecular weight compounds as they are of higher structural complexity and more easily entrapped in the polymeric matrix due to their large size. [4][5][6][7][8][9] For effective target removal and rebinding, the binding sites should be located at the surface of the MIP (surface-imprinting). [3,7,10,11] This is accomplished by pre-assembling the template on the surface followed by the electrosynthesis of a polymer layer with thicknesses in the lower nanometer range, [12][13][14][15][16][17][18][19] comparable with the size of the protein. For oriented pre-assembly, rather unspecific ligands, such as charged self-assembled monolayers (SAMs) facilitating electrostatic attraction of the template or boronic acid derivatives for the capture of glycoproteins [20] as well as specific ligands, like inhibitors or substrates, [21] aptamers [22] or fragments of the protein itself (epitopes) [23][24][25][26][27] have been successfully applied. ...
A new approach for synthesizing a “vectorially” imprinted polymer (VIP) is presented for the microbial cytochrome P450cam enzyme. A surface attached binding motif of a natural reaction partner of the target protein, putidaredoxin (Pdx), is the anchor to the underlying transducer. The 15 amino acid peptide anchor, which stems from the largest continuous amino acid chain within the binding site of Pdx was modified: (i) internal cysteines were replaced by serines to prevent disulfide bond formation; (ii) 2 polyethylene glycol units were attached to the N‐terminus as a spacer region; and (iii) an N‐terminal cysteine was added to allow the immobilization on the gold electrode surface. Immobilization on GCE was achieved via an N‐(1‐pyrenyl)maleimide (NPM) cross‐linker. In this way oriented immobilization of P450cam was accomplished by binding it to a peptide‐modified gold or glassy carbon electrode (GCE) prior to the electrosynthesis of a polymer nanofilm around the immobilized target. This VIP nanofilm enabled reversible oriented docking of P450cam as it is indicated by the catalytic oxygen reduction via direct electron transfer between the enzyme and the underlying electrode. Catalysis of oxygen reduction by P450cam bound to the VIP‐modified GCE was used to measure rebinding to the VIP. The “mild” coupling of an oxidoreductase with the electrode may be appropriate for realizing electrode‐driven substrate conversion by instable P450 enzymes without the need of NADPH.
... (ii) Alternatively, the target can be adsorbed at the transducer surface prior polymerization [13] ( Figure 1B). Besides direct adsorption of proteins [14,15], deposition of proteinenanoparticle conjugates can be also used, for example, by nanosphere lithography [16] to generate surfaceimprinted polymer layers. (iii) Oriented binding of the target prior polymerization via site-specific anchors, for example, charged selfassembled monolayers (SAMs), boronic acid derivatives [17], aptamers [18], or inhibitors [19], which allows the formation of more uniform cavities in the MIPs ( Figure 1C). ...
Electrochemical synthesis and signal generation dominate among the almost 1200 articles published annually on protein-imprinted polymers. Such polymers can be easily prepared directly on the electrode surface, and the polymer thickness can be precisely adjusted to the size of the target to enable its free exchange. In this architecture, the molecularly imprinted polymer (MIP) layer represents only one 'separation plate’; thus, the selectivity does not reach the values of ‘bulk’ measurements. The binding of target proteins can be detected straightforwardly by their modulating effect on the diffusional permeability of a redox marker through the thin MIP films. However, this generates an ‘overall apparent’ signal, which may include nonspecific interactions in the polymer layer and at the electrode surface. Certain targets, such as enzymes or redox active proteins, enables a more specific direct quantification of their binding to MIPs by in situ determination of the enzyme activity or direct electron transfer, respectively.
... Subsequently, this MOF was removed resulting in electrode surface coated with a porous MIP film. Moreover, preliminary protein template immobilization on the nanomold surface results in location of all molecular cavities on the surface of the deposited MIP (Scheme 2a and b) (Lautner et al., 2011;Menaker et al., 2009). Ice crystals seem to be a perfect candidate for the nanomold. ...
Molecularly imprinted polymers (MIPs) are tailor made recognition materials that can mimic biological receptors. If used as recognition units for chemosensors fabrication, they outperform natural receptors with their durability, chemical stability, and low production costs. Novel techniques of MIP deposition as thin films, surface development, and introduction of additional properties are very much demanded in terms of selective and sensitive chemosensors fabrication. Therefore, in recent years a particular attention has been paid to syntheses of nanostructured MIP films and MIP nanoparticles. The present brief review surveys novel achievements in the field of MIP nanostructures and their application for determination of protein analytes.
... An interesting approach to construct such devices is to incorporate surface imprinted polymers (SIPs), acting as biomimetic cell receptors into sensor platforms [7]. SIPs can be made via several approaches and have been imprinted with various templates including proteins [8][9][10], yeast [11,12], viruses [13,14], bacteria [15,16], and mammalian cells [17,18]. SIP-covered electrodes are typically combined with low-cost, user-friendly readout platforms based on microgravimetric [19][20][21], electrochemical [22][23][24], optical [25,26], and thermal transducer principles [27][28][29]. ...
Previous studies have shown that selective synthetic cell receptors can be produced by cell imprinting on polymer layers. However, knowledge on the fundamental detection mechanisms remains limited. In this article, while using yeast cells (Saccharomyces cerevisiae) as model cells, the factors influencing cellular recognition by surface-imprinted polymers (SIPs) are studied by means of spectroscopic and microscopy techniques and a transducer platform based on interfacial thermal transport, the so-called heat-transfer method (HTM). These analyses indicate that cell imprinting creates selective binding sites on the surface of the SIP layer in the form of binding cavities that match the cells in shape and size. Also, we show that phospholipid moieties are incorporated into the SIP cavities during imprinting, while membrane proteins do not seem to be transferred. More importantly, we demonstrate that the incorporated phospholipids significantly enhance cell adhesion to the SIP, and thus play a significant role in the cell-SIP binding mechanism. Furthermore, the hydrophobicity of the SIP layer was found to be considerably higher when compared with a non-imprinted polymer layer (NIP), an effect that could not be attributed to the presence of cavities on the surface of the SIP layer. Therefore, we suggest that the role of phospholipids in the SIP recognition mechanism is mediated by long range hydrophobic forces.
... However, structures obtained were far from those of inverse opals (Li et al., 2013) or resulted merely in one half-layer inverted opals (Bognar et al., 2013). Despite numerous difficulties, which still remain to be solved, nano-and microstructured MIP films gain more and more interest (Bompart et al., 2012;Hu et al., 2008;Lautner et al., 2011;Linares et al., 2009;Zdunek et al., 2013). ...
Nanostructured artificial receptor materials with unprecedented hierarchical structure for determination of human serum albumin (HSA) are designed and fabricated. For that purpose a new hierarchical template is prepared. This template allowed for simultaneous structural control of the deposited molecularly imprinted polymer (MIP) film on three slenght scales. A colloidal crystal templating with optimized electrochemical polymerization of 2,3’-bithiophene enables deposition of an MIP film in the form of an inverse opal. Thickness of the deposited polymer film is precisely controlled with the number of current oscillations during potentiostatic deposition of the imprinted poly(2,3’-bithiophene) film. Prior immobilization of HSA on the colloidal crystal allows formation of molecularly imprinted cavities exclusively on the internal surface of the pores. Furthermore, all binding sites are located on the surface of the imprinted cavities at locations corresponding to positions of functional groups present on the surface of HSA molecules due to of prior derivatization of HSA molecules with appropriate functional monomers. This synergistic strategy results in a material with superior recognition performance. Integration of the MIP film as a recognition unit with a sensitive extended-gate field-effect transistor (EG-FET) transducer leads to highly selective HSA determination in the femtomolar concentration range.
... Methods to fabricate these types of biosensors include surface initiated atom radical polymerization (ATRP), amine coupling of MIP nanoparticles, photopolymerization of films using water compatible cross-linker and monomers, and amine coupling of MIP-NPs (Altintas et al., 2016;Jing et al., 2016;. A number of MIP-based SPR biosensors for detection of food analytes have been reported in the literature (Lautner et al., 2011;Matsui et al., 2009). Yao et al. reported the use of a MIP based SPR sensor for the detection of pesticide residues (Yao et al., 2013). ...
... In the "top-down" approach of surface imprinting the target protein is attached to a support or mold, which is removed after the formation of the polymer layer. Stereochemically complementary cavities are left behind on the inner surfaces [53][54][55]. ...
Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either “evolution in the test tube” of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the “biological” degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application.
... For this, the imprinting process should mimic "enzyme" for the molecular recognition in order to orient synthetic protocol from organic phase to aqueous phase to render water compatible biomimetic MIP receptor. Despite some attempts undertaken in the recent past to obtain water compatible MIPs for small and macromolecular imprints [10][11][12][13][14][15][16][17][18][19], water compatibility of MIPs still remains to be a challenge. To ensure better water compatibility in this work, we propose to use a water-soluble functional monomer and a cross-linker for synthesizing an imprinted receptor selective for the target (5,6-dihydrouracil, UH 2 ) analyte. ...
