KEYWORDS: Systems modeling, Switches, Printing, Pathogens, Natural surfaces, Modulation, In vitro testing, Digital micromirror devices, Digital Light Processing, Control systems
Cellular studies using model in vitro systems limit environmental complexities that may obscure input-outcome relationships. To address the need for more relevant model systems, we have developed a platform for interfacing biomaterials with cultured cells with high spatiotemporal control. Pulsed near-infrared light is used to create protein-based cellular landscapes in a direct-write, multiphoton photo-crosslinking process patterned using a dynamic mask. High-resolution 3D environments are created either in advance of cellular application, or in the presence of viable cells, yielding dynamically controllable enclosures and surfaces for organizing cellular communities that more accurately reproduce mechanical, chemical, and convective properties of native environments.
In this review of our work, we describe the application of multiphoton-excited fluorescence as a detection strategy for biological molecules fractionated in micrometer-diameter electrophoresis channels. By tightly focusing a modelocked titanium:sapphire laser beam at the outlet of such channels, spectroscopically similar components can be differentiated in analysis times that range from milliseconds to minutes. Moreover, the ability to excite different chromophores through the combined energies of different numbers of photons (e.g., two and three near-infrared quanta) provides a means to analyze species that are spectroscopically diverse. Finally, we demonstrate that multiphoton photochemistry can be used as a rapid 'photoderivatization' technique for hydroxyindoles and potentially other biological species, in some cases significantly improving the mass detectability of these analytes.
A micromachined fluidic sensor array for the rapid characterization of multiple analytes in solution has been developed. A simple micromachined fluidic structure for this biological and chemical agent detection system has been designed and fabricated, and the system has been tested. Sensing occurs via optical changes to indicator molecules that are attached to polymeric microspheres (beads). A separate charged-coupled- device (CCD) is used for the simultaneous acquisition of the optical data from the selectively arranged beads in micromachined etch cavities. The micromachined bead support structure has been designed to be compatible wit this hybrid optical detection system. The structure consists of four layers: cover glass, micromachined silicon, dry film photoresist, and glass substrate. The bottom three layers are fabricated first, and the beads are selectively placed into micromachined etch cavities. Finally, the cover glass is applied to confine the beads. This structure utilizes a hydrophilic surface of the cover glass to draw a liquid sample into the sensor array without moving components, producing a compact, reliable, and potentially low-cost device. We have initially characterized fluid flow through a complete chip, showing complete filling of the sample chamber in approximately 2 seconds. The test results show that this system may be useful in micro total analysis systems ((mu) - TAS), especially in single-use biomedical applications.
The rational design of small molecules for the selective complexation of analytes has reached a level of sophistication such that there exists a high degree of prediction. An effective strategy for transforming these hosts into sensors involves covalently attaching a fluorophore to the receptor which displays some fluorescence modulation when analyte is bound. Competition methods, such as those used with antibodies, are also amenable to these synthetic receptors, yet there are few examples. In our laboratories, the use of common dyes in competition assays with small molecules has proven very effective. For example, an assay for citrate in beverages and an assay for the secondary messenger IP3 in cells have been developed. Another approach we have explored focuses on multi-analyte sensor arrays with attempt to mimic the mammalian sense of taste. Our system utilizes polymer resin beads with the desired sensors covalently attached. These functionalized microspheres are then immobilized into micromachined wells on a silicon chip thereby creating our taste buds. Exposure of the resin to analyte causes a change in the transmittance of the bead. This change can be fluorescent or colorimetric. Optical interrogation of the microspheres, by illuminating from one side of the wafer and collecting the signal on the other, results in an image. These data streams are collected using a CCD camera which creates red, green and blue (RGB) patterns that are distinct and reproducible for their environments. Analysis of this data can identify and quantify the analytes present.
Steven Savoy, John Lavigne, J. Yoo, John Wright, Marc Rodriguez, Adrian Goodey, Bridget McDoniel, John McDevitt, Eric Anslyn, Jason Shear, Andrew Ellington, Dean Neikirk
A micromachined sensor array has been developed for the rapid characterization of multi-component mixtures in aqueous media. The sensor functions in a manner analogous to that of the mammalian tongue, using an array composed of individually immobilized polystyrene-polyethylene glycol composite microspheres selectively arranged in micromachined etch cavities localized o n silicon wafers. Sensing occurs via colorimetric or fluorometric changes to indicator molecules that are covalently bound to amine termination sites on the polymeric microspheres. The hybrid micromachined structure has been interfaced directly to a charged-coupled-device that is used for the simultaneous acquisition of the optical data from the individually addressable `taste bud' elements. With the miniature sensor array, acquisition of data streams composed of red, green, and blue color patterns distinctive for the analytes in the solution are rapidly acquired. The unique combination of carefully chosen reporter molecules with water permeable microspheres allows for the simultaneous detection and quantification of a variety of analytes. The fabrication of the sensor structures and the initial colorimetric and fluorescent responses for pH, Ca+2, Ce+3, and sugar are reported. Interface to microfluidic components should also be possible, producing a complete sampling/sensing system.
Nonlinear excitation of fluorophores through molecular absorption of two or three near infrared photons from the tightly focused femtosecond pulses of a mode-locked laser offers the cellular biologist an unprecedented panoply of biomolecular indicators for microscopic imaging and cellular analysis. Measurements of the two-photon excitation spectra of more than twenty ultra-violet and visible absorbing fluorophores from 690 to 1050 nm reveal useful cross sections for near infrared excitation, providing an artist's palette of emission markers and chemical indicators for living biological preparations. Measurements of three-photon fluorophore excitation spectra now define alternative windows of relatively benign wavelength to excite deeper UV fluorophores. The three-photon excitation spectrum of the amino acid tryptophan, measured 700-900 nm, delivers native fluorescence for imaging and assay of proteins and neurotransmitter sin living tissues. The inherent optical sectioning capabilities of focused nonlinear excitation provides 3D resolution for imaging and avoids out of focus background. Here, we describe the characteristics of the measured nonlinear excitation spectra and define the resulting features of nonlinear microscopy for biological imaging.
Capillary zone electrophoresis is fast becoming one of the most sensitive separation schemes for sampling complex microenvironments. A unique detection scheme is developed in which a charge-coupled device (CCD) detects laser induced fluorescence from an axially illuminated electrophoresis capillary. The fluorescence from an analyte band is measured over a several centimeter section of the capillary, greatly increasing the observation time of the fluorescently tagged band. The sensitivity of the system is in the 1-8 X 10-20 mol range for derivatized amino acids and peptides. Subattomole quantities of bag cell neuropeptides collected from the giant marine mollusk Aplysia californica can be measured.
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