A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Nanoreactors based on DNAzyme-functionalized magnetic nanoparticles activated by magnetic field.

A new biomimetic nanoreactor design, MaBiDz, is presented based on a copolymer brush in combination with superparamagnetic nanoparticles. This cellular nanoreactor features two species of magnetic particles, each functionalized with two components of a binary deoxyribozyme system. In the presence of a target mRNA analyte and a magnetic field, the nanoreactor is assembled to form a biocompartment enclosed by the polymeric brush that enables catalytic function of the binary deoxyribozyme with enhanced kinetics. MaBiDz was demonstrated here as a cellular sensor for rapid detection and imaging of a target mRNA biomarker for metastatic breast cancer, and its function shows potential to be expanded as a biomimetic organelle that can downregulate the activity of a target mRNA biomarker.

Magnetic Field-Activated Sensing of mRNA in Living Cells.

Detection of specific mRNA in living cells has attracted significant attention in the past decade. Probes that can be easily delivered into cells and activated at the desired time can contribute to understanding translation, trafficking and degradation of mRNA. Here we report a new strategy termed magnetic field-activated binary deoxyribozyme (MaBiDZ) sensor that enables both efficient delivery and temporal control of mRNA sensing by magnetic field. MaBiDZ uses two species of magnetic beads conjugated with different components of a multicomponent deoxyribozyme (DZ) sensor. The DZ sensor is activated only in the presence of a specific target mRNA and when a magnetic field is applied. Here we demonstrate that MaBiDZ sensor can be internalized in live MCF-7 breast cancer cells and activated by a magnetic field to fluorescently report the presence of specific mRNA, which are cancer biomarkers.

DNA Computing Systems Activated by Electrochemically-triggered DNA Release from a Polymer-brush-modified Electrode Array.

An array of four independently wired indium tin oxide (ITO) electrodes was used for electrochemically stimulated DNA release and activation of DNA-based Identity, AND and XOR logic gates. Single-stranded DNA molecules were loaded on the mixed poly(N,N-di-methylaminoethyl methacrylate) (PDMAEMA)/poly-(methacrylic acid) (PMAA) brush covalently attached to the ITO electrodes. The DNA deposition was performed at pH 5.0 when the polymer brush is positively charged due to protonation of tertiary amino groups in PDMAE-MA, thus resulting in electrostatic attraction of the negatively charged DNA. By applying electrolysis at -1.0 V(vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase near the electrode surface. The process resulted in recharging the polymer brush to the negative state due to dissociation of carboxylic groups of PMAA, thus repulsing the negatively charged DNA and releasing it from the electrode surface. The DNA release was performed in various combinations from different electrodes in the array assembly. The released DNA operated as input signals for activation of the Boolean logic gates. The developed system represents a step forward in DNA computing, combining for the first time DNA chemical processes with electronic input signals.

An Enzyme-based 1:2 Demultiplexer Interfaced with an Electrochemical Actuator.

An enzyme-based 1:2 demultiplexer is designed in a flow system composed of three cells where each one is modified with a different enzyme: hexokinase, glucose dehydrogenase and glucose-6-phosphate dehydrogenase. The Input signal activating the biocatalytic cascade is represented by glucose, while the Address signal represented by ATP is responsible for directing the Input signal to one of the output channels, depending on the logic value of the Address. The biomolecular 1:2 demultiplexer is extended to include two electrochemical actuators releasing entrapped DNA molecules in the active output channel. The modular design of the system allows for easy exchange and extension of the functional elements. The present demultiplexer can be easily integrated in various biomolecular logic systems, including different logic gates based on the enzyme- or DNA-based reactions, as well as containing different chemical actuators, for example, with a biomolecular release function.

AFM study shows prominent physical changes in elasticity and pericellular layer in human acute leukemic cells due to inadequate cell-cell communication.

Biomechanical properties of single cells in vitro or ex vivo and their pericellular interfaces have recently attracted a lot of attention as a potential biophysical (and possibly prognostic) marker of various diseases and cell abnormalities. At the same time, the influence of the cell environment on the biomechanical properties of cells is not well studied. Here we use atomic force microscopy to demonstrate that cell-cell communication can have a profound effect on both cell elasticity and its pericellular coat. A human pre-B p190BCR/ABL acute lymphoblastic leukemia cell line (ALL3) was used in this study. Assuming that cell-cell communication is inversely proportional to the distance between cells, we study ALL3 cells in vitro growing at different cell densities. ALL3 cells demonstrate a clear density dependent behavior. These cells grow very well if started at a relatively high cell density (HD, >2 × 105 cells ml-1) and are poised to grow at low cell density (LD, <1 × 104 cells ml-1). Here we observe ∼6× increase in the elastic (Young's) modulus of the cell body and ∼3.6× decrease in the pericellular brush length of LD cells compared to HD ALL3 cells. The difference observed in the elastic modulus is much larger than typically reported for pathologically transformed cells. Thus, cell-cell communication must be taken into account when studying biomechanics of cells, in particular, correlating cell phenotype and its biophysical properties.

