Review pubs.acs.org/acssensors
A Review on Synthetic Receptors for Bioparticle Detection Created by Surface-Imprinting TechniquesFrom Principles to Applications Kasper Eersels,*,† Peter Lieberzeit,‡ and Patrick Wagner† †
KU Leuven, Soft-Matter Physics and Biophysics Section, Department of Physics and Astronomy, Celestijnenlaan 200 D, B-3001 Leuven, Belgium ‡ University of Vienna, Faculty of Chemistry, Department of Physical Chemistry, Währinger Straße 38, A-1090 Vienna, Austria
ABSTRACT: The strong affinity of biological receptors for their targets has been studied for many years. Noncovalent interactions between these natural recognition elements and their ligands form the basis for a broad range of biosensor applications. Although these sensing platforms are usually appreciably sensitive and selective, certain drawbacks are associated with biological receptors under nonphysiological conditions in terms of temperature, pH, or ionic strength. Therefore, there are considerable efforts to mimic such molecular interactions with robust, synthetic receptors. Molecular imprinting is the bestknown technique to obtain antibody mimics by synthesizing a polymer matrix in the presence of a template species, such as molecules or larger aggregates. Extraction of the template results in sterically and functionally adapted binding cavities in or on a porous matrix. Although in principle possible, the detection of larger bioparticles such as proteins, microorganisms, or cells remains challenging when using the classical MIP concept. To tackle inherent difficulties, extending the concept of molecular imprinting toward surface imprinting is a promising approach: Here, binding cavities are formed directly on the surface of a crosslinked polymer layer, thus facilitating the removal of the templates. This article reviews the main surface-imprinting techniques and focuses on the implementation of surface-imprinted polymers (SIPs) into various biomimetic sensors and related applications. In addition, we provide an outlook on emerging research on surface imprinting and the development of biomimetic tools for diagnostic purposes. KEYWORDS: surface imprinted polymers, biomimetic sensors, macromolecular bioparticle detection, noncovalent interactions, medical diagnostics, biotechnology
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FROM NATURAL RECEPTORS TO SYNTHETIC RECEPTORS Ever since L. C. Clark, Jr., introduced the idea of combining enzymes with oxygen electrodes in 1962,1,2 research on the integration of natural receptors with sensor platforms has become increasingly popular. The high affinity of these receptors for their ligands allows creating devices with remarkable selectivity and sensitivity.3 Not in the least, advances in the fields of microelectronics and biomolecular chemistry have brought about biosensors for various fields including environmental monitoring, agriculture, food safety, and medical diagnostics.4 The key element of a biosensor is the biological receptor that enables detecting targets selectively at concentration levels, which are defined by a specific application. As alternatives to enzymes,5,6 biosensors may also utilize other natural receptors including DNA,7 aptamers,8,9 antibodies,10 and whole cells.11,12 The binding of molecules to receptors results in measurable physical or chemical changes at the interface, that are converted © 2016 American Chemical Society
into analytical signals by a transducer. Popular transducers are electrochemical techniques such as impedance spectroscopy,13 cyclic voltammetry,14 electronic field effects,15 potentiometry,16 and amperometry.17 Other options include optical,18 microgravimetric,19 or thermal read-out principles.20 Although the combination of natural receptors with label-free transducers allows for the successful implementation of a plethora of sensor devices, biological receptors also have inherent drawbacks: Their chemical and physical stability and shelf life are limited, which holds especially for antibodies (e.g., immunoglobins) and enzymes. Additionally, their synthesis or isolation and purification from nature is usually expensive and labor-intensive. Finally, their compatibility with most transducer surfaces is limited, necessitating the design of adequate linker layers.21,22 These disadvantages can be overcome by Received: September 13, 2016 Accepted: September 15, 2016 Published: September 15, 2016 1171
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ACS Sensors working with so-called antibody mimics: Robust, synthetic receptors that are able to bind their targets with an affinity comparable to their natural counterparts. Molecularly imprinted polymers (MIPs) are currently the most widespread approach, which is described in earlier reviews.23−25 MIPs selectively bind their targets in a reversible manner via functional nanocavities and are superior in terms of their longterm thermal and chemical stability.26,27 The synthesis of MIPs, especially when bulk-polymerization routes are used, is comparatively fast, low-cost, and straightforward.28 For MIP synthesis, the selected type of target molecule (template) is dissolved in an appropriate solvent (the “porogen”) in the presence of optimized ratios of functional monomers, a crosslinker and an initiator. After polymerization, the template molecules are extracted, creating a MIP that is able to rebind the target selectively.29 A major application field of bulkpolymerized MIPs is the detection of compounds with a low molecular weight, and several transducers were successfully employed including impedance spectroscopy,30 microbalances,31 and thermal detection.32 MIPs were also synthesized for macromolecular structures such as viruses,33,34 DNA,35 and proteins.36−38 However, the extraction of these nano- to micrometer-sized templates after polymerization is far from trivial, necessitating alternative strategies. Surface-imprinted polymers (coined “SIPs”) are the solution to this and they are the topic on which our review will primarily focus.
