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International audience; By means of Silicon Nano Tweezers (SNTs) the effects on the mechanical properties of λ-phage DNA during interaction with calf thymus nucleosome to form an artificial chromatin analog were measured. At a concentration of 100 nM, a nucleosome solution induced a strong stiffening effect on DNA (1.1 N m −1). This can be compared to the effects of the histone proteins, H1, H2A, H3 where no changes in the mechanical properties of DNA were observed and the complex of the H3/H4 proteins where a smaller increase in the stiffness is observed (0.2 N m −1). Para-sulphonato-calix[4]arene, SC4, known for epigenetic activity by interacting specifically with the lysine groups of histone proteins, was studied for its effect on an artificial chromatin. Using a microfluidic SNT device, SC4 was titrated against the artificial chromatin, at a concentration of 1 mM in SC4 a considerable increase in stiffness, 15 N m −1 , was observed. Simultaneously optical microscopy showed a physical change in the DNA structure between the tips of the SNT device. Electronic and Atomic Force microscopy confirmed this structural rearrangement. Negative control experiments confirmed that these mechanical and physical effects were induced neither by the acidity of SC4 nor through nonspecific interactions of SC4 on DNA. With the recent and better knowledge on how gene regulation occurs and after intense debate in the academic community 1 , epigenetics has been commonly defined as "the study of changes in gene function that are mitoti-cally and/or meiotically heritable and that do not entail a change in DNA sequence" 2. Over the past years, epigenetics has been shown to play a central role in many different key functions at the cellular level, including differentiation, replication, and gene transcription 3. In a larger context, its contribution in physiological disorders has been revealed in cardio vascular illnesses, mental disorders and various cancers 4. Despite its inheritable character, epigenetics paradoxically possesses a reversible nature 5 that can be illustrated through its two main covalent modifications either directly on DNA with the methylation of cytosine nucleic bases or indirectly with various post translational modifications on proteins interacting with DNA such as histone proteins or other proteins called writers, erasers or readers 6. New therapeutic treatments are expected to target and reverse these modifications by inactivating the enzymes responsible for the deregulation of specific genes 7. Some inhibitors have been already approved by FDA, such as Azacitidine and Decitabine as DNA methyltrans-ferase inhibitors for the treatment of myelodysplastic syndrome 8 and Vorinostat and Romidepsin as inhibitors of histone deacetylase for the treatment of peripheral T-cell lymphoma 9. A new class of DNA artificial binders that directly block the methyllysine reading functions of reader enzymes in charge of conveying the methylation signal downstream has recently arisen as promising epigenetic drugs 10 .

International audience; We introduced a MEMS device to perform direct measurements on electrical and mechanical properties of single cancer cells. Working in a continuous flow format permits high-throughput measurements. The device design allows us to define fixed or movable electrodes as part of the side-walls of an embedded microchannel. The stable air-liquid interface at micro-sized gaps between the electrodes and the channel wall provide flow continuity without causing any leakage problems. This unified processing approach allows us to define optimized electrode pattern configuration for both electrical and mechanical characterization areas. Moreover, the movable electrode is in direct contact with passing cells which enables direct sensing during compression. The device can perform both electrical and mechanical measurements on single cells in a continuous flow as demonstrated and also validated by optical control measurements. The proposed method aims at providing high-throughput biophysical cytometry, in an optics- and marker-free way, for cancer cell evaluation.

oral; International audience; We report a method for combining confocal microscopy with single-cell mechanical characterization. This method allows investigating the effect of subcellular deformation (e.g. membranes, cytoplasm, cytoskeleton and organelles as endoplasmic reticulum, mitochondria, and nucleus) on cell mechanical properties (e.g. stiffness and viscosity). Such a method is essential to choose biologically relevant mechanical properties of malignant cells as diagnostic cancer biomarkers. Using MEMS tweezers, we captured live single cancer cells and performed subcellular imaging during compression assays for mechanical measurements to obtain the deformation-dependent cell properties.

