Visible and infra-red spectral filters based on arrays of uncoupled identical waveguides having coherently modulated cross sections offer many unique advantages, such as independence of the spectral position of the Bragg-resonance based transmission or reflection features on the angle of light incidence. The resulting spectral filters are mechanically and optically stable, do not degrade over time, and offer superior transmittance for use as band pass, narrow band pass, band blocking, short pass and long pass filters. Such filters are useful for a wide variety of applications including but not limited to biomedical analysis systems, spectroscopy and optical communications systems.

Generally, optical filters and coatings are passive components whose basic function is to define or improve the performance of optical systems. There are many types of optical filters and they are used for a broad range of different applications. One common type of optical filter is a sunglass lens. Polarized sunglass lenses filter out light with a certain direction of polarization in addition to reducing the sun's intensity. Applications of optical filters and coatings can be diverse as in anti-glare computer screens, colored glass, sighting devices, and electrical spark imagers—to name just a few.
Some optical filters are specialized for different wavelength ranges of light because of limitations in available materials that are optically transparent in the range of interest. For example, many applications and instruments require optical filters that can be used to tune the optical behavior of light in the near infrared, mid infrared or far infrared wavelength range (i.e., at frequencies of radiant energy that are generally below the frequencies of visible light). Some example applications for such filters include far- and mid-IR focal-plane arrays for military applications, chemical sensing, astronomy, wavelength division multiplexing in optical communications, space observations to name a few.
Much work has been done in the past to develop useful optical filters and coatings for different wavelength ranges. Widely spread filter types include: absorption-based filters (i.e., filters where the rejection of light is caused by absorption in filter material) and interference-based filters (i.e., filters where the rejection of light is caused by reflectance from multiple layers composing the filter). Detailed discussion of such filters can be found in for example Macleod H. A., Thin-Film Optical Filters, 3rd ed., Institute of Physics Publishing, 2001.
Exemplary Absorption Type Filters
Absorption filters generally consist of a thin film or slide of material that has an absorption feature (band or edge) at the required wavelength or incorporates an optically excitable material, such as a color center. Filters that utilize semiconductor material can serve as an illustrative example of an edge absorption filter (more particularly long-pass filters). Semiconductors are known to have an absorption band that extends to some characteristic wavelength, which corresponds to the bandgap energy of a particular semiconductor. The transmission/rejection edge could be made very sharp for a semiconductor layer thicknesses above 100 μm. Absorption band edge of different semiconductors and semiconductor composites can vary from ˜500 nm for gallium phosphate (GaP) and aluminum arsenate (AlAs) to more than 2 μm for indium arsenate (InAs) and InSb. The absorption band edge can be smoothly tuned by adjusting the semiconductor composition (for example, AlxGa1-xAs absorption band edge tunes quite linearly from 2.1 eV for x=1 to 1.4 for x=0), long-pass filters can be obtained with a reasonably sharp edge for any wavelength at the ˜500 to 2400 nm range). However, at least some semiconductor-based long-pass filters have a significant disadvantage—i.e., high reflection losses caused by the high refractive index of semiconductor materials. Such a problem is usually solved by antireflection coatings of both semiconductor surfaces in the case of semiconductor wafer (or slide) used or, in the case of thin film semiconductor material, of the top and between the substrate and the layer of semiconductor material. However, such an approach can also have some significant disadvantages While the absorption edge shape and position of uncoated semiconductor film does not depend on angle of incidence (only the degree of an absorption value changes), both the absorption edge shape and position of antireflection-coated semiconductor film depend on angle of incidence. Hence, the filter can in at least some cases be used effectively only for some limited angular range. In addition, semiconductor absorption edges show strong temperature dependence. It also should be noted that semiconductor-based absorption filter cannot be used as a band-pass or narrow-band pass filtering, since the absorption bands of semiconductors are generally wide.
Other materials that are sometimes used to form absorption filters include colored-glass filters (for example, Schott glass filter). Such colored-glass filters generally operate through the process of ionic absorption of inorganic material, dispersed uniformly through the glass slide or through the absorptive scattering of crystallites formed within the glass. Such filters generally offer fairly wide design freedom in terms of absorption band position and can be arranged either in short-pass, long-pass or band-blocking forms. The significantly lower refractive index of such filters as compared to semiconductor filters make the reflection losses lower. The temperature dependence of the rejection band edge position of such filters is also lower than that of semiconductors. However, such filters can also suffer from considerable limitations. For example, the transmission through the transparency range of such filters is usually not very uniform. Therefore, such filters in some cases cannot tolerate high power and/or are not well-suited for narrow bandpass filters.
Exemplary Interference Type Filters
Generally, the basis of interference filters is a Fabry-Perot interferometer. A Fabry-Perot (FP) interferometer can be imagined as a thin film having two flat surfaces that are parallel to each other and coated with relatively high-reflectance coatings. In practice, a different realization of Fabry-Perot interferometer is common—the so-called Fabry-Perot etalon, which consists of two flat plates separated by a distance d and aligned parallel to each other with a high degree of accuracy. The separation is usually maintained by a spacer ring made of quartz or Invar, and the inner surfaces of the two plates are usually coated to enhance their reflections. The spectral dependence of the transmittance through an FP interferometer contains peaks and valleys. The peaks of transmittance are known as fringes. It can be proven that the maximum values of transmittance at the peaks reach unity if the Fabry-Perot interferometer is constructed from nonabsorptive materials.
The classic design of a high-reflectance coating is based on alternating quarter-wave layers of two different materials. The high reflectance in a quarter-wave layer stack takes place because the light beams, reflected from all the interfaces in the multilayer, are in phase when they reach the front surface where constructive interference of all the reflected waves occurs. As with the Fabry-Perot etalon, the reflectance spectrum of such a multilayer contains multiple reflection peaks corresponding to the phase-matching conditions of the reflected waves at the different interfaces. However, these reflection maximums are considerably wider than that of the Fabry-Perot etalon. The width of the high reflectance plateau depends on the refractive index contrast between the low and high refractive index materials that compose the high reflectance multilayer. As follows from the above discussion, the high-reflectance dielectric multilayer can be constructed to have very high reflectance over a wide range of wavelengths. However, such multilayers can have some disadvantages. One possible disadvantage is that the high reflectance zone of such a reflector, although it can be made wide, is still limited. Moreover, since the reflectance peaks are located where the waves reflected from each interface in multilayer are in phase, the wavelength positions for the reflectance peaks may strongly depend on the angle of incidence, similar to the Fabry-Perot etalon case.
A quarter-wave stack can be considered as the basic type of interference edge filter. The transmission spectrum of the quarter-wave stack contains alternating low- and high-reflectance zones and, hence, alternative high- and low-transmittance zones. Such a filter can be used as a long-pass filter or as a short-pass filter. Edge wavelengths can be tuned by changing the wavelength at which the stack is quarter-wave. Such an edge filter will be suitable for relatively narrow-band applications, that is, when the width of the rejection zone is greater than the spectral width of light to be eliminated. For all other cases, the required elimination of all wavelengths shorter than (or longer than) a particular value requires a different filter design.
Perhaps the simplest design of a narrowband-pass filter is the Fabry-Perot filter discussed previously. However, the spectral pass-band shape of the Fabry-Perot filter is triangular. In addition, the original design generally may require two precisely aligned and spaced high-flatness plates, which may not be practical for many applications. Therefore, narrowband-pass filters are usually made in slightly modified form with respect to a Fabry-Perot etalon. A Fabry-Perot thin film filter is a thin film assembly consisting of a dielectric layer bounded by either two metallic reflecting layers or by two multilayer dielectric reflectors. The realization of a Fabry-Perot filter with metallic reflective layers is called a metal-dielectric Fabry-Perot filter, while a Fabry-Perot filter with two dielectric multilayer reflectors is called an all-dielectric Fabry-Perot filter.
The metal-dielectric Fabry-Perot filter is perhaps the simplest realization of a narrowband-pass filter. In such filters, the dielectric layer, surrounded by metal reflection layers, serves as a spacer in the Fabry-Perot etalon. and therefore, is called a spacer layer. The metallic reflective layers must provide reasonably high reflectivity at the surface while keeping losses as low as possible. For the visible region of the spectrum, silver is an optimal metal, while for ultraviolet and deep ultraviolet aluminum is the preferred material. However, other metals can be used as well.