In this work, a novel water compatible molecularly imprinted polymer was synthesized in bead shape by the precipitation polymerization technique. Immobilization of such imprinted beads on the surface of quartz crystal microbalance (QCM) was made through Au-S links using the “surface-grafting to” approach. The imprinted beads revealed a superiority in terms of recapture ability of analyte in the large concentration range and sensitivity as compared to traditionally imprinted monolith films. The piezoelectric analysis of target analyte (5,6-dihydrouracil) with imprinted polymer beads was found to be highly selective and sensitive, without any cross-reactivity and false-positives, in real samples. A perfect linearity between the frequency shift and the analyte concentration was observed in the range 0.03–1.75 μM, with detection limits varying between 0.003–0.006 μM (S/N = 3) [cf., imprinted film modified QCM: 0.04–1.32 μM, limit of detection 0.01 μM (S/N = 3)]. The endogenous 5,6-dihydrouracil level was also obtained in the blood plasma sample which may help exploring concerned dihydropyrimidine dehydrogenase activity, on the basis of concentration ratio [dihydrouracil]/[uracil] in cancer patients undergoing chemotherapy with 5-fluorouracil supplementation.
... For effective removal of the target molecule from the polymer and accessibility for rebinding, the binding sites should be located on the surface of the polymers. Therefore, so-called surface imprinting techniques have been developed, where the template is imprinted in ultra-thin polymer films [9,[12][13][14]. Uniform orientation of the templates during polymerization is a prerequisite for homogeneous binding sites [12,15]. ...
Natural evolution has created biopolymers on the basis of amino acids and nucleotides showing high chemical selectivity and catalytic power. Molecular recognition by antibodies and catalytic conversion of the substrate molecules by enzymes take place in so called paratopes or catalytic centres of the macromolecule which comprise typically 10-15 amino acids. The concerted interaction between the reaction partners result in affinities down to nanomolar concentrations for the antigen binding and approaches one million turnovers per second in enzyme-catalyzed reactions.
... One of the major promises of nanoplasmonics for sensing [1][2][3] has been the miniaturization of the sensing elements made of metal nanostructures to allow for the sensing and detection with wide applications in biomedical diagnostics, drug discovery, and environmental sensing of a single or very few objects with nanoscale dimensions [4][5][6][7]. Propagating surface plasmon resonance (SPR) on a noble metal thin film is sensitive to a change in refractive index near the metal surface and causes a resonance angle or wavelength shift. ...
We predict a novel multispectral spatial and frequency selective sensing based on a plasmonic sub-diffraction-limited (< λ/4) nanostructure. Via combining the strong plasmon near-field coupling effects, particle plasmon resonances (PPRs) and their hybridization by the metallic cross-shaped antennas (CSAs), a total of seven resonances with the minimum bandwidth < 7 nm are obtained in the visible and near-infrared region. Remarkably distinct biosensing behaviors at different spatial locations and different resonant wavelengths are simultaneously achieved, suggesting a new impressive sensing motif. High-quality biosensing with the maximal sensitivity (S = 1134 nm/RIU), figure of merit (FoM∼71.4), and high contrast ratio of spectral intensity difference (ΔR = 44.2%) can be attained by detection a slight refractive index change of a thin biomolecular layer. These unique features of the proposed sensing motif could provide a powerful approach to develop desirable plasmon sensors with simultaneous multispectral spatial and frequency selective sensing, and hold potential applications in the high-integrated components for the high-performance plasmonic biosensing, detection and imaging.
... Lautner et al. [46] prepared avidin-imprinted microstructures to bind avidin. The dissociation constants were found in the submicromolar range (125 nM). ...
In the past five years, significant progress has been made in preparation of various molecularly imprinted polymer (MIP)-based materials for applications in bioassays and biotransformation. This chapter reviews the important advances in these two fields. The first part mainly focuses on the development of various MIP-based bioassays that convert the rebinding of template to the imprinted cavities into measurable luminescent signals, including fluorescence, phosphorescence, Raman scattering, diffraction, and the like. In addition, MIP-based bioassays that are measured by surface plasmon resonance or quartz crystal microbalance are also discussed. In the following part, representative biotransformation reactions that make use of MIPs are summarized. In the last part of this chapter, some remaining challenges are briefly discussed for further development of the two fields.
Graphical Abstract
Phycocyanin, a macromolecular protein known for its robust fluorescence, proves to be highly suitable for verifying the successful deposition of imprinted layers. In this study, an acid-propelled magnetic micromotor was successfully fabricated by utilizing surface imprinting and self-propelled nanomotor technology to achieve selective loading and capture of targets such as phycocyanin for future applications in environmental monitoring and precision drug delivery in vivo. This micromotor features a distinct recognition layer achieved through a template electrodeposition method. The outermost imprint layer of the micromotor was meticulously crafted using poly(3,4-ethylenedioxythiophene)/poly(sodium-4-styrenesulfonate) in the presence of a template, while the Pt layer serves as the supportive foundation, the Ni layer acts as the magnetic guidance component, and the innermost layer consists of metal Zn. In acidic environments, the Zn reacts to generate bubbles, which propels the micromotor's motion. The micromotor was comprehensively characterized using techniques such as scanning electron microscopy. Findings highlight the exceptional self-propulsion of the Zn-based micromotor, which is a fusion of molecular imprinting and micromotor technologies. This innovative design achieves an impressive maximum velocity of approximately 100 μm s-1, as well as commendable magnetic steering performance. Furthermore, the micromotor demonstrates the ability to imprint target protein through the imprint layer, enabling selective recognition and capture for transport of specific phycocyanin. In vitro cytotoxicity tests have also demonstrated that the micromotors are non-toxic to cells. This breakthrough concept offers a novel avenue for realizing targeted capture and transport of specific nutrients within the human gastric environment.
To address the lack of functional monomer diversity for the electrosynthesis of protein‐selective molecularly imprinted polymers (MIPs), we introduce a new concept able to lead to a new class of functional monomers. This is based on conjugating an electropolymerizable monomer unit (umbelliferone) to an amino acid for closer mimicking of protein‐based natural affinity ligands such as antibodies. As the first representative of this class of monomers an aspartate‐umbelliferone (Asp‐UMB) conjugate was synthesized and here we provide the proof for its suitability to generate highly affine MIPs for proteins by epitope imprinting. As model we used a heptapeptide (GFNCYFP) stemming from the receptor binding domain (RBD) of the SARS‐CoV‐2 spike protein to generate epitope imprinted polymers able to recognize the parent RBD protein.
Inspired by the recognition mechanism of biological molecules, molecular imprinting techniques (MITs) are imparted with numerous merits like excellent stability, recognition specificity, adsorption properties, and easy synthesis processes, and thus broaden the avenues for convenient fabrication protocol of bio‐inspired molecularly imprinted polymers (MIPs) with desirable functions to satisfy the extensive demands of biomedical applications. Herein, the recent research progress made with respect to bio‐inspired imprinting materials is discussed in this review. First, the underlying mechanism and basic components of a typical molecular imprinting procedure are briefly explored. Then, emphasis is put on the introduction of diverse MITs and novel bio‐inspired imprinting materials. Following these two sections, practical applications of MIPs in the field of biomedical science are focused on. Last but not least, perspectives on the remaining challenges and future development of bio‐inspired imprinting materials are presented.
In this study, a novel fibrous-like molecularly imprinted polymer film (F-MIP)-based QCM sensor was developed for the detection of methyl-4-chlorophenoxyacetic acid (MCPA). An electrospinning process was used to create a poly(N-tert-butylacrylamide) (TBAm) nanofiber membrane that would be used as the master mold for the creation of a polydimethylsiloxane (PDMS) replica mold. Further, this replicated mold was used for the fabrication of an F-MIP film on a gold-coated quartz crystal (QC) substrate via microcontact imprinting. One of the well-known functional monomers, methacrylic acid (MAA), and ethylene glycol dimethacrylate were used as functional monomer and crosslinker for the F-MIP films, respectively. The imprinted polymer network from the MIP precursor solution including the MCPA template was formed via photopolymerization during mold contact on the QC substrate. The diameter of the poly(TBAm) nanofiber used as a membrane mold controlled the fibrous morphology on the MIP film, which was investigated using field emission scanning electron microscopy. The binding behaviors of MCPA on the MIP-QCM sensor were evaluated by measuring the change in resonant frequency (Δf) values of F-MIP/NIP films with analyte adsorption in MCPA solution with a concentration range of 0.02–200 ng/mL. Furthermore, the sensing responses on the film were used to calculate the limit of detection, the limit of qualification, and the imprinting factor. The selectivity of the films was determined by comparing their sensing responses to similar herbicides. Despite the effect of nonspecific adsorption on both films due to the functional groups on MAA monomer, the imprinted film exhibited higher sensing properties and amplified sensing responses due to the fibrous-like MIP film’s increased surface area. As a result, this sensor is expected to be a better option than another analytical method for detecting herbicides.
This article describes biomimetic sensors, a class of chemical sensing and biosensing devices that adapt synthetic components that mimick the function of biomacromolecules such as antibodies and biological receptors. The biomimetic sensors offer unique advantages as they often exhibit higher stability and/or they can be manufactured with lower costs in comparison to their biological counterparts. This article aims to introduce the previously reported works on biomimetic sensors with a particular focus on the transducers being used for signal readout, types of biomimetic components being used, and how each biomimetic components are prepared and coupled with the transducers.