Figure 1. CLSM image of ethidium bromide-stained Listeria cells attached to a microcapsule (a) and stained Listeria imprints (b). The SEM micrographs show partially embedded S. aureus cells (c) and an S. aureus SIP (d). Figure adapted with permission from ref 39, Copyright 1997 John Wiley & Sons.
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ON THE ORIGINS OF SURFACE IMPRINTING: THE PIONEERS The concept of cell-mediated lithography for functional patterning of polymer surfaces was introduced by Alexander and Vulfson in 1997.39 In this work, they describe the formation of micron-sized bacterial imprints by a self-assembly process: For that purpose, they stirred bacterial suspensions (Listeria monocytogene cells and Staphylococcus aureus served as templates) in a two-phase system containing water-soluble amine monomers and diacid chloride in a dispersed organic phase. The template bacteria associated at the interface between the aqueous and organic phases. The two reactants formed a polyamide in the organic phase, which immobilized the bacteria at the phase boundary. Upon formation of the polyamide layer, the microcapsules were converted into polymer beads by photopolymerization of a diacrylate in the organic phase. The beads were treated with perfluoropolyether to prevent cell attachment to the unreacted areas of the polyamine shell. The bacteria were finally removed from the surface by acidic hydrolysis, leaving behind micrometer-sized lithographic imprints matching the dimensions of the template bacteria. The imprinted polymer surfaces were characterized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM); see Figure 1. The rebinding capacity was not assessed in this study, which aimed primarily at a novel, cell-based lithographic concept. In 1999, Shi and co-workers demonstrated a second approach for synthesizing surface imprinted polymer layers, based on molding thin polymer films with various types of proteins.40 Template proteins were adsorbed onto mica substrates and coated with a 1−5-nm-thick disaccharide film by spin-casting. Polar residues on the proteins reacted with hydroxyl groups of the sugar molecules by hydrogen bonding, inhibiting protein degradation during the rest of the procedure. Then, hexafluoropropylene films were cast over the protected protein layer by radiofrequency glow-discharge (RFGD)
plasma deposition. This layer was fixated on a microscope slide using epoxy resin, allowing removal of the mica substrate. Finally, protein-imprinted nanocavities were created by removing the template proteins by rinsing in an alkaline solution. The cavities matched the size and shape of the template proteins and displayed also functional complementarity with the templates due to the spatial arrangement of hydroxyl groups in the disaccharide layer. The film surface was analyzed with atomic force microscopy (AFM) and rebinding of various proteins (e.g., bovine serum albumin, BSA) to the SIP was verified by fluorescence microscopy. The integration of SIPs into biomimetic sensor platforms was introduced by Dickert et al. in 2001.41,42 Honeycomb-like yeast imprints were prepared by stamping template cells into a polyurethane film deposited before on a quartz crystal microbalance (QCM) chip; see Figure 2a. Polyurethane layers were prepolymerized in tetrahydrofuran (THF) using 4′,4′-diisocyanato diphenylmethane (4′,4′ DPDI) and bisphenol A as monomers and phloroglucinol as crosslinker. The gel was applied onto screen-printed QCM sensor chips by spin coating. In parallel, glass slides were coated with monolayers of dry yeast cells by drop casting a yeast-cell suspension onto the slides and removing the excess fluid by spinning. These “stamps” were gently pressed onto the sensor chips overnight until the polyurethane was fully cured. Removal of the stamps and washing off remaining yeast cells with hot water resulted in micron-scaled cavities on the polyurethane surface, matching the template cells in size and shape as seen in the AFM image of Figure 2a. Finally, the rebinding capacity of the SIP and selectivity toward other yeast strains were assessed microgravimetrically (see Figure 2b), which demonstrates the potential of SIPs for cell detection and identification. The work of these pioneers has launched an entirely new research field, focusing on the synthesis of surface-imprinted 1172
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Figure 2. Scheme for preparing a yeast-imprinted polyurethane layer on a QCM chip by cell stamping and AFM imaging of imprinted and nonimprinted polyurethane surfaces (a). QCM selectivity test exposing a S. cerevisiae-imprinted SIP to both target and competitor cells. This analysis clearly shows that the sensor is able to selectively detect its target (b). Figure reproduced with permission from ref 42, Copyright 2001 John Wiley & Sons.