Biophysical differences between chronic myelogenous leukemic quiescent and proliferating stem/progenitor cells.

The treatment of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder has improved recently, but most patients have not yet been cured. Some patients develop resistance to the available tyrosine kinase treatments. Persistence of residual quiescent CML stem cells (LSCs) that later resume proliferation is another common cause of recurrence or relapse of CML. Eradication of quiescent LSCs is a promising approach to prevent recurrence of CML. Here we report on new biophysical differences between quiescent and proliferating CD34+ LSCs, and speculate how this information could be of use to eradicate quiescent LSCs. Using AFM measurements on cells collected from four untreated CML patients, substantial differences are observed between quiescent and proliferating cells in the elastic modulus, pericellular brush length and its grafting density at the single cell level. The higher pericellular brush densities of quiescent LSCs are common for all samples. The significance of these observations is discussed.

Bioelectronic Interface Connecting Reversible Logic Gates Based on Enzyme and DNA Reactions.

It is believed that connecting biomolecular computation elements in complex networks of communicating molecules may eventually lead to a biocomputer that can be used for diagnostics and/or the cure of physiological and genetic disorders. Here, a bioelectronic interface based on biomolecule-modified electrodes has been designed to bridge reversible enzymatic logic gates with reversible DNA-based logic gates. The enzyme-based Fredkin gate with three input and three output signals was connected to the DNA-based Feynman gate with two input and two output signals-both representing logically reversible computing elements. In the reversible Fredkin gate, the routing of two data signals between two output channels was controlled by the control signal (third channel). The two data output signals generated by the Fredkin gate were directed toward two electrochemical flow cells, responding to the output signals by releasing DNA molecules that serve as the input signals for the next Feynman logic gate based on the DNA reacting cascade, producing, in turn, two final output signals. The Feynman gate operated as the controlled NOT gate (CNOT), where one of the input channels controlled a NOT operation on another channel. Both logic gates represented a highly sophisticated combination of input-controlled signal-routing logic operations, resulting in redirecting chemical signals in different channels and performing orchestrated computing processes. The biomolecular reaction cascade responsible for the signal processing was realized by moving the solution from one reacting cell to another, including the reacting flow cells and electrochemical flow cells, which were organized in a specific network mimicking electronic computing circuitries. The designed system represents the first example of high complexity biocomputing processes integrating enzyme and DNA reactions and performing logically reversible signal processing.

Diffusion of Oligonucleotides from within Iron-Cross-Linked, Polyelectrolyte-Modified Alginate Beads: A Model System for Drug Release.

An analytical model to describe diffusion of oligonucleotides from stable hydrogel beads is developed and experimentally verified. The synthesized alginate beads are Fe(3+) -cross-linked and polyelectrolyte-doped for uniformity and stability at physiological pH. Data on diffusion of oligonucleotides from inside the beads provide physical insights into the volume nature of the immobilization of a fraction of oligonucleotides due to polyelectrolyte cross-linking, that is, the absence of a surface-layer barrier in this case. Furthermore, the results suggest a new simple approach to measuring the diffusion coefficient of mobile oligonucleotide molecules inside hydrogels. The considered alginate beads provide a model for a well-defined component in drug-release systems and for the oligonucleotide-release transduction steps in drug-delivering and biocomputing applications. This is illustrated by destabilizing the beads with citrate, which induces full oligonucleotide release with nondiffusional kinetics.

Substance Release Triggered by Biomolecular Signals in Bioelectronic Systems.

A new approach to bioelectronic Sense-and-Act systems was developed with the use of modified electrodes performing sensing and substance-releasing functions. The sensing electrode was activated by biomolecular/biological signals ranging from small biomolecules to proteins and bacterial cells. The activated sensing electrode generated reductive potential and current, which stimulated dissolution of an Fe(3+)-cross-linked alginate matrix on the second connected electrode resulting in the release of loaded biochemical species with different functionalities. Drug-mimicking species, antibacterial drugs, and enzymes activating a biofuel cell were released and tested for various biomedical and biotechnological applications. The studied systems offer great versatility for future applications in controlled drug release and personalized medicine. Their future applications in implantable devices with autonomous operation are proposed.