Food safety is the prime area of concern that builds trust. With the prevailing advancements, it has become facile to ensure safety in almost all aspects. Technology has grown from tedious lab techniques to modern chromatographic techniques and immunoassays, progressed with more precise and rapid sensing through the advent of Biosensors. Biosensors provide an automated technology by presenting superfast, nondestructive and cost-effective detection in food analysis. SPR biosensor is an optical biosensor known for its versatility and has wider applications in food testing and analysis. It has an optical system for excitation and interrogation of surface plasmons, and a biomolecular recognition element to detect and seize the target analyte present in a sample. The optical signal detects the binding analyte, on the recognition element, which results in a change in refractive index at the surface and modifies the surface plasmons' propagation constant. SPR aids in label-free detection of various components such as adulterants, antibiotics, biomolecules, genetically modified foods, pesticides, insecticides, herbicides, microorganisms and microbial toxins in food and assures safety. The distinct advancements of SPR in food analysis have been found and discussed. The review also provides knowledge on the advantages and the key challenges encountered by SPR.
Patterned conducting polymer films with unique structures have promising prospects for application in various fields, such as actuation, water purification, sensing, and bioelectronics. However, their practical application is hindered because of the limitations of existing construction methods. Herein, a strategy is proposed for the superhydrophobic-substrate-assisted construction of free-standing 3D microcavity-patterned conducting polymer films (McPCPFs) at micrometer resolution. Easy-peeling and nondestructive transfer properties are achieved through electrochemical polymerization along the solid/liquid/gas triphase interface on micropillar-structured substrates. The effects of the wettability and geometrical parameters of the substrates on the construction of McPCPFs are systematically investigated in addition to the evolution of the epitaxial growth along the triphase interface at different polymerization times. The McPCPFs can be easily peeled from superhydrophobic surfaces using ethanol because of weak adhesion and nondestructively transferred to various substrates taking advantage of the capillarity. Furthermore, sensitive light-driven McPCPF locomotion on organic liquid surfaces is demonstrated. Ultimately, a facile strategy for the construction of free-standing 3D microstructure-patterned conducting polymer films is proposed, which can improve productivity and applicability of the films in different fields and expand the application scope of superwettable interfaces.
As importance of early diagnosis, prognosis, and clinical monitoring in improving survival rate of lung cancer is increasing, the advance of effective diagnostic method for tumor marker is crucial. Herein, linear poly(ε-caprolactone)-b-poly(ethylene oxide) (PCL-PEO) copolymer with hydrophilic property is applied to detect CYFRA 21-1 in label-free electrochemical immunoassay. 4-(2-trimethylsilylethynyl)benzoic acid (TEB) as “bridge” bond first connects initial antibody to the surface of gold electrode. Then one side of CYFRA 21-1 antigen can recognize with initial antibody of electrode interface, and the other side hybridize specifically secondary antibody grafted PCL-PEO copolymer. So far we have constructed a complete immunoassay for CYFRA 21-1 detection using electrochemical impedance spectroscopy. The sensitivity and selectivity of analytical method depend on “Au–C” bonding, specific recognition of “antibody-antigen-antibody sandwich”, and PCL-PEO copolymer. Under optimal conditions, the analytical method for quantitative detection of CYFRA 21-1 manifests a broad linear range (1 pg/mL – 10 ng/mL) and a low detection limit (0.125 pg/mL, about 3.47 × 10⁻¹⁵ M). Furthermore, according to the analysis results in clinical samples, the constructed strategy also shows satisfactory sensitivity and anti-interference. It indicates that the immunoassay has broad promising applications for expecting to be applied in practical clinical prevention and monitoring.
Molecularly imprinted polymers (MIPs) are biomimetic materials that garnered great interest in many fields including biomedicine. MIPs can mimic physiologic recognition units such as antibodies and receptors giving a resourceful platform for biomedical applications. They possess attractive advantages due to their high specificity and selectivity, stability under critical conditions, long shelf life, and their economic efficiency compared to routinely used molecules such as antibodies, proteins, and aptamers. MIPs can be prepared for targets lacking any available recognizing molecules. MIPs have been used for many applications such as biosensors, nanocarriers, and drug delivery systems, diagnostic and imaging. Of interest to the current chapter, the use of MIPs as nanoprobes for targeting, diagnosis, and treatments are discussed. An overview of the synthesis methods is summarized while a comprehensive highlight of MIP-based probing applications is presented. MIPs utilization is still in its infancy, and given the positive results seen through the current research present in the field, we believe that many interesting discoveries will be uncovered in the next future.
To elucidate the secretary function of immune cells, we develop a nanoplasmonic circular interferometric biosensor based on intensity interrogation for label-free and dynamic sensing of molecular secretion from a small population of cells. Exceptional sensitivity is achieved through coupling free light and surface plasmon polariton (SPPs) waves, which generates a constructive and deconstructive interference pattern with high contrast and narrow linewidth when illuminated by white light. Alternatively, by adopting a narrow-band LED source and a CCD camera, the transmission intensity of multiple sensing units can be monitored simultaneously with a simple collinear optical setup. This intensity-modulated sensing platform yields a resolution of 4.1×10⁻⁵ refractive index unit (RIU) with a high temporal resolution of 1s and a miniaturized footprint as small as 9.8×9.8 µm² for a single sensing unit. By integrating the signals from multiple sensor units, the resolution of a 12×12 sensor array was found to reach 7.3×10⁻⁶ RIU. We apply this sensor array to detect matrix metalloproteinase 9 (MMP-9) secretion from human monocytic cells, THP-1, at different time points after lipopolysaccharide (LPS) simulation and the results are in good agreement with enzyme-linked immunosorbent assay (ELISA) tests, but without the need for labeling. The spatial, temporal and mass resolutions of the sensor array are found to exceed other label-free technologies. These biomolecular arrays, incorporated in a microfluidic sensor platform, hold great potential for the study the dynamics and interplay of cell secretion signals and achieve a better understanding of single cell functions.
There is great interest in the preparation of synthetic receptor-based recognition units for cheap, robust, economic, and selective chemical sensors. Molecular imprinting provides the technology to prepare these synthetic units. There are still more and more syntheses of artificial molecular recognition constructs using analytes or their close structural analogues as templates for molecular imprinting. Stability of complexes of these constructs with the target analytes are often similar to those of biological receptors. Therefore, subsequent polymerization of these complexes results in molecularly imprinted polymers (MIPs) that have a selectivity close to that revealed by natural receptors.
The book summarizes the latest developments and applications of molecular imprinting for selective chemical sensing with each chapter devoted to different analytical applications of molecularly imprinted polymers. Specific chapters include: designing of molecular cavities aided by computational modelling, application of molecularly imprinted polymers for separation as well as sensing of pharmaceuticals and nucleotides.
The book is suitable for academics, postgraduates, and industrial researchers active in analytical chemistry, synthetic organic chemistry, molecular recognition, electrochemistry, and spectroscopy.
A chemical sensor based on molecularly imprinted polymer (MIP) and quartz crystal microbalance (QCM) to detect amoxicillin (AMO) antibiotics in aqueous samples was developed. The thin film of AMO-MIP was generated electrochemically from meta-phenylenediamine (mPD) directly on the QCM transducer. Pre-polymerization complex formation between the template (AMO) and the monomer molecules (mPD) was confirmed by a combination of computational modeling and spectroscopic studies. The electrodeposition process was carefully studied to allow for the selection of the optimal parameters for stable AMO-MIP film deposition. The AMO-MIP QCM sensor showed a significantly better sensitivity and affinity than the reference film displaying more than seven times relative adsorption capacity and a limit of detection down to 0.2 nM. Likewise, the sensor demonstrates good selectivity to the target analyte (AMO) than the other non-templated molecules and remain sensitive to the target even after a prior exposure to other interferents that may be present within the same environment. This remarkable result in the analysis of amoxicillin on QCM sensor without employing any signal amplification methodology demonstrates an important step towards the fabrication of MIP-based environmental sensor.
The rapidly emerging field of organic bioelectronics exploits the functional versatility of conducting polymers to transduce biological recognition events into electronic signals. For the majority of biosensors or biomedical devices, immobilization of a biorecognition element is a critical step to improve the biotic/abiotic interface. In this work, a simple strategy is described to construct large-area all-plastic poly(3,4-ethylenedioxythiophene) (PEDOT) electrodes displaying carbohydrate biorecognizable motifs. First, the method involves the preparation of PEDOT-poly(allylamine) composites through supramolecular interactions. It is demonstrated by Raman, X-ray spectroscopy and cyclic voltammetry that the PEDOT-poly(allylamine) ratio and the film electoactivity can be easily controlled. Then, carbohydrate motifs are covalently anchored to the primary amine groups by a straightforward route using divinylsulfone chemistry. The recognition-driven assembly of the lectin concanavalin A (Con A) and the glycoenzyme glucose oxidase (GOx) onto mannosylated surfaces is demonstrated by surface plasmon resonance spectroscopy. Furthermore, the bioelectrocatalytic glucose detection mediated by the assembled enzyme is studied for all-plastic and gold electrodes. Interestingly, the synergistic combination of conducting polymers and recognition-directed assembly leads to a 2.7-fold enhancement of the bioelectrocatalitic signal. Finally, it is proved that Con A/GOx nanoarchitectures can be constructed onto PEDOT platforms using the layer-by-layer technique.