Microtubule-associated protein (MAP) tau is a well-established hallmark of a large group of age related neurodegenerative diseases collectively called tauopathies. Under pathological conditions the equilibrium of tau binding to the MTs is perturbed, either by misregulation in the expression levels of specific tau isoforms or by MAPT gene mutations. Preclinical detection of such misregulated tau proteins in cerebrospinal fluid (CSF) is desirable for differential diagnosis and effective prognosis of neurodegeneration. Conventional tau protein detection methods utilize tau isoform-specific antibodies. Such immuno-based protocols, including enzyme-linked immunosorbent assay (ELISA) and Western blots have appropriate sensitivity and specificity, but often show high variability and are time consuming. Here, we established a non-immuno tau protein detection method utilizing microtubule (MT)-kinesin “gliding”assay. All the six tau isoforms expressed in the human brain (0N3R, 1N3R, 2N3R, 0N4R, 1N4R and 2N4R) and five MAPT gene mutants (V248L, G272V, P301L, V337M and R406W) were studied. The landing rate, binding density and gliding velocity of MTs with respect to each tau type were determined and are proposed as tau detection parameters. The detection parameters depicted the type of tau bound to the MTs. Furthermore, MT landing rate and density were found to be superior to gliding velocity in differentiating tau isoforms and mutants. The 3R vs. 4R isoforms, their admixtures, wild vs. mutant 2N4R and specific mutants were differentiated. Our data show that MT-kinesin gliding assay provides a convenient, lab-on-a-chip (LOC) compatible and antibody-free protocol for tau protein analysis.

International audience; The couple Calix[4]arene-1,3-O-diphosphorous acid (C4diP) and zinc ions (Zn 2+) acts as a synergistic DNA binder. Silicon NanoTweezer (SNT) measurements show an increase in the mechanical stiffness of DNA bundles by a factor of >150, at Zn 2+ to C4diP ratios above 8, as compared to Zinc alone whereas C4diP alone decreases the stiffness of DNA. Electroanalytical measurements using 3D printed devices demonstrate a progression of events in the assembly of C4diP on DNA promoted by zinc ions. A mechanism at the molecular level can be deduced in which C4diP initially coordinates to DNA by phosphate-phosphate hydrogen bonds or in the presence of Zn 2+ by Zn 2+ bridging coordination of the phosphate groups. Then, at high ratios of Zn 2+ to C4diP, interdigitated dimerization of C4diP is followed by cross coordination of DNA strands through Zn 2+ /C4diP inter-strand interaction. The sum of these interactions leads to strong stiffening of the DNA bundles and increased inter-strand binding. DNA is one of the key bio-polymers in life, encoding the information required to build organisms. Defects in DNA can occur either via natural mutations or through external damage 1. Such defects are the cause of a vast range of diseases, from Huntington's disease to cancer, although some mutations may be beneficial 2. In recent years, the rise of artificial nanostructures based on DNA often coupled to metal ions, has been pioneered by Seeman 3. Artificial DNA binders present a great interest in the biomedical field 4. They can be used in various ways, such as the transportation of DNA through the cell membrane in transfection or gene therapy 5 for blocking a specific DNA sequence to avoid the transcription of a specific gene 6 or arresting DNA replication in cancer treatment 7. The calix[n]arenes, are well known for their ability to interact with a wide range of biomolecules including amino acids, nucleotides, surfactants, proteins and DNA 8 , leading to a wide panoply of biological applications 9. Cationic calix[n]arene derivatives have been reported to be efficient DNA binders 10. However, such derivatives present several drawbacks in biology, including toxicity due to the destabilization of cellular membranes possessing negative potential or non-specific electrostatic aggregation by negatively charged DNA 11. Protein ligands of DNA (finger zinc proteins, transcription factors, nucleases) interact mainly through conjoint coordination

Micro and nano systems (MNS) and Nano scaled devices, that are capable of handling fluids and to interact with DNA and proteins enable bio analysis at the “ultimate” molecular level and are prone to be coupled to IC Technology. This paper includes recent developments in this area, aimed at illustrating the diversity and potential of the MNS approach: (1) micromachined tweezers with sharp tips successfully captured a bundle of DNA molecules, allowing real-time observation of DNA degradation dynamics under therapeutic irradiations; (2) a simple method to fabricate FET silicon nanowires using only standard micro-electromechanical system (MEMS) processes is able to perform molecular level medical diagnosis; (3) a bio motor system, composed by microtubules and kinesin, reconstructed on a chip, can distinguish normal and abnormal tau-proteins related to Alzheimer’s disease; and (4) an advanced concept of molecular programing in which real molecular interactions emulate partial different equations and are able to solve complex systems such as a predator–prey ecosystem.

