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We present a new technique for high throughput screening of tumor cells in a sensitive nanodevice that has the potential to quickly identify a cell population that has begun the rapid protein synthesis and mitosis characteristic of cancer cell proliferation. Currently, pathologists rely on microscopic examination of cell morphology using century-old staining methods that are labor-intensive, time-consuming and frequently in error. New micro-analytical methods for automated, real time screening without chemical modification are critically needed to advance pathology and improve diagnoses. We have teamed scientists with physicians to create a microlaser biochip (based upon our R&D award winning bio- laser concept) which evaluates tumor cells by quantifying their growth kinetics. The key new discovery was demonstrating that the lasing spectra are sensitive to the biomolecular mass in the cell, which changes the speed of light in the laser microcavity. Initial results with normal and cancerous human brain cells show that only a few hundred cells -- the equivalent of a billionth of a liter -- are required to detect abnormal growth. The ability to detect cancer in such a minute tissue sample is crucial for resecting a tumor margin or grading highly localized tumor malignancy.
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In photomedicine in some of cases radiation delivery to local zones through optical fibers can be changed for the direct placing of tiny optical sources like semiconductor microlasers or light diodes in required zones of ears, nostrils, larynx, nasopharynx cochlea or alimentary tract. Our study accentuates the creation of optoelectronic microdevices for local phototherapy and functional imaging by using reflected light. Phototherapeutic micromodule consist of the light source, microprocessor and miniature optics with different kind of power supply: from autonomous with built-in batteries to remote supply by using pulsed magnetic field and supersmall coils. The developed prototype photomodule has size (phi) 8X16 mm and work duration with built-in battery and light diode up several hours at the average power from several tenths of mW to few mW. Preliminary clinical tests developed physiotherapeutic micrimodules in stomatology for treating the inflammation and in otolaryngology for treating tonsillitis and otitis are presented. The developed implanted electro- optical sources with typical size (phi) 4X0,8 mm and with remote supply were used for optical stimulation of photosensitive retina structure and electrostimulation of visual nerve. In this scheme the superminiature coil with 30 electrical integrated levels was used. Such devices were implanted in eyes of 175 patients with different vision problems during clinical trials in Institute of Eye's Surgery in Moscow. For functional imaging of skin layered structure LED arrays coupled photodiodes arrays were developed. The possibilities of this device for study drug diffusion and visualization small veins are discussed.
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Porous silicon nanostructures are ideal hosts for sensor applications because of their large internal surface area, which implies strong adsorbate effects. The average pore size can be easily adjusted to accommodate either small or large molecular species. When porous silicon is fabricated into a structure consisting of two high reflectivity multilayer mirrors separated by an active layer, a microcavity is formed. Multiple narrow and visible luminescence peaks are observed with a full width at half the maximum value of 3 nm. These multiple peak microcavity resonators are very sensitive structures. Any slight change in the effective optical thickness induces a change in the reflectivity spectra, causing a shift in the interference peaks. We demonstrate the usefulness of this microcavity resonator structure as a biosensor. Biosensors are devices that exploit the powerful recognition capability of bioreceptors. We have fabricated a DNA biosensor based on a porous silicon multiple peak microcavity structure. An initial strand of DNA is first immobilized in a porous silicon substrate and then subsequently exposed to its complementary DNA strand. Shifts in the luminescence spectra are observed and detected for DNA concentrations less than 1 (mu) M. When exposed to a non- complementary DNA strand, no shifts are observed. A detailed study on the selectivity and sensitivity issues of porous silicon microcavity biosensors is presented.
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A micro fluidic device capable of detecting the abundance of bacteria in an environmental solution is described. The micro channels are made of poly(dimethylsioxane) (PDMS) elastomer integrated with fused silica capillaries coated with Aluminum. The detection of specific bacteria is based on molecular probes (beacons) that emit a fluorescent signal only when hybridized to the target. This method allows hybridization in solution, without immobilization, and avoids washing of the unbound probes. By marking 16S rDNA oligonucleotide probes (different genetic sequences) with different color dyes, and detecting the spectral intensity of light in the micro- channel, different micro-organisms can be detected in one sample. Miniaturization of the analytic device allows the use of small quantities of RNA molecules, as target molecules, and improves the detection limits. Future devices should incorporate a parallel array of micro-channels, and enable fast and parallel processing of the molecular signals.