Molecularly imprinted polymers (MIPs) have been prepared mostly for low-molecular-weight biomarkers and drugs but also for a spectrum of proteins. As compared with antibodies, MIPs have higher chemical and thermal stability, and they can be regenerated for repeated measurements. Electrochemical methods dominate the read-out of MIP sensors. Many protein MIPs have been tested in artificial urine or spiked semi-synthetic plasma, and point-of-care detection of marker proteins e.g. for cardiac, cancer, Alzheimer’s disease or virus infections is the prospective aim.
In the following chapter, the preparation and analytical performance of a broad spectrum of MIP sensors for protein biomarker are presented. The examples are grouped according to the respective diseases. For the majority of biomarkers, different approaches of sensor preparation and signal read-out can be compared.
Molecularly imprinted polymers (MIPs) rendered selective solely by the imprinting with protein templates lacking of distinctive properties to facilitate strong target-MIP interaction are likely to exhibit medium to low template binding affinities. While this prohibits the use of such MIPs for applications requiring the assessment of very low template concentrations, their implementation for the quantification of high-abundance proteins seems to have a clear niche in the analytical practice. We investigated this opportunity by developing a polyscopoletin-based MIP nanofilm for the electrochemical determination of elevated human serum albumin (HSA) in urine. As reference for low abundance protein ferritin-MIPs were also prepared by the same procedure. Under optimal conditions, the imprinted sensors gave a linear response to HSA in the concentration range of 20-100 mg/dm³, and to ferritin in the range of 120-360 mg/dm³. While as expected the obtained limit of detection was not sufficient to determine endogenous ferritin in plasma, the HSA-sensor was successfully employed to analyse urine samples of patients with albuminuria. The results suggest that MIP-based sensors may be applicable for quantifying high abundance proteins in a clinical setting.
A new ultra sensing molecularly imprinted polymer beads modified pencil graphite electrode was fabricated, with the help of the inverse suspension polymerization technique, for ascertaining the adequate supplementation of dacarbazine in the cancer treatment. The inverse suspension polymerization technique was beneficial in obtaining surface imprinted polymer-based electrocatalytic nanospheres with narrow size distribution. These nanospheres were found to be superior to the corresponding microspheres and planar films, in terms of electrode kinetics and sensitivity, with the differential pulse anodic stripping voltammetric transduction. Herein, multiwalled carbon nanotubes functionalized ester links were invoked in between the imprinted nanospheres and the pencil graphite electrode surface to secure a stable coating and better electrodics. The proposed electrochemical sensor showed the imprinting factor and the analyte adsorption coefficient as high as 24.3 and 1.06×10⁹ L mol⁻¹, respectively. Furthermore, 16-fold and 4-fold faster electron transfer kinetics were observed with the imprinted nanospheres than the corresponding imprinted planar film and the microspheres based electrodes, respectively. The limits of detection [0.02 (aqueous), 0.02 (plasma), 0.01 (urine), and 0.03 ng mL⁻¹ (pharmaceutics), (3σ, RSD ≤ 0.23%)] of dacarbazine, realized with the imprinted polymer nanospheres, were free from any cross-reactivity and false-positive complications in aqueous, blood plasma, urine, and pharmaceutical samples.
Quantitative determination of biomacromolecules has great significance. Quartz crystal microbalance sensors are favored by their sensibility while their selectivity was limited. Molecularly imprinted polymer has great selectivity performance. By combining the advantages of quartz crystal microbalance and molecularly imprinted polymer, this work established an epitope molecularly imprinted polymer based quartz crystal microbalance sensor. Using the epitope of human serum albumin as the template, the epitope molecularly imprinted polymer (EMIP) was successfully synthesized. Then a simple coating method was applied to fabricate the epitope molecularly imprinted polymer based quartz crystal microbalance sensor (EMIP-QCM). The EMIP-QCM sensor had good sensibility and selectivity to the target human serum albumin. The linear range was from 0.050 μg mL⁻¹ to 0.500 μg mL⁻¹ and the detection limit was calculated to be 0.026 μg mL⁻¹. In addition, it could be used in real sample analysis and had good accuracy and reproducibility. This method proposed a general and simple way of establishing quartz crystal microbalance sensors for sensitive determination of biomacromolecules.
Biosensing and diagnostics have attracted significant attention within the field of modern analytical chemistry due to their high sensitivity and specificity, and they have been successfully used in many practical application fields such as health care, food safety, and environmental control. This chapter presents a detailed summary of the research activity in the field of molecular imprinting technique (MIPs) for biomimetic sensing and diagnostics. MIPs are prepared by the MIPs, which can easily lead to the formation of specific nanosized cavities by means of template-directed synthesis. MIPs have been extensively studied as promising alternatives to biological receptors within the field of analytical chemistry for the purposes of sensing and diagnostics mainly in two research directions, that is, MIP-based immunoassays and biomimetic chemosensors. The chapter discusses the MIP-based chemosensors based on their transduction formats, which mainly can be classified into electrochemical, fluorescent, surface plasmon resonance (SPR), and quartz crystal microbalance (QCM) sensors.
This book addresses in an integrated manner all the critical aspects for building the next generation of biorecognition platforms - from biomolecular recognition to surface fabrication. The most recent strategies reported to create surface nano and micropatterns are thoroughly analyzed. This book contains descriptions of the types of molecules immobilized at surfaces that can be used for specific biorecognition, how to immobilize them, and how to control their arrangement and functionality at the surface. Small molecules, peptides, proteins and oligonucleotides are at the core of the biorecognition processes and will constitute a special part of this book. The authors include detailed information on biological processes, biomolecular screening, biosensing, diagnostic and detection devices, tissue engineering, development of biocompatible materials and biomedical devices. © Springer International Publishing Switzerland 2015. All rights reserved.
As introduced in Chap. 1 many biomolecules are involved in molecular recognition processes. These molecules include proteins, peptides, and nucleic acids. In the current chapter we introduce in detail the different recognition molecules found in nature. The chapter analyzes the recognition processes that they mediate and the key aspects of such recognition, including affinity and specificity. Finally, we hint about the tools that can be used in order to modify and expand the natural biorecognition diversity. The chapter aims to provide an overview of the biomolecular complexity and the array of biorecognition functions available in nature or by design, focusing mostly into the two major recognition moieties: proteins and peptides and nucleic acids. We cover some of the biorecognition pairs that are used in the applications described in the following book chapters.
Molecularly imprinted polymers (MIPs) with specific molecular recognition property for target analytes have attracted great attention in food safety filed. Molecularly imprinted technology (MIT) have been widely employed to produce stable, robust and cheap MIP materials which possess selective binding sites for recognition of target analytes in food, such as pesticide, veterinary drugs, mycotoxins, illegal drugs and so on. In this review, the recent developments of MIPs in various applications for food safety, including sample preparation, chromatographic separation, sensing and immunoassay etc, have been summarized. We particularly discuss the problems and advancements in these applications, as well as the attempts conducted for the improvement.
The synergistic effect of combining molecular imprinting and surface acoustic wave (SAW) technologies for the selective and label-free detection of sulfamethizole as a model antibiotic in aqueous environment was demonstrated. A molecularly imprinted polymer (MIP) for sulfamethizole (SMZ) selective recognition was prepared in the form of a homogeneous thin film on the sensing surfaces of SAW chip by oxidative electropolymerization of m-phenylenediamine (mPD) in the presence of SMZ, acting as a template. A special attention was paid to the rational selection of the functional monomer using computational and spectroscopic approaches. SMZ template incorporation and its subsequent release from the polymer was supported by IR microscopic measurements. Precise control of thicknesses of SMZ-MIP and respective non-imprinted reference films (NIP) was achieved by correlating the electrical charge dosage during electrodeposition with spectroscopic ellipsometry measurements in order to ensure accurate interpretation of label-free responses originating from the MIP-modified sensor. The fabricated SMZ-MIP films were characterized in terms of their binding affinity and selectivity toward the target by analyzing the binding kinetics recorded using SAW system. The SMZ-MIPs had SMZ binding capacity approximately more than eight times higher than the respective NIP and were able to discriminate among structurally similar molecules i.e. sulfanilamide and sulfadimethoxine. The presented approach for the facile integration of a sulfonamide antibiotic-sensing layer with SAW technology allowed observing the real-time binding events of the target molecule at nanomolar concentration levels and could be potentially suitable for cost effective fabrication of a multianalyte chemosensor for analysis of hazardous pollutants in aqueous environment.
A novel and simple protein molecularly imprinted electrochemical sensor (MIPs/CS/IL-GR/GCE) based on chitosan/ionic liquid-graphene modified glassy carbon electrode (CS/IL-GR/GCE) was fabricated via electrochemical polymerization, which could be used for the sensitive and selective detection of bovine serum albumin (BSA). The synergistic effects of chitosan, ionic liquid and graphene nanocomposites improved the electrochemical response and the sensitivity of the sensor. Scanning electron microscope (SEM), cyclic voltammetry (CV), electrochemical impedance spectrum (EIS) and differential pulse voltammery (DPV) were used to characterize the sensor and investigate the electrochemical response of the sensor. The prepared MIPs/CS/IL-GR/GCE exhibited a linear relationship between the changes of current response and the logarithms of BSA concentrations in the range from 1.0 × 10-10 to 1.0 × 10-4 g/L (R = 0.996) with a detection limit of 2 × 10-11 g/L. Moreover, the fabricated sensor possessed a high selectivity, good reproducibility, excellent stability and acceptable recovery, which indicating potential application in clinical field.