Figure 3. Scheme of the self-assembly technique for SIP synthesis. The template is mixed with the polymerization mixture containing monomers, cross-linker(s), and initiators. The SIP forms through interaction of the template with monomers during polymerization. After template extraction, microcavities on the SIP surface are able to rebind targets selectively.
polymer films43−48 and their use in various analytical applications.49−51 This review aims at providing a survey of the various surface-imprinting methods developed over the last 20 years, the integration of SIPs into biomimetic sensing platforms, and also their potential in a variety of biotechnological applications.
noncovalent interactions in a self-assembly process. After template extraction, the binding cavities are able to rebind the target specifically. Although some templates may end up in the bulk of the polymer, only binding cavities at the surface are accessible for rebinding. These polymers are usually deposited as thin layers on sensor chips or as shells around nano- or microbeads. Brown et al. used an imprinting technique that fits this scheme for detecting lysozyme using imprinted porous silica scaffolds.52 The scaffolds were made in a two-step sol−gel process with γ-aminopropyltriethoxysilane (APS) as functional monomer, tetraethoxysilane (TEOS) as cross-linker, sodium dodecyl sulfate (SDS) as foaming agent, and water−ethanol as cosolvents. The protein lysozyme was added to the APS
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SURFACE IMPRINTING APPROACHES Self-Assembly. SIPs created by self-assembly typically follow the strategy shown in Figure 3. In the same way as “classical” molecular imprinting, templates are brought into a solvent containing monomers, cross-linkers, and, if applicable, an initiator. During cross-linking of the (co-)polymer, the template interacts with the monomers through covalent or 1173
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groups (−OH, −COOH, −NH2, and others) on the surface of the templates during cross-linking. After curing, the polymer is peeled off and the template particles are removed, resulting in a flexible, imprinted polymer layer with a high areal density of imprinted sites. As an example, DePorter et al. imprinted polyacrylamide hydrogels with HeLa cells (an immortal cervical-cancer cell line) using a molding approach.67 The cells were grown on a tissue culture plate onto which a solution was poured containing acrylamide (monomer), bis(acrylamide) (cross-linker), tetramethylethylene-diamine (TEMED, initiator), and ammonium persulfate (APS initiator). The mixture was cross-linked at room temperature for 20 min after which the hydrogel was removed from the culture plate. Any cells or cell debris remaining on the SIP surface were removed by washing the gel in sodium hydroxide, sodium dodecyl sulfate (SDS), and eventually phosphate buffered saline (PBS). The HeLa-imprinted hydrogels displayed a considerably improved attachment and growth of HeLa cells as compared to nonimprinted hydrogels and hydrogels imprinted with other cervical-cancer cell lines. Qi et al. presented a similar approach for synthesizing bacteria-imprinted SIPs by molding:68 They coated indium tin oxide (ITO) substrates with a nanocomposite film of chitosan (CS) and reduced graphene oxide sheets (RGSs) by electrodeposition. Sulfate-reducing bacteria (SRB) were immobilized on this composite layer by physical adsorption. Then, a 1 wt % chitosan solution was applied to this layer and electropolymerized. The SRB templates were removed from the chitosan layer by rinsing with acetone and deionized water. This cell-imprinted layer was able to bind SRBs selectively as shown with impedance spectroscopy. Generating SIPs Directly on a Solid Surface. Although the molding approach is relatively straightforward, it is not trivial to obtain a seamless joint between peeled-off polymer layers and sensor-chip surfaces. Therefore, several groups have proposed a modified imprinting strategy in which SIP synthesis starts directly at the surface to be functionalized. The templates are immobilized on a solid substrate (sensor chip or particles) and the polymer is grown right from the surface, partially engulfing the templates and creating a core−shell structure as illustrated in Figure 5. Shiomi et al. used a similar approach to synthesize hemoglobin SIP layers on silica beads. First, hemoglobin was coupled to the beads using glutaraldehyde. Then, the beads were coated with a polymer shell containing 