The mechanical properties of cells have been suggested as alternative biomarkers; however, there is no practical high-throughput and high information content measurement technique applicable for non-adherent cells. To address this problem, we propose a MEMS-based device with an embedded microfluidic channel. Cells flow inside the channel individually and position directly at a sensing area. Then, developed LabVIEW program applies compression cycles to the cell and detects the mechanical properties of the cell in real-time. At the same time, the electrical measurements are performed. Due to direct measurements, we can simplify the experimental setup and improve the throughput compared to the conventional high information content techniques, e.g. AFM. This technique leads to a promising alternative way to identify non-adherent rare cells, e.g. circulating tumor cells and cancer stem cells.

This study combines the high-throughput capabilities of microfluidics with the sensitive measurements of microelectromechanical systems (MEMS) technology to perform biophysical characterization of circulating cells for diagnostic purposes. The proposed device includes a built-in microchannel that is probed by two opposing tips performing compression and sensing separately. Mechanical displacement of the compressing tip (up to a maximum of 14 &micro, m) and the sensing tip (with a quality factor of 8.9) are provided by two separate comb-drive actuators, and sensing is performed with a capacitive displacement sensor. The device is designed and developed for simultaneous electrical and mechanical measurements. As the device is capable of exchanging the liquid inside the channel, different solutions were tested consecutively. The performance of the device was evaluated by introducing varying concentrations of glucose (from 0.55 mM (0.1%) to 55.5 mM (10%)) and NaCl (from 0.1 mM to 10 mM) solutions in the microchannel and by monitoring changes in the mechanical and electrical properties. Moreover, we demonstrated biological sample handling by capturing single cancer cells. These results show three important capabilities of the proposed device: mechanical measurements, electrical measurements, and biological sample handling. Combined in one device, these features allow for high-throughput multi-parameter characterization of single cells.

Intracellular transport is affected by the filament network in the densely packed cytoplasm. Biophysical studies focusing on intracellular transport based on microtubule–kinesin system frequently use in vitro motility assays, which are performed either on individual microtubules or on random (or simple) microtubule networks. Assembling intricate networks with high flexibility requires the manipulation of 25 nm diameter microtubules individually, which can be achieved through the use of pick-and-place assembly. Although widely used to assemble tiny objects, pick-and-place is not a common practice for the manipulation of biological materials. Using the high-level handling capabilities of microelectromechanical systems (MEMS) technology, tweezers are designed and fabricated to pick and place single microtubule filaments. Repeated picking and placing cycles provide a multilayered and multidirectional microtubule network even for different surface topographies. On-demand assembly of microtubules forms crossings at desired angles for biophysical studies as well as complex networks that can be used as nanotransport systems.

This study presents a practical method to perform mechanical characterization of cells using Silicon Nano Tweezers (SNT). Integrated with a microfluidic device, SNT tips were inserted in a channel via a side opening. Cells to be characterized were injected to an inlet of the microfluidic channel and withdrawn by a vacuum pump connected to an outlet. Mechanical properties of a cell, e.g. diameter, stiffness and viscous losses, were analyzed after capturing between the SNT tips (a sensing tip and a compressing tip). After characterization, the captured cell was released to capture another cell. Repeated capturing and characterization steps allow us to test several cells and distinguish fixed and live SUM-159-PT cancer cells based on their mechanical properties.

The killing of tumor cells by ionizing radiation beams in cancer radiotherapy is currently based on a rather empirical understanding of the basic mechanisms and effectiveness of DNA damage by radiation. By contrast, the mechanical behaviour of DNA encompassing sequence sensitivity and elastic transitions to plastic responses is much better understood. A novel approach is proposed here based on a micromechanical Silicon Nanotweezers device. This instrument allows the detailed biomechanical characterization of a DNA bundle exposed to an ionizing radiation beam delivered here by a therapeutic linear particle accelerator (LINAC). The micromechanical device endures the harsh environment of radiation beams and still retains molecular-level detection accuracy. In this study, the first real-time observation of DNA damage by ionizing radiation is demonstrated. The DNA bundle degradation is detected by the micromechanical device as a reduction of the bundle stiffness, and a theoretical model provides an interpretation of the results. These first real-time observations pave the way for both fundamental and clinical studies of DNA degradation mechanisms under ionizing radiation for improved tumor treatment.

