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Molded Nanowell Electrodes for Site-Selective Single Liposome Arrays
Advanced Materials
Abstract
A study was conducted to demonstrate the construction of functional lipid vesicles (FLV), using nanowell-based electrochemical biosensors. Three key factors were considered, to achieve a nanowell-based (NW) electrochemical biosensor with FLVs, capturing FLVs at predefined locations without nonspecific binding, stabilizing and fictionalizing the captured FLVs, and quantitatively interpreting signals from affinity binding. A nanowell array electrode, with controlled geometry and density was also prepared, to capture individual liposomes without nonspecific binding. The nanowells were fabricated on gold electrodes, using dewetting-assisted nanomolding, with a polyethylene glycol (PEG) copolymer. The nanomolding process also involved the contact of a nanopatterned polymeric mold on the PEG surface, by drop dispensing and forming well-defined NW arrays, with exposed gold substrate.
... [2][3][4] In addition, microarrays, which are spatially ordered arrays in discrete spots on a solid matrix, have enabled rapid and high-throughput screening of bioanalytes in a quantitative manner. [9][10][11][12] Despite increasing demands for such miniaturized bioanalytical systems, they still have obstacles for practical applications partly due to their relatively complicated and expensive operation schemes. For example, current microuidic devices frequently require external tubing and pumps along with an additional integration to the device for the overall miniaturization and portable uses. ...
... 69 If a poor solvent is used (contact angle >90 ), on the other hand, the process can be mediated by dewetting in such a way that the hydrogel in the void region recedes downwards until exposure of the substrate. 11,70 Using this concept, very small nanowells ($50 nm) of polyethylene glycol (PEG) hydrogels were achieved on a glass substrate and used as reservoirs for nanoarrays of single lipid vesicles. 11 4 Bioanalytical manipulation using stimuliresponsive hydrogel patterns Bioanalytical devices require controlled transport, adsorption, or release of biosamples for achieving timely analytic reactions at desired locations. ...
... 11,70 Using this concept, very small nanowells ($50 nm) of polyethylene glycol (PEG) hydrogels were achieved on a glass substrate and used as reservoirs for nanoarrays of single lipid vesicles. 11 4 Bioanalytical manipulation using stimuliresponsive hydrogel patterns Bioanalytical devices require controlled transport, adsorption, or release of biosamples for achieving timely analytic reactions at desired locations. [2][3][4][5] To this end, stimuli-responsive hydrogel patterns can be useful for manipulating various bioanalytes in microuidics and microarrays in a spatio-temporal fashion. ...
In this review, we highlight the properties, functions and applications of stimuli-responsive hydrogel patterns in bioanalytical applications. Stimuli-responsive hydrogel patterns can be realized by well-established micro- and nanofabrication technologies such as photolithography and micromolding, and are currently adopted as active components for manipulation of flow and biosamples in microchannel and microarray systems. We overview the properties of stimuli-responsive hydrogel materials and their fabrication methods along with some representative examples in microfluidics and microarrays.
... Therefore, to fabricate more efficient and reliable biosensors, it is essential to develop a method to immobilize probe antibodies with a more uniform coverage and in a geometric arrangement that minimizes nonspecific bindings. Strategies to overcome this problem involve specific chemistries that limit nonspecific binding, but these approaches do not eliminate nonspecific adsorption entirely (28). ...
... The nanowell array biosensors have shown great promise for providing highly sensitive label-free detection by blocking nonspecific bindings without introducing chemical or biological reagents (27)(28)(29)(30). It has also been shown that nanowell array electrodes benefit from rapid detection of biomolecules with higher reproducibility, because these sensors can reduce mass transfer limitations (31,32). ...
A non-faradaic label-free cortisol sensing platform is presented using a nanowell array design, in which the two probe electrodes are integrated within the nanowell structure. Rapid and low volume (≤5 μl) sensing was realized through functionalizing nanoscale volume wells with antibodies and monitoring the real-time binding events. A 28-well plate biochip was built on a glass substrate by sequential deposition, patterning, and etching steps to create a stack nanowell array sensor with an electrode gap of 40 nm. Sensor response for cortisol concentrations between 1 and 15 μg/dl in buffer solution was recorded, and a limit of detection of 0.5 μg/dl was achieved. Last, 65 human serum samples were collected to compare the response from human serum samples with results from the standard enzyme-linked immunosorbent assay (ELISA). These results confirm that nanowell array sensors could be a promising platform for point-of-care testing, where real-time, laboratory-quality diagnostic results are essential.
... This sensing technique can benefit from the high salt concentration in the samples in the case that a higher conductivity environment is more sensitive to the impedance altering due to the protein binding (Xie et al. 2020). Nanowell array biosensors offer significant advantages for highly sensitive, label-free detection by preventing nonspecific binding without the use of chemical or biological reagents, along with rapid and more reproducible biomarker detection due to their ability to reduce mass transfer limitations (Seo et al. 2018;Kim et al. 2008). Nevertheless, there are still several obstacles when employing nanowell impedance sensors in the clinical study. ...
Wearable and implantable biosensors have rapidly entered the fields of health and biomedicine to diagnose diseases and physiological monitoring. The use of wired medical devices causes surgical complications, which can occur when wires break, become infected, generate electrical noise, and are incompatible with implantable applications. In contrast, wireless power transfer is ideal for biosensing applications since it does not necessitate direct connections between measurement tools and sensing systems, enabling remote use of the biosensors. In addition, wireless sensors eliminate the need for a battery or energy harvester, reducing the size of the sensor. As far as we are aware, this is the first report ever describing a new method for wireless readout of a label-free electronic biosensor for detecting protein biomarkers. Our results reveal that we are able to successfully detect target protein and corresponding antibodies within this wireless setup. We are able to distinguish target protein in purified samples from a blank PBS sample as a negative control by tracking gradual changes in impedance at the input of the transmitter (P-value = 0.00788). We also demonstrate real-time wireless quantification of cytokines within rheumatoid arthritis patient serum samples (P-value = 0.00891). A Fine Gaussian Support Vector Machine is also used to differentiate protein from negative controls with the highest accuracy from a dataset of 54 experiments.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10544-024-00728-9.
... It has been revealed that nanowell-based biosensors exhibit significant potential for delivering remarkably sensitive label-free detection by preventing nonspecific bindings, all without the need for chemical or biological reagents. Additionally, prior research indicates that the electrodes in nanowell-based biosensors offer the advantage of swiftly detecting biomolecules with enhanced reproducibility, owing to their capability to alleviate mass transfer constraints [9][10][11][12][13]. Thus, nanowell-based biosensors offer a solution to the previously mentioned drawbacks of impedance-based biosensors. ...
Nanowell-based impedance-based label-free biosensors have demonstrated significant advantages in sensitivity, simplicity, and accuracy for detecting cancer biomarkers and macromolecules compared to conventional impedance-based biosensors. Although nanowell arrays have previously been employed for biomarker detection, a notable limitation exists in the photolithography step of their fabrication process, leading to a reduced efficiency rate. Historically, the diameter of these nanowells has been 2 μm. To address this issue, we propose alternative geometries for nanowells that feature larger surface areas while maintaining a similar circumference, thereby enhancing the fabrication efficiency of the biosensors. We investigated three geometries: tube, spiral, and quatrefoil. Impedance measurements of the samples were conducted at 10 min intervals using a lock-in amplifier. The study utilized interleukin-6 (IL-6) antibodies and antigens/proteins at a concentration of 100 nM as the target macromolecules. The results indicated that tube-shaped nanowells exhibited the highest sensitivity for detecting IL-6 protein, with an impedance change of 9.55%. In contrast, the spiral, quatrefoil, and circle geometries showed impedance changes of 0.91%, 0.95%, and 1.62%, respectively. Therefore, the tube-shaped nanowell structure presents a promising alternative to conventional nanowell arrays for future studies, potentially enhancing the efficiency and sensitivity of biosensor fabrication.