3-aminopropyltrimethoxysilane (APTMS) and propyltrimethoxysilane (PTMS). Hemoglobin was removed from the silane matrix by washing with oxalic acid and distilled water and binding capacity and cross-selectivity were assessed with the Bradford method. The SIP-coated particles had a substantially higher selectivity for hemoglobin than bulk MIPs synthesized from the same compounds. Cumbo and co-workers employed a similar technique, in this case for the detection of small RNA viruses.70 Virions were immobilized onto aminopropyltriethoxysilane (APTES)-modified silica nanoparticles (Si-NP). After binding the virions, a silsesquioxane layer was grown from the surface of the Si-NPs. The organosilanes were chosen in a way to mimic the lateral chains of amino acids, thereby improving target− receptor interaction during cross-linking. The rebinding capacity and selectivity were studied using an enzyme-linked immunosorbent assay (ELISA). Inhibitor-assisted surface imprinting is based on a similar approach, where the selectivity of SIPs is enhanced by immobilizing inhibitor molecules onto the substrate prior to
solution prior to mixing it with TEOS. In addition, they imprinted a SIP with a peptide comprising 16 terminal amino acids of lysozyme in the same way for a direct comparison between protein and epitope imprinting. Both the peptide and the protein SIP were able to recognize the native protein specifically and to distinguish between lysozyme and RNase. This nicely demonstrates the feasibility of the epitope approach, which circumvents the difficult extraction of whole-protein templates. In addition to proteins, polymers have been imprinted with even bigger bioparticles by the self-assembly technique. Wang et al. imprinted self-assembled thiol monolayers (SAMs) with both proteins and poliovirus particles.53 The templates were dissolved in deionized water and mixed with a solution of alkanethiols in water. This mixture was applied to a gold substrate for 2 h after which the template was released by rinsing with deionized water. Recently, bacteria (Escherichia coli) were used as templates for imprinting polymer microspheres.54 The bacteria were mixed with N-acrylchitosan in water and this solution was mixed with an oil phase in order to create microspheres. After cross-linking the chitosan matrix and removal of the template bacteria, fluorescence microscopy analysis confirmed that the microbeads could distinguish between rod-shaped E. coli and spherical Micrococcus luteus. Several groups have developed modifications to the surfaceimprinting scheme of Figure 3 by adding external forces to enhance the self-assembly process: For instance, Tokonami et al. developed a concept for imprinting polypyrrole (PPy) layers with bacteria using electropolymerziation.55 QCM electrodes were immersed in a solution containing both the monomer and the template. The SIP was formed by applying a positive potential over the substrate. The template bacteria were removed from the PPy film by reversing the potential. Sen and co-workers proposed an alternative technique to prepare bacteria-imprinted layers using spray-drying as a driving force for self-assembly.56 An overview of various surface-imprinting techniques based on self-assembly, without claiming to be exhaustive, is given in Table 1. Molding Technique. Typically, molding techniques as sketched in Figure 4, start with immobilizing the template particles on a solid substrate. In parallel, a suitable polymer mixture is prepared and deposited on top of the template layer. The polymer engulfs the template and interacts with functional Table 1. Overview of Surface Imprinting Techniques Using the Self-Assembly Approach templatea Bacteria BVL glycoprotein gp51 FNP 1 Lysozyme Poliovirus HIV-1RP gp 41 Rhodamine B Cyclophilin 18
reference(s)
comments
Amide, silica, Py, Chi Py
monomers
39, 54−57 58
Ref 55 EP EP
A, AA Silane, siloxane, A, Ph, MAA Alkenethiols Dopamine Silica A
59 52, 60−63 53 64 65 66
SD
BVL: bovine leukemia virus; FNP 1: flavivirus nonstructural protein 1; HIV-1RP: human immunodeficiency virus-1 related protein; Py: pyrrole; Chi: chitosan; A: acrylamide; AA: acrylic acid; Ph: phenol; MAA: methacrylic acid; EP: electropolymerization, SD: spray drying. a
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Figure 4. General scheme of the molding approach for SIP synthesis. The polymer is deposited on top of a template-covered substrate. After crosslinking, the polymer is peeled off, creating surface cavities that are complementary with the template.