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Cristina R. Rusu, Ronny van't Oever, Meint J. de Boer, Henri V. Jansen, Erwin Berenschot, Miko C. Elwenspoek, Martin L. Bennink, Johannes Sake Kanger, Bart G. de Grooth, et al.
Proceedings Volume Micro- and Nanotechnology for Biomedical and Environmental Applications, (2000) https://doi.org/10.1117/12.379579
We have developed a micromachined flow cell consisting of a flow channel integrated with micropipettes. The flow cell is used in combination with an optical trap set-up (optical tweezers) to study mechanical and structural properties of (lambda) -DNA molecules. The flow cell was realized using silicon micromachining including the so-called buried channel technology to fabricate the micropipettes, the wet etching of glass to create the flow channel, and the powder blasting of glass to create the fluid connections. The volume of the flow cell is 2 (mu) l. The pipettes have a length of 130 micrometer, a width of 5 - 10 micrometer, a round opening of 1 micron and can be processed with different shapes. Using this flow cell we stretched single molecules ((lambda) -DNA) showing typical force-extension curves also found with conventional techniques.
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Novel designs of integrated fluidic microchips allow separations, chemical reactions, and calibration-free analytical measurements to be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples. This technology lends itself to applications such as clinical diagnostics, including tumor marker screening, and environmental sensing in remote locations. Lab-on-a-Chip based systems offer many *advantages over traditional analytical devices: They consume extremely low volumes of both samples and reagents. Each chip is inexpensive and small. The sampling-to-result time is extremely short. They perform all analytical functions, including sampling, sample pretreatment, separation, dilution, and mixing steps, chemical reactions, and detection in an integrated microfluidic circuit. Lab-on-a-Chip systems enable the design of small, portable, rugged, low-cost, easy to use, yet extremely versatile and capable diagnostic instruments. In addition, fluids flowing in microchannels exhibit unique characteristics ('microfluidics'), which allow the design of analytical devices and assay formats that would not function on a macroscale. Existing Lab-on-a-chip technologies work very well for highly predictable and homogeneous samples common in genetic testing and drug discovery processes. One of the biggest challenges for current Labs-on-a-chip, however, is to perform analysis in the presence of the complexity and heterogeneity of actual samples such as whole blood or contaminated environmental samples. Micronics has developed a variety of Lab-on-a-Chip assays that can overcome those shortcomings. We will now present various types of novel Lab- on-a-Chip-based immunoassays, including the so-called Diffusion Immunoassays (DIA) that are based on the competitive laminar diffusion of analyte molecules and tracer molecules into a region of the chip containing antibodies that target the analyte molecules. Advantages of this technique are a reduction in reagents, higher sensitivity, minimal preparation of complex samples such as blood, real-time calibration, and extremely rapid analysis.
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In this paper, we present the forward (optical gating) and inverse (reconstruction) approaches to obtain high-resolution imaging in tissue-like media under single-photon (1-p) and two-photon (2-p) excitation. Effective point spread functions (EPSF) for fluorescence microscopic imaging are introduced for fast image modeling and reconstruction. It is demonstrated that a deeper penetration depth can be achieved under 2-p excitation due to the use of a longer illumination wavelength and the non-linear dependence of the fluorescence on excitation intensity. The fundamental difference between 1-p and 2-p fluorescence imaging is that the penetration depth is limited by the degradation in image resolution in 1-p fluorescence, while the penetration depth is limited by the loss in signal strength in 2-p fluorescence imaging. Based on the EPSF that derived in the simulation, image reconstruction using deconvolution methods can partially recover the resolution loss.