... To evaluate potential applications of the site-selective deposition of single functional lipid vesicle (FLVs), we tested streptavidin binding to biotin on the FLVs surface. This coupling is widely studied for development of biosensor for its strong affinity (dissociation constant, K d = 10 −15 M) [72][73][74]. To claim that NW electrode can act as a restrictive surface for bioassays, the binding event should block the electron transfer to NW electrode (so called the shielding effect). ...
Nanostructured biosensors have pioneered biomedical engineering by providing highly sensitive analyses of biomolecules. The nanowell array (NWA)-based biosensing platform is particularly innovative, where the small size of NWs within the array permits extremely profound sensing of a small quantity of biomolecules. Undoubtedly, the NWA geometry of a gently-sloped vertical wall is critical for selective docking of specific proteins without capillary resistances, and nanoprocessing has contributed to the fabrication of NWA electrodes on gold substrate such as molding process, e-beam lithography, and krypton-fluoride (KrF) stepper semiconductor method. The Lee group at the Mara Nanotech has established this NW-based biosensing technology during the past two decades by engineering highly sensitive electrochemical sensors and providing a broad range of detection methods from large molecules (e.g., cells or proteins) to small molecules (e.g., DNA and RNA). Nanosized gold dots in the NWA enhance the detection of electrochemical biosensing to the range of zeptomoles in precision against the complementary target DNA molecules. In this review, we discuss recent innovations in biomedical nanoengineering with a specific focus on novel NWA-based biosensors. We also describe our continuous efforts in achieving a label-free detection without non-specific binding while maintaining the activity and stability of immobilized biomolecules. This research can lay the foundation of a new platform for biomedical nanoengineering systems.
... In this study, we developed an highly sensitive immunosensor for quantitative detection of stress-induced-phosphoprotein-1 [STIP-1] using wafer-scale nanowell array (NWA) electrodes. In our previous work, we optimized NWA electrode based amperometric detection of molecular binding events by controlling the molecular binding sites (Kim et al., 2005a,b;Lee et al., 2006;Kim et al., 2008). Here, we used EIS with our NWA to detect antigens with low concentrations. ...
We have reported that nanowell array (NWA) can enhance electrochemical detection of molecular binding events by controlling the binding sites of the captured molecules. Using NWA biosensor based amperometric analysis, we have detected biological macromolecules such as DNA, protein or aptamers at low concentrations. In this research, we developed an impedimetric immunosensor based on wafer-scale NWA for electrochemical detection of stress-induced-phosphoprotein-1 (STIP-1). In order to develop NWA sensor through the cost-effective combination of high-throughput nanopattern, the NWA electrode was fabricated on Si wafer by krypton-fluoride (KrF) stepper semiconductor process. Finally, 12,500,000 ea nanowell with a 500nm diameter was fabricated on 4×2 mm(2) substrate. Next, by using these electrodes, we measured impedance to quantify antigen binding to the immunoaffinity layer. The limit of detection (LOD) of the NWA was improved about 100-fold compared to milli-sized electrodes (4×2 mm(2)) without an NWA. These results suggest that wafer-scale NWA immunosensor will be useful for biosensing applications because their interface response is appropriate for detecting molecular binding events.
... For example, preliminary experimental and theoretical models have been reported based on vesicle recognition by biotin-avidin and electrostatic interactions (Farbman-Yogev et al, 1998; Walker et al, 1997 and Chiruvolu et al, 1994). We propose an alternative protocol, which may be more easily accessible, namely, first patterning the vesicles onto a planar surface and then fusing these pre-organized vesicles with a second vesicle population to complete the reactions (Lee et al, 2008; Christensen et al, 2007; Kim et al, 2008). The first step has been achieved by some smart designs, e.g. ...
... Scanning probe techniques include nanografting, dip-pen lithography, conductive AFM, and other direct-write methods [25][26][27][28][29][30] Nano-patterned positive photoresist Figure 1: Schematic of the interference lithography process used to create submicron protein arrays. lithography, decal transfer, and nanomolding [31][32][33][34]. Selfassembly techniques include particle lithography, polymerassisted templating and DNA-assisted assembly [3,35,36]. ...
Understanding cellular interactions with material surfaces at the micro- and nanometer scale is
essential for the development of the next generation of biomaterials. Several techniques have been
used to create micro- and nanopatterned surfaces as a means of studying cellular interactions with a
surface. Herein, we report the novel use of interference lithography to create a large (4 cm2) array of 33 nm deep channels in a gold surface, to expose an antireflective coating on a silicon wafer at the bottom
of the gold channels. The fabricated pores had a diameter of 140–350 nm separated by an average pitch
of 304–750 nm, depending on the fabrication conditions. The gold surface was treated with 2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethanol to create protein-resistant areas. Fibronectin was
selectively adsorbed onto the exposed antireflective coating creating nanometer-scale cell adhesive
domains. A murine osteoblast cell line (MC3T3-E1) was seeded onto the surfaces and was shown to
attach to the fibronectin domains and spread across the material surface.
Biosensors that can analyze a single drop of biological fluid can overcome the limitation such as extraction volume from humans or animals, ethical problems, time and cost. In this work, we have developed a highly sensitive electrochemical biosensor based on a nanowell array (NWA) for the detection of alkaline phosphatase (ALP), a serum indicator of bone formation. The size of electrode is 2 × 1 mm2 and has over ten million nanowells (400 nm diameter) arranged uniformly on the electrode surface. For detecting ALP, Anti-ALP was immobilized and oriented on the NWA surface using a self-assembled monolayer and protein G. Electrochemical impedance spectroscopy (EIS) was used to determine the amount of ALP in 10 µL of sample. The impedance was calibrated with ALP concentration. The NWA has a linear dynamic range from 1 pg/mL to 100 ng/mL with a limit of detection (LOD) at 12 pg/mL. We used the sensor to measure the ALP in real mouse serum from 4, 10, and 20 weeks old mice and compared the results to the standard photometric assay. This work demonstrates the potential of electrochemical NWA sensors to analyze a single drop of real body fluid sample and can be developed for broad applications.
