Biochips for Cell Biology by Combined Dip‐Pen Nanolithography and DNA‐Directed Protein Immobilization

A general methodology for patterning of multiple protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (DPN) patterning of DNA oligonucleotides which are then used as capture strands for DNA‐directed immobilization (DDI) of oligonucleotide‐tagged proteins. This study reports the development and optimization of PEG‐based liquid ink, used as carrier for the immobilization of alkylamino‐labeled DNA oligomers on chemically activated glass surfaces. The resulting DNA arrays have typical spot sizes of 4–5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent DNA‐streptavidin (DNA‐STV) conjugates bearing ligands recognized by cells. To this end, biotinylated epidermal growth factor (EGF) is coupled to the DNA‐STV conjugates, the resulting constructs are hybridized with the DNA arrays and the resulting surfaces used for the culturing of MCF‐7 (human breast adenocarcinoma) cells. Owing to the lateral diffusion of transmembrane proteins in the cell's plasma membrane, specific recruitment and concentration of EGF receptor can be induced specifically at the sites where the ligands are bound on the solid substrate. This is a clear demonstration that this method is suitable for precise functional manipulations of subcellular areas within living cells.     

DNA‐SMART: Biopatterned Polymer Film Microchannels for Selective Immobilization of Proteins and Cells

A novel SMART module, dubbed “DNA‐SMART” (DNA substrate modification and replication by thermoforming) is reported, where polymer films are premodified with single‐stranded DNA capture strands, microthermoformed into 3D structures, and postmodified with complementary DNA‐protein conjugates to realize complex biologically active surfaces within microfluidic devices. As a proof of feasibility, it is demonstrated that microchannels presenting three different proteins on their inner curvilinear surface can be used for selective capture of cells under flow conditions.     

Biopebble Containers: DNA‐Directed Surface Assembly of Mesoporous Silica Nanoparticles for Cell Studies

The development of methods for colloidal self‐assembly on solid surfaces is important for many applications in biomedical sciences. Toward this goal, described is a versatile class of mesoporous silica nanoparticles (MSN) that contain on their surface various types of DNA molecules to enable their self‐assembly into micropatterned surface architectures useful for cell studies. Monodisperse dye‐doped MSN are synthesized by biphase stratification and functionalized with an aptamer oligonucleotide that serves as gatekeeper for the triggered release of encapsulated molecular cargo, such as fluorescent dye rhodamine B or the anticancer drug doxorubicin. One or two additional types of oligonucleotides are installed on the MSN surface to enable DNA‐directed immobilization on solid substrates bearing patterns of complementary capture oligonucleotides. It is demonstrated that this strategy can be used for efficient self‐assembly of microstructured surface architectures, which not only promote the adhesion and guidance of cells but also are capable of affecting the fate of adhered cells through triggered release of their cargo. It is believed that this approach is useful for diverse applications in tissue engineering and nanobio sciences.     

Discontinuous Nanoporous Membranes Reduce Non‐Specific Fouling for Immunoaffinity Cell Capture

The microfluidic isolation of target cells using adhesion‐based surface capture has been widely explored for biology and medicine. However, high‐throughput processing can be challenging due to interfacial limitations such as transport, reaction, and non‐specific fouling. Here, it is shown that antibody‐functionalized capture surfaces with discontinuous permeability enable efficient target cell capture at high flow rates by decreasing fouling. Experimental characterization and theoretical modeling reveal that “wall effects” affect cell–surface interactions and promote excess surface accumulation. These issues are partially circumvented by reducing the transport and deposition of cells near the channel walls. Optimized microfluidic devices can be operated at higher cell concentrations with significant improvements in throughput.     

Configurable Low‐Cost Plotter Device for Fabrication of Multi‐Color Sub‐Cellular Scale Microarrays

The construction and operation of a low‐cost plotter for fabrication of microarrays for multiplexed single‐cell analyses is reported. The printing head consists of polymeric pyramidal pens mounted on a rotation stage installed on an aluminium frame. This construction enables printing of microarrays onto glass substrates mounted on a tilt stage, controlled by a Lab‐View operated user interface. The plotter can be assembled by typical academic workshops from components of less than 15 000 Euro. The functionality of the instrument is demonstrated by printing DNA microarrays on the area of 0.5 cm² using up to three different oligonucleotides. Typical feature sizes are 5 μm diameter with a pitch of 15 μm, leading to densities of up to 10⁴–10⁵ spots/mm². The fabricated DNA microarrays are used to produce sub‐cellular scale arrays of bioactive epidermal growth factor peptides by means of DNA‐directed immobilization. The suitability of these biochips for cell biological studies is demonstrated by specific recruitment, concentration, and activation of EGF receptors within the plasma membrane of adherent living cells. This work illustrates that the presented plotter gives access to bio‐functionalized arrays usable for fundamental research in cell biology, such as the manipulation of signal pathways in living cells at subcellular resolution.     