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Transmission ultrasound is not in widespread use, partially because of the time and expense of acquiring the data. We are addressing this problem with an optically parallel ultrasound sensor. The core of the sensor is a thin silicon nitride membrane patterned with gold to create 'acoustic pixels' over a large area. Each acoustic pixel vibrates at the frequency of the acoustic excitation. The thin membrane, supported by short walls over an optical substrate, with one side immersed in the ultrasound medium and the supported side exposed to air, flexes when an ultrasound pressure wave encounters it. This flexing causes the air gap between the optical substrate and the membrane to change. The change in the air gap modulates the reflection of an optical beam by frustrated total internal reflection. By strobing the optical beam, the deflection of the membrane can be detected and measured at any point through the acoustic period. Acquiring a sequence of images allow us to extract the relative pressure phase and amplitude. Proof of principle experiments have shown that we can build this sensor, and we are currently using a small aperture version to examine simple test objects.
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Biological applications of MEMS technology (bioMEMS) is of increasing interest in the development of miniature and portable instrumentation for cell-based microassays and sensor applications. A major bioMEMS challenge is the physical incorporation of living cells into sensors and diagnostic devices and creation of the environmental conditions conducive for organization of differentiated cells into tissue-like structures. Our work towards these goals is illustrated by a tissue-based bioassay system we are developing based on a miniature cross-flow bioreactor constructed from of an array of cell-filled microchannels integrated into an environmentally-controlled polymer microfluidics manifold. We describe our microchannel array and manifold manufacturing methods and report on the in vitro culture of cell populations in the bioreactor.
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Microfabrication technology is implemented to realize a fluidic microinstrument for the study of endothelial cell elongation and cell responsiveness to fluid flow. The microinstrument contains arrays of microchannels, 30 - 300 micrometer wide, that are fabricated by deep reactive ion etching (DRIE) of silicon and anodic bonding to glass. Silicon fluidic input/output modules, also micromachined in silicon, provide modular connections between the microchannels and off- chip devices for flow monitoring and control. Image analysis of cells cultured in microchannels shows that the cells become progressively more elongated as channel width decreases. When subjected to a fluid shear stress of 2 N/m2, cuboidal cells grown in 200 micrometer wide microchannels progressively align and elongate in the direction of flow.
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Arrays of microgrooves (groove width; 2, 3, 4, 5, 6, 7, 8, 10, 12, and 14 micrometer, groove interval; width x3, x10, and x20, one size and interval per chip) each connecting a center well and a side edge of a silicon substrate were created by photolithography and anisotropic wet etching. A penetrating hole was made by sand blast at the substrate center for the access to the center well. By tightly covering the substrate surface with a glass plate, the microgroove arrays were converted to microchannel arrays having one ends open at the side edges of the substrate. These microchannel arrays were used for cell trapping for microinjection and also used for emulsification. Poplar (Populus alba) protoplasts were used for the test of cell trapping. Cells showed a very large variation in size and irregularity in shape, and, furthermore, the protoplast preparation contained a number of cell membrane fragments and chloroplasts. Despite the cell size and shape variations and obstruction by the admixtures, many cells could be trapped by aspiration at the channel ends because of their openness to the outside free space and also their large multiplicity in parallel. The free space outside the side of the substrate allowed a free manipulation of a glass micropipette under microscopic observation using transmitted illumination. The microscopic observation direction nearly perpendicular to the movement directions of the micropipette further allowed the movement of the pipette tip nearly always in focus. These led to an easy pointing and puncturing. In addition, the cell trapping points in a line made successive approach to adjacent cells easier. Soybean oil containing 1.5 wt% polyoxyethylene(20)sorbitan monoolete as a surfactant was forced to flow into physiological saline filling the outside of the substrate through the microchannels. Regularly sized oil particles were created by this process with a variation coefficient (S.D./mean) 16% of their diameter. This variation, which is larger than those (minimum 1 - 2%) obtained in our previous trials using our previous microchannel arrays, appeared to be attributable to an irregularity of the channel ends due to microchipping by saw cutting. As an advantage over the previous ones, the present microchannel arrays allowed an easy collection of the created oil particles and also an easy change of the composition of the suspending fluid during the process. The substrate side surface is thus indicated to be useful for interfacing structures or devices microfabricated in the main substrate surface, which may be covered with a glass plate, with conventional or hand-operated tools or processes outside the substrate.