The degree of light absorption is perhaps the biggest disadvantage of metal-dielectric narrowband-pass filters. Although absorption during a single reflection from thin metal film is small and absorption during transmission also can be minimized by using thin metal films, in a Fabry-Perot cavity absorption is greatly enhanced due to multiple reflections of the transmitted light. Metal-dielectric Fabry-Perot filters have the same dependence of the transmittance peak wavelength as the Fabry-Perot cavity. In addition to this disadvantage, the absorbance of such a filter strongly depends on the angle of incidence. Such filters are usually used in applications where other filters, such as all-dielectric Fabry-Perot filters, are prohibited by either cost or other factors, such as the inability to function in the deep UV.
As was discussed above, in the all-dielectric Fabry-Perot filter the metallic reflecting layers are replaced by high-reflectance dielectric multilayers. Two different cases of such filters can be considered: {Air| H L H L . . . H L H H L H . . . L H L H| Substrate} and {Air| H L H L . . . L H L L H L . . . L H L H| Substrate}, where H indicates the higher index of refraction and L indicates the lower index of refraction. The refractive indices of the layers adjacent to air and the substrate should be high to maximize the reflection from the multilayer. The transmission spectrum of an all-dielectric Fabry-Perot will be a narrow maximum within a broad minimum. The width of the maximum and the transmittance at the maximum will depend upon the reflectivities of the two multilayer stacks.
The central position of the transmittance peak in the all-dielectric Fabry-Perot filters is generally the same as for air cavity Fabry-Perot filters. However, the effect of variations in angle of incidence can be more severe for narrowband-pass filters than that of bandpass, band edge or multilayer reflectors due to a generally narrow transmittance peak. Shifts of the central position of the transmittance peak as strong as 800% in terms of the transmittance peak half width at just a 30 degrees tilt are not uncommon. Such a strong angular dependence of the transmittance spectra of all-dielectric Fabry-Perot filters causes strong dependence of the transmittance spectra on the convergence (or divergence) of the incident beam. Hence, all-dielectric Fabry-Perot filters are often suitable only for plane-parallel or slightly convergent or divergent beams, which causes additional complexity in the optical designs employing such filters.
As was discussed above, the transmitted spectral shape of the all-dielectric Fabry-Perot filter is generally not ideal. For many filter purposes, a nearly rectangular shape of the transmittance spectra is desired. In addition, the maximum achievable rejection in the rejection zone of the filter and the bandwidth of the transmission zone are related. That is, for a given rejection factor, the bandwidth value of the filter is predetermined if the refractive indices of the layers in the filter are fixed. The solution of this problem was found in using multiple-cavity filter designs.
Exemplary Multi-Cavity Interference Filter Designs
Perhaps the simplest type of multiple cavity filter is a double-cavity filter. Such a filter has the structure of {Air|reflector|half-wave spacer|reflector|half-wave spacer|reflector|Substrate}. Such a structure can have some advantages with respect to a single-cavity design. However, for some applications such as dense wavelength division multiplexing (DWDM), a better spectral shape may be needed. The important criteria in high-performance, narrowband-pass filters are steeper edges and a flatter top on the transmission peak. For two-cavity filter designs, the peaks at both sides of pass band (so-called "rabbit's ears") are prominent. In this case the number of cavities needed can be considerably more than two to reduce the "rabbit's ears."
Although at normal incidence the advantages of multiple-cavity filters are generally strong, the effects of variations of angle of incidence and beam divergence on the transmittance spectra can be more severe for multiple-cavity, all-dielectric Fabry-Perot filters than for single cavity filters. This occurs because the rectangular shape of the pass band of the multiple-cavity filter is due to phase matching between the light waves reflected from the different reflector stacks in the multiple-cavity structure. The phase-matched conditions hold only for a distinct angle and wavelength. Unlike single-cavity filters, where the transmittance peak generally experiences a wavelength shift when illuminated at non-normal angles without significant perturbation of its shape, in multiple-cavity, all-dielectric Fabry-Perot filters the shape of the transmittance band generally changes dramatically with variations in the angle of incidence. The flat top of the multiple-cavity filter at normal incidence frequently resolves into separate narrow transmittance peaks related to the interference between the waves reflected from different reflector stacks within the multiple-cavity multilayer structure. Hence, multiple cavity, all-dielectric Fabry-Perot filters can become unusable at incident angles more than 3 to 5 degrees from normal incidence. Such a property is important in DWDM filters where several hundred layers may be required to produce flat topped transmittance bands with bandwidths narrower than 1 nm. Additional precise mechanical alignment may solve this problem, but with additional complexity and resultant additional cost.
In addition, multiple-cavity, all-dielectric Fabry-Perot filters generally require the incident beam to be highly collimated. The shape of the pass band of such filters degrades significantly even for Gaussian beams, the convergence (or divergence) angle of which is about 10–15 degrees. Hence, multiple-cavity, all-dielectric Fabry-Perot filters generally can require not only precise mechanical alignment to ensure normal incidence of the beam, but also a high degree of collimation. Several other significant disadvantages exist with multiple-cavity, all-dielectric Fabry-Perot filters. These disadvantages may include the presence of long-wave pass bands (i.e., wavelength-limited rejection bands) and significant difficulties in manufacturing such filters for short wavelength spectral ranges (deep and far ultraviolet). For the applications that require the useful filter properties in the UV ranges, multiple-cavity metal dielectric filters are usually used. In particular, it has been found that, in addition to the disadvantages of an all-dielectric Fabry-Perot filter such as the relationship between pass-band bandwidth and maximum obtainable rejection and the resultant triangular shape of the pass band, the single-cavity metal-dielectric Fabry-Perot filters exhibit increased losses with decrease of the pass-band bandwidth due to the losses in the metal.
In multiple-cavity, metal-dielectric Fabry-Perot filters, this problem is usually solved by an induced-transmission design. This phenomenon serves as the basis of such filters so that it is possible to match metal layers and dielectric spacer thicknesses such that, for a given wavelength and angle of incidence, the localization of the light in the metal layers during transmission is minimal at the same time it is maximized inside the dielectric layers. Using such a design, it is still usually not practical or possible to achieve perfect transmission. However, the transmission can be made somewhat greater than 50%, combined with near square pass band shape and simultaneously good control of rejection and pass band bandwidth. Multiple-cavity metal-dielectric filters can, however, have some significant disadvantages. In addition to an angular shift of the wavelength position of the pass band due to absorption in metal layer, such filters may not be suitable for high-power applications. The temperature dependence of the optical performance of such filters also can be the strongest among all interference-based filters.
Exemplary Spectral Filter Implementations
Various different designs for spectral filters are known. Among them it worthwhile to mention ultraviolet optical filter disclosed in Lehmann et al., Appl. Phys. Lett. V 78, N. 5, January 2001. The filter configuration of Lehmann et al., Appl. Phys. Lett. V 78, N. 5, January 2001 is based on the spectral filtering of light in an array of leaky waveguides in the form of pores in Macroporous Silicon ("MPSi"). One such an illustrative method of optical filter manufacturing consists of forming a freestanding macropore array from N-doped Si wafer in fluoride-containing electrolyte under certain backside illumination conditions. Precise control over the pore distribution across the surface of the wafer may be possible if preliminary patterning of the silicon wafer surface with regularly distributed depressions (so-called "etch pits") is performed. A method of manufacturing such filters by forming of free-standing macropore arrays from n-doped Si wafer can be found for example in U.S. Pat. No. 5,262,021 issued to V. Lehmann et al. Nov. 16, 1993. Lehmann also discloses the use of such arrays as optical filters. However, it appears that the method of removing the macroporous layer from the Si wafer, as disclosed in U.S. Pat. No. 5,262,021, will result in the second surface of the macroporous layer being inherently rough, causing higher losses due to scattering. Lehmann seems to use the MPSi layer without any further modifications. Thus, while such filters exhibit some short-pass filtering, the transmission spectral shape through them may be unusable for commercial applications due to the wide blocking edge.