Molecularly imprinted polymer (MIP)-based synthetic receptors integrated with Surface Acoustic Wave (SAW) sensing platform were applied for the first time for label-free protein detection. The ultrathin polymeric films with surface imprints of immunoglobulin G (IgG-MIP) were fabricated onto the multiplexed SAW chips using an electrosynthesis approach. The films were characterized by analyzing the binding kinetics recorded by SAW system. It was revealed that the capability of IgG-MIP to specifically recognize the target protein was greatly influenced by the polymer film thickness that could be easily optimized by the amount of the electrical charge consumed during the electrodeposition. The thickness-optimized IgG-MIPs demonstrated imprinting factors towards IgG in the range of 2.8-4, while their recognition efficiencies were about 4 and 10 times lower toward the interfering proteins, IgA and HSA, respectively. Additionally, IgG-MIP preserved its capability to recognize selectively the template after up to four regeneration cycles. The presented approach of the facile integration of the protein-MIP sensing layer with SAW technology allowed observing the real-time binding events of the target protein at relevant sensitivity levels and can be potentially suitable for cost effective fabrication of a biosensor for analysis of biological samples in multiplexed manner.
Functional monomers are electropolymerized in the presence of the target analyte to create polymer layers that mimic the binding sites of biopolymers, e.g., antibodies, enzymes, or nucleic acids. In a biomimetic sensor for proteins the molecularly imprinted polymer (MIP) must be in close proximity to the surface of the signal-generating electrode. By using electropolymerization, thin MIP films can be obtained directly on the surface of the transducer. Nanosphere litography, template synthesis in microporores, and photolitography allow to prepare surface-confined cavities for the template analyte, e.g., strepavidin. Hybrid materials consisting of a molecularly imprinted layer on top of a self assembled monolayer exhibit increased affinity and specificity for the target analyte, e.g., lectins. Furthermore, spatial integration of enzymatic conversion of the analyte and recognition of the product by a MIP-sublayer allows interference-free measurements of drugs, e.g., aminopyrine.
Due to their size and difficulty to obtain cost/effective biological or synthetic receptors (e.g. antibodies or aptamers, respectively), organic toxic compounds (e.g. less than 1 kDa) are generally challenging to be detected using simple platforms such as biosensors. This study reports on the synthesis and characterization of a novel multifunctional composite material, magnetic silica beads/graphene quantum dots/molecularly imprinted polypyrrole (mSGP). mSGP is engineered to specifically and effectively capture and signal small molecules due to the synergy among chemical, magnetic and optical properties combined with molecular imprinting of tributyltin (291 Da), a hazardous compound, selected as a model analyte. Magnetic and selective properties of the mSGP composite can be exploited to capture and pre-concentrate the analyte onto its surface and its photoluminescent graphene quantum dots, which are quenched upon analyte recognition, are used to interrogate the presence of the contaminant. This multifunctional material enables a rapid, simple and sensitive platform for small molecule detection even in complex mediums such as seawater without any sample treatment.
Effective recognition of enzymatically active tetrameric acetylcholinesterase (AChE) is accomplished by a hybrid nanofilm composed of a propidium-terminated self-assembled monolayer (Prop-SAM) which binds AChE via its peripheral anionic site (PAS) and an ultrathin electrosynthesized molecularly imprinted polymer (MIP) cover layer of a novel carboxylate-modified derivative of 3,4-propylenedioxythiophene. The rebinding of the AChE to the MIP/Prop-SAM nanofilm covered electrode is detected by measuring in situ the enzymatic activity. The oxidative current of the released thiocholine is dependent on the AChE concentration from ≈0.04 × 10−6 to 0.4 × 10−6m. An imprinting factor of 9.9 is obtained for the hybrid MIP, which is among the best values reported for protein imprinting. The dissociation constant characterizing the strength of the MIP-AChE binding is 4.2 × 10−7m indicating the dominant role of the PAS-Prop-SAM interaction, while the benefit of the MIP nanofilm covering the Prop-SAM layer is the effective suppression of the cross-reactivity toward competing proteins as compared with the Prop-SAM. The threefold selectivity gain provided by i) the “shape-specific” MIP filter, ii) the propidium-SAM, iii) signal generation only by the AChE bound to the nanofilm shows promise for assessing AChE activity levels in cerebrospinal fluid.
Here we introduce microelectrospotting as a new approach for preparation of protein-selective molecularly imprinted polymer microarrays on bare gold SPR imaging chips. During electrospotting both the gold chip and the spotting tip are electrically connected to a potentiostat as working and counter electrodes, respectively. The spotting pin encloses the monomer-template protein cocktail that upon contacting the gold surface is in-situ electropolymerized resulting in surface confined polymer spots of ca. 500µm diameter. By repeating this procedure at preprogrammed locations for various composition monomer-template mixtures microarrays of nanometer-thin surface-imprinted films are generated in a controlled manner. We show that the removal and rebinding kinetics of the template and various potential interferents to such microarrays can be monitored in real-time and multiplexed manner by SPR imaging. The proof of principle for microelectrospotting of electrically insulating surface-imprinted films is made by using scopoletin as monomer and ferritin as protein template. It is shown that microelectrospotting in combination with SPR imaging can offer a versatile platform for label-free and enhanced throughput optimization of the molecularly imprinted polymers for protein recognition and for their analytical application.
Copyright © 2015 Elsevier B.V. All rights reserved.
Synthetic materials capable of selectively recognizing proteins are important in separations, biosensors and the development of biomedical materials. The technique of molecular imprinting creates specific recognition sites in polymers by using template molecules. Molecular recognition is attributed to binding sites that complement molecules in size, shape and chemical functionality. But attempts to imprint proteins have met with only limited success. Here we report a method for imprinting surfaces with protein-recognition sites. We use radio-frequency glow-discharge plasma deposition to form polymeric thin films around proteins coated with disaccharide molecules. The disaccharides become covalently attached to the polymer film, creating polysaccharide-like cavities that exhibit highly selective recognition for a variety of template proteins, including albumin, immunoglobulin G, lysozyme, ribonuclease and streptavidin. Direct imaging of template recognition is achieved by patterning a surface at the micrometre scale with imprinted regions.
Molecular imprinting is an important tool for generating synthetic receptors with specific recognition sites. The resulting artificial receptor has extensive applications in chromatographic stationary phases, solid phase extraction, catalysis, drug delivery and sensors. The synthesis of molecularly imprinted polymers (MIPs) specific for proteins has been proved challenging due to a number of inherent problems in protein imprinting but potentially rewarding work. Hence, this review discusses recent advances in various synthetic protocols developed to overcome the obstacles, focusing on their advantages, disadvantages and potential future direction. In addition, the case of the selection of the functional monomers has also been reviewed here.
A sensor system based on the optical phenomenon of surface plasmon resonance (SPR), which employs either photothermal deflection spectroscopy (PDS) or a photodiode array (PDA) for detection, was developed to use molecularly imprinted (MI) polymethacrylic acid - ethylene glycol dimethacrylates (PMAA-EDMA) as the sensing element. The MI polymers were first processed by Soxhlet extraction to remove the print molecules (theophylline, caffeine, and xanthine), yielding the specific anti-polymers. Each anti-polymer was layered over a silver film to serve as the analysis surface for the molecularly imprinted sorbent assay (MIA) of one target drug. This surface was exposed for 60 min to an aqueous standard drug solution, dried in air, and the uptake of the print molecule into the anti-polymer was monitored by shifts in the SPR angle theta(r) (and hence the SPR-PDS signal measured at constant theta). The linear dynamic range of the MIA was found to extend up to 6 mg/mL, with a concentration detection limit estimated at 0.4 mg/mL for theophylline in aqueous solution. A cross-reactivity study of the anti-theophylline and anti-caffeine polymers, using eight other drugs structurally similar to theophylline and caffeine, showed none or very slight shifts in theta(r). This implies that the anti-polymers were selective only for their original print molecules and had no affinity for the other drug molecules. Similar molecular recognition characteristics were observed for the anti-xanthine polymer.
Molecular imprinting can be used to prepare heterogeneous catalysts which mimic their homogeneous counterparts.
A method for micro-contact imprinting CRP has been developed. An analogue, O-(-4-nitrophenylphosphoryl)choline, of the templates natural ligand, phosphorylcholine, was used as the functional monomer. A series of non-imprinted polymers made without template, but with varying cross-linking agents, were made in order to produce a control polymer with minimal non-specific recognition.The affinity of the imprinted polymers for the competing proteins, lysozyme and albumin, was examined. Their respective relative affinities, with 1.04μg/cm2 of the template protein bound to the imprinted surface as reference, were CRP/albumin=4.0 and CRP/lysozyme=346. Measurement of the film thickness showed it to be approximately 10μm.A competitive binding experiment showed the CRP imprinted material to retain good selectivity for its template when jointly incubated with human serum albumin and CRP. Using the same method we were able to form a micro-contact imprint of human serum albumin which demonstrated relatively good recognition for its own template 2.66μg/cm2 compared with 0.27μg/cm2 for CRP.