Figure 5. Scheme of a molding approach for synthesizing SIPs directly on a solid substrate, in this case, microparticles. The polymer engulfs the immobilized templates partially while forming a shell around the substrate. After template removal, the imprints are in immediate contact with the solid support.
the imprinting procedure.71−73 Zhang et al. introduced the technique in 2013 for the synthesis of a SIP for trypsin, an enzyme involved in diabetes: First, the trypsin-inhibitor benzamidine was adsorbed on silica microspheres. After binding of trypsin to the inhibitors, an acrylamide-based polymer was molded around the templates. Releasing the templates resulted in SIP particles with controlled enzymeinhibiting properties superior to those of free inhibitor molecules. Table 2 summarizes examples of SIP synthesis using molding techniques. The Stamping Technique. The stamping technique is the third major approach for synthesizing SIPs and the concept is illustrated schematically in Figure 6. The first synthesis step typically consists of applying a semicured, still viscous oligomer layer onto a solid substrate, e.g., by spin coating. In parallel, a stamp is covered with a monolayer of template particles. This stamp is pressed gently onto the oligomer-coated substrate, enabling the template particles to indent the viscous oligomer layer. During cross-linking, the forming polymer matrix interacts with functional groups on the surface of the template particles. After curing the polymer, the stamp is removed and
the template particles are washed off, leaving behind persistent nano- to microscale indentations that are able to rebind target particles. As described in On the Origins of Surface Imprinting: The Pioneers, Dickert and Hayden used the stamping technique originally in 2001 to create yeast-cell imprints in thin polyurethane (PU) layers.42 Researchers of the same team, including author PL of the present article, extended the concept during the following years toward imprinting of PU layers with proteins,87 lipoproteins,88 bacteria,89 viruses,87 pollen,90 and human erythrocytes.91 We note that bioparticles can be fragile with respect to the conditions of the stamping technique and living cells especially may be mechanically squeezed under pressure. Therefore, a stampless imprinting technique was also developed, using sedimentation by gravity in order to imprint erythrocytes into polyvinylpyrrolidone (PVP) layers.92 Jenik and co-workers proposed an interesting refinement to the original stamping technique in order to improve the reproducibility of the concept by preparing so-called “masterstamps”.91 To create the master stamp, polyurethane or PDMS layers were imprinted as described above. These SIPs were cast 1175
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monomersa
reference(s)
Hemoglobin HeLa cells Chondrocytes Cytochrome C Proteins SRB Algae
Silanes A, NO 81 PDMS Scopoletin MAA Chi EVAL
69 67, 74 75 76 40, 77 68 78−80
PSA R. sphaeroides Viruses S. aureus Trypsin Lysozyme Pepsin
MAA, MA EVAL Silanes, A Silica A, HEMA MA, HEMA APBA
81 82, 83 70, 84, 85 86 71 72 73
comments
Electropolymerization Ref 77 Solid phase synthesis Molding/stamping combination Solid phase synthesis
Electropolymerization
Figure 7. Contact-AFM image of an epoxy layer (master stamp) featuring positive cell replicas of “donut-like” human erythrocytes at its surface.92 The replica was obtained by filling a SIP layer with SU8 resin. Figure adopted with permission from ref 92, Copyright 2009 Elsevier.
a A: acrylamide, NO 81: Nordland optical adhesive, PDMS: polydimethysiloxane, MAA: methacrylic acid, SRB: sulfate-reducing bacteria, Chi: chitosan, EVAL: ethylene-co-vinyl alcohol, PSA: porcine serum albumine, MA: methacrylamide, HEMA: 2-hydroxyethyl methacrylate, APBA: aminophenylboronic acid.