International audience; Micro-electromechanical systems (MEMS) tweezers developed for the trapping and characterization of bio molecules such as DNA are also capable of cell handling, which will enable further application involving single cell characterization. However, cellular adhesion to Si tweezers surfaces can inhibit the release of cells after capture and analysis. To allow for high-throughput cell handling, the surface modification process for probe sidewalls was optimized. The hydrophilicity of sample silicon wafers modified with various chemical coatings was measured to identify the optimal process. The surface modification was then applied to MEMS probes and the feasibility of cell capture/release was evaluated.

Direct transport powered by motor proteins can alleviate the challenges presented by miniaturization of microfluidic systems. There have been several recent attempts to build motor-protein-driven transport systems based on simple capturing or transport mechanisms. However, to achieve a multifunctional device for practical applications, a more complex sorting/transport system should be realized. Herein, the proof of concept of a sorting device employing selective capture of distinct target molecules and transport of the sorted molecules to different predefined directions is presented. By combining the bottom-up functionality of biological systems with the top-down handling capabilities of micro-electromechanical systems technology, highly selective molecular recognition and motor-protein-based transport is integrated in a microfluidic channel network.

International audience; Monitoring biological reactions using the mechanical response of macromolecules is an alternative approach to immunoassays for providing real-time information about the underlying molecular mechanisms. Although force spectroscopy techniques, e.g. AFM and optical tweezers, perform precise molecular measurements at the single molecule level, sophisticated operation prevent their intensive use for systematic biosensing. Exploiting the biomechanical assay concept, we used micro-electro mechanical systems (MEMS) to develop a rapid platform for monitoring bio/chemical interactions of bio macromolecules, e.g. DNA, using their mechanical properties. The MEMS device provided real-time monitoring of reaction dynamics without any surface or molecular modifications. A microfluidic device with a side opening was fabricated for the optimal performance of the MEMS device to operate at the air-liquid interface for performing bioassays in liquid while actuating/sensing in air. The minimal immersion of the MEMS device in the channel provided long-term measurement stability (>10 h). Importantly, the method allowed monitoring effects of multiple solutions on the same macromolecule bundle (demonstrated with DNA bundles) without compromising the reproducibility. We monitored two different types of effects on the mechanical responses of DNA bundles (stiffness and viscous losses) exposed to pH changes (2.1 to 4.8) and different Ag + concentrations (1 μM to 0.1 M). Mechanical properties of molecules are used to investigate a wide range of biological and chemical mechanisms varying from nucleic acid conformation to enzymatic reaction kinetics and from cell manipulation to motor protein operation 1. Unlike some of the other well-established methods that are often limited to smaller sized molecules , e.g. nuclear magnetic resonance (NMR) 2 and surface plasmon resonance (SPR) 3 , force spectroscopy techniques , e.g. optical tweezers 4,5 , magnetic tweezers 6-8 and AFM 9,10 , provide sensitive measurements on mechanical properties spanning six orders of magnitude in length (10 −10-10 −4 m) including macromolecules 1,11,12. These techniques provide information at the single molecule level to reveal novel properties of various different molecules. However, low throughput, surface modification, high-level operational skills and complicated calibra-tion/setup procedures exclude these techniques for rapid routine tests that are essential to provide statistically significant biomechanical information especially for clinical studies 13. As a result, a rapid, practical, time-and cost-efficient, automated and preferably portable method is beneficial as a complementary or alternative method to the conventional techniques to be employed even by non-specialists. MEMS technology presents certain advantages to cover the mentioned requirements for routinely performed assays on mechanical responses of macromolecules. MEMS, capable of conducting real-time mechanical sensing with electrical readouts associated with low noise and high stability measurements 14 , allows low

Tau protein is a well-established biomarker for a group of neurodegenerative diseases collectively called tauopathies. So far, clinically relevant detection of tau species in cerebrospinal fluid (CSF) cannot be achieved without immunological methods. Recently, it was shown that different tau isoforms including the ones carrying various types of mutations affect microtubule (MT)–kinesin binding and velocity in an isoform specific manner. Here, based on these observations, we developed a microfluidic device to analyze tau mutations, isoforms and their ratios. The assay device consists of three regions: a MT reservoir which captures MTs from a solution to a kinesin-coated surface, a microchannel which guides gliding MTs, and an arrowhead-shaped collector which concentrates MTs. Tau-bound fluorescently labeled MTs (tau-MTs) were assayed, and the increase in fluorescence intensity (FI) corresponding to the total number of MTs accumulated was measured at the collector. We show that our device is capable of differentiating 3R and 4R tau isoform ratios and effects of point mutations within 5 minutes. Furthermore, radially oriented collector regions enable simultaneous FI measurements for six independent assays. Performing parallel assays in the proposed device with minimal image processing provides a cost-efficient, easy-to-use and fast tau detection platform.