Most solid-state biosensor platforms require a specific immobilization chemistry and a bioconjugation strategy separately to tether sensory molecules to a substrate and attach specific receptors to the sensory unit, respectively. We developed a mussel-inspired universal conjugation method that enable both surface immobilization and bioconjugation at the same time. By incorporating dopamine or catechol moiety into self-signaling polydiacetylene (PDA) liposomes, the PDA liposomes can be immobilized to a wide range of substrates, without any substrate modification. Moreover, receptor molecules having specificity toward target molecules can also be attached to the immobilized PDA liposome layer without any chemical modification. We applied our mussel-inspired coating method to a droplet-array biosensor, exploiting hydrophilic nature of PDA liposomes coated on the hydrophobic polytetrafluoroethylene (PTFE) surface, and demonstrated selective and sensitive detection of vascular endothelial growth factor (VEGF) down to 10 nM
Methods based on surfactant-responsive controlled release systems of cargoes from nanocontainers have been developed for bioanalytical applications, but most were utilized for drug delivery and a few reports were focused on immunoassays. Herein we design an in situ amplified immunoassay protocol for high-efficient detection of aflatoxins (aflatoxin B1, AFB1 used in this case) based on surfactant-responsive cargo release from glucose-encapsulated liposome nanocarriers with sensitivity enhancement. Initially, biotinylated liposome nanocarrier encapsulated with glucose was synthesized using a reverse-phase evaporation method. Thereafter, the nanocarrier was utilized as the signal-generation tag on capture antibody-coating microplate through classical biotin-avidin linkage after reaction with biotinylated detection antibody. Upon addition of buffered surfactant (1X PBS-Tween 20 buffer) into the medium, the surfactant immediately hydrolyzed the conjugated liposome, and released the encapsulated glucose from the nanocarriers, which could be quantitatively determined by using a low-cost personal glucometer (PGM). The detectable signal increased with the increment of target analyte. Under the optimal conditions, the assay could allow PGM detection toward target AFB1 as low as 0.6pgmL(-1) (0.6ppt). Moreover, the methodology also showed good reproducibility and high specificity toward target AFB1 against other mycotoxins and proteins, and was applicable for quantitatively monitoring target AFB1 in the complex systems, e.g., naturally contaminated/spiked peanut samples and serum specimens, with the acceptable results. Taking these advantages of simplification, low cost, universality and sensitivity, our design provides a new horizon for development of advanced immunoassays in future point-of-care testing.
This article reviews the recent progress in nanowell array biosensors that use the label-free detection protocol, and are detected in their natural forms. These nanowell array biosensors are fabricated by nanofabrication technologies that should be useful for developing highly sensitive and selective also reproducible biosensors. Moreover, electrochemical method was selected as analysis method that has high sensitivity compared with other analysis. Finally, highly sensitive nanobiosensor was achieved by combining nanofabrication technologies and classical electrochemical method. Many examples are mentioned about the sensing performance of nanowell array biosensors will be evaluated in terms of sensitivity and detection limit compared with other micro-sized electrode without nanowell array.
Nanoparticles (NPs) are considered a promising tool in both diagnosis and therapeutics. Theranostic NPs possess the combined properties of targeted imaging and drug delivery within a single entity. While the categorization of theranostic NPs is based on their structure and composition, the pharmacokinetics of NPs are significantly influenced by the physicochemical properties of theranostic NPs as well as the routes of administration. Consequently, altered pharmacokinetics modify the pharmacodynamic efficacy and toxicity of NPs. Although theranostic NPs hold great promise in nanomedicine and biomedical applications, a lack of understanding persists on the mechanisms of the biodistribution and adverse effects of NPs. To better understand the diagnostic and therapeutic functions of NPs, this review discusses the factors that influence the pharmacokinetics, pharmacodynamics and toxicology of theranostic NPs, along with several strategies for developing novel diagnostic and therapeutic modalities.
Immobilization of bioanalytes (e.g., protein, lipid membrane, and cells) within a microfludic channel is a useful strategy in diverse biological analysis including biosensor, diagnosis, and biochemical reactions. Such microfluidic systems can offer miniaturized platforms with distinct advantages such as reduced use of samples or reagents and increased resolution. In particular, polymeric microfluidic devices (e.g., polydimethylsiloxane (PDMS)) bound to a substrate have been widely used. However the non-specific adsorption of bioanalytes is serious problem for microfluidic biological assays, especially for dilute samples. To overcome such limitations, a simple and widely applicable microfluidic channels combined with polyethylene glycol (PEG) microwells have been developed. In this review, we summarize the methods and application of PEG microwells with a particular emphasis on integrated microfluidic systems. The assembly between PEG microwell and microchannel enables precise delivery and manipulation of the biosamples, leading to development of miniaturized diagnostic assays, microreactors, and multiple screening platforms for tissue engineering and cell biology.
Maintaining the intrinsic features of mesophases is critically important when employing phospholipid self-assemblies to mimic biomembranes. Inorganic solid surfaces provide platforms to support, guide, and analyze organic self-assemblies, but impose upon them a tendency to form well-ordered phases not often found in biomembranes. To address this, we measured mesophase formation in a thiolate self-assembled monolayer (SAM) of diacyl phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) on Au(111), and provide thermodynamic analysis on the mixing behavior of inequivalent DPPTE acyl chains. Our work has uncovered three fundamental issues that enable mesophase formation: (1) Elimination of templating effects of the solid surface, (2) Weakening intermolecular and molecule-substrate interactions in adsorbates, and (3) Equilibrium through entropy-driven self-assembly. Thus, our work provides a more holistic understanding of phase behavior, from liquid phases to mesophases to highly crystalline phases, in organic self-assemblies on solid surfaces, which may extend their applications in nanodevices and to the wider fields of biology and medicine.
In recent years, a new paradigm of nanobiomedical devices combining miniaturization and integration has been exploited in areas such as combinational chemistry, biotechnology, engineering, proteomics and clinical diagnostics. One of the critical issues in the development of nanobiomedical system is how to differentiate signal-to-noise ratio per very small amount of signal. Biocompatible integrated nanopattern requires the fabrication of appropriately designed nanomatrix for high sensitivity homogenous assays, which are capable of ultimately mimic the physiological environment. We reported the nanomatrix geometry of a well-oriented nanowell array derived from nanofabrication technology which can easily be employed for digital detection with a high S/N ratio, miniaturization, integrated assays and single molecule analysis. In this present, we describe a nano(submicro) array of tethered lipid bilayer raft membranes comprising a biosensing platform.
Soft lithographic method is gaining in popularity for the manufacture of low-cost micro and sub-microstructures. For many applications, it is desirable to fabricate structures with different lateral size as well as different height, i.e. hierarchic structures, on the same substrate. This is difficult for the normal microfabrication method, particularly for photolithography method which is the main methods for IC industry. Here we demonstrate a simple yet cost-effective process to form the hierarchical structures by capillary force lithography (CFL). In our process, the simple principle that the time needed for the fluid to fill up the cavity in the mold is different if the contact angle of the mold or the width of the cavity in the mold is different. Based on this principle, two methods are developed to achieve hierarchical structures by CFL. One is to selectively modify the PDMS mold by UV or Oxygen plasma treatment to change its wettability in different areas. The other is to make patterns with variable dimensions in the mold. By using the PDMS mold with wettability selectively modified or with patterns with different lateral dimensions, hierarchical structures of convex/concave microlens array or stripes with convex/concave surfaces have been manufactured successfully.
The two most common methods to grow single crystals are the evaporation of solvent and the diffusion of one kind of solvent to solution. Evaporation-induced self-assembly has been widely used to make nanostructures, but to date there is no report on the nanostructures from solvent diffusion-induced self-assembly. In this work, different morphologies of porphyrin self-assembly were achieved by controlling the diffusion rate of water into organic solution. When water was mixed and homogenized with the porphyrin solution immediately, AFM measurements showed that porphyrin nanoparticles approximately 250 nm formed. On the other hand, when porphyrin DMF solution was diffused into water slowly, long nanotubes up to several hundred micrometers in length were observed. In particular, porphyrin macroscopic nanowell-array films, which may consisted of vertically-aligned porphyrin nanotube arrays, were obtained by diffusing water vapor into porphyrin organic solution. The methodology and results reported here may provide a novel, simple and efficient approach to obtain well-defined self-assembly structures.