applications. Two authors of this article (K.E. and P.W.), e.g., coupled cell-imprinted polyurethane layers to a novel, thermal transducer platform, which will be addressed in Thermal Sensing: The Heat-Transfer Method (HTM).93,94 In the meantime, other polymers have also been successfully used to create SIPs with the stamping method. As an example, Schirhagl et al. imprinted thin PDMS layers with cyanobacteria for cell-separation purposes and the SIPs allowed distinguishing between different cyanobacterial strains.96 Table 3 provides an overview on the wide variety of biological targets for which SIPs have been prepared using the stamping technique. Miniemulsion Polymerization. In contrast to the molding and stamping techniques, miniemulsion polymerization is not primarily intended to functionalize solid sensor chips with a SIP layer on top. Instead, miniemulsion polymerization is a colloidal approach, resulting in submicron-scale spherical particles with a narrow size distribution and a comparatively
with the single-component epoxy SU8−2025 and, after removal of the mold, the SU8-layers featured positive template replicas on their surface. Figure 7 shows a master stamp with welldefined three-dimensional erythrocyte replicas. The SIPs prepared by using the polymer-based master stamps were able to rebind the original cell type and to discriminate between yeast and erythrocytes. It is still unclear whether the affinity for a certain cell type involves chemical cues when using plastic cell replicas, but the pure fact that it works is promising: This way, SIPs can be prepared without the need for culturing new template cells and the concept may eventually enable largerscale SIP production by roll-to-roll processing. From 2010 on, the work by Dickert et al. inspired other researchers to synthesize similar SIPs for various bioanalytical
Figure 6. Scheme of the stamping approach for creating SIPs. The template is immobilized onto a stamp made of, e.g., silicones. In parallel, a solid surface, serving later as a sensor chip, is coated with a semicured polymer layer. Pressing the stamp onto the polymer layer enables the template particles to deform the polymer and to interact with it during cross-linking. 1176
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IMPLEMENTATION OF SIP-BASED BIOMIMETIC SENSOR PLATFORMS In recent years, SIPs have been combined with several transducer platforms for developing numerous sensing applications, mainly in a bioanalytical context. This chapter aims at giving an overview of the read-out platforms that have been mainly used until today. Each platform has its own benefits and drawbacks, which will be discussed in a balanced way. Finally, this section reports on various sensor applications in terms of their selectivity, sensitivity, and performance in complex matrices. Microgravimetric Detection: Quartz Crystal Microbalance (QCM). Microgravimetric transducer platforms are based on measuring changes in the resonance frequency of piezoelectric crystals in response to mass or dissipation changes at the solid−liquid (or solid−gas) interface. For biosensing purposes, the quartz crystal microbalance (QCM) is the most common type of a mass-sensitive transducer. More details on QCMs and their working principle can be found in the literature.118,119 It is worthwhile mentioning that these devices are sensitive not only to mass loading but also to the viscoelastic properties of the interface, which can be quantified from the dissipation signal. QCM platforms can range from high-cost, ultrasensitive commercial devices that monitor changes in frequency and dissipation up to high-order overtones to low-cost, homemade instruments. Dickert et al. combined such a homemade QCM with thin polyurethane layers imprinted with yeast cells synthesized by the stamping approach as described earlier in On the Origins of Surface Imprinting: The Pioneers.41,42 In order to assess the SIP specificity on a single chip and to compensate for any fluctuations in temperature or viscosity, Dickert’s team used quartz chips with at least two independent, sensitive spots. This allows for differential measurements using a functional electrode (SIP-electrode) and a reference channel (NIP electrode) in parallel. The design displayed an excellent specificity, as no significant change in frequency was observed on the NIP electrode throughout the yeast-recognition measurements. In contrast to that, the SIP-electrode showed a frequency drop by about 5 Hz/ng toward yeast. Selectivity was assessed using solutions containing either S. cerevisiae or one of three other yeast strains. In more recent research, similar platforms were developed for the detection of proteins,87 lipoproteins,88 bacteria,89 viruses,87 pollen,90 and red blood cells.91 The sensitivity of the device was remarkably high with a limit of detection (LoD) down to 3−5 ng for