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The use of microfabricated and micromachined substrates can aid in the creation of design two- and three-dimensional microstructures, scaffolds, and platforms for cell culture and tissue engineering. These platforms may offer several advantages for in vitro cell culture by providing (1) well controlled microarchitectures, (2) spatial localization of different cell populations, and (3) biochemically modified substrates to promote selective protein and/or cell attachment. The development of methodologies to create microfabricated tissue engineering constructs using traditional natural and polymeric biomaterials may allow us to engineer highly controlled interfaces in order to better understand and modulate cell behavior and have the capability to provide: more physiologically relevant models of cell and tissues in vitro. This paper describes the use of microfabricated cell culture platforms to study cardiac myocytes.
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Two mechanisms can be applied initially for the high resolution patterning on radiation-assisted functionalized polymer surfaces: hydrophobicity-controlled patterning; and chemical linkage. The present contribution assesses the merits and drawbacks of these mechanisms in terms of resolution and contrast of protein/peptide features. Two microlithographic materials (an acrylate-based system and a diazo-naphto-quinone one); and two lithographic methods (e-beam and optical) have been used to test the merits of the protein patterning mechanisms. The hydrophobicity-controlled patterning produces sharp images but with multiple defects, whereas chemical linkage produces defect-free images but with a decreased contrast. The benefits of a third mechanism for protein confinement, namely bimolecular specific recognition, was explored in the view of the possible fabrication of artificial molecular motors.
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The ability of biomaterial surfaces to regulate cell behavior requires control over surface chemistry and microstructure. One of the greatest challenges with silicon biomedical microdevices is to improve biocompatibility, which may be achieved by modifying the exposed silicon surface with thin or adsorbed films. In this study, films of silicon nitride, doped polysilicon, undoped polysilicon, as well as RGD peptide adsorbed surfaces were incubated with fibroblasts for a period of four days. Results demonstrate that RGD adsorbed surfaces encourage the greatest cell proliferation, followed by undoped polysilicon and unmodified (control) surfaces. Protein adsorption studies using fibrinogen and albumin, two proteins implicated in cellular adhesion and surface activity, reveal greatest protein adsorption on low stress silicon nitride surfaces, followed by undoped polysilicon and unmodified surfaces. This finding may compliment the differential cellular binding found on modified and unmodified silicon surfaces. The thickness of adsorbed albumin and fibrinogen was measured by ellipsometry and compared to contact angle measurements of non-adsorbed surfaces. Moreover, silicon surfaces coupled with a synthetic RGD peptide, as characterized with XPS and atomic force microscopy, display enhanced cell proliferation and bioactivity. Understanding the biological response to thin films will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic silicon-based microdevices.
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Michael D. Pocha, Henry E. Garrett, Rajesh R. Patel, Leslie M. Jones III, Michael C. Larson, Mark A. Emanuel, Steven W. Bond, Robert J. Deri, R. F. Drayton, et al.
Proceedings Volume Micro- and Nanotechnology for Biomedical and Environmental Applications, (2000) https://doi.org/10.1117/12.379570
At Lawrence Livermore National Laboratory, we have extensive experience with the design and development of miniature photonic systems which require novel packaging schemes. Over the years we have developed silicon micro-optical benches to serve as a stable platform for precision mounting of optical and electronic components. We have developed glass ball lenses that can be fabricated in-situ on the microbench substrate. We have modified commercially available molded plastic fiber ribbon connectors (MT) and added thin film multilayer semiconductor coatings to create potentially low-cost wavelength combiners and wavelength selective filters. We have fabricated both vertical-cavity and in-plane semiconductor lasers and amplifiers, and have packaged these and other components into several miniature photonics systems. For example, we have combined the silicon optical bench with standard electronic packaging techniques and our custom-made wavelength-selective filters to develop a four-wavelength wavelength-division-multiplexing transmitter module mounted in a standard 120-pin ceramic PGA package that couples light from several vertical-cavity-surface-emitting-laser arrays into one multimode fiber-ribbon array. The coupling loss can be as low as 2 dB, and the transmitters can be operated at over 1.25 GHz. While these systems were not designed for biomedical or environmental applications, the concepts and techniques are general and widely applicable.