Macroporous silicon layers with modulated pore diameters throughout the pore depth is disclosed in, for example, U.S. Pat. No. 5,987,208 issued to U. Gruning and V. Lehmann et al. Nov. 16, 1999 or J. Schilling et al., Appl. Phys. Lett. V 78, N. 9, February 2001. These structures may not exhibit advantageous properties such as independence of the spectral response of the filter on the angle of incidence for at least two reasons. First—the structure of these filters (i.e. hexagonal array of pores) may not be suitable to act as an array of waveguides, so the filtering may be directly affected by the angle of incidence of light on the structure. Second, the pore modulation period, pore array period and the Bragg wavelength seem to be chosen so that the light, while traveling through such structure effectively averages the dielectric properties of the structure (similar to what happens in microporous silicon-based filters). The resulting optical behavior will therefore likely resemble that of ordinary interference filters.
FIG. 1 is a diagrammatic perspective view of an exemplary prior art freestanding MPSi uniform pore array section wherein the pores form a uniform cubic lattice. The FIG. 1 exemplary prior art spectral filter consists of air- or vacuum-filled macropores 1.2 starting from the 1st surface 1.3 of the filter wafer and ending at the 2nd surface 1.4 of the filter wafer host 1.1. The macropores 1.2 are disposed such that an ordered uniform macropore array is formed (the ordering may be a key attribute). The pore ends are open at both first and second surfaces of the silicon wafer 1.1. Since silicon is opaque in the deep UV, UV, and visible and part of the near IR wavelength ranges, light can pass through the structure shown in FIG. 1 only through the pores. As shown in FIG. 2, the silicon absorption coefficient k is very high at wavelengths below ˜400 nm and moderately high at wavelengths below ˜900 nm, which blocks all radiation coming through the silicon having a thickness of 50 micrometers or more.
Since pore diameters of 100 nm to 5000 nm are comparable with the wavelength of light and due to the high aspect ratios possible in MPSi structures ((usually above 30), the transmission through such a macroporous structure at wavelengths below about 700 nm takes place through leaky waveguide modes. In such leaky waveguide modes, the cores of the leaky waveguides are air or vacuum-filled, while the reflective walls of the leaky waveguides are the pore walls. This can be seen in FIG. 2 by the near-metallic behavior of the refractive index n and absorption coefficient k of silicon at wavelengths below ˜370 nm. Hence, MPSi material can be considered as an ordered array of leaky waveguides. By means of the high absorption of the walls, each leaky waveguide pore can be considered to be independent of the others in the visible, UV and deep UV spectral ranges if they are separated by silicon walls with thicknesses >20–100 nm.
In the near IR and IR wavelength ranges, the nature of the transmission through the filter of FIG. 1 changes. This happens because silicon becomes less opaque at 700–900 nm and becomes transparent at wavelengths starting approximately from 1100 nm. Light at these wavelengths can pass through the MPSi structure of FIG. 1 not only through the pores, but also through the silicon host. Due to the porous nature of the silicon host, the transmission of light propagating at the angles close to the perpendicular directions to the surface of the MPSi structure occurs through waveguide modes confined in the silicon host for the wavelengths comparable to the pitch of the pore array. As a high refractive index material, silicon can support waveguide modes if surrounded by a lower refractive index material (air or vacuum).
Since close packing of the pores is essential for efficient transmission through the filter of FIG. 1, such a structure can be considered to some approximation in the near IR and IR wavelength ranges as an array of Si waveguides in an air host. For the light propagating at oblique angles to the axes of the waveguides, a non-waveguiding channel of transmission through the structures of FIG. 1 arises. However, such transmission is accomplished by strong reflection and scattering and in the far field of the filter and in many cases such a transmission channel can be neglected. In the near field, however, such a transmission channel usually should be taken into account. When the wavelength of light becomes much larger than the pore array pitch, the light starts interacting with the MPSi layer as if it were a single layer of uniform material having its dielectric constants averaged through the pores and the host. As an illustration, for a square array of pores with 4 micrometer pitch, transmission takes place starting approximately at a wavelength of 20 micrometers.
For purposes of certain analysis, only the waveguide channel of transmission through the MPSi structure of FIG. 1 can be considered. The non-waveguide transmission channel may be neglected. As will be shown below, the latter transmission channel can be completely suppressed.
To take absorption-based losses into account, the optical loss coefficient, α, having dimensions cm−1, will be used to characterize the optical transmission. The amount of light still remaining in the pore leaky waveguide or Si host waveguide after it travels a length l is proportional to exp(−α(λ)l), and the light remaining in the MPSi layer at the distance l from the first MPSi layer interface is equal to I0 P(λ) exp (−α(λ)l), where I0 is the initial intensity of the light entering the pore and P(λ) is the coupling efficiency at the first MPSi interface. The optical loss coefficient is, in turn, a function of pore size, geometry, distribution, and wavelength. It is also dependent upon the smoothness of the pore walls. Roughness in the walls introduces another source of light absorption, i.e., scattering, which is proportional to the roughness to wavelength ratio.
An illustrative, numerically calculated spectral dependence of loss coefficients for the prior art MPSi filter of FIG. 1 is given in FIG. 3. The pore array is of cubic symmetry and is made up of 1×1-micrometer vacuum-filled pores in this example. It follows from this illustrative plot that for the chosen pore array dimensions, transmission through pore leaky waveguides is dominant up to about 700 nm and the transmission through the silicon host waveguides is dominant starting from about 800 nm. At 700–800 nm, both transmission mechanisms compete with each other. The increase of the losses through leaky waveguides with increasing wavelength is due to both the reduction of the reflection coefficient of silicon and to the redistribution of the leaky waveguide modes over the pore cross-sections. The modal field penetration into the silicon host material, as well as the optical losses, increase with the wavelength.
Depending on pore size and pore array geometry, leaky waveguides in the deep UV, UV, VIS spectral ranges and waveguides in the near IR and IR spectral ranges can be either single mode (i.e., supporting only the fundamental mode) or multimode (higher order modes are also supported). The amount of light remaining at the distance l into the pore from the first MPSi filter surface can be estimated asI0{ΣPi,jLW(λ)exp(−αi,jLW(λ)l)+ΣPi,jW(λ)exp(−αi,jW(λ)l)}
where the i,j are the mode order indices, introduced as follows: i=j=0 corresponds to the fundamental mode and so on; Pi,jW(λ) is the coupling efficiency into i,j-th waveguide mode, Pi,jLW(λ) is the coupling efficiency into i,j-th leaky waveguide mode and αi,jW(λ) and αi,jLW(λ) are loss coefficients of i,j-th waveguide and leaky waveguide modes respectively. The summation should be done over all the modes supported by the given pore structure.
The leaky waveguide mode losses increase very quickly with increase of mode order, while waveguide mode losses do not change much. For both leaky waveguide and waveguide modes, the coupling coefficient Pi,j(λ) is the highest for the fundamental mode and quickly decreases with increasing mode order.
There are other parameters affecting prior art MPSi filter performance. These include the coupling efficiency of incident light into waveguide or leaky waveguide modes at the first MPSi wafer interface and the out coupling from the waveguide or leaky waveguide modes to transmitted light at the second MPSi wafer interface. If a plane-parallel beam of light is incident on the MPSi interface, the coupling efficiency to the leaky waveguide fundamental mode can be roughly estimated as:
            P                    (        λ        )              ≈          S              S        uc              ,where S is the area of pores 1.2 in FIG. 1, while Suc is the area of a MPSi array unit cell (which can be introduced for ordered MPSi arrays only). In other words, to a good approximation, P00LW(λ)˜p in the UV spectral range, where p is the porosity of an MPSi filter near the first MPSi wafer interface. For the waveguide transmission (i.e. for Near IR or IR wavelength ranges), the formula for the coupling efficiency, P00W(λ), can also be simplified to:
                    P        00        W                          (        λ        )              ≈                            4                                                              n                Si                                                          (                λ                )                                      ·                          n              I                                                            (                                                            n                  Si                                                                  (                  λ                  )                                            +                              n                I                                      )                    2                    ·                                    S            uc                    -                      S            p                                    S          uc                      ,where nSi(λ) is the refractive index of silicon at the wavelength λ and n1 is the refractive index of the medium from where light is incident on MPSi layer. For the most common case of the latter being air or vacuum, this formula can be rewritten as
            P      00      W                  (      λ      )        ≈                    4                                      n            Si                                          (            λ            )                                                (                                                    n                Si                                                          (                λ                )                                      +            1                    )                2              ·                                        S            uc                    -                      S            p                                    S          uc                    .      In other words, to some approximation, P0LW(λ)≈p and P0W(λ)≈0.69(1−p), where p is porosity of MPSi layer. It should be noted that for the exemplary filter of FIG. 1, the approximation given above for the waveguide case (i.e., for near IR and IR wavelength ranges) is not as good as for the leaky waveguide case (deep UV, UV and VIS spectral ranges) due to strong cross-coupling between neighboring waveguides and due to the presence of the previously described non-waveguiding channel of transmission. This cross-coupling is not taken into account by the approximation set forth above.