A nanowell array electrode-based electrochemical quantitative system without amplification was developed and applied for the detection of H5N1 target DNA. An 18-mer probe was immobilized on a nanowell array electrode with a diameter of 500 nm, which was coated with streptavidin and a self-assembly monolayer (SAM). The surface properties of probe DNA hybridization with complementary target DNA were characterized using atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The AFM image shows that the depth of nanowell was reduced from 200 nm to 15 nm due to the formation of a DNA hybridization complex on the streptavidin/SAM structure. Differences in charge transfer resistance (deltaR(ct)) in EIS upon hybridization of the probe DNA with complementary target DNA were analyzed and used for the quantitation of H5N1 DNA. This approach shows that the quantitative analysis of H5N1 DNA ranging from 1 pM to 1 microM DNA is possible on a nanowell array electrode.
We describe the nanometrics geometry of a well-oriented nanowell (NW) array derived from nanofabrication technology which can easily be employed for digital detection with a high S/N ratio, miniaturization, integrated assays and single molecule analysis. In this geometry, the depth and width of the nanosized well can be adjusted to allow for only one or a few biomolecules to enter inside the nanowell and become attached with the self-assembled modified gold surface.
The self-assembly of neutral chiral porphyrins in aqueous solution was studied by absorption, fluorescence, circular dichroism (CD) spectroscopy, and optical and electron microscopy. Different supramolecular structures of the porphyrins were obtained by changing the diffusion rate of water into the porphyrin organic solution. When water was mixed and homogenized with the porphyrin solution immediately, the absorbance at the both sides of absorption Soret band increased along with hypochromism, indicating that J- and H-type self-assembly occurred simultaneously, and AFM measurements showed that porphyrin nanoparticles approximately 250 nm formed. On the other hand, the significant red-shift of the absorption Soret band and very long porphyrin microtubes observed by optical microscopy clearly demonstrated that J-type intermolecular packing increased significantly when porphyrin solution was slowly diffused into water. These observations indicate that the J- and H-type self-assembly pathways are kinetic and thermodynamic, respectively, and different self-assembly structures could be obtained by controlling the kinetic and thermodynamic pathways, which was also supported by theoretical calculations. In particular, porphyrin macroscopic nanowell-array films, which morphology is ideal for organic photovoltaic devices and biosensors, were obtained by diffusing water vapor into porphyrin organic solution. The methodology and results reported here may provide a novel, simple, and efficient approach to obtain well-defined self-assembly nanostructures.
This Feature Article aims to provide an in-depth overview of the recently developed molding technologies termed capillary force lithography (CFL) that can be used to control the cellular microenvironment towards cell and tissue engineering. Patterned polymer films provide a fertile ground for controlling various aspects of the cellular microenvironment such as cell–substrate and cell–cell interactions at the micro- and nanoscale. Patterning thin polymer films by molding typically involves several physical forces such as capillary, hydrostatic, and dispersion forces. If these forces are precisely controlled, the polymer films can be molded into the features of a polymeric mold with high pattern fidelity and physical integrity. The patterns can be made either with the substrate surface clearly exposed or unexposed depending on the pattern size and material properties used in the patterning. The former (exposed substrate) can be used to adhere proteins or cells on pre-defined locations of a substrate or within a microfluidic channel using an adhesion-repelling polymer such as poly(ethylene glycol) (PEG)-based polymer and hyaluronic acid (HA). Also, the patterns can be used to co-culture different cells types with molding-assisted layer-by-layer deposition. In comparison, the latter (unexposed substrate) can be used to control the biophysical surrounding of a cell with tailored mechanical properties of the material. The surface micropatterns can be used to engineer cellular and multi-cellular architecture, resulting in changes of the cell shape and the cytoskeletal structures. Also, the nanoscale patterns can be used to affect various aspects of the cellular behavior, such as adhesion, proliferation, migration, and differentiation.
The mesoporous inorganic–organic SiO2–TiO2–PEG hybrid resin systems with various useful functionalities were directly synthesized by a binary sol–gel reaction of TEOS–TiCl4 and the subsequent chelation with a chatecholic compound (dihydroxy benzene), dihydroxy-m-benzenedisulfonic acid disodium
salt, on the surface Ti ion of the ordered mesoporous SBA-15 network structure, respectively. Moreover, the hybrid resins
consisting of polyethylene glycol and a silane coupling agent exhibited the controlled wettability, excellent coating processibility
on various substrates with strong abrasion resistance. Furthermore, the transparent and low viscous resin showed the successful
performance to fabricate various nanoscale patterns with the feature size down to 170nm by imprint lithography. Based on
the excellent patternability, nanofluidics with 100nm of the narrowest dimension channel height was fabricated to employ
a capillary electrophoresis for separation DNAs without gel matrix. In addition, the presence of sulfonic acid in the resin
also showed the solid acid catalytic performance. These results reveal that the developed hybrid materials are very useful
as an imprint resin as well as versatile microchemical applications.
KeywordsMesoporous SiO2–TiO2–PEG resin–Chelation–Hydrophilicity–Imprint stamp–Nanofluidics–Solid acid catalyst
Although the SiOx nanoparticles were previously reported to electrochemically nucleate from the bottoms of the pore array formed in the AAO/Ti/Si system, new applications of anodic aluminum oxide templates, i.e., successive nucleation and a rodlike growth of SiOx nanoparticles, were observed utilizing the subsequent annealing technique after the nanoparticle precipitation. Tailoring the pore bottom profile by doping type and level of wafers also critically affected to nucleate the SiOx nanoparticles from the pore bottoms. By anodization, as previously reported, only one nanoparticle per each pore was generally precipitated from the pyramid-shaped Si-containing TiOx nanopillars; however, subsequent annealing under a low pressure of hydrogen enabled the successive precipitation of nanoparticles. Annealing under atmospheric pressure with H2 and N2 resulted in the rodlike growth of a single nanoparticle without successive nanoparticle precipitation.
Si nano-well arrays, with precisely controlled undercut Si sidewall profiles and flat bottomed pockets, enable uniform nanoscale pattern transfer from resists to metal deposits without degradation of the initial lithographic resolution, as verified by the formation of arrays of Au nano-dots with 10 nm diameter. An additional functionality of the Si nano-wells as local nano-reactors, where the patterned material is enclosed in a Si pocket during high temperature reaction, is demonstrated by thermally inducing a phase transformation of the as-deposited A1 phase of FePt nano-dots to the high coercivity, chemically ordered L1(0) phase.
We present new methods that enable the fabrication of multiscale, multicomponent protein-patterned surfaces and multiscale topologically structured surfaces by exploiting the merits of two well-established techniques: capillary force lithography (CFL) and microscope projection photolithography (MPP) based on a protein-friendly photoresist. We further demonstrate that, when hierarchically organized micro- and nanostructures were used as a cell culture platform, human colon cancer cells (cell line SW480) preferentially adhere and migrate onto the area with nanoscale topography over the one with microscale topography. These methods will provide many exciting opportunities for the study of cellular responses to multiscale physicochemical cues.