An alternative method of micro/nano-transport has been achieved by using motor proteins. Microtubules on a kinesin-coated surface have potential to act as a nano-transport system. When microtubules are used as carriers, either cargo or cargo linkers are attached on the microtubule surface. Such cargo attachments can significantly affect kinesin motion. To deal with the difficulty caused by molecular attachment to the microtubule surface, the cargo loading and transport mechanism should be separated. In this work, we propose to use micromachined needles as cargo carriers which then can be transported on microtubules. Because of the separation of needle functionalization and transport mechanism, functionalization of the needles can proceed without any effect on the microtubule structure, significantly increasing the possible types of cargo. We have fabricated silicon needles in mass numbers using a simple and effective method and have shown that the microtubule-needle composites are transported without affecting the kinesin activity.

Massively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were digested by a restriction enzyme, XhoI, for DNA analysis. One end of the lambdaDNA was modified with biotin and the other end was modified with digoxin molecules by fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was attached to a biotinylated microtubule via avidin-biotin biding and the DNA was stretched by a kinesin-based gliding assay. A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method of performing molecular surgery at the single-molecule scale.

Microelectromechanical systems (MEMS) have become an invaluable technology to advance the development of point-of-care (POC) devices for diagnostics and sample analyses. MEMS can transform sophisticated methods into compact and cost-effective microdevices that offer numerous advantages at many levels. Such devices include microchannels, microsensors, etc., that have been applied to various miniaturized POC products. Here we discuss some of the recent advances made in the use of MEMS devices for POC applications.

We report the successful demonstration of an on-chip tau detection system based on the difference in landing rate and binding density of microtubules (MTs) on a kinesin surface. Tau detection device comprises of a MT reservoir, channel and collector region with an overhang structure. We assayed MTs decorated with three tau types in the kinesin coated device. Since the increase in fluorescence intensity (FI) at the collector regions reflected the type of tau decorated on MTs, thus by measuring the FI we were able to distinguish wild 3R, 4R and P301L mutant tau.

We examine the effect of physiological cations Na+, K+, Mg2+, and Ca2+ on the mechanical properties of bundles of λ-phage DNA using silicon nanotweezers (SNTs). Integrating SNTs with a microfluidic device allows us to perform titration experiments while measuring the effect in real-time. The results show that only for Mg2+ and in particular, at the intra-nuclear concentration (100 mM), the interaction occurs.

Tau protein is a biomarker for neurodegeneration. The microtubule (MT)-tau binding affinity varies according to the type of tau isoform and their degree of phosphorylation. We have utilized the difference in binding affinity of tau protein to MT to be evaluated by kinesin motor protein based MT gliding system, in order to detect and differentiate tau isoforms and their mutants. Evaluation parameters are landing rate and density of MTs on a kinesin-coated surface, and their strong correlation enables us to measure only landing rate to distinguish 2N3R and 2N4R (tau isoforms) and mutants. Secondly; we designed and fabricated a microstructure to detect tau-attached MTs, which is composed of a reservoir, parallel channels and collectors. Increase of fluorescent intensity by accumulation of MTs over time was successfully detected at the collector areas. Sensitive and rapid MT-kinesin based detection of tau isoforms (3R/4R) and mutated tau proteins on a microchip format will aid in differential diagnosis and early detection of neurodegenerative condition such as Alzheimer disease (AD).

Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction. Optimization of the intramolecular nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equilibrium between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-molecule localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resolution imaging of microtubules in live cells for up to 1 h.