The use of organic silane monomers in the preparation of substrate-selective polymers by molecular imprinting is described. Silanes are allowed to polymerize on the surface of porous silica particles in aqueous solution. The resulting polysiloxane copolymer becomes covalently anchored to silanol groups of the original silica. such preparations retain the rigidity of the silica matrix and can therefore be used in high-performance liquid chromatography. Polysiloxane copolymers imprinted with the dyes rhodanile blue or safranine O showed preferential binding of the respective compound. The observed recognition is believed to occur because cavities containing specific binding groups for the dyes at defined positions are developed during the polymerization procedure. In this context the synthesis of a new silane, boronatesilane, was carried out. This compound was included in the monomer mixture used for the preparation of a polysiloxane-coated silica showing affinity for the glycoprotein transferrin. Organic silanes were also used for entrapment of enzymes, resulting in block polymers, which after fragmentation yielded relatively high recoveries of enzyme activity. Alternatively, the entrapment/polymerization was allowed to proceed on the surface of porous silica, in analogy with the imprinting procedure, resulting in entrapped enzyme preparations with high mechanical stability.
Synthetic materials capable of specifically recognition proteins are important in bioseparation and biosensors. In this study, bovine serum albumin-imprinted polyacrylamide gel beads were synthesized via inverse-phase seed suspension polymerization, using high-density crosslinked gel beads as core, low-density crosslinked polyacrylamide gel as imprinting shell. The surface of gel bead had a large quantity of well-distributed macropores, which were suited to let the proteins pass in and out. The selectivity test showed that imprinting gel beads exhibited good recognition for template proteins, as compared to the control protein. We consider the formation of multiple hydrogen bonds and complementary shape between the imprinting cavities and the template proteins are the two factors that lead to the imprinting effect. The imprinting beads had quick adsorption rate and possessed improved regeneration property in comparison with those prepared directly via inverse-phase suspension polymerization.
Molecularly imprinted polyurethanes are presented as sensitive coatings for the detection of polycyclic aromatic hydrocarbons in water. These sensor layers were combined with fluorescence and mass-sensitive transducers. Imprinting based on van der Waals interactions allows detection of these analytes even without any pronounced functionality. The geometry of the imprint molecule determines the selectivity of the sensor layer. In varying the size of template molecules from anthracene up to 1,12-benzoperylene, selectivity is tuned to a distinct analyte. The enrichment factor of up to approximately 107 renders detection down to the ppt range possible with hardly any matrix effect by humic acids.
The construction and operation of fiber-optic sensing devices based on molecularly imprinted polymers are reported. Fiber-optic detection of an amino acid derivatized with a fluorescent labeling group (dansyl-L-phenyl-alanine) is demonstrated. The advantages of using molecularly imprinted polymers as artificial recognition systems in sensor technology are discussed.
A new approach to sample enrichment and analyte determination is reported. An imprinted dispersion polymer capable of molecular recognition of pentamidine (PAM), a drug used for the treatment of AIDS-related pneumonia, was used in solid-phase extraction in order to selectively retain PAM from a dilute solution. At a physiological concentration (30 nM) this gave an enrichment factor of 54 using a PAM-selective polymer whereas the enrichment factor on a benzamidine- (BAM-) imprinted reference polymer was only 14. The high selectivity of the polymer allowed the drug to be detected directly in the desorption step, thus eliminating the need for a successive chromatographic analysis. In this way,; PAM could be enriched and directly analyzed when present in low concentration in a urine sample.
This article reports a novel method to significantly enhance the conductivity of conducting poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) films through a treatment with aqueous solutions of various salts, such as copper(II) chloride. Conductivity enhancement by a factor of about 700 was observed. Many salts were investigated, and the conductivity enhancement depended on the softness parameter of cations and the concentration of the salts in solution. A salt like copper(II) chloride or indium chloride, whose cation has positive softness parameter, could enhance the conductivity of the PEDOT:PSS film by 2 orders in magnitude, while other salt like sodium chloride or magnesium chloride, whose cation has negative softness parameter, gave rise to negligible effect on the conductivity. The mechanism for the conductivity enhancement was studied by various characterizations. It is attributed to PSS loss from the PEDOT:PSS film, and conformational change of PEDOT chains resulted from the salt-induced charge screening between PEDOT and PSS.
Over the past two decades, molecularly imprinted polymers (MIPs) have attracted broad interest from scientists engaged in sensor development. This attention can be explained by the serious potential advantages of using MIPs in place of natural receptors and enzymes such as their superior stability, low cost and easy preparation. This review encompasses recent achievements in molecular imprinting related to the area of sensor technology. Since electrochemical biosensors dominate the market and due to specific requirements of this journal, the emphasis of this review will be on the development of electrochemical MIP sensors. The problems associated with application of imprinted polymers in sensors are highlighted and possible solutions indicated. The commercial potential of MIP-based sensors is analyzed in the expectation that they can offer improved performance in the analytical market place.
A study was conducted to demonstrate surface-imprinted conducting polymers (SIP) microrods for selective protein recognition. Precisely sized cylindrical pores of a track-etch polycarbonate membrane (PCM) filter served as sacrificial microreactor for the synthesis. It was observed that native membranes were hydrophobic in nature and absorbed protein molecules, offering a method for fixing the target protein onto the pore walls by simple physical adsorption. Conducting polymer rods of poly-3,4-ethylenedioxythiophene (PEDOT) doped with polystyrene sulfonate (PSS) were electrochemically grown in the microreactors positioned on the surface of a gold electrode. The PCM was easily removed by dissolution in chloroform after polymerizing the microrods in the pores. The removal of the sacrificial material resulted in the formation of microrods confined to the surface of the gold electrodes ossessing the imprint of the target protein on their surface.
In a previous paper we presented preliminary experiments aimed at the preparation of gel particles with the property to recognize
selectively some particular protein (hemoglobin, cytochrome C, transferrin) [1]. Using the same method we show in this article
that human growth hormone, ribonuclease and myoglobin from horse can also be adsorbed specifically, indicating that the method
may be universal or at least applicable to a great number of proteins. A gel with specific adsorption of three model proteins
was synthesized in order to demonstrate that the beds can be employed to remove (traces of) several proteins contaminating
a sample (“negative purification”). The degree of selective recognition is high, to judge from the fact that myoglobin from
horse, but not that from whale, was adsorbed onto a column designed to bind specifically the former protein. This selectivity
is noteworthy, since these two proteins have similar amino acid sequences and 3-D structures. The method for the synthesis
of the specific gels involves polymerization of appropriate monomers (for instance acrylamide and its derivatives) in the
presence of the protein to be adsorbed specifically, granulation of the gel formed, packing a column with the gel particles,
washing the column to remove the protein and finally application of the sample for selective adsorption of the protein. The
approach resembles that used for entrapment (immobilization) of proteins for affinity chromatography and that for molecular
imprinting, with the distinct difference that the monomer composition is quite different and thereby the binding mechanism.
This mechanism is discussed, for instance, in terms of (1) a new classification system for chromatographic beds based on the
number of bonds between the solute and the matrix and the strength of each bond and (2) “non-specific bonds” (these bonds
are often harmful in conventional chromatography, but we have used them to advantage). In this classification system the selective
recognition is characterized by a large number of weak bonds. Therefore, so-called functional monomers are not used for the
preparation of the gels because they often are charged and, accordingly, give rise to strong electrostatic interactions, i.e.
the beds behave to some extent as ion-exchangers. In most experiments we have used a polyacrylamide gel with large pores to
facilitate diffusion of proteins into and out of the gel granules. When used in chromatography these soft gels (which can
be used repeatedly) allow only rather low flow rates. This problem can be overcome by a new approach to prepare the granules.
Potential applications of the selective beds are discussed, as well as future improvements.
Molecular imprinting is a promising technique for the preparation of synthetic polymers of predetermined specificity. Functional monomers are copolymerized with crosslinkers in the presence of the desired molecule, the imprint molecule. The use of these polymers as chiral stationary phases is discussed. Other applications, such as antibody-mimics, enzyme-like catalysts and sensors, are also focused upon.
The resonance shifts (i.e., the resonance angle and the reflectance minimum) in surface plasmon resonance (SPR) curves due to the complex refractive index and/or thickness variations of dielectric films were investigated. For both, nonabsorbing and absorbing dielectrics, the resonance angle shifts linearly with the refractive index and/or thickness variations. The reflectance minimum of the nonabsorbing dielectric does not change as the resonance angle shifts. For an absorbing dielectric, the direction of the reflectance change depends strongly on the magnitude of the absorption and thickness of the metal film. The reflectance minimum of the sensor with a thin metal film decreases before increasing while that of the sensor with a thick metal film continuously increases as the absorption of the dielectric film increases. The phenomena were theoretically explained based on the SPR-generated evanescent field at the metal/dielectric interface associated with the optical properties of the sensor architecture.