A free-radical-polymerizable SSQ/PEG blend with direct patternability has been proposed as an ideal nonfouling material for nanostructure-based biomedical applications. Cured SSQ/PEG networks show an UV transparency of >90% at 365 nm, high resistance to organic/aqueous solutions, hydrophilicity and Young's moduli of 1.898-2.815 GPa. SSQ/PEG patterns with 25-nm linewidths, 25-nm spacing, and an aspect ratio of 4:1 were directly fabricated on transparent substrates by UV embossing, and cured SSQ/PEG networks with long-term stability under chemical, thermal, and biological stress showed strong resistance to the nonspecific adsorption of biomolecules. These characteristics may offer a new strategy for the development of a number of medical nanodevice applications such as labs-on-a-chip.
Sensitive and selective biosensors for high-throughput screening are having an increasing impact in modern medical care. The establishment of robust protein biosensing platforms however remains challenging, especially when membrane proteins are involved. Although this type of proteins is of enormous relevance since they are considered in >60% of the pharmaceutical drug targets, their fragile nature (i.e., the requirement to preserve their natural lipid environment to avoid denaturation and loss of function) puts strong additional prerequisites onto a successful biochip. In this review, the leading approaches to create lipid membrane-based arrays towards the creation of membrane protein biosensing platforms are described. Liposomes assembled in micro- and nanoarrays and the successful set-ups containing functional membrane proteins, as well as the use of liposomes in networks, are discussed in the first part. Then, the complementary approaches to create cell-mimicking supported membrane patches on a substrate in an array format will be addressed. Finally, the progress in assembling free-standing (functional) lipid bilayers over nanopore arrays for ion channel sensing will be reported. This review illustrates the rapid pace by which advances are being made towards the creation of a heterogeneous biochip for the high-throughput screening of membrane proteins for diagnostics, drug screening, or drug discovery purposes.
Solvent-assisted decal transfer lithography (DTL) enables the formation of well-defined micro-/nanostructures over a large area (similar to 4 in. wafer) by combining irreversible oxygen bonding and anisotropic swelling of poly(dimethoxylsiloxane) (PDMS). Such swelling-induced stress gradient allows for cohesion failure of the skin layer upon removal of the stamp, leaving behind a highly uniform layer (similar to 100 nm).
New mesoporous silicate-titania resin systems hybridized with 4,5-dihydroxy-m-benzenedisulfonic acid and poly(ethylene glycol)-dimethacrylate component were developed. These inorganic-organic hybrid resins were found to reveal highly controlled ionic and hydrophilic surface with excellent durability and adhesion onto various substrates. The resin films revealed high resistance to nonspecific adsorption of fibrinogen and to adherence by several bacterial pathogens such as Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. Furthermore, excellent biocompatibility of the developed resins was proved by both HEp-2 cell adhesion in vitro and subcutaneous implantation in mice. The inorganic-organic hybrid resins are strongly promising for biomedical applications including biomedical devices and biosensors.
Bioinspired masks, created by merging sol-gel chemistry with biotemplating, were used as local chemical reactors to grow aligned arrays of gold nanoparticle-like wires.
Lipid rafts are cholesterol- and sphingolipid-rich domains that function as platforms for signal transduction and other cellular processes. Tethered lipid bilayers have been proposed as a promising model to describe the structure and function of cell membranes. We report a nano(submicro) array of tethered lipid bilayer raft membranes (tLBRMs) comprising a biosensing platform. Poly(vinyl alcohol) (PVA) hydrogel was directly patterned onto a solid substrate, using ultraviolet-nanoimprint lithography (UV-NIL), as an inert barrier to prevent biofouling. The robust structures of the nanopatterned PVA hydrogel were stable for up to three weeks in phosphate-buffered saline solution despite significant swelling (100% in height) by hydration. The PVA hydrogel strongly restricted the adhesion of vesicles, resulting in an array of highly selective hydrogel nanowells. tLBRMs were not formed by direct vesicle fusion, although raft vesicles containing poly(ethylene glycol) lipopolymer were selectively immobilized on gold substrates patterned with PVA hydrogel. The deposition of tLBRM nano(submicro) arrays was accomplished by a mixed, self-assembled monolayer-assisted vesicle fusion method. The monolayer was composed of a mixture of 2-mercaptoethanol and poly(ethylene glycol) lipopolymer, which promoted vesicle rupture. These results suggest that the fabrication of inert nanostructures and the site-selective modification of solid surfaces to induce vesicle rupture may be essential in the construction of tLBRM nano(submicro) arrays using stepwise self-assembly.
Novel nanomaterials for bioassay applications represent a rapidly progressing field of nanotechnology and nanobiotechnology. Here, we present an exploration of single-walled carbon nanotubes as a platform for investigating surface-protein and protein-protein binding and developing highly specific electronic biomolecule detectors. Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins, is overcome by immobilization of polyethylene oxide chains. A general approach is then advanced to enable the selective recognition and binding of target proteins by conjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. This scheme, combined with the sensitivity of nanotube electronic devices, enables highly specific electronic sensors for detecting clinically important biomolecules such as antibodies associated with human autoimmune diseases.
The design and performance of a filter holder which enables convenient preparation of volumes of up to a milliliter of large, unilamellar vesicles formed by extrusion (LUVETs) from multilamellar vesicles (MLVs) are described. The filter holder provides for back-and-forth passage of the sample between two syringes, a design that minimizes filter blockage, eliminates the need to change filters during LUVET preparation and reduces preparation time to a few minutes. Replicas of slam-frozen LUVETs in the electron microscope are unilamellar and reasonably homogeneous with an average diameter close to the pore size of the filters used to extrude them. Extrusion per se does not destabilize the vesicles, which trapped a fluorescent dye only when they were disrupted on freeze-thawing and during the first extrusion when most of the MLVs were apparently converted to LUVETs.
Scientific and practical applications of supported lipid-protein bilayers are described. Membranes can be covalently coupled to or separated from solids by ultrathin layers of water or soft polymer cushions. The latter systems maintain the structural and dynamic properties of free bilayers, forming a class of models of biomembranes that allow the application of a manifold of surface-sensitive techniques. They form versatile models of low-dimensionality complex fluids, which can be used to study interfacial forces and wetting phenomena, and enable the design of phantom cells to explore the interplay of lock-and-key forces (such as receptor-ligand binding) and universal forces for cell adhesion. Practical applications are the design of (highly selective) receptor surfaces of biosensors on electrooptical devices or the biofunctionalization of inorganic solids.
Boron-doped silicon nanowires (SiNWs) were used to create highly sensitive, real-time electrically based sensors for biological
and chemical species. Amine- and oxide-functionalized SiNWs exhibit pH-dependent conductance that was linear over a large
dynamic range and could be understood in terms of the change in surface charge during protonation and deprotonation. Biotin-modified
SiNWs were used to detect streptavidin down to at least a picomolar concentration range. In addition, antigen-functionalized
SiNWs show reversible antibody binding and concentration-dependent detection in real time. Lastly, detection of the reversible
binding of the metabolic indicator Ca2+ was demonstrated. The small size and capability of these semiconductor nanowires for sensitive, label-free, real-time detection
of a wide range of chemical and biological species could be exploited in array-based screening and in vivo diagnostics.
Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 microg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein-protein binding, DNA hybridization, and DNA-protein interactions, as well as drug discovery.