Imparting Designer Biorecognition Functionality to Metal–Organic Frameworks by a DNA‐Mediated Surface Engineering Strategy

Surface functionality is an essential component for processing and application of metal–organic frameworks (MOFs). A simple and cost‐effective strategy for DNA‐mediated surface engineering of zirconium‐based nanoscale MOFs (NMOFs) is presented, capable of endowing them with specific molecular recognition properties and thus expanding their potential for applications in nanotechnology and biotechnology. It is shown that efficient immobilization of functional DNA on NMOFs can be achieved via surface coordination chemistry. With this strategy, it is demonstrated that such porphyrin‐based NMOFs can be modified with a DNA aptamer for targeting specific cancer cells. Furthermore, the DNA–NMOFs can facilitate the delivery of therapeutic DNA (e.g., CpG) into cells for efficient recognition of endosomal Toll‐like receptor 9 and subsequent enhanced immunostimulatory activity in vitro and in vivo. No apparent toxicity is observed with systemic delivery of the DNA–NMOFs in vivo. Overall, these results suggest that the strategy allows for surface functionalization of MOFs with different functional DNAs, extending the use of these materials to diverse applications in biosensor, bioimaging, and nanomedicine.     

Nano‐Dissection and Sequencing of DNA at Single Sub‐Nuclear Structures

The relative positioning of gene loci within a mammalian nucleus is non‐random and plays a role in gene regulation. Some sub‐nuclear structures may represent “hubs” that bring specific genetic loci into close proximity where co‐regulatory mechanisms can operate. The identification of loci in proximity to a shared sub‐nuclear structure can provide insights into the function of the associated structure, and reveal relationships between the loci sharing a common association. A technique is introduced based on the nano‐dissection of DNA from thin sections of cells by high‐precision nano‐tools operated inside a scanning electron microscope. The ability to dissect and identify gene loci occupying a shared site at a single sub‐nuclear structure is demonstrated here for the first time. The technique is applied to the nano‐dissection of DNA in vicinity of a single promyelocytic leukemia nuclear body (PML NB), and reveals novel loci from several chromosomes that are confirmed to associate at PML NBs with statistical significance in a cell population. Furthermore, it is demonstrated that pairs of loci from different chromosomes congregate at the same nuclear body. It is proposed that this technique is the first that allows the de novo determination of gene loci associations with single nuclear sub‐structures.     

A framework for capturing and analyzing the failures due to system/component interactions

To keep up with the speed of globalization and growing customer demands for more technology‐oriented products, modern systems are becoming increasingly more complex. This complexity gives rise to unpredictable failure patterns. While there are a number of well‐established failure analysis (physics‐of‐failure) models for individual components, these models do not hold good for complex systems as their failure behaviors may be totally different. Failure analysis of individual components does consider the environmental interactions but is unable to capture the system interaction effects on failure behavior. These models are based on the assumption of independent failure mechanisms. Dependency relationships and interactions of components in a complex system might give rise to some new types of failures that are not considered during the individual failure analysis of that component. This paper presents a general framework for failure modes and effects analysis (FMEA) to capture and analyze component interaction failures. The advantage of the proposed methodology is that it identifies and analyzes the system failure modes due to the interaction between the components. An example is presented to demonstrate the application of the proposed framework for a specific product architecture (PA) that captures interaction failures between different modules. However, the proposed framework is generic and can also be used in other types of PA. Copyright © 2007 John Wiley & Sons, Ltd.     

Specific Capture of Peptide‐Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide‐Binding Assay in Live Cells

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H‐2K^b, into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H‐2K^b and hold it at the cell surface. This patterned receptor enables a novel peptide‐binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.     