yeast, bacteria, pollen, and proteins; 105 cells/mL for viruses; and 107 cells/mL for blood cells. While most QCM-based biomimetic sensor platforms were tested in buffer, Schirhagl et al. introduced a SIP-based platform for selective detection of proteins and viruses in complex matrices using artificial antibodies.101 Their sensor proved suitable to detect allergenic sesame protein in bread samples and extracts from sesame and brazil nut in concentrations as low as 500 ng/mL which, in terms of selectivity, is quite remarkable given the complex nature of the samples under study. Furthermore, the identification of human rhinovirus (HRV) particles in spiked blood plasma samples with a LoD of 1014 viruses/mL was demonstrated. It has to be noted that bacteria were detected in plasma samples that were spiked with HRV but not analogue viral species. Tokonami et al. tested their bacteria-imprinted polypyrrole films in spiked apple juice
Table 3. Overview of Biological Targets/Templates for Which SIPs Have Been Prepared Using Stamping Techniques or Modifications Thereof template types Yeast, bacteria Viruses, pollen, mammalian cells, proteins Erythrocytes Viruses, proteins E. coli Proteins, viruses Influenza A virus Yeast, bacteria Proteins Bacteria, viruses
monomersa
reference(s)
MDI MDI
41, 42, 87 88−91, 93−95 92, 97 98, 99 100
PVP MAA/PVP PVP/PG/ DPDI MAA/PVP A/MAA/ PVP HEMA DMAEMA PDMS
comments
Sedimentation
101 102 103, 104 105 96, 106−108
a
MDI: diisocyanato diphenylmethane, PVP: polyvinylpyrrolidone, MAA: methacrylic acid, PG: phloroglucinol, DPDI: diphenylmethane-4,4-diisocyanate, A: acrylamide, HEMA: hydroxyethyl methacrylate, DMAEMA: dimethylamino ethyl methacrylate, PDMS: polydimethylsiloxane.
large functional surface area. The concept was introduced in 2002 by Vaihinger et al., who imprinted methacrylic acid-based polymers with the amino acids D- and L-boc-phenylaline anilid (BFA).109 Hereby, SDS was dissolved in water and acted as a surfactant while hexadecane was used as hydrophobic agent to prevent Ostwald ripening of the miniemulsion. The polymer phase, dispersed in the water phase, created small reactors that led to uniform BFA-imprinted polymer beads that were able to discriminate between D- and L-boc-phenylaline anilid as confirmed in a UV/vis-spectroscopy rebinding experiment. Tan et al. applied miniemulsion polymerization for imprinting of the protein ribonuclease A.110 Batch rebinding experiments in combination with high-performance liquid chromate-graphy (HPLC) demonstrated that the particles were able to selectively distinguish between the template and bovine serum albumin in a mixed protein solution. In 2015, the selectivity of miniemulsion particles synthesized for pepsin detection was tested by Pluhar et al. using batch rebinding analysis and gel electrophoresis.111 Imprinted particles and nonimprinted reference particles were made in a two-step process and their performance was analyzed in the presence of the competitor proteins BSA and β-lactoglobulin. The dissociation constant of imprinted particles was 7.94 μM for pepsin with a binding capacity of 0.72 μM per m2. An overview of imprinted miniemulsion particles for various targets is given in Table 4. Table 4. Overview of Surface Imprinting Methods Using Miniemulsion Techniques template
monomersa
reference(s)
Amino acids Proteins Viruses Proteins Peptides
MAA MAA MAA MAA/HEMA/AEMA/APTMA A
109 110, 112, 113 114 111, 115 116, 117
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a
MAA: methacrylic acid, HEMA: hydroxyethyl methacrylate, AEMA: 2-aminoethyl methacrylate hydrochloride, APTMA: (3acrylamidopropyl)trimethylammonium chloride, A: acrylamide. 1177
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compartment, serving as a measuring chamber. The temperature of the copper backside contact T1 was kept constant at 37 °C. The temperature inside the liquid measuring chamber T2 was measured by a second thermocouple. This way, it is possible to monitor the thermal resistance (Rth) of the solid− liquid interface over time by analyzing the ratio of the temperature difference ΔT = T1 − T2 and the input power P according to Rth = ΔT/P. Rebinding of target cells to the SIP layer lead to a measurable increase in Rth. This becomes plausible considering the poor thermal conductivity of cellmembrane materials, predicted earlier by molecular-dynamics simulations.122 In 2013, it was shown that HTM can be combined with SIPs for detecting human cancer cells.93 The selectivity of the platform was assessed by exposing the SIP to a solution containing either target or analogue cells. The platform was able to distinguish between tumor cells and healthy blood cells as well as between different cancer cell lines in buffer (see Figure 9). This illustrates the principal potential of the device for detecting circulating tumor cells (CTCs), but the low concentration of CTCs (