At the second interface of the MPSi filter, the light from waveguide ends (leaky or not, as applicable) is emitted with a divergence governed by the numerical aperture, NA, and wavelength. In the far field, the destructive and constructive interference of all light sources in the form of leaky waveguide or waveguide ends takes place. In the case of an ordered MPSi array, this leads to a number of diffraction orders that are defined by the pore array geometry (i.e. by the relationship between pore size, pore-to-pore distance) and the wavelength of the light. For most applications of optical filters, only light outcoupled into the 0th-diffraction order is of interest. However, some applications are not sensitive to the outcoupling of light to higher diffraction orders. For instance, when the filter is directly mounted on the top of a photodetector or a detector array, only the near field behavior is important. In other cases, the main source of outcoupling losses is the redistribution of light into higher diffraction orders. Such losses are sensitive to both wavelength and pore array geometry. They are more pronounced at short wavelengths due to the higher number of diffraction orders.
It should be noted that outcoupling losses can be completely suppressed for any given wavelength if the MPSi array period is less than or equal to that wavelength. For instance, for a 1550 nm wavelength that is important for optical communications, this will require a pore array period on the order of 1550 nm or less and pore diameters of about 300–1000 nm.
The exemplary prior art spectral filter structure of FIG. 1 cannot be used as a band-pass or narrow band-pass filter in the near IR or IR since the structure of FIG. 1 passes the light above the absorption band of silicon uniformly and does not offer any means to select a band for passing or blocking. In order for it to serve as a band-pass or narrow bandpass filter, some improvements in its design must be made.
Exemplary New Spectral Filter Designs
We provide in one non-limiting illustrative exemplary arrangement, an improved IR filter configuration based on a substantially uniform array of waveguides made of porous semiconductor (where the pores are straight and non-branching). Pore cross sections are either modulated at least along part of their depth while other parts are left unmodulated, or the entire pore depth can be modulated. The pore walls may be covered by at least one layer of transparent material. The pores may be filled or partially coated by a layer of absorptive or reflective material.
Such spectral filters can be used for band-pass, narrow-band pass or band blocking spectral filtering, and provide significant advantages. Exemplary advantages of particular implementations include, but are not limited to: p1 Omnidirectionality, i.e., absence of the spectral shape dependence of transmission (for transmission type optical filters) or reflection (for reflection type optical filters) on the angle of light incidence within the acceptance angles of the filter.                Manufacturability (i.e. ability to fabricate such filters relatively simply and inexpensively compared to the other filter configurations known by those skilled in the art).        The absence of delamination and other structural deficiencies.
Exemplary non-limiting configurations are based on the formation of a large number of identical, mutually de-coupled (at least having cross-coupling coefficients small enough that cross-coupling between neighbor waveguides can be neglected) waveguides arranged with respect to each other such that the transmission through the array at the operational wavelengths of such a filter is possible mostly or only through at least one of the waveguide modes of the assembly of waveguides. The transmission and reflection spectra of each of said waveguides is wavelength dependent due to well known Bragg phenomena occurring in the parts of the waveguides that are made to have modulated pore diameters (and through this, modulated waveguide cross-sections). Coherent modulation, meaning periodical modulation with a single period of the waveguide cross sections along the depths of the waveguides can be used. The far-field transmission spectrum of such a spectral filter (and, in the case of pores coated or filled by said reflective or absorptive material, the near field spectrum also) is determined by the transmission spectrum of each leaky waveguide and by the coupling/outcoupling efficiencies at the first and second surfaces of such a spectral filter. In addition, one or both broad faces of the filter made up of waveguide ends separated by pores can be covered by antireflective structure such as, for example, an antireflection layer or an antireflective dielectric multilayer coating. These coatings, covering the broad faces of the non-pore material between the pores that comprises waveguide ends, provide higher coupling and outcoupling efficiencies within the desired spectral band of the filter.
In one exemplary illustrative non-limiting implementation, said waveguide array is formed in a semiconductor wafer in the form of wafer host separated by the channels going through the wafer (pores). Such a structure can be fabricated, for example, by forming a layer of porous semiconductor by means of electrochemical etching of a single crystal semiconductor wafer as deeply as necessary. The un-etched remainder of the wafer may be either subsequently removed or left as a supportive base for the waveguide array. The semiconductor host (in the form of "islands" between the pores), which is transmissive at wavelengths above the band edge of the particular semiconductor material, will serve as waveguides, while the pores formed by such a process will insure a low level of coupling between the waveguides.
The previously mentioned modulation of the cross sections of the waveguides can be achieved through modulating the pore diameters along their depths by modulating the electrochemical etching parameters during electrochemical etching process. For example, the parameters available for modulation include the current density, illumination intensity or others known to those skilled in the art. Said semiconductor material can be silicon (p-type doped or n-type doped), gallium arsenide, indium phosphide, or any other material transparent over some wavelength band, which can be shown to form non-branching pores during electrochemical etching in a suitable electrolyte and under suitable conditions. The covering of the walls of the waveguides can be achieved by partial thermal oxidation of a semiconductor (principally silicon), or by depositing a dielectric single layer or multilayer onto the pore walls by Chemical Vapor Deposition or by any other deposition, sputtering, evaporation or growth process known to those skilled in the art. Covering the substrate or wafer surface (or surfaces) between the pores by an antireflective structure can be accomplished by directional deposition techniques, such as physical vapor deposition, magnetron sputtering, thermal or electron beam evaporation, ion assisted ion plating or any other technique known to those skilled in the art. If the filter structure is too fragile for its intended use (which can be the case if the unetched part of the wafer is removed after the electrochemical process), the porous layer can be reinforced by sealing between two plates of a material that is transparent over the transparency wavelength range of the porous filter. Such plates can be, for instance, of glass, silica, CaF2 or any other transparent dielectric known to those skilled in the art.
In one exemplary non-limiting illustrative implementation, at least one optically transparent layer covering the pore (channel) walls may have a refractive index lower than that of the silicon and may serve as a cladding of said waveguides, designed to substantially minimize cross-coupling between neighboring waveguides and to mechanically reinforce said spectral filter.
In another exemplary non-limiting illustrative implementation, the pores can be disposed across the broad surfaces of the wafer or substrate with a predetermined pattern having predetermined symmetry (for example, cubic or hexagonal). Alternatively, said pores can be disposed at a predetermined pattern that exhibits more complex (advanced) symmetry. The pores may have circular or near-square cross-sections. The pores (and through that the silicon island waveguides) can be made to have tapered ends at the at least one first or second surface of said filter, or to taper uniformly or non-uniformly along their entire lengths. At the narrow end of the taper, the pore lateral cross-section can be gradually decreased when approaching the near surface of the filter substrate in order to increase the coupling and/or outcoupling efficiency to improve the transmittance through the filter. The tapering of the pores in this manner effectively gradually increases the waveguide lateral cross-section
In a further exemplary non-limiting illustrative implementation, pores can be filled (partially or completely) by a material that is absorptive or reflective in the transparency wavelength range of the filter configuration to suppress cross-coupling between neighboring waveguides even further. This may increase the propagation losses of the waveguide modes. The use of a transparent pore wall coating as a cladding of silicon waveguides may be used to reduce said propagation losses while keeping cross-coupling suppressed. Said absorptive or reflective material can be metal, metal alloy or any other absorptive or reflective material in the operational spectral band of the filter known to those skilled in the art. Pore filling can be accomplished by electro-plating, electroless plating, chemical vapor deposition, injection molding, dye casting, capillary absorption of a liquid (melted metal) into the pores or by any other method known to those skilled in the art. Removal of excessive material from either one or both surfaces of the spectral filter after the pore filling may be required and can be accomplished through chemical etching, reactive ion etching, chemical-mechanical polishing, mechanical polishing or by any other method known to those skilled in art.