Nanotentacle‐Structured Magnetic Particles for Efficient Capture of Circulating Tumor Cells

Circulating tumor cells (CTCs) have attracted considerable attention as promising markers for diagnosing and monitoring the cancer status. Despite many technological advances in isolating CTCs, the capture efficiency and purity still remain challenges that limit clinical practice. Here, the construction of “nanotentacle”‐structured magnetic particles using M13‐bacteriophage and their application for the efficient capturing of CTCs is demonstrated. The M13‐bacteriophage to magnetic particles followed by modification with PEG is conjugated, and further tethered monoclonal antibodies against the epidermal receptor 2 (HER2). The use of nanotentacle‐structured magnetic particles results in a high capture purity (>45%) and efficiency (>90%), even for a smaller number of cancer cells (≈25 cells) in whole blood. Furthermore, the cancer cells captured are shown to maintain a viability of greater than 84%. The approach can be effectively used for capturing CTCs with high efficiency and purity for the diagnosis and monitoring of cancer status.     

Antibody‐Free Hydrogel with the Synergistic Effect of Cell Imprinting and Boronate Affinity: Toward the Selective Capture and Release of Undamaged Circulating Tumor Cells

The selective and highly efficient capture of circulating tumor cells (CTCs) from blood and their subsequent release without damage are very important for the early diagnosis of tumors and for understanding the mechanism of metastasis. Herein, a universal strategy is proposed for the fabrication of an antibody‐free hydrogel that has a synergistic effect by featuring microinterfaces obtained by cell imprinting and molecular recognition conferred by boronate affinity. With this artificial antibody, highly efficient capture of human hepatocarcinoma SMMC‐7721 cells is achieved: as many as 90.3 ± 1.4% (n = 3) cells are captured when 1 × 10⁵ SMMC‐7721 cells are incubated on a 4.5 cm² hydrogel, and 99% of these captured cells are subsequently released without any loss of proliferation ability. In the presence of 1000 times as many nontarget cells, namely, leukaemia Jurkat cells, the SMMC‐7721 cells can be captured with an enrichment factor as high as 13.5 ± 3.2 (n = 3), demonstrating the superior selectivity of the artificial antibody for the capture of the targeted CTCs. Most importantly, the SMMC‐7721 cells can be successfully captured even when spiked into whole blood, indicating the great promise of this approach for the further molecular characterization of CTCs.     

Functionalized DNA Enables Programming Exosomes/Vesicles for Tumor Imaging and Therapy

Exosomes serve as significant information carriers that regulate important physiological and pathological processes. Herein, functionalized DNA is engineered to be a hinge that anchors quantum dots (QDs) onto the surface of exosomes, realizing a moderate and biocompatible labeling strategy. The QDs‐labeled exosomes (exosome–DNA–QDs complex) can be swiftly engulfed by tumor cells, indicating that exosome–DNA–QDs can be applied as a specific agent for tumor labeling. Furthermore, the engineered artificial vesicles of M1 macrophages (M1mv) are constructed via a pneumatic liposome extruder. The results reveal that the individual M1mv can kill tumor cells and realize desirable biological treatment. To reinforce the antitumor efficacy of M1mv and the specificity of drug release, a target‐triggered drug delivery system is constructed to realize a specific microRNA‐responded delivery system for visual therapy of tumors. These strategies facilitate moderate labeling and functionalization of exosomes/vesicles and construct artificial drug‐delivery vesicles that simultaneously possess biological treatment and chemotherapy functions, and thus have the potential to serve as a new paradigm for tumor labeling and therapy.     

Renewable Time‐Responsive DNA Circuits

DNA devices have been shown to be capable of evaluating Boolean logic. Several robust designs for DNA circuits have been demonstrated. Some prior DNA‐based circuits are use‐once circuits since the gate motifs of the DNA circuits get permanently destroyed as a side effect of the computation, and hence cannot respond correctly to subsequent changes in inputs. Other DNA‐based circuits use a large reservoir of buffered gates to replace the working gates of the circuit and can be used to drive a finite number of computation cycles. In many applications of DNA circuits, the inputs are inherently asynchronous, and this necessitates that the DNA circuits be asynchronous: the output must always be correct regardless of differences in the arrival time of inputs. This paper demonstrates: 1) renewable DNA circuits, which can be manually reverted to their original state by addition of DNA strands, and 2) time‐responsive DNA circuits, where if the inputs change over time, the DNA circuit can recompute the output correctly based on the new inputs, that are manually added after the system has been reset. The properties of renewable, asynchronous, and time‐responsiveness appear to be central to molecular‐scale systems; for example, self‐regulation in cellular organisms.     