Dip-pen nanolithography was used to construct arrays of proteins with 100- to 350-nanometer features. These nanoarrays exhibit
almost no detectable nonspecific binding of proteins to their passivated portions even in complex mixtures of proteins, and
therefore provide the opportunity to study a variety of surface-mediated biological recognition processes. For example, reactions
involving the protein features and antigens in complex solutions can be screened easily by atomic force microscopy. As further
proof-of-concept, these arrays were used to study cellular adhesion at the submicrometer scale.
An ultrasensitive method for detecting protein analytes has been developed. The system relies on magnetic microparticle probes with antibodies that specifically bind a target of interest [prostate-specific antigen (PSA) in this case] and nanoparticle probes that are encoded with DNA that is unique to the protein target of interest and antibodies that can sandwich the target captured by the microparticle probes. Magnetic separation of the complexed probes and target followed by dehybridization of the oligonucleotides on the nanoparticle probe surface allows the determination of the presence of the target protein by identifying the oligonucleotide sequence released from the nanoparticle probe. Because the nanoparticle probe carries with it a large number of oligonucleotides per protein binding event, there is substantial amplification and PSA can be detected at 30 attomolar concentration. Alternatively, a polymerase chain reaction on the oligonucleotide bar codes can boost the sensitivity to 3 attomolar. Comparable clinically accepted conventional assays for detecting the same target have sensitivity limits of approximately 3 picomdar, six orders of magnitude less sensitive than what is observed with this method.
We describe highly sensitive, label-free, multiplexed electrical detection of cancer markers using silicon-nanowire field-effect devices in which distinct nanowires and surface receptors are incorporated into arrays. Protein markers were routinely detected at femtomolar concentrations with high selectivity, and simultaneous incorporation of control nanowires enabled discrimination against false positives. Nanowire arrays allowed highly selective and sensitive multiplexed detection of prostate specific antigen (PSA), PSA-alpha1-antichymotrypsin, carcinoembryonic antigen and mucin-1, including detection to at least 0.9 pg/ml in undiluted serum samples. In addition, nucleic acid receptors enabled real-time assays of the binding, activity and small-molecule inhibition of telomerase using unamplified extracts from as few as ten tumor cells. The capability for multiplexed real-time monitoring of protein markers and telomerase activity with high sensitivity and selectivity in clinically relevant samples opens up substantial possibilities for diagnosis and treatment of cancer and other complex diseases.
Stamp deformation can affect the dimensional stability of the microcontact printing process. We consider limitations imposed due to reversible deformation of a single stamp. Detailed analyses of several modes of stamp deformation have been carried out. Stability criteria have been obtained for both vertical and lateral collapse of surface relief features, including buckling. The shape change of surface features imposed by surface tension has been analyzed, and the corresponding internal stresses are given in closed form. The residual stresses induced by chemical and thermal shrinkage when the elastomeric stamp is bonded to a stiff substrate are analyzed. In addition, the relation between applied load and displacement of a stamp supported by a stiff substrate is given in closed form. Contact stresses between the stamp and substrate have been analyzed both analytically and numerically by a finite element method. The role of adhesion in determining the contact area is clarified. The effect of surface roughness on the contact mechanics has been studied, and closed form solutions have been obtained for surface asperities that are periodically distributed. The contact mechanics of stamps with smooth relief features has been studied, and the dependence of contact area on the work of adhesion and the applied pressure is given in closed form. The force required to separate the stamp from the substrate has been estimated using a fracture mechanics approach. The stability and contact mechanics results are summarized by a stability and contact map.
A novel method of patterning cells and proteins with a copolymer comprised of poly-anchoring groups is presented. As such, the technique allows for control over surface topography and surface molecules. The potential use of the technique for the development of improved biosensors and analytical tools is an area of active research.
Microcontact printing has been used to prepare patterned self-assembled monolayers (SAMs) of cholesterylpolyethylenoxy thiol. These patterned SAMs were used as supports for the formation of integral supported lipid bilayers. Biofunctionality was confirmed by addition of valinomycin ionophores and gramicidin ion channels. Impedance spectroscopy of the resulting bilayer structures, incorporating valinomycin and the gramicidin, showed the expected ion selectivity.
We present a general survey of the preparation, the behaviour and the characterization of liposomes, and their versatility as analytical tools. Advances on the design of artificial liposomes have allowed manipulation of their features (size, lamellarity, resistance, fusion capability and encapsulation efficiency), which have given rise to a wide range of procedures to encapsulate or internalise a variety of reagents. These approaches have been used to study solute-membrane interactions and to improve sensitivity and/or selectivity in different analytical methods. We discuss the advantages and the limitations of the most recent applications of liposomes in chromatography, capillary electrophoresis, immunoassays, sensors and microfluidic systems.
We report on the fabrication of microstructures of poly (ethylene glycol) (PEG) using a soft molding technique. When a patterned poly (dimethylsiloxane) stamp is placed on a wet PEG film, the polymer in contact with the stamp spontaneously moves into the void space as a result of capillary action. Three types of microstructures are observed with the substrate surface completely exposed: a negative replica of the stamp, a two-dimensional projection of the simple cubic structure, and a two-dimensional projection of the diamond structure. A molding process is responsible for the first type and a dewetting process for the final two. A phase diagram is constructed based on the effects of molecular weight and concentration, which shows that mobility and confinement play a crucial role in determining the particular type of microstructure obtained. The PEG microstructure could be used as a lithographic resist in fabricating electronic devices and a resistant layer for preventing nonspecific adsorption of proteins or cells. © 2003 American Institute of Physics.
T Cells, B cells, and other specialized cells in the immune system are activated by clustering of antigenic receptors on their plasma membrane surfaces; rat basophilic leukemia (RBL) mast cells which express cell surface immunoglobulin E (IgE) receptors and operate in the allergic response serve as a prototypic model. To investigate signaling pathways of the cellular response, a versatile system is being developed for presenting antigens on micron scale patterns of lipid bilayers that are prepared with a polymer-based wet lift-off method. A supported lipid bilayer is formed on a silicon substrate after photolithographic patterning of a polymer and before the polymer is mechanically peeled away in solution. This forms well-defined bilayer patterns that can contain haptenated and fluorescent lipids and can be used to simulate a biological substrate for cellular receptor engagement and response. For this model system, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (ammonium salt) (DNP-cap-DPPE) was used for specific binding of anti-DNP IgE. IgE's bound to mast cell surface receptors (FcεRI) aggregate and cluster over the patterned features if they are significantly smaller than the cells. For patterned features that are cell sized or larger, cells adhere to the silicon surface with receptors polarizing toward the edge of the patterned lipid. RBL cells with specifically engaged IgE-receptors are activated to undergo striking morphological changes that can be analyzed with fluorescence or scanning electron microscopy. This novel method for patterning antigen-functionalized lipids provides spatial control down to micron resolution for the antigenic stimulus while retaining the features of a plasma membrane substrate, including dynamics and variable composition. This method offers an alternative to microcontact printing with some enhanced capabilities for cellular immunological studies.
A study of confinement of lipid bilayers within a prespecified region by employing patterned self-assembled monolayers (SAMs) was carried out. The edge of the thiol support serves as a self-limiting boundary for the bilayer and enables deposition of dissimilar lipid layers on different thiol supports adjacent to each other. The results revealed that it is possible to reduce feature size without loss of discrimination.