The far IR spectral range can be quite important for many applications such as the nonlimiting examples of astronomy and chemical analyses. Silicon, Ge, III-V compound semiconductors or other materials known to permit ordered pore array formation through electro-chemical etching, however, are not transparent over the whole spectral range of interest. Hence, some modifications of the spectral filter design may be made to serve these applications. According to another exemplary illustrative non-limiting implementation, an improved IR filter configuration based on a substantially uniform array of waveguides is made of free-standing porous semiconductor with straight and non-branching pores. The pore cross sections are either modulated at least along part of the depths while other parts are left unmodulated, or the entire depths can be modulated. The pores are filled with a material that is transparent within the spectral range of interest (nonlimiting examples of such materials include ZnSe, CdTe and thallium iodide). The pore walls may be covered by at least one layer of transparent material that is different from the material filling the pores completely (having a smaller refractive index) prior to said filling of the pores. In this exemplary illustrative implementation, the filled pores will act as waveguides. The material completely filling the pores acts as a waveguide core, while the material covering pore walls (if any) serves as a waveguide cladding. The porous semiconductor matrix can be oxidized before filling the pores to reduce its refractive index and, through that, reduce the cross coupling between neighboring waveguides. Unlike the previously described exemplary illustrative implementation, the ordering of the pore array (and through that of the waveguide array) is not strictly required—only the uniformity of the pore size is needed. However, ordering still can be an advantageous feature.
According to a further exemplary illustrative non-limiting implementation, the first, the second or both surfaces of said filter wafer may be coated with an antireflective structure after said pore filling to suppress coupling and outcoupling losses. Said antireflective coating can be a single layer antireflective coating, or, alternatively, can be made in the form of a multilayer antireflective coating and can be deposited through chemical or physical vapor deposition or by any other technique known to those skilled in the art. Said pore filling can be accomplished by chemical vapor deposition, injection molding, dye casting, capillary absorption of a liquid into the pores or by any other method known to those skilled in the art.
The resulting exemplary non-limiting illustrative filters can have the advantages of stability. They do not exhibit delamination problems and offer remarkable stability over wide range of temperatures and large temperature gradients. They also offer transmittance comparable to that of prior art narrow bandpass, bandpass and band blocking filters combined with the new advantage of omnidirectionality. Omnidirectionality is meant here to be the independence of the spectral position of the reflection band, transmission valley or transmission edge on the angle of light incidence. Such filters are useful for a wide variety of applications, including applications where currently available filter systems cannot provide acceptable performance (e.g., a variety of analytical devices, wavelength division multiplexing, astronomical instrumentation, spectroscopy, and others uses).

Spectral filter for green and longer wavelengths


The present invention addresses the problem of providing a super-liquid-repellent coating film that has improved abrasion resistance. Provided as a means for solving the problem is a coating film that has a fluorine atom content of 1 to 60 wt %, the coating film having a surface that has an average surface roughness Ra of 0.5 to 20, a surface area ratio of 1.7 to 5, a contact angle with water of 150° or more, a contact angle with n-hexadecane of 80° or more, and a surface that has a contact angle with water of 150° or more after being rubbed 100 times with a PET film under a load of 100 g.

Various super-liquid-repellent coating films have been proposed.
For example, there has been proposed a method for imparting super liquid repellency by chemically bonding a fluoroalkylsilane compound to a surface to be treated, the surface having formed thereon a cured coating film comprising silica particles etc. (Patent Literature (PTL) 1).
A method for forming a coating film by hydrolyzing alkoxysilane while the alkoxysilane, perfluoroalkylsilane, and silica particles are in a mixed state has also been proposed (PTL 2).
Further, a coating film obtained by copolymerizing particles having a radically polymerizable group on the surface and a fluorine-containing monomer has also been proposed (Non-patent Literature (NPL) 1).

Super-liquid-repellent coating film and curable composition for forming super-liquid-repellent coating film


A high pressure apparatus and related methods for processing supercritical fluids are disclosed. In certain embodiments, the present apparatus includes a capsule, a heater, at least one ceramic ring or multiple rings, optionally, with one or more scribe marks and/or cracks present. In certain embodiments, the apparatus has a metal sleeve containing each ceramic ring. The apparatus also has a high strength enclosure, end flanges with associated insulation, and a power control system. In certain embodiments, a high pressure apparatus is constructed such that the diametric annular gap between the outer diameter of the heater and the ceramic ring is selected to provide radial load-bearing contact above a particular temperature and pressure. In certain embodiments, the apparatus is capable of accessing pressures of 0.2 GPa to 2 GPa and temperatures of 400° C. to 1200° C.

The present disclosure relates generally to techniques for processing materials in supercritical fluids. More specifically, embodiments of the disclosure include techniques for controlling parameters associated with a material processing a capsule disposed within a high pressure apparatus enclosure. Merely by way of example, the disclosure can be applied to growing crystals of GaN, AlN, InN, InGaN, AlGaN, and AlInGaN, and others for manufacture of bulk or patterned substrates. Such bulk or patterned substrates can be used for a variety of applications including optoelectronic devices, lasers, light emitting diodes, solar cells, photoelectrochemical water splitting and hydrogen generation devices, photodetectors, integrated circuits, and transistors, among other devices.
Supercritical fluids are used to process a wide variety of materials. A supercritical fluid is often defined as a substance beyond its critical point, i.e., critical temperature and critical pressure. A critical point represents the highest temperature and pressure at which the substance can exist as a vapor and liquid in equilibrium. In certain supercritical fluid applications, the materials being processed are placed inside a pressure vessel or other high pressure apparatus. In some cases it is desirable to first place the materials inside a container, liner, or capsule, which in turn is placed inside the high pressure apparatus. In operation, the high pressure apparatus provides structural support for the high pressures generated within the container or capsule holding the materials. The container, liner, or capsule provides a closed/sealed environment that is chemically inert and impermeable to solvents, solutes, and gases that may be involved in or generated by the process.
Scientists and engineers have been synthesizing crystalline materials using high pressure techniques. As an example, synthetic diamonds are often made using high pressure and temperature conditions. Synthetic diamonds are often used for industrial purposes but can also be grown large enough for jewelry and other applications. Scientists and engineers also use high pressure to synthesize complex materials such as zeolites, which can be used to filter toxins and the like. Moreover, geologists have also used high pressure techniques to simulate conditions and/or processes occurring deep within the earth's crust. High pressure techniques often rely upon supercritical fluids, herein referred to as SCFs.
Supercritical fluids provide an especially ideal environment for growth of high quality crystals in large volumes and low costs. In many cases, supercritical fluids possess the solvating capabilities of a liquid with the transport characteristics of a gas. Thus, on the one hand, supercritical fluids can dissolve significant quantities of a solute for recrystallization. On the other hand, the favorable transport characteristics include a high diffusion coefficient, so that solutes may be transported rapidly through the boundary layer between the bulk of the supercritical fluid and a growing crystal, and also a low viscosity, so that the boundary layer is very thin and small temperature gradients can cause facile self-convection and self-stirring of the reactor. This combination of characteristics enables, for example, the growth of hundreds or thousands of large α-quartz crystals in a single growth run in supercritical water.
Supercritical fluids also provide an attractive medium for synthesis of exotic materials, such as zeolites, for solvent extractions, as of caffeine from coffee, and for decomposition and/or dissolution of materials that are relatively inert under more typical conditions, such as biofuels and toxic waste materials.
In some applications, such as crystal growth, the pressure vessel or capsule also includes a baffle plate that separates the interior into different chambers, e.g., a top half and a bottom half. The baffle plate typically has a plurality of random or regularly spaced holes to enable fluid flow and heat and mass transfer between these different chambers, which hold the different materials being processed along with a supercritical fluid. For example, in typical crystal growth applications, one portion of the capsule contains seed crystals and the other half contains nutrient material. In addition to the materials being processed, the capsule contains a solid or liquid that forms the supercritical fluid at elevated temperatures and pressures and, typically, also a mineralizer to increase the solubility of the materials being processed in the supercritical fluid. In other applications, for example, synthesis of zeolites or of nanoparticles or processing of ceramics, no baffle plate may be used for operation. In operation, the capsule is heated and pressurized toward or beyond the critical point, thereby causing the solid and/or liquid to transform into the supercritical fluid. In some applications the fluid may remain subcritical, that is, the pressure or temperature may be less than the critical point. However, in all cases of interest here, the fluid is superheated, that is, the temperature is higher than the boiling point of the fluid at atmospheric pressure. The term "supercritical" will be used throughout to mean "superheated", regardless of whether the pressure and temperature are greater than the critical point, which may not be known for a particular fluid composition with dissolved solutes.