Selective Capture and Quick Detection of Targeting Cells with SERS‐Coding Microsphere Suspension Chip

Circulating tumor cells (CTCs) captured from blood fluid represent recurrent cancers and metastatic lesions to monitor the situation of cancers. We develop surface‐enhanced Raman scattering (SERS)‐coding microsphere suspension chip as a new strategy for fast and efficient capture, recovery, and detection of targeting cancer cells. Using HeLa cells as model CTCs, we first utilize folate as a recognition molecule to be immobilized in magnetic composite microspheres for capturing HeLa cells and attaining high capturing efficacy (up to 95%). After capturing cells, the composite microsphere, which utilizes a disulfide bond as crosslinker in the polymer shell and as a spacer for linking folate, can recycle 90% cells within 20 min eluted by glutathion solution. Taking advantage of the SERS with fingerprint features, we characterize captured/recovered cells with the unique signal of report‐molecule 4‐aminothiophenol through introducing the SERS‐coding microsphere suspension chip to CTCs. Finally, the exploratory experiment of sieving cells shows that the magnetic composite microspheres can selectively capture the HeLa cells from samples of mixed cells, indicating that these magnetic composite microspheres have potential in real blood samples for capturing CTCs.     

Electronic Activation of a DNA Nanodevice Using a Multilayer Nanofilm

A method to control activation of a DNA nanodevice by supplying a complementary DNA (cDNA) strand from an electro‐responsive nanoplatform is reported. To develop functional nanoplatform, hexalayer nanofilm is precisely designed by layer‐by‐layer assembly technique based on electrostatic interaction with four kinds of materials: Hydrolyzed poly(β‐amino ester) can help cDNA release from the film. A cDNA is used as a key building block to activate DNA nanodevice. Reduced graphene oxides (rGOs) and the conductive polymer provide conductivity. In particular, rGOs efficiently incorporate a cDNA in the film via several interactions and act as a barrier. Depending on the types of applied electronic stimuli (reductive and oxidative potentials), a cDNA released from the electrode can quantitatively control the activation of DNA nanodevice. From this report, a new system is successfully demonstrated to precisely control DNA release on demand. By applying more advanced form of DNA‐based nanodevices into multilayer system, the electro‐responsive nanoplatform will expand the availability of DNA nanotechnology allowing its improved application in areas such as diagnosis, biosensing, bioimaging, and drug delivery.     

DNA Polymer Brush Patterning through Photocontrollable Surface‐Initiated DNA Hybridization Chain Reaction

The fabrication of DNA polymer brushes with spatial resolution onto a solid surface is a crucial step for biochip research and related applications, cell‐free gene expression study, and even artificial cell fabrication. Here, for the first time, a DNA polymer brush patterning method is reported based on the photoactivation of an ortho‐nitrobenzyl linker‐embedded DNA hairpin structure and a subsequent surface‐initiated DNA hybridization chain reaction (HCR). Inert DNA hairpins are exposed to ultraviolet light irradiation to generate DNA duplexes with two active sticky ends (toeholds) in a programmable manner. These activated DNA duplexes can initiate DNA HCR to generate multifunctional patterned DNA polymer brushes with complex geometrical shapes. Different multifunctional DNA polymer brush patterns can be fabricated on certain areas of the same solid surface using this method. Moreover, the patterned DNA brush surface can be used to capture target molecules in a desired manner.     

An Acidic‐Microenvironment‐Driven DNA Nanomachine Enables Specific ATP Imaging in the Extracellular Milieu of Tumor

Extracellular ATP is an emerging target for cancer treatment because it is a key messenger for shaping the tumor microenvironment (TME) and regulating tumor progression. However, it remains a great challenge to design biochemical probes for targeted imaging of extracellular ATP in the TME. A TME‐driven DNA nanomachine (Apt‐LIP) that permits spatially controlled imaging of ATP in the extracellular milieu of tumors with ultrahigh signal‐to‐background ratio is reported. It operates in response to the mild acidity in the TME with the pH (low) insertion peptide (pHLIP) module, thus allowing the specific anchoring of the structure‐switching signaling aptamer unit to the membrane of tumor cells for “off–on” fluorescence imaging of the extracellular ATP. Apt‐LIP allows for acidity driven visualization of different extracellular concentrations of exogenous ATP, as well as the monitoring of endogenous ATP release from cells. Furthermore, it is demonstrated that Apt‐LIP represents a promising platform for the specific imaging of the extracellular ATP in both primary and metastatic tumors. Ultimately, since diverse aptamers are obtained through in vitro selection, this design strategy can be further applied for precise detection of various extracellular targets in the TME.     