Towards early detection of cervical cancer: Fractal dimension of AFM images of human cervical epithelial cells at different stages of progression to cancer.

We used AFM HarmoniX modality to analyse the surface of individual human cervical epithelial cells at three stages of progression to cancer, normal, immortal (pre-malignant) and carcinoma cells. Primary cells from 6 normal strains, 6 cancer, and 6 immortalized lines (derived by plasmid DNA-HPV-16 transfection of cells from 6 healthy individuals) were tested. This cell model allowed for good control of the cell phenotype down to the single cell level, which is impractical to attain in clinical screening tests (ex-vivo). AFM maps of physical (nonspecific) adhesion are collected on fixed dried cells. We show that a surface parameter called fractal dimension can be used to segregate normal from both immortal pre-malignant and malignant cells with sensitivity and specificity of more than 99%. The reported method of analysis can be directly applied to cells collected in liquid cytology screening tests and identified as abnormal with regular optical methods to increase sensitivity. FROM THE CLINICAL EDITOR: Despite cervical smear screening, sometimes it is very difficult to differentiate cancers cells from pre-malignant cells. By using AFM to analyze the surface properties of human cervical epithelial cells, the authors were able to accurately identify normal from abnormal cells. This method could augment existing protocols to increase diagnostic accuracy.

A bioelectronic system for insulin release triggered by ketone body mimicking diabetic ketoacidosis in vitro.

A bioelectronic system composed of two modified electrodes, one activated in the presence of ketone bodies, a biomarker of diabetic ketoacidosis, and another releasing insulin upon receiving a signal, was designed and tested in vitro to operate as a Sense-and-Act device. The functional integration of biomarker-sensing and insulin-releasing electrodes represents a step to a theranostic system with autonomous operation.

Bridging the Two Worlds: A Universal Interface between Enzymatic and DNA Computing Systems.

Molecular computing based on enzymes or nucleic acids has attracted a great deal of attention due to the perspectives of controlling living systems in the way we control electronic computers. Enzyme-based computational systems can respond to a great variety of small molecule inputs. They have the advantage of signal amplification and highly specific recognition. DNA computing systems are most often controlled by oligonucleotide inputs/outputs and are capable of sophisticated computing as well as controlling gene expressions. Here, we developed an interface that enables communication of otherwise incompatible nucleic-acid and enzyme-computational systems. The enzymatic system processes small molecules as inputs and produces NADH as an output. The NADH output triggers electrochemical release of an oligonucleotide, which is accepted by a DNA computational system as an input. This interface is universal because the enzymatic and DNA computing systems are independent of each other in composition and complexity.

Enzyme-based logic gates switchable between OR, NXOR and NAND Boolean operations realized in a flow system.

The enzyme-based system performing a biocatalytic cascade reaction was realized in a flow device and was used to mimic Boolean logic operations. Chemical inputs applied to the system resulted in the activation of additional reaction steps, allowing the reversible switch of the logic operations between OR, NXOR and NAND gates for processing of two other biomolecular inputs.

Activation of a biocatalytic electrode by removing glucose oxidase from the surface--application to signal triggered drug release.

A biocatalytic electrode activated by pH signals was prepared with a multilayered nanostructured interface including PQQ-dependent glucose dehydrogenase (PQQ-GDH) directly associated with the conducting support and glucose oxidase (GOx) located on the external interface. GOx was immobilized through a pH-signal-cleavable linker composed of an iminobiotin/avidin complex. In the presence of GOx, glucose was intercepted at the external interface and biocatalytically oxidized without current generation, thus keeping the electrode in its nonactive state. When the pH value was lowered from pH 7.5 to 4.5 the iminobiotin/avidin complex was cleaved and GOx was removed from the interface allowing glucose penetration to the electrode surface where it was oxidized by PQQ-GDH yielding a bioelectrocatalytic current, thus switching the electrode to its active state. This process was used to trigger a drug-mimicking release process from another connected electrode. Furthermore, the pH-switchable electrode can be activated by biochemical signals logically processed by biocatalytic systems mimicking various Boolean gates. Therefore, the developed switchable electrode can interface biomolecular computing/sensing systems with drug-release processes.

If cell mechanics can be described by elastic modulus: study of different models and probes used in indentation experiments.