Although somewhat effective for conventional crystal growth, drawbacks exist with conventional processing vessels. As an example, processing capabilities for conventional steel hot-wall pressure vessels (e.g., autoclaves) are typically limited to a maximum temperature of about 400 Degrees Celsius and a maximum pressure of 0.2 GigaPascals (GPa). Fabrication of conventional pressure vessels from nickel-based superalloys allows for operation at a maximum temperature of about 550 degrees Celsius and a maximum pressure of about 0.5 GPa. Therefore, these conventional hot-wall pressure vessels are often inadequate for some processes, such as the growth of gallium nitride crystals in supercritical ammonia that often require pressures and temperatures that extend significantly above this range in order to achieve growth rates above about 2-4 microns per hour. In addition, nickel-based superalloys are very expensive and are difficult to machine, limiting the maximum practical size and greatly increasing the cost compared to traditional steel pressure vessels.
Attempts have been made to overcome the drawbacks of conventional pressure vessels. D'Evelyn et al., U.S. Publication No. 2003/0140845A1, indicates a so-called zero-stroke high pressure apparatus adapted from the type of belt apparatus used for synthesis of diamond using high pressure and high temperature. Cemented tungsten carbide, however, is used as the die material, which is relatively expensive and is difficult to manufacture in large dimensions. In addition, the use of a hydraulic press to contain the apparatus increases the cost and further limits the maximum volume. Finally, the use of a pressure transmission medium surrounding the capsule used to contain the supercritical fluid reduces the volume available within the hot zone for processing material.
D'Evelyn et al., U.S. Publication No. 2006/0177362A1, indicates several types of apparatus with capability for pressures and temperatures well in excess of that of conventional autoclaves and with improved scalability relative to the zero-stroke press apparatus described above. A series of wedge-shaped radial ceramic segments are placed between a heater which surrounds a capsule and a high strength enclosure, in order to reduce both the pressure and temperature to which the inner diameter of the high strength enclosure is exposed compared to the corresponding values for the capsule. Fabrication and use of these ceramic wedge-shaped radial segments, however, can be difficult and expensive. These and other limitations of conventional apparatus may be described throughout the present specification.
From the above, it is seen that techniques for improving a high pressure apparatus for crystal growth is highly desirable.

This patent is subject to a terminal disclaimer.

High pressure apparatus and method for nitride crystal growth


A valve having secondary spring force containment includes a valve body featuring an opening with a valve closure member sized to close the opening. A stem has a first end connected to the valve closure member. A proximal spring seat is positioned adjacent to the valve body, A distal spring seat is mounted on the second end of the stem. A spring is positioned between the proximal and distal spring seats so that the valve closure member is urged into a position where the valve body opening is closed. A hollow rod is attached between the proximal and distal spring seats to provide secondary spring force containment with the stem passing through the hollow rod.

As is known in the art, tanks containing fluids under pressure are often provided with pressure relief valves. Such tanks may exist, for example, on railroad tank cars. Pressure relief valves typically use a compression spring to maintain the valve in a closed condition. When the pressure in the tank exceeds a pre-determined level, the spring force holding the valve in the closed condition is overcome and the valve opens so that fluid from the tank is vented to ambient so as to lower the pressure within the tank. An example of such a pressure relief valve is presented in U.S. Pat. No. 3,131,718 to Mingrone.
Pressure relief valves that are spring powered typically have a stem axially aligned with the motion of the valve closure member. This stem typically passes through the center of a coil spring and is typically used to set the compression spring at a correct height (or extension if a tension spring is used to achieve the force for the desired pre-determined venting pressure. In the event that the spring is not compressed before removing the component used to set the spring, such as during disassembly of the valve for service, repair or replacement, the spring can release stored energy in unpredictable directions. In the theoretical scenario of stein breakage or other component failure, the spring force can also be released in the unpredictable directions.
A need therefore exists for a device that reduces or eliminates the risk of accidental release of spring forces in a pressure relief valve assembly.

Pressure relief valve and secondary spring force containment assembly


The present invention relates to recombinant N-glycosylated proteins, comprising one or more introduced N-glycosylated optimized amino acid sequence(s), nucleic acids encoding these proteins as well as corresponding vectors and host cells. In addition, the present invention is directed to the use of said proteins, nucleic acids, vectors and host cells for preparing medicaments. Furthermore, the present invention provides methods for producing said proteins.

N-linked protein glycosylation is an essential and conserved process occurring in the endoplasmic reticulum of eukarotic organisms. It is important for protein folding, oligomerization, stability, quality control, sorting and transport of secretory and membrane proteins (Helenius, A., and Aebi, M. (2004). Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019-1049).
Protein glycosylation has a profound influence on the antigenicity, the stability and the half-life of a protein. In addition, glycosylation can assist the purification of proteins by chromatography, e.g. affinity chromatography with lectin ligands bound to a solid phase interacting with glycosylated moieties of the protein. It is therefore established practice to produce many glycosylated proteins recombinantly in eukaryotic cells to provide biologically and pharmaceutically useful glycosylation patterns.
Only within recent years it was demonstrated that a bacterium, the food-borne pathogen Campylobacter jejuni, can also N-glycosylate its proteins (Szymanski, et al. (1999). Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol. Microbiol. 32, 1022-1030). The machinery required for glycosylation is encoded by 12 genes that are clustered in the so-called pgl locus. Disruption of N-glycosylation affects invasion and pathogenesis of C. jejuni but is not lethal as in most eukaryotic organisms (Burda P. and M. Aebi, (1999). The dolichol pathway of N-linked glycosylation. Biochim Biophys Acta 1426(2):239-57). It is possible to reconstitute the N-glycosylation of C. jejuni proteins by recombinantly expressing the pgl locus and acceptor glycoprotein in E. coli at the same time (Wacker et al. (2002). N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298, 1790-1793).
European Patent Application No. 03 702 276.1 (European Patent 1 481 057), an earlier invention of the present inventors, teaches a procaryotic organism into which is introduced a nucleic acid encoding for (i) specific glycosyltransferases for the assembly of an oligosaccharide on a lipid carrier, (ii) a recombinant target protein comprising a consensus sequence "N-X-S/T", wherein X can be any amino acid except proline, and (iii) an oligosaccharyl transferase of C. jejuni (OTase) that covalently links said oligosaccharide to the consensus sequence of the target protein. Said procaryotic organism produces N-glycans with a specific structure which is defined by the type of the specific glycosyltransferases.
Even though the presence of the known N-glycosylation consensus sequence in a protein does allow for the N-glycosylation of recombinant target proteins in procaryotic organisms comprising the oligosaccharyl transferase (OTase) of C. jejuni, the N-glycosylation of some target proteins is often inefficient.
The object of the present invention is to provide proteins as well as means and methods for producing such proteins having an optimized efficiency for N-glycosylation that can be produced in procaryotic organisms in vivo. Another object of the present invention aims at the more efficient introduction of N-glycans into recombinant proteins for modifying antigenicity, stability, biological, prophylactic and/or therapeutic activity of said proteins. A further object is the provision of a host cell that efficiently displays recombinant N-glycosylated proteins of the present invention on its surface.
In a first aspect the present invention provides a recombinant N-glycosylated protein, comprising one or more of the following N-glycosylated optimized amino acid sequence(s):                D/E-X-N-Z-S/T (SEQ ID NO:7), (optimized consensus sequence)wherein X and Z may be any natural amino acid except Pro, and wherein at least one of said N-glycosylated partial amino acid sequence(s) is introduced.
It was surprisingly found that the introduction of specific partial amino acid sequence(s) (optimized consensus sequence(s)) into proteins leads to proteins that are efficiently N-glycosylated by the oligosaccharyl transferase (OST, OTase) from Campylobacter spp., preferably C. jejuni, in these introduced positions.
The term "partial amino acid sequence(s)" as it is used in the context of the present invention will also be referred to as "optimized consensus sequence(s)". The optimized consensus sequence is N-glycosylated by the oligosaccharyl transferase (OST, OTase) from Campylobacter spp., preferably C. jejuni, much more efficiently than the regular consensus sequence "N-X-S/T" known in the prior art.