Sequentially Site‐Specific Delivery of Apoptotic Protein and Tumor‐Suppressor Gene for Combination Cancer Therapy

Nanocarrier‐mediated codelivery of multiple anticancer drugs is a potential strategy for enhanced efficacy of combination cancer treatment by unifying differential pharmacokinetic properties and maintaining an optimal ratio of drug cargoes. However, a programmable codelivery system is highly desired to deliver different therapeutics to their specific sites of action to pursue maximized combinational effect. Herein a liposome‐based nanoassembly (p53/C‐rNC/L‐FA) is developed for intracellular site‐specific delivery of an apoptotic protein cytochrome c (CytoC) and a plasmid DNA encoding tumor‐suppressing p53 protein (p53 DNA). p53/C‐rNC/L‐FA consists of an acid‐activated fusogenic liposomal membrane shell modified with folic acid (L‐FA) and a DNA/protein complex core assembled by the p53 DNA, protamine and CytoC‐encapsulated redox‐responsive nanocapsule (C‐rNC). Intratumoral and intraendosomal acidities promote membrane fusion between liposome and biomembrane, resulting in release of the encapsulated p53/C‐rNC complex into the cytoplasm. The cytoplasmic reduction causes degradation of C‐rNC with release of CytoC that induces tumor cell apoptosis. The p53 DNA is transported into the nucleus by the aid of the cationic protamine and thus generates expression of the p53 protein that enhances apoptosis combined with CytoC. p53/C‐rNC/L‐FA is demonstrated to significantly induce tumor cell apoptosis and inhibit tumor growth in the orthotopic breast tumor mouse model.     

Reduced Graphene Oxide‐Functionalized High Electron Mobility Transistors for Novel Recognition Pattern Label‐Free DNA Sensors

We designed and constructed reduced graphene oxide (rGO) functionalized high electron mobility transistor (HEMT) for rapid and ultra‐sensitive detection of label‐free DNA in real time. The micrometer sized rGO sheets with structural defects helped absorb DNA molecules providing a facile and robust approach to functionalization. DNA was immobilized onto the surface of HEMT gate through rGO functionalization, and changed the conductivity of HEMT. The real time monitor and detection of DNA hybridization by rGO functionalized HEMT presented interesting current responses: a “two steps” signal enhancement in the presence of target DNA; and a “one step” signaling with random DNA. These two different recognition patterns made the HEMT capable of specifically detecting target DNA sequence. The working principle of the rGO functionalized HEMT can be demonstrated as the variation of the ambience charge distribution. Furthermore, the as constructed DNA sensors showed excellent sensitivity of detect limit at 0.07 fM with linear detect range from 0.1 fM to 0.1 pM. The results indicated that the HEMT functionalized with rGO paves a new avenue to design novel electronic devices for high sensitive and specific genetic material assays in biomedical applications.     

Magneto‐Controllable Capture and Release of Cancer Cells by Using a Micropillar Device Decorated with Graphite Oxide‐Coated Magnetic Nanoparticles

Aiming to highly efficient capture and analysis of circulating tumor cells, a micropillar device decorated with graphite oxide‐coated magnetic nanoparticles is developed for magneto‐controllable capture and release of cancer cells. Graphite oxide‐coated, Fe₃O₄ magnetic nanoparticles (MNPs) are synthesized by solution mixing and functionalized with a specific antibody, following by the immobilization of such modified MNPs on our designed micropillar device. For the proof‐of‐concept study, a HCT116 colorectal cancer cell line is employed to exam the capture efficiency. Under magnetic field manipulation, the high density packing of antibody‐modified MNPs on the micropillars increases the local concentration of antibody, as well as the topographic interactions between cancer cells and micropillar surfaces. The flow rate and the micropillar geometry are optimized by studying their effects on capture efficiency. Then, a different number of HCT116 cells spiked in two kinds of cell suspension are investigated, yielding capture efficiency >70% in culture medium and >40% in blood sample, respectively. Moreover, the captured HCT116 cells are able to be released from the micropillars with a saturated efficiency of 92.9% upon the removal of applied magnetic field and it is found that 78% of the released cancer cells are viable, making them suitable for subsequent biological analysis.