In nature, peptides bind metals reproducibly and selectively via specific amino acid sequences, producing nanocrystals in controlled sizes and morphologies. A novel method of growing uniform nanocrystals directly on biological nanotubes by immobilizing synthetic Ni-mineralizing peptides on the nanotubes is reported (see Figure). Control of the Ni nanocrystal size is a function of pH, enabling tunability of magnetic properties in the resulting nanotube.
The use of rigiflex lithography as an effective method of transferring the bilayer to a substrate was described. Rigiflex lithography allows to physically transfer nanostructures to a substrate at a pressure level that was reduced by almost an order of magnitude. It also allows to pattern nanostructures by a roller bilayer-transfer technique (BLT) with a cylindrical roller, suitable for high throughput. The temperature needed for the transfer is moderate enough to permit the step-and-repeat technique.
The wet photolithography method was used for micropatterning fluid phospholipid bilayers. This approach involved spatially directed photodegradation of bilayer lipids using patterned deep-UV illumination resulting in the creation of optically defined patterns of fluid bilayers submerged in the aqueous phase. It extended into the aqueous phase popular methods of light-directed synthesis for designing peptides or DNA sequences on planar supports in the dry state. The results show that in conjugation with multiple patterning and backfilling cycles, new constructs can be used for high throughput proteomics, membrane-protein arrays, and aqueous-phase materials synthesis.
This article reports the theory and analytical applications of thin lipid films. Recent advances of electrochemical devices based on lipid membranes have lead to reports of construction of biosensors for environmental and food applications, and may provide opportunities for commercial fabrication. The methods of formation of lipid membranes on various supports including metals (silver, gold, stainless steel), agar, conducting polymers and ultrafiltration membranes have provided stabilization of lipid films with a diversity of analytical applications in real samples. Methods of immobilization and incorporation of various functional macromolecules are summarized. Several examples of the application of various methods for study of physical properties of supported bilayer lipid membranes are described. Applications of lipid-based biosensors in analytical chemistry for determination of compounds are demonstrated, including a diversity of chemical compounds such as environmental pollutants (ammonia and carbon dioxide, cyanide ions, etc.) and food toxins (aflatoxin M1and direct detection of toxin in real samples such as milk and milk preparations). Methods for application of liposomes as a sensing system are also summarized.
Carbon nanotubes, compounded into plastic create bulk electrodes, form the basis of a novel sensor for biological assays. The nanotubes function as both a solid phase and working electrode for generation of electrochemiluminescence, the detection method used in this system. The Figure shows a scanning electron microscopy image of a composite used for an immunoassay. The bright spots in the image are 10 nm diameter gold colloids covalently attached to antibodies. The inset shows a region of a composite with no gold colloids.
Model membranes, based on thin organic films containing an even, random distribution of functionalized amphiphilic lipid molecules, are reported. The monolayers are stabilized by polymerization and can be transferred to solid substrates using the Langmuir-Blodgett technique, allowing structures such as that shown in the Figure to be produced.
The synthesis of a poly(ethylene glycol) (PEG)-grafted surface-reactive random copolymer and its self-assembled structure on Si/SiO2 substrates for construction of nonbiofouling surfaces are reported. The copolymer, poly(TMSMA-r-PEGMA), which is comprised of an "anchor part" (trimethoxysilane) and a "function part" (PEG), was synthesized by a radical polymerization reaction. The copolymer spontaneously formed monolayers on Si/SiO2 wafers with average thicknesses of 11 Å. Tapping mode atomic force microscopy (AFM) revealed that the surface of the polymer monolayers was smooth with an average roughness of 1.3 Å (root-mean-square). The protein resistance of the polymer monolayers on Si/SiO2 wafers was evaluated using insulin, lysozyme, and fibrinogen. For all tested proteins, the polymer monolayers showed significant reduction (up to 98%) in nonspecific protein adsorption compared to the unmodified Si/SiO2 wafers. In addition, cell adhesion as probed using 3T3 fibroblasts was significantly reduced on the polymer-coated glass substrates in comparison to unmodified glass substrates.
A method for direct patterning of lipid bilayer membranes on the surface of colloidal-silica particles by in-site UV photochemical lithography was analyzed. Highly parallel generation of micrometer-resolution membrane patterns was achieved with biocompatible processes. The direct patterning technique enabled the presentation of biomolecules in a controllably asymmetric manner. The results show that the homogeneity of membrane patterns over the surface of the colloidal particle suggests the achievement of 3D photolithography.
Enhanced fluorescence emission is detected from interacting protein pairs of dichlorotriazinylaminofluoresceinstreptavidin and biotinylated bovine serum albumin (see figure) that are adsorbed onto periodically spaced, square-patterned ZnO nanostructures. This florescence-enhancement capability of nanoscale ZnO and its potential easy integration into biosensor arrays may allow ultrasensitive protein detection, which is needed in the areas of biomedical research and in large-scale testing and screening.
Liposomes are microparticulate lipoidal vesicles which are under extensive investigation as drug carriers for improving the delivery of therapeutic agents. Due to new developments in liposome technology, several liposome-based drug formulations are currently in clinical trial, and recently some of them have been approved for clinical use. Reformulation of drugs in liposomes has provided an opportunity to enhance the therapeutic indices of various agents mainly through alteration in their biodistribution. This review discusses the potential applications of liposomes in drug delivery with examples of formulations approved for clinical use, and the problems associated with further exploitation of this drug delivery system.
During the last decade, avidin-biotin technology has become a commercially viable tool for research, medical and industrial applications. From the beginning, mediation via the avidin-biotin complex was proposed for affinity-based separations. This particular application, however, has been slow in gaining acceptance. One of the reasons is that the strength of binding between avidin and biotin is sometimes inappropriate for the desired affinity system. Another problem involves certain "undesirable" structural properties in the avidin molecule which may lead to high levels of "non-specific" binding. Recent progress in understanding the molecular requirements for binding biotin may eventually lead to the design of avidin-like proteins which will exhibit preferred recognition properties according to the desired application.
Potassium ferrocyanide Is encapsulated In the aqueous cavity of spherical phosphollpid bilayer vesicles (liposomes) at concentrations of approximately 104 molecules/liposome. Physical parameters and stability of these structures are determined by electrochemical and spectroscopic methods. The electroactive marker ions (ferrocyanide) are released from within the liposome by either the addition of surfactant or the complement lysis of the membrane. The classical complement pathway is an antigen/antibody-specific reaction that occurs when an antigen-sensitized liposome immunospecifically binds with a corresponding antibody in the presence of certain serum proteins (complement). The release of encapsulated ferrocyanide is monitored by differential pulse vottammetry. Preliminary Investigations with an ion-exchange polymer modified electrode demonstrate the ability to preconcentrate the released marker at the electrode surface as well as the necessity of a polymer film to protect the surface from fouling during serum-mediated lysis.
The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques.
Lithographically patterned grids of photoresist, aluminum oxide, or gold on oxidized silicon substrates were used to partition
supported lipid bilayers into micrometer-scale arrays of isolated fluid membrane corrals. Fluorescently labeled lipids were
observed to diffuse freely within each membrane corral but were confined by the micropatterned barriers. The concentrations
of fluorescent probe molecules in individual corrals were altered by selective photobleaching to create arrays of fluid membrane
patches with differing compositions. Application of an electric field parallel to the surface induced steady-state concentration
gradients of charged membrane components in the corrals. In addition to producing patches of membrane with continuously varying
composition, these gradients provide an intrinsically parallel means of acquiring information about molecular properties such
as the diffusion coefficient in individual corrals.