The concept of a reconstructed microtubule kinesin-based transport system was originally introduced for studies of underlying biophysical mechanisms of intracellular transport and its potential applications in bioengineering at micro- and nanoscale levels. However, several technically challenging shortcomings prohibit its use in practical applications. One of them is the propensity of microtubules to bind various protein molecules creating “roadblocks” for kinesin molecule movement and subsequently preventing efficient delivery of the molecular cargo. The interruption in kinesin movement strictly depends on the specific type of “roadblock”, i.e. the microtubule associated protein (MAP). Therefore, we propose to use the “roadblock” effect as a molecular sensor that may be used for functional characterization of particular MAPs with respect to their role in MT-based transport and associated pathologies, such as neurodegeneration. Here, we applied a kinesin-based assay using a suspended MT design (sMT assay) to functionally characterize known MAP tau protein isoforms and common mutations found in familial frontotemporal dementia (FTD). The proposed sMT assay is compatible with an on-chip format and may be used for the routine characterization of MT associated molecules applicable to diagnostics and translational research.

A microfluidics device to detect microtubule-binding proteins was proposed using microtubule-kinesin based system. To provide “proof of principle”, attachment of microtubule associated tau protein was detected via the change in kinesin velocity. The proposed device is capable of multiplex parallel assay of several experimental samples by using the same kinesin-coated beads moving in the same channel with immobilized microtubules. The results showed that the device successfully detected the hindered kinesin motion due to tau attachment on microtubule (MT) surface.

Microtubule (MT) based intraneuronal transport deficiency is directly linked to neurodegeneration. Hence, the development of a reliable and sensitive in vitro approach permitting efficient analysis of MT-based transport is essential for our understanding of the underlying molecular mechanisms that may lead to novel therapeutic approaches for treating neurodegenerative diseases. Here, based on previously developed reconstructed MT–kinesin assay, we propose its “suspended” modification that shows higher sensitivity and lower experimental variability.

A molecular detection system was realized using the change in the characteristics of the kinesin-coated bead motion along protein-attached microtubules. A hybrid bio/microfluidics device with a microtubule immobilized main channel and protein-injected sub-channels allowed us to compare different protein attachments and several control experiments with the same experimental conditions. As kinesin/microtubule system is an essential component of neurons, the proposed device is a promising tool for diagnostics of several neurodegenerative diseases.

Despite the benefits of miniaturized devices, handling of tiny amount of molecules became a great challenge. Direct transport, similar to the one in intracellular transport, is a way to cope with the problem of transporting tiny amount of materials. Using motor proteins, i.e. kinesin, and the corresponding rail structures, i.e. microtubules, provides powerful building blocks for a nanotransport system. However, it is extremely difficult to construct a complex transport network to allow arbitrary design. Here, we have developed a MEMS-based molecular handling system for the pick-and-place assembly of individual microtubules. The technology enabled us to build a bio/MEMS hybrid system composed of multidirectional microtubule networks along which kinesin molecules move. Using MEMS tweezers to manipulate a single microtubule enables precise positioning and full control in functional orientation and stacking order. As a result, kinesin-coated beads can be transported along the route specifically assembled for them to follow. Direct transport of target molecules can be achieved by loading the beads with the molecules to carry. “Bottom-up” functionalities of biomaterials are incorporated with micro fluidic devices by the handling capabilities of the “top-down” approach.

We have utilized a cytoskeleton, microtubule (MT), as a template for conductive nanowires. Biotin-labeled MTs were coated with streptavidin-labeled nanogold (NG) particles. Labeling of two modified MTs, biotin-labeled and NG-coated MTs, were evaluated by fluorescent and scanning electron microscopy, which verified the specific binding of NG particles on biotin-labeled MTs. Then, NG-coated MTs were further enlarged by catalytic deposition for better conductivity. Electrical characterization proved that only the enhanced NG-coated MTs have conductivity with dependency on MT and NG concentrations.

In a motor protein system (kinesin/microtubule), individual microtubules were captured and relocated to build a complex nano-scale transport system. Micro tweezers, attached to a micromanipulator arm, were used to capture polarity marked microtubules. Precise relocation of those microtubules provided an oriented track along which kinesin-coated beads move according to the microtubule polarities.

Artificial nanotransport systems inspired by intracellular transport processes have been investigated for over a decade using the motor protein kinesin and microtubules. However, only unidirectional cargo transport has been achieved for the purpose of nanotransport in a microfluidic system. Here, we demonstrate bidirectional nanotransport by integrating kinesin and dynein motor proteins. Our molecular system allows microtubule orientation of either polarity in a microfluidic channel to construct a transport track. Each motor protein acts as a nanoactuators that transports microspheres in opposite directions determined by the polarity of the oriented microtubules: kinesin-coated microspheres move toward the plus end of microtubules, whereas dynein-coated microspheres move toward the minus end. We demonstrate both unidirectional and bidirectional transport using kinesin- and dynein-coated microspheres on microtubules oriented and glutaraldehyde-immobilized in a microfluidic channel. Tracking and statistical analysis of microsphere movement demonstrate that 87-98% of microspheres move in the designated direction at a mean velocity of 0.22-0.28 microm/s for kinesin-coated microspheres and 0.34-0.39 microm/s for dynein-coated microspheres. This bidirectional nanotransport goes beyond conventional unidirectional transport to achieve more complex artificial nanotransport in vitro.