Here we investigated the question whether cells, being highly heterogeneous objects, could be described with the elastic modulus (effective Young's modulus) in a self-consistent way. We performed a comparative analysis of the elastic modulus derived from the indentation data obtained with atomic force microscopy (AFM) on human cervical epithelial cells (both normal and cancerous). Both sharp (cone) and dull (2500-nm radius sphere) AFM probes were used. The indentation data were processed through different elastic models. The cell was approximated as a homogeneous elastic medium that had either 1), smooth hemispherical boundary (Hertz/Sneddon models) or 2), the boundary covered with a layer of glycocalyx and membrane protrusions ("brush" models). Consistency of these approximations was investigated. Specifically, we tested the independence of the elastic modulus of the indentation depth, which is assumed in these models. We demonstrated that only one model showed consistency in treating cells as a homogeneous elastic medium, namely, the brush model, when processing the indentation data collected with the dull AFM probe. The elastic modulus demonstrated strong depth dependence in all models: Hertz/Sneddon models (no brush taken into account), and when the brush model was applied to the data collected with sharp conical probes. We conclude that it is possible to describe the elastic properties of the cell body by means of an effective elastic modulus, used in a self-consistent way, when using the brush model to analyze data collected with a dull AFM probe. The nature of these results is discussed.

Model system for targeted drug release triggered by immune-specific signals.

A new sense-and-act system was realized by integrating a biocatalytic/bioaffinity electrode responding to immune signals represented by an antibody and a polymer-modified electrode loaded with drug-mimicking species. The release of the drug-mimicking species was achieved specifically in response to a signal antibody, thus demonstrating for the first time an immune-induced drug-releasing process. The present approach promises new options for future applications in controlled drug release and personalized medicine.

Majority and minority gates realized in enzyme-biocatalyzed systems integrated with logic networks and interfaced with bioelectronic systems.

Biocatalytic reactions operating in parallel and resulting in reduction of NAD(+) or oxidation of NADH were used to mimic 3-input majority and minority logic gates, respectively. The substrates corresponding to the enzyme reactions were used as the input signals. When the input signals were applied at their high concentrations, defined as logic 1 input values, the corresponding biocatalytic reactions were activated, resulting in changes of the NADH concentration defined as the output signal. The NADH concentration changes were dependent on the number of parallel reactions activated by the input signals. The absence of the substrates, meaning their logic 0 input values, kept the reactions mute with no changes in the NADH concentration. In the system mimicking the majority function, the enzyme-biocatalyzed reactions resulted in a higher production of NADH when more than one input signal was applied at the logic 1 value. Another system mimicking the minority function consumed more NADH, thus leaving a smaller residual output signal, when more than one input signal was applied at the logic 1 value. The performance of the majority gate was improved by processing the output signal through a filter system in which another biocatalytic reaction consumed a fraction of the output signal, thus reducing its physical value to zero when the logic 0 value was obtained. The majority gate was integrated with a preceding AND logic gate to illustrate the possibility of complex networks. The output signal, NADH, was also used to activate a process mimicking drug release, thus illustrating the use of the majority gate in decision-making biomedical systems. The 3-input majority gate was also used as a switchable AND/OR gate when one of the input signals was reserved as a command signal, switching the logic operation for processing of the other two inputs. Overall, the designed majority and minority logic gates demonstrate novel functions of biomolecular information processing systems.

A biocatalytic cascade with several output signals--towards biosensors with different levels of confidence.

The biocatalytic cascade based on enzyme-catalyzed reactions activated by several biomolecular input signals and producing output signal after each reaction step was developed as an example of a logically reversible information processing system. The model system was designed to mimic the operation of concatenated AND logic gates with optically readable output signals generated at each step of the logic operation. Implications include concurrent bioanalyses and data interpretation for medical diagnostics.

Enzymatic filter for improved separation of output signals in enzyme logic systems towards 'sense and treat' medicine.

The major challenge for the application of autonomous medical sensing systems is the noise produced by non-zero physiological concentrations of the sensed target. If the level of noise is high, then a real signal indicating abnormal changes in the physiological levels of the analytes might be hindered. Inevitably, this could lead to wrong diagnostics and treatment, and would have a negative impact on human health. Here, we report the realization of a filter system implemented to improve both the fidelity of sensing and the accuracy of consequent drug release. A new filtering method was tested in the sensing system for the diagnosis of liver injury. This sensing system used the enzymes alanine transaminase (ALT) and aspartate transaminase (AST) as the inputs. Furthermore, the output of the sensing system was designed to trigger drug release, and therefore, the role of the filter in drug release was also investigated. The drug release system consists of beads with an iron-cross-linked alginate core coated with different numbers of layers of poly-l-lysine. Dissolution of the beads by the output signals of the sensing system in the presence and absence of the filter was monitored by the release of rhodamine-6G dye encapsulated in the beads, mimicking the release of a real drug. The obtained results offer a new view of the problem of noise reduction for systems intended to be part of sense and treat medical devices.