Various aspects of the application of liposomes as a label in immunoassays are reviewed. Methods for the preparation of liposomes, from the basic film method to the more advanced dehydration-rehydration method, are discussed. Furthermore, the markers used in liposome labels, as well as the methods to conjugate liposomes to antigens or antibodies, are summarized. Liposome immunoassays are applied as homogeneous or heterogeneous assays. Homogeneous assays often rely on the lytic activity of complement on antibody-associated liposomes. Another group of homogeneous assays utilizes the inhibitory action of antibodies on the activity of conjugates of mellitin (a bee venom protein) with a hapten. Free mellitin conjugates are able to lyse liposomes effectively. Heterogeneous liposome immunoassays, performed either competitively or non-competitively, resemble more closely standard enzyme linked immunosorbent assays, with the enzyme being replaced by a liposome label. Washing steps are used to separate antigen-specifically bound liposomes from unbound liposomes. All bound liposomes are lysed with a detergent, giving an instantaneous amplification. Flow-injection liposome immunoassays and liposome immunosensors are also described as examples of other possible immunoassay formats.
A direct-write “dip-pen” nanolithography (DPN) has been developed to deliver collections of molecules in a positive printing
mode. An atomic force microscope (AFM) tip is used to write alkanethiols with 30-nanometer linewidth resolution on a gold
thin film in a manner analogous to that of a dip pen. Molecules are delivered from the AFM tip to a solid substrate of interest
via capillary transport, making DPN a potentially useful tool for creating and functionalizing nanoscale devices.
Metalloproteins and enzymes can be immobilized on SWNTs of different surface chemistry. The combination of high surface area, robust immobilization and inherent nanotube electrochemical properties is of promising application in bioelectrochemistry.
Patterning techniques that rely on high-resolution elastomeric elements such as stamps, molds, and conformable photomasks are operationally simple methods for nanofabrication that may find applications in areas such as molecular and organic electronics. The resolution of these "soft" lithographic procedures is often limited by the mechanical properties of the elastomers. We introduce here a chemically modified poly(dimethylsiloxane) material that is designed and optimized specifically for soft lithography, particularly in the nanometer regime. We demonstrate its use for nanopatterning tasks that are challenging with the commercially available elastomers that have been used in the past.
Investigations of ligand-receptor binding between bivalent antibodies and membrane-bound ligands are presented. The purpose of these studies was to explore binding as a function of hapten density in a two-dimensionally fluid environment. A novel microfluidic strategy in conjunction with total internal reflection fluorescence microscopy was designed to achieve this. The method allowed binding curves to be acquired with excellent signal-to-noise ratios while using only minute quantities of protein solution. The specific system investigated was the interaction between anti-DNP antibodies and phospholipid membranes containing DNP-conjugated lipids. Binding curves for ligand densities ranging from 0.1 to 5.0 mol % were obtained. Two individual dissociation constants could be extracted from the data corresponding to the two sequential binding events. The first dissociation constant, K(D1), was 2.46 x 10(-)(5) M, while the second was K(D2) = 1.37 x 10(-)(8) mol/m(2). This corresponded to a positively cooperative binding effect with an entropic difference between the two events of 62.3 +/- 2.7 J/(mol.K). Furthermore, the percentage of monovalently and bivalently bound protein was determined at each ligand density.
Supported planar bilayers have been used extensively in immunology to study molecular interactions at interfaces as a model for cell-cell interaction. Examples include Fc receptor-mediated adhesion and signaling and formation of the immunological synapse between T cells and antigen-presenting cells. The advantage of the supported planar bilayer system is control of the bilayer composition and the optical advantages of imaging the cell-bilayer or bilayer-bilayer interface by various types of trans-, epi- and total internal reflection illumination. Supported planar bilayers are simple to form by liposome fusion and recent advances in micro- and nanotechnology greatly extend the power of supported bilayers to address key questions in immunology and cell biology.
In the coming decade, the ability to sense and detect the state of biological systems and living organisms optically, electrically and magnetically will be radically transformed by developments in materials physics and chemistry. The emerging ability to control the patterns of matter on the nanometer length scale can be expected to lead to entirely new types of biological sensors. These new systems will be capable of sensing at the single-molecule level in living cells, and capable of parallel integration for detection of multiple signals, enabling a diversity of simultaneous experiments, as well as better crosschecks and controls.
Scanning probe lithography (SPL) is applied to pattern fluid lipid membranes on a solid borosilicate substrate. Grids of metal lines, prepatterned onto the substrate by electron beam lithography, serve to partition the supported membrane into an array of isolated fluid pixels. By toggling the pH of the surrounding solution, the effect of the probe tip on the membrane can be regulated. Alkaline conditions favor membrane removal, while neutral pH favors membrane deposition. Arbitrary membrane patterns with spatial dimensions limited by the underlying grid size can be constructed by sequential SPL membrane removal followed by refill with a different membrane type. In the present study, bilayers of unique composition fill 1 x 1 mum corrals and were positioned 100 nm apart.
An original electrochemical immunosensor has now been developed that is based upon the spontaneous immobilization of biotinylated, functional lipid vesicles (FLVs) on a polymeric resist layer. An electrode was fabricated utilizing a form of electron-beam (e-beam) that has been used to fabricate 200 nm (nanoscale) wells in the resist layer covering of the gold electrode. The stability of adhered FLVs upon the nanowell (NW) electrode was observed by atomic force microscopy (AFM). From these observations, we were able to determine that the assembled FLVs primarily adhered as individual molecules, that is, without the aggregation or fusion noted in earlier designs. Additionally, these immobilized FLVs demonstrated clearly defined redox activity in electrochemical measurements. Streptavidin, biotinylated capture antibody, and target proteins were consequently injected in order to set up the immunoassay environment. Electrochemical immunoassay experimentation was performed on the NW array electrode with model proteins, such as human serum albumin (HSA) and carbonic anhydrase from bovine (CAB). We observed a notable current decrease, following the redox path, interrupted by the target HSA, indicating the binding of the capture antibody with the target antigen. On the basis of these results, we propose a new type of immunosensor incorporating this mechanism of spontaneous immobilization of FLVs.
Individual M13 viruses were spatially confined within wells fabricated from nanomolding of a PEG-based random copolymer. The viruses were selectively adhered to the region pretreated with an antibody against the virus, resulting in individual virus arrays. The polymer surface was found to be highly resistant to the attachment of the virus (approximately 0.02 microm-2), approximately 2 orders of magnitude lower than that on a bare silicon surface. The physical height of the template provided an additional barrier to the attachment of the virus due to entropic penalty in bending of a semi-flexible M13 virus. The effects of pattern size and barrier height were investigated, revealing that a certain critical height is needed to ensure successful confinement within the template for a given pattern size.
Ink flowing from a pen: Direct-write dippen nanolithography of His-tagged proteins (ubiquitin and thioredoxin) has been used to generate biologically active protein nanoarrays with feature sizes as small as 80 nm on nickel oxide surfaces without the need for an applied electric field. The protein molecules in this system seem to diffuse from the Ni-coated atomic force microscopy (AFM) tips to the Ni-coated substrate (see diagram).
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