Artificial nanotransport inspired by intracellular transport processes has been investigated for over a decade using the motor protein kinesin and microtubules to achieve unidirectional cargo transport. Here, we demonstrate simultaneous bidirectional nanotransport of microspheres by integrating the oppositely directed motor proteins, kinesin and dynein, with an oriented microtubule track. Our statistical analysis demonstrates that 87-98% of microspheres move in the designated direction at mean velocity 0.22-0.26 mum/s (kinesin-coated microspheres) and 0.34-0.39 mum/s (dynein-coated microspheres).

A bio-hybrid microsystem for sorting and carrying target molecules by bio-functional molecules is presented. Different target molecules, biotin-4-fluorescein and Rabbit anti-mouse IgG labeled with Alexa Fluor 568, were selectively attached on different beads (Streptavidin-coated and protein A-coated beads). Target molecules were separately transported by a motor protein system (kinesin on beads / microtubules on chip) without any liquid manipulation proving the feasibility of multiple target molecules on multiple beads sorter.

The present paper describes the use of Indium-Tin oxide microelectrodes for manipulating microtubules in solution through interaction with electric fields. The experimental set-up that is described here is a platform for both investigating the fundamental dielectric properties of microtubules and developing methods to exert control over their behaviour in solution. Direct applications of such methods can be found in the development of microsystems integrating nano-machines based on motor proteins such as kinesin or dynein. Electro-rotation of microtubules in solution is presented as an example of interaction between microtubules and electric field.

We demonstrate the active transport of liquid cargos in the form of oil-in-water emulsion droplets loaded on kinesin motor proteins moving along oriented microtubules. We analyze the motility properties of the kinesin motors (velocity and run length) and find that the liquid cargo in the form of oil droplets does not alter the motor function of the kinesin molecules. This work provides a novel method for handling only a few molecules/particles encapsulated inside the oil droplets and represents a key finding for the integration of kinesin-based active transport into nanoscale lab-on-a-chip devices. We also investigate the effect of the diameter of the droplets on the motility properties of the kinesin motors. The velocity is approximately constant irrespective of the diameter of the droplets whereas we highlight a strong increase of the run length when the diameter of the droplets increases. We correlate these results with the number of kinesin motors involved in the transport process and find an excellent agreement between our experimental result and a theoretical model.

Microtubules are filamentous proteins that act as a substrate for the translocation of motor proteins. As such, they may be envisioned as a scaffold for the self-assembly of functional materials and devices. Physisorption, self-assembly and combing are here investigated as a potential prelude to microtubule-templated self-assembly. Dense films of self-assembled microtubules were successfully produced, as well as patterns of both dendritic and non-dendritic bundles of microtubules. They are presented in the present paper and the mechanism of their formation is discussed.

A motor protein system, kinesin/microtubule, transported target molecules specifically attached on bead surfaces. The target molecule, biotin-4-fluorescein, was specifically immobilized on streptavidin-coated beads, on which kinesin molecules were also attached. The target molecules were successfully transported along microtubules immobilized on a glass surface. The activity of kinesin was not disturbed by co-existing target molecules. The result has significant implication to sort out any target molecule selectively from a sample solution by a nano-scale transport system driven by the kinesin/microtubule system.

A hybrid bio-electrical system for direct transportation and electrofusion of oil droplets is proposed. A motor protein system, kinesin/microtubule, was used to transport kinesin-coated oil droplets along microtubules oriented and immobilized on a glass surface. The attachment between the biotinylated-kinesin and the functionalized oil droplet was achieved using the biotin-streptavidin binding. Fusion of the oil droplets was accomplished by applying AC voltage on the electrodes placed inside a flow cell. Together with encapsulation of particles/reactants within oil droplets, this method may lead to the fabrication of a nanoscale reactor.