Molecular imprinting involves the synthesis of polymers in the presence of a template to produce complementary binding sites with specific recognition ability. The technique has been successfully applied as a measurement and separation technology, producing a uniquely robust and antibody-like polymeric material. Low molecular weight molecules have been extensively exploited as imprint templates, leading to significant achievements in solid-phase extraction, sensing and enzyme-like catalysis. By contrast, macromolecular imprinting remains underdeveloped, principally because of the lack of binding site accessibility. In this review, we focus on the most recent developments in this area, not only covering the widespread use of biological macro-templates but also highlighting the emerging use of synthetic macro-templates, such as dendrimers and hyperbranched polymers.
"Plastic replicas" of natural antibodies are accessible by molecular imprinting procedures. The first step involves the synthesis of molecularly imprinted polymer (MIP) particles templated with human immunoglobulin. They then can be utilized as templates for a stamp-based surface imprinting procedure that results in polymer surface structures exactly mimicking the initial globulin. Compared to their natural counterparts, these artificial antibodies show improved selectivity and sensitivity on quartz crystal microbalance sensors. (Figure Presented).
(Figure Presented) An ultrasensitive SPR-detection of the explosive hexahydro-1,3,5-trinitro-1,3,5triazine (RDX) is achieved by composites of Kemp's acid molecularly imprinted Au nanoparticles crosslinked by bisaniline units on a Au surface (see figure).
We report that simple, synthetic organic polymer nanoparticles (NPs) can capture and clear a target peptide toxin in the bloodstream of living mice. The protein-sized polymer nanoparticles, with a binding affinity and selectivity comparable to those of natural antibodies, were prepared by combining a functional monomer optimization strategy with molecular-imprinting nanoparticle synthesis. As a result of binding and removal of melittin by NPs in vivo, the mortality and peripheral toxic symptoms due to melittin were significantly diminished. In vivo imaging of the polymer nanoparticles (or "plastic antibodies") established that the NPs accelerate clearance of the peptide from blood and accumulate in the liver. Coupled with their biocompatibility and nontoxic characteristics, plastic antibodies offer the potential for neutralizing a wide range of biomacromolecules in vivo.
Specific detection of virus strains by affinity-based bioassays is often limited by the availability of ligands able to differentiate among close homologues of virus coat proteins. As viruses are prone to mutation, the ligand generation should, in addition, be fast enough to allow rapid identification of new varieties. These two criteria are difficult to be fulfilled by antibodies; however, they open up opportunities for aptamer-based detection. Here we report on the feasibility of selectively detecting the apple stem pitting virus (ASPV) coat proteins (PSA-H, MT32) using original DNA aptamers. Surface plasmon resonance (SPR) imaging was used together with aptamer-modified sensor chips to optimize the aptamer immobilization for highest sensitivity and to characterize the aptamer-virus coat protein binding. Different parameters affecting this binding, such as the aptamer flanking, surface coverage, and type of spacer molecules, were identified and their influence was determined. A direct label-free method is proposed for assessing the ASPV based on the detection of the respective virus coat proteins in plant extracts.
A simple method for the preparation of core-shell micro/nanostructured magnetic molecularly imprinted polymers (MIPs) for protein recognition is described. Magnetic MIPs were synthesized by copolymering gamma-aminopropyltrimethoxysilane and tetraethyl orthosilicate at the surface of Fe(3)O(4) nanospheres, which were directly covalently bound with template molecule, bovine hemoglobin (BHb), through imine bond. Transmission electron microscopy and scanning electron microscopy images showed that the Fe(3)O(4) nanospheres with diameter about 50-150 nm were coated with the MIPs layer with average thickness about 10 nm, which enabled the magnetic MIPs to have a sensitive and fast magnetic response. The proximity between the thickness of MIPs layer and the spatial size of BHb indicated that the imprinted sites almost situated at the surface of magnetic MIPs, leading a rapid adsorption saturation within 1 h. And the adsorption amounts of magnetic MIPs toward BHb were estimated to be 10.52 mg/g at pH 6.5, which was 4.6 times higher than that of magnetic nonmolecularly imprinted polymers. Meanwhile, the result of selective test showed that the magnetic MIPs had an excellent recognition capacity to BHb compared to the other nontemplate proteins. Except for the spatial size complementary between BHb and the binding sites in magnetic MIPs, the electrostatic interaction also was proven to be an important factor for recognizing the imprinting molecule.
Hepatitis B surface antibody (HBsAb) imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-L-tyrosine methyl ester) (PHEMAT) film on the surface plasmon resonance (SPR) sensor chip was prepared for diagnosis of HBsAb in human serum. Gold SPR chip surface was modified with allyl mercaptane and, then, HBsAb-imprinted PHEMAT film was formed on the chip surface. Surface characterization of the non-modified, allyl mercaptane modified and HBsAb-imprinted PHEMAT SPR chips were investigated with contact angle, atomic force microscopy (AFM). Kinetic studies were performed using HBsAb positive human serum. In order to determine the kinetic and binding constants, Scatchard, Langmuir, Freundlich and Langmuir-Freundlich models were applied to experimental data. Scatchard curve shows that HBsAb imprinted SPR chip has some surface heterogeneity, SPR chip obeyed the Langmuir adsorption model. The maximum detection limit was 208.2 mIU/mL. K(A) and K(D) values are 0.015 mIU/mL and 66.0 mL/mIU, respectively. Control experiments of the SPR chip were performed using non-immunized, HBsAb negative serum. The control experiment results show that SPR chip does not give any noticeable response to HBsAb negative serum.
The aim of the present work was to investigate the feasibility of employing the molecular imprinting polymer technique for detecting the mycotoxin zearalenone using a surface plasmon resonance (SPR) transducer. The molecularly imprinted polypyrrole (MIPPy) film was prepared by electropolymerization of pyrrole onto the bare Au chip in the presence of a template zearalenone molecule. The MIPPy-SPR sensor exhibited a linear response in the range of 0.3-3000 ng/mL (R (2) = 0.993) for detection of zearalenone. The selectivity efficiencies of zearalenone and other structurally related analogues were 1.0 and 0.15-0.27, respectively. The limit of detection and average recovery of blank corn matrix spiked with 30 ng/g zearalenone were 0.3 ng/g and 89%, respectively, and these were found to be comparable to those obtained by enzyme-linked immunosorbent assay. These results suggest that a combination of SPR sensing with MIPPy film is a promising alternative method for the detection of zearalenone.
The high affinity of the noncovalent interaction between biotin and streptavidin forms the basis for many diagnostic assays
that require the formation of an irreversible and specific linkage between biological macromolecules. Comparison of the refined
crystal structures of apo and a streptavidin:biotin complex shows that the high affinity results from several factors. These
factors include the formation of multiple hydrogen bonds and van der Waals interactions between biotin and the protein, together
with the ordering of surface polypeptide loops that bury the biotin in the protein interior. Structural alterations at the
biotin binding site produce quaternary changes in the streptavidin tetramer. These changes apparently propagate through cooperative
deformations in the twisted beta sheets that link tetramer subunits.
A new technique for coating microtitre plates with molecularly imprinted polymers (MIP), specific for low-molecular weight analytes (epinephrine, atrazine) and proteins is presented. Oxidative polymerization was performed in the presence of template; monomers: 3-aminophenylboronic acid (APBA), 3-thiopheneboronic acid (TBA) and aniline were polymerized in water and the polymers were grafted onto the polystyrene surface of the microplates. It was found that this process results in the creation of synthetic materials with antibody-like binding properties. It was shown that the MIP-coated microplates are particularly useful for assay development. The high stability of the polymers and good reproducibility of the measurements make MIP coating an attractive alternative to conventional antibodies or receptors used in enzyme linked immunosorbent assay (ELISA).
A technique for coating microplate wells with molecularly imprinted polymers (MIPs) specific for proteins is presented. 3-Aminophenylboronic acid was polymerized in the presence of the following templates: microperoxidase, horseradish peroxidase, lactoperoxidase, and hemoglobin, via oxidation of the monomer by ammonium persulfate. This process resulted in the grafting of a thin polymer layer to the polystyrene surface of the microplates. Imprinting resulted in an increased affinity of the polymer toward the corresponding templates. The influence of the washing procedure, template concentration, and buffer pH on the polymer affinity was analyzed. It was shown that the stabilizing function of the support and spatial orientation of the polymer chains and template functional groups are the major factors affecting the imprint formation and template recognition. Easy preparation of the MIPs, their high stability, and their ability to recognize small and large proteins, as well as to discriminate molecules with small variations in charge, make this approach attractive and broadly applicable in biotechnology, assays and sensors.
The use of biomimetic receptor systems capable of binding target molecules with affinities and specificities on a par with natural receptors is drawing significant interest. A review is made on recent advances and developments in the molecular imprinting area. Focus is on the application of molecularly imprinted polymers in sensors.
The development of new and efficient catalysts plays a central role in chemical research. The progress in synthetic work, both scientifically and technically, depends greatly on the quality of the catalysts. This review presents the status and the problems in the preparation of enzyme-like catalysts made by imprinting.