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In recent years, microspectrometer systems fabricated by the LIGA technology for use in the visible wavelength range increasingly made their way to the market. For spectral analysis in the infrared range, a highly transmissive hollow waveguide has now been developed and demonstrated successfully. In combination with linear detector arrays, the hollow waveguide microspectrometer allows to design portable near infrared spectrometer systems. Such a system has now been applied to evaluate the spectrum from 0.9 micrometer to 1.15 micrometer in second order and from 1.15 micrometer to 1.75 micrometer in first order. Due to its outer dimensions of 54 X 36 X 7 mm3 and a low power consumption, it can be integrated in portable spectral analysis systems. It exhibits a high sensitivity (NEP below 11 pW) due to a fiber-optical entrance with a fiber core diameter of 300 micrometer. An indium-gallium-arsenide linear detector array based on a novel setup concept is incorporated in the system. Furthermore, units for preamplification of the signal and 16-bit AD conversion are contained. In the present paper, the setup and fabrication of the whole microspectrometer system, its optical features and the detector-specific solutions are described. The fabrication process which is based on molded polymer parts is presented. Using the experimental results of the electro- optical tests and the polymer spectra measured, the performance of the system will be demonstrated.
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A long range translation actuator designed for optic and robotic applications is presented. Specifically, the microstage is designed to operate as the moving mirror in a miniature version of a traditional Michelson Fourier transform spectrometer. The translational microstage utilizes an electromagnetic actuation mechanism to realize linear translation of centimeters of precision travel. Motion is constrained in the normal and lateral directions using silicon dovetail microjoints. The electromagnetic actuation is based on macro linear synchronous motor design using a linear array of microcoils. Microcoils are arranged in a 3-phase configuration to enable both velocity and direction control. The electromagnetic force is characterized by finite element computer simulations to develop the input signal for translational travel at constant velocity. Optical position detection was used to measure the translation with time. Operation was demonstrated at various drive frequencies.
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The determination of ammonia has been studied using a compact diode laser/fiber optic colorimetric spectrometer combined with a flow injection cell. This system is mainly based on the diode laser at 638 nm. The flow rate and the molar concentration of reagents for indophenol formation in flows were specified through the experiment of optimization. The relationship between the colorimetric absorption signal and the concentration of injected ammonia was found to be linear over the three orders of magnitude indicating a practical dynamic range of the system. In the linear region, a correlation coefficient was 0.9996 and the detection limit was 4 (mu) g/L. This spectrometer as an ammonia analyzer is appropriate for environmental and industrial monitoring applications.
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In analytical chemistry so-called 'capillary waveguide cells' are used as effective optical cells with a low sample volume and a high optical pathlength. In this paper we will demonstrate that the concept of liquid core guiding, when the test liquid is really used as the optical core, is the most effective one. Guiding and coupling properties of different capillaries are investigated for different diameters of fibers and capillaries. Examples for using a liquid core guide cell as a biosensor is discussed as well as an example for 'fill once' (mu) l capillary cell.
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The technique of dynamic ATR-leaky mode spectroscopy is demonstrated for polymer films on 50 nm Ag layers. For the vapor series R OH with R equals H,CH3, C2 H5 and the BTX compounds benzene, toluene and the 3 xylene isomers two different ways of obtaining selective sensor detection are described. The first approach uses the specific sensitivity. Each vapor has a specific molecular polarizability which causes a specific sensitivity. The second approach takes advantage of the dynamic method. The specific diffusion of the particular molecule in the polymer matrix can be evaluated. In this context the characteristic diffusion of the 3 isomers of xylene are investigated.
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