In general, the term "recombinant N-glycosylated protein" refers to any heterologous poly- or oligopeptide produced in a host cell that does not naturally comprise the nucleic acid encoding said protein. In the context of the present invention this term refers to a protein produced recombinantly in any host cell, e.g. an eukaryotic or prokaryotic host cell, preferably a procaryotic host cell, e.g. Escherichia ssp., Campylobacter ssp., Salmonella ssp., Shigella ssp., Helicobacter ssp., Pseudomonas ssp., Bacillus ssp., more preferably Escherichia coli, Campylobacter jejuni, Salmonella typhimurium etc., wherein the nucleic acid encoding said protein has been introduced into said host cell and wherein the encoded protein is N-glycosylated by the OTase from Campylobacter spp., preferably C. jejuni, said transferase enzyme naturally occurring in or being introduced recombinantly into said host cell.
In accordance with the internationally accepted one letter code for amino acids the abbreviations D, E; N, S and T denote aspartic acid, glutamic acid, asparagine, serine, and threonine, respectively. Proteins according to the invention differ from natural or prior art proteins in that one or more of the optimized consensus sequence(s) D/E-X-N-Z-S/T (SEQ ID NO:7) is/are introduced and N-glycosylated. Hence, the proteins of the present invention differ from the naturally occurring C. jejuni proteins which also contain the optimized consensus sequence but do not comprise any additional (introduced) optimized consensus sequences.
The introduction of the optimized consensus sequence can be accomplished by the addition, deletion and/or substitution of one or more amino acids. The addition, deletion and/or substitution of one or more amino acids for the purpose of introducing the optimized consensus sequence can be accomplished by chemical synthetic strategies well known to those skilled in the art such as solid phase-assisted chemical peptide synthesis. Alternatively, and preferred for larger polypeptides, the proteins of the present invention can be prepared by standard recombinant techniques.
The proteins of the present invention have the advantage that they may be produced with high efficiency and in any procaryotic host comprising a functional pgl operon from Campylobacter spp., preferably C. jejuni. Preferred alternative OTases from Campylobacter spp. for practicing the aspects and embodiments of the present invention are Campylobacter coli and Campylobacter lari (see Szymanski, C. M. and Wren, B. W. (2005). Protein glycosylation in bacterial mucosal pathogens. Nat. Rev. Microbiol. 3: 225-237). The functional pgl operon may be present naturally when said procaryotic host is Campylobacter spp., preferably C. jejuni. However, as demonstrated before in the art and mentioned above, the pgl operon can be transferred into cells and remain functional in said new cellular environment.
The term "functional pgl operon from Campylobacter spp., preferably C. jejuni" is meant to refer to the cluster of nucleic acids encoding the functional oligosaccharyl transferase (OTase) of Campylobacter spp., preferably C. jejuni, and one or more specific glycosyltransferases capable of assembling an oligosaccharide on a lipid carrier, and wherein said oligosaccharide can be transferred from the lipid carrier to the target protein having one or more optimized amino acid sequence(s): D/E-X N-Z-S/T (SEQ ID NO:7) by the OTase. It to be understood that the term "functional pgl operon from Campylobacter spp., preferably C. jejuni" in the context of this invention does not necessarily refer to an operon as a singular transcriptional unit. The term merely requires the presence of the functional components for N-glycosylation of the recombinant protein in one host cell. These components may be transcribed as one or more separate mRNAs and may be regulated together or separately. For example, the term also encompasses functional components positioned in genomic DNA and plasmid(s) in one host cell. For the purpose of efficiency, it is preferred that all components of the functional pgl operon are regulated and expressed simultaneously.
It is important to realize that only the functional oligosaccharyl transferase (OTase) should originate from Campylobacter spp., preferably C. jejuni, and that the one or more specific glycosyltransferases capable of assembling an oligosaccharide on a lipid carrier may originate from the host cell or be introduced recombinantly into said host cell, the only functional limitation being that the oligosaccharide assembled by said glycosyltransferases can be transferred from the lipid carrier to the target protein having one or more optimized consensus sequences by the OTase. Hence, the selection of the host cell comprising specific glycosyltransferases naturally and/or incapacitating specific glycosyltransferases naturally present in said host as well as the introduction of heterologous specific glycosyltransferases will enable those skilled in the art to vary the N-glycans bound to the optimized N-glycosylation consensus site in the proteins of the present invention.
As a result of the above, the present invention provides for the individual design of N-glycan-patterns on the proteins of the present invention. The proteins can therefore be individualized in their N-glycan pattern to suit biological, pharmaceutical and purification needs.
In a preferred embodiment, the proteins of the present invention may comprise one but also more than one, preferably at least two, preferably at least 3, more preferably at least 5 of said N-glycosylated optimized amino acid sequences.
The presence of one or more N-glycosylated optimized amino acid sequence(s) in the proteins of the present invention can be of advantage for increasing their antigenicity, increasing their stability, affecting their biological activity, prolonging their biological half-life and/or simplifying their purification.
The optimized consensus sequence may include any amino acid except proline in position(s) X and Z. The term "any amino acids" is meant to encompass common and rare natural amino acids as well as synthetic amino acid derivatives and analogs that will still allow the optimized consensus sequence to be N-glycosylated by the OTase. Naturally occurring common and rare amino acids are preferred for X and Z, X and Z may be the same or different.
It is noted that X and Z may differ for each optimized consensus sequence in a protein according to the present invention.
The N-glycan bound to the optimized consensus sequence will be determined by the specific glycosyltransferases and their interaction when assembling the oligosaccharide on a lipid carrier for transfer by the OTase. Those skilled in the art can design the N-glycan by varying the type(s) and amount of the specific glycosyltransferases present in the desired host cell.
N-glycans are defined herein as mono-, oligo- or polysaccharides of variable compositions that are linked to an ε-amide nitrogen of an asparagine residue in a protein via an N-glycosidic linkage. Preferably, the N-glycans transferred by the OTase are assembled on an undecaprenol-pyrophosphate lipid-anchor that is present in the cytoplasmic membrane of gram-negative or positive bacteria. They are involved in the synthesis of O antigen, O polysaccharide and peptidoglycan (Bugg, T. D., and Brandish, P. E. (1994). From peptidoglycan to glycoproteins: common features of lipid-linked oligosaccharide biosynthesis. FEMS Microbiol Lett 119, 255-262; Valvano, M. A. (2003). Export of O-specific lipopolysaccharide. Front Biosci 8, s452-471).
In a preferred embodiment, the recombinant protein of the present invention comprises one or more N-glycans selected from the group of N-glycans from Campylobacter spp., preferably C. jejuni, the N-glycans derived from oligo- and polysaccharides transferred to O antigen forming O polysaccharide in Gram-negative bacteria or capsular polysaccharides from Gram-positive bacteria, preferably: P. aeruginosa O9, O11; E. coli O7, O9, O16, O157 and Shigella dysenteriae O1 and engineered variants thereof obtained by inserting or deleting glycosyltransferases and epimerases affecting the polysaccharide structure.
In a further preferred embodiment the recombinant protein of the present invention comprises two or more different N-glycans.
For example, different N-glycans on the same protein can prepared by controlling the timing of the expression of specific glycosyltransferases using early or late promoters or introducing factors for starting, silencing, enhancing and/or reducing the promoter activity of individual specific glycosyltransferases. Suitable promoters and factors governing their activity are available to those in the art routinely and will not be discussed further.
There is no limitation on the origin of the recombinant protein of the invention. Preferably said protein is derived from mammalian, bacterial, viral, fungal or plant proteins. More preferably, the protein is derived from mammalian, most preferably human proteins. For preparing antigenic recombinant proteins according to the invention, preferably for use as active components in vaccines, it is preferred that the recombinant protein is derived from a bacterial, viral or fungal protein.
In a further preferred embodiment the present invention provides for recombinant proteins wherein either the protein and/or the N-glycan(s) is (are) therapeutically and/or prophylactically active. The introduction of at least one optimized and N-glycosylated consensus sequence can modify or even introduce therapeutic and/or prophylactic activity in a protein. In a more preferred embodiment it is the protein and/or the N-glycan(s) that is (are) immunogenically active. In this case the introduced N-glycosylation(s) may have a modifying effect on the proteins biological activity and/or introduce new antigenic sites and/or may mask the protein to evade degrading steps and/or increase the half-life.