Molecularly imprinted polymers (MIPs) are synthetic polymers with a predetermined selectivity for a given analyte, or group of structurally related compounds, that make them ideal materials to be used in separation processes. In this sense, it is not surprising that the first applications of MIPs were as tailor-made chiral stationary phases in liquid chromatography. However, peak broadening and tailing, especially of the more retained enantiomers, were observed. Accordingly, this paper gives an overview of the attempts carried out during the recent years to improve the chromatographic performance of MIPs in liquid chromatography and capillary electrophoresis as well as the more recent applications. We conclude that MIPs are very promising materials to be used as selective stationary phases in chromatography although further developments are necessary in order to fully exploit their potential.
A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000. The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000. Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months.
Molecularly imprinted polymer gel with embedded gold nanoparticle was prepared on a gold substrate of a chip for a surface plasmon resonance (SPR) sensor for fabricating an SPR sensor sensitive to a low molecular weight analyte. The sensing is based on swelling of the imprinted polymer gel that is triggered by an analyte binding event within the polymer gel. The swelling causes greater distance between the gold nanoparticles and substrate, shifting a dip of an SPR curve to a higher SPR angle. The polymer synthesis was conducted by radical polymerization of a mixture of acrylic acid, N-isopropylacrylamide, N,N'-methylenebisacrylamide, and gold nanoparticles in the presence of dopamine as model template species on a sensor chip coated with allyl mercaptan. The modified sensor chip showed an increasing SPR angle in response to dopamine concentration, which agrees with the expected sensing mechanism. Furthermore, the gold nanoparticles were shown to be effective for enhancing the signal intensity (the change of SPR angle) by comparison with a sensor chip immobilizing no gold nanoparticles. The analyte binding process and the consequent swelling appeared to be reversible, allowing one the repeated use of the presented sensor chip.
In this paper, we present a technique for the preparation of polymer nanowires with the protein molecule imprinted and binding sites at surface. These surface imprinting nanowires exhibit highly selective recognition for a variety of template proteins, including albumin, hemoglobin, and cytochrome c. This recognition may be through a multistep adsorption, with the specificity conferred by hydrogen bonding and shape selectivity. Due to the protein imprinted sites are located at, or close to, the surface; these imprinted nanowires have a good site accessibility toward the target protein molecules. Furthermore, the large surface area of the nanowires results in large protein molecule binding capacity of the imprinted nanowires.
Surface plasmon resonance spectroscopy (SPR) was used to measure the adsorption kinetics and isotherms of dansylated amino acids onto surface-confined molecularly imprinted polymer films (MIP-Fs) and the corresponding non-imprinted polymer control films (NIP-Fs). The surface-confined polymer films were grafted from flat gold surfaces using atom transfer radical polymerization (ATRP). This approach allowed uniform nanothin films to be grown, thereby ensuring that the amino acids see a uniform surface during adsorption. N,N'-Didansyl-l-cystine (DDC) and didansyl-l-lysine (DDK) were used as the template molecules to form the MIP-Fs. Adsorption kinetics data were analyzed using single- and dual-site Langmuir adsorption models. It was found that, within the experimental measurement range, adsorption isotherm data were well described by any of four isotherm models: Langmuir, dual-site Langmuir, Freundlich, or Langmuir-Freundlich (LF). The relatively high heterogeneity index values regressed using the Freundlich and LF isotherms suggest the formation of fairly homogeneous MIP-Fs; although Scatchard analysis reveals binding site heterogeneity does exist. Selectivity studies showed that the MIP-Fs display cross-reactivity between DDC and DDK; nevertheless, MIP-Fs prepared against one template showed selectivity for that template. Solution pH and polymer layer thickness were studied as independent parameters to determine their impacts on amino acid adsorption, as monitored by SPR.
The performance of molecularly imprinted polymers (MIPs) is of interest to researchers in the field of analytical chemistry, and in the pharmaceutical and food industries. Because the choice of the functional monomer(s) plays a key role in the selectivity of a MIP, the synthesis of an effective, tight-binding MIP can be difficult and time-consuming, involving the evaluation of the binding performance of MIPs of many different compositions. In this study, we report an express method combining molecular imprinting and microcontact printing techniques to prepare a polymer thin film as an artificial antibody. In addition to the microcontact printing technique, isothermal titration of monomers to proteins stamps was investigated to screen the functional monomer for MIPs. Finally, the importance of the choice of cross-linking monomers in MIPs was studied, and these studies suggest that monomers containing an optimal length PEG spacer give higher imprinting effectiveness. Several model antigens (lysozyme, ribonuclease A and myoglobin) were adsorbed on a cover glasses that were pretreated with hexamethyldisilazane (HMDS). These protein stamps were then contacted with different monomer solutions (cross-linking monomers) on a glass slide substrate. Photopolymerization yielded the molecularly imprinted polymer. This technique, analogous to microcontact printing, allows for the rapid, parallel synthesis of MIPs of different compositions, and requires very small volumes of monomers (ca. 4 microL). The technique also avoids potential solubility problems with the molecular targets. Of several cross-linking monomers screened, tetraethyleneglycol dimethacrylate (TEGDMA) gave the most selective lysozyme binding, while polyethyleneglycol 400 dimethacrylate (PEG400DMA) were most selective for ribonuclease A and myoglobin.
The application of the molecular imprinting technology in the design of new drug delivery systems (DDS) and devices useful in closely related fields, such as diagnostic sensors or biological traps, is receiving increasing attention. Molecular imprinting technology can provide polymeric materials with the ability to recognize specific bioactive molecules and with a sorption/release behaviour that can be made sensitive to the properties of the surrounding medium. In this review, an introduction to the imprinting technology presenting the different approaches in preparing selective polymers of different formats is given, and the key factors involved in obtaining of imprinted binding sites in materials useful for pharmaceutical applications are analysed. Examples of DDS based on molecularly imprinted polymers (MIPs) can be found for the three main approaches developed to control the moment at which delivery should begin and/or the drug release rate; i.e., rate-programmed, activation-modulated or feedback-regulated drug delivery. This review seeks to highlight the most remarkable advantages of the imprinting technique in the development of new efficient DDS as well as to point out some possibilities of adapting the synthesis procedures to create systems compatible with both the relative instable drug molecules, especially of peptide nature, and the sensitive physiological tissues with which MIP-based DDS would enter into contact when administered. The prospects for future development are also analysed.
Self-organized receptor layers are synthesized by molecular imprinting methods directly on pre-coated 10 MHz quartz-crystal microbalances (QCMs). The surface-imprinting is performed by three methods using amorphous, crystalline and solubilized trypsin, respectively, as templates. These attempts allowed us to compare imprinting results obtained with templating proteins in the dry state as well as in aqueous solution. All methods are generally applicable for surface imprinting of thin films. The biomimetic sensor layers allow selective enzyme enrichment on the imprinted electrode with detection limits as low as 100 ng ml(-1) and response times of a few minutes. Solution-based polymer imprinting with native trypsin as template resulted in the highest specific enzyme recognition, which even allowed us to distinguish denatured trypsin from the native form.
Molecularly imprinted polymers (MIPs) selective for lysozyme were prepared on SPR sensor chips by radical co-polymerization with acrylic acid and N,N'-methylenebisacrylamide. Gold-coated SPR sensor chips were modified with N,N'-bis(acryloyl)cystamine, on which MIP thin films were covalently conjugated. The presence of NaCl during the polymerization and the re-binding tests affected the selectivity and the optimization of NaCl concentration in the pre-polymerization mixture and the re-binding buffer could enhance the selectivity in the target protein sensing. When the lysozyme-imprinted polymer thin films were prepared in the presence of 40 mM NaCl, the selectivity factor (target protein bound/reference protein bound) of MIP in the re-binding buffer containing 20 mM NaCl was 9.8, meanwhile, that of MIP in the re-binding buffer without NaCl was 1.2. A combination of SPR sensing technology with protein-imprinted thin films is a promising tool for the construction of selective protein sensors.
We report on a new sensor strategy that we have termed protein imprinted xerogels with integrated emission sites (PIXIES). The PIXIES platform is completely self-contained, and it achieves analyte recognition without a biorecognition element (e.g., antibody). The PIXIES relies upon sol-gel-derived xerogels, molecular imprinting, and the selective installation of a luminescent reporter molecule directly within the molecularly imprint site. In operation the templated xerogel selectively recognizes the target analyte, the analyte binds to the template site, and binding causes a change in the physicochemical properties within the template site that are sensed and reported by the luminescent probe molecule. We report the PIXIES analytical figures of merit for and compare these results to a standard ELISA. For human interleukin-1 the PIXIES-based sensor elements exhibited the following analytical figures of merit: (i) approximately 2 pg/mL detection limits; (ii) <2 min response times; (iii) >85 selectivity; (iv) <6% R.S.D. long term drift over 16 weeks of ambient storage; (v) >95% reversibility after more than 25 cycles; and (vi) >85% recoveries on spiked samples.
This article gives the recent developments in molecular imprinting for proteins. Currently bio-macromolecules such as antibodies and enzymes are mainly employed for protein recognition purposes. However, such bio-macromolecules are sometimes difficult to find and/or produce, therefore, receptor-like synthetic materials such as protein-imprinted polymers have been intensively studied as substitutes for natural receptors. Recent advances in protein imprinting shown here demonstrate the possibility of this technique as a future technology of protein recognition.
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