The recombinant proteins of the present invention can be efficiently targeted to the outer membrane and/or surface of host cells, preferably bacteria, more preferably gram-negative bacteria. For assisting the surface display and/or outer membrane localisation it is, preferred that the recombinant protein of the invention further comprises at least one polypeptide sequence capable of targeting said recombinant protein to the outer membrane and/or cell surface of a bacterium, preferably a gram-negative bacterium.
In a preferred embodiment the recombinant protein of the invention is one, wherein said targeting polypeptide sequence is selected from the group consisting of type II signal peptides (Paetzel, M., Karla, A., Strynadka, N. C., and Dalbey, R. E. 2002. Signal peptidases. Chem Rev 102: 4549-4580.) or outer membrane proteins (reviewed in Wernerus, H., and. Stahl, S. 2004. Biotechnological applications for surface-engineered bacteria. Biotechnol Appl Biochem 40: 209-228.), preferably selected from the group consisting of the full length protein or the signal peptides of OmpH1 from C. jejuni, JIpA from C. jejuni, outer membrane proteins from E. coli, preferably OmpS, OmpC, OmpA, OprF, PhoE, LamB, Lpp'OmpA (a fusion protein for surface display technology, see Francisco, J. A., Earhart, C. F., and Georgiou, G. 1992. Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci USA 89: 2713-2717.), and the Inp protein from Pseudomonas aeruginosa.
In a different aspect, the present invention relates to a nucleic acid encoding a recombinant protein, according to the invention. Preferably, said nucleic acid is a mRNA, a DNA or a PNA, more preferably a mRNA or a DNA, most preferably a DNA. The nucleic acid may comprise the sequence coding for said protein and, in addition, other sequences such as regulatory sequences, e.g. promoters, enhancers, stop codons, start codons and genes required to regulate the expression of the recombinant protein via the mentioned regulatory sequences, etc. The term "nucleic acid encoding a recombinant protein according to the invention" is directed to a nucleic acid comprising said coding sequence and optionally any further nucleic acid sequences regardless of the sequence information as long as the nucleic acid is capable of producing the recombinant protein of the invention in a host cell containing a functional pgl operon from Campylobacter spp., preferably C. jejuni. More preferably, the present invention provides isolated and purified nucleic acids operably linked to a promoter, preferably linked to a promoter selected from the group consisting of known inducible and constitutive prokaryotic promoters, more preferably the tetracycline promoter, the arabinose promoter, the salicylate promoter, lac-, trc-, and tac promotors (Baneyx, F. (1999). Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10, 411-421; Billman-Jacobe, H. (1996). Expression in bacteria other than Escherichia coli. Curr Opin Biotechnol 7, 500-504.). Said operably linked nucleic acids can be used for, e.g. vaccination.
Furthermore, another aspect of the present invention relates to a host cell comprising a nucleic acid and/or a vector according to the present invention. The type of host cell is not limiting as long as it accommodates a functional pgl operon from C. jejuni and one or more nucleic acids coding for recombinant target protein(s) of the present invention. Preferred host cells are prokaryotic host cells, more preferably bacteria, most preferably those selected from the group consisting of Escherichia ssp., Campylobacter ssp., Salmonella ssp., Shigella ssp., Helicobacter ssp., Pseudomonas ssp., Bacillus ssp., preferably Escherichia coli, more preferably E. coli strains Top10, W3110, CLM24, BL21, SCM6 and SCM7 (Feldman et al., (2005). Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. Proc. Natl. Acad. Sci. USA 102, 3016-3021; Alaimo, C., Catrein, I., Morf, L., Marolda, C. L., Callewaert, N., Valvano, M. A., Feldman, M. F., Aebi, M. (2006). Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides. EMBO Journal 25, 967-976) and S. enterica strains SL3261 (Salmonella enterica sv. Typhimurium LT2 (delta) aroA, see Hoiseth, S. K., and Stocker, B. A. 1981, Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291:238-239), SL3749 (Salmonella enterica sv. Typhimurium LT2 waaL, see Kaniuk et al., J. Biol. Chem. 279: 36470-36480) and SL3261ΔwaaL.
In a more preferred embodiment the host cell according to the invention is one that is useful for the targeting to the outer membrane and/or surface display of recombinant proteins according to the invention, preferably one, wherein said host cell is a recombinant gram-negative bacterium having:
i) a genotype comprising nucleotide sequences encoding for                a) at least one natural or recombinant specific glycosyltransferase for the assembly of an oligosaccharide on a lipid carrier,        b) at least one natural or recombinant prokaryotic oligosaccharyl transferase (OTase) from Campylobacter spp., preferably C. jejuni,         c) at least one recombinant protein according to the invention, preferably a protein further comprising a targeting polypeptide, and
ii) a phenotype comprising a recombinant N-glycosylated protein according to the invention that is located in and/or on the outer membrane of the gram-negative bacterium.
The host cell for the above embodiment is preferably selected from the group consisting of Escherichia ssp., Campylobacter ssp., Shigella ssp, Helicobacter ssp. and Pseudomonas ssp., Salmonella ssp., preferably E. coli, more preferably E. coli strains Top10, W3110, CLM24, BL21, SCM6 and SCM7, and S. enterica strains SL3261, SL3749 and SL3261ΔwaaL. (see Hoiseth, S. K., and Stocker, B. A. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291: 238-239), SL3749 (Kaniuk, N. A., Vinogradov, E., and. Whitfield, C. 2004. Investigation of the structural requirements in the lipopolysaccharide core acceptor for ligation of O antigens in the genus Salmonella: WaaL "ligase" is not the sole determinant of acceptor specificity. J Biol Chem 279: 36470-36480).
Because preferred proteins of the present invention may have a therapeutic or prophylactic activity by themselves and/or due to the introduced N-glycosylation sites, they can be used for the preparation of a medicament. The type of protein for practicing the invention is not limited and, therefore, proteins of the invention such as EPO, IFN-alpha, TNFalpha, IgG, IgM, IgA, interleukins, cytokines, viral and bacterial proteins for vaccination like C. jejuni proteins such as HisJ (Cj0734c), AcrA (Cj0367c), OmpH1 (Cj0982c), Diphteria toxin (CRM197), Cholera toxin, P. aeruginosa exoprotein, to name just a few, and having introduced therein the optimized N-glycosylated consensus sequence are useful for preparing a medicament (Wyszynska, A., Raczko, A., Lis, M., and Jagusztyn-Krynicka, E. K. (2004). Oral immunization of chickens with avirulent Salmonella vaccine strain carrying C. jejuni 72Dz/92 cjaA gene elicits specific humoral immune response associated with protection against challenge with wild-type Campylobacter. Vaccine 22, 1379-1389).
In addition, the nucleic acids and/or vectors according to the invention are also useful for the preparation of a medicament, preferably for use in gene therapy.
Moreover, a host cell according to the invention, preferably one that has a phenotype comprising an N-glycosylated recombinant protein of the invention that is located in and/or on the outer membrane of a bacterium, preferably a gram-negative bacterium, more preferably one of the above-listed gram-negative bacteria, is particularly useful for the preparation of a medicament.
More preferably, a protein of the invention is used for the preparation of a medicament for the therapeutic and/or prophylactic vaccination of a subject in need thereof.
In a more preferred embodiment the present invention relates to the use of a nucleic acid and/or a vector according to the invention for the preparation of a medicament for the therapeutic and/or prophylactic vaccination of a subject in need thereof, preferably by gene therapy.
The host cells of the invention displaying said N-glycosylated recombinant proteins are particularly useful for preparing vaccines, because the displayed N-glycosylated proteins are abundantly present on the host cell's surface and well accessible by immune cells, in particular their hydrophilic N-glycans, and because the host cells have the added effect of an adjuvant, that, if alive, may even replicate to some extent and amplify its vaccination effects.
Preferably, the host cell for practicing the medical aspects of this invention is an attenuated or killed host cell.
Another advantage of the use of the inventive host cells for preparing medicaments, preferably vaccines, is that they induce IgA antibodies due to the cellular component.
Preferably, said host cells are used according to the invention for inducing IgA antibodies in an animal, preferably a mammal, a rodent, ovine, equine, canine, bovine or a human.
It is preferred that said subject in need of vaccination is avian, mammalian or fish, preferably mammalian, more preferably a mammal selected from the group consisting of cattle, sheep, equines, dogs, cats, and humans, most preferably humans. Fowls are also preferred.
A further aspect of the present invention relates to a pharmaceutical composition, comprising at least one protein, at least one nucleic acid, a least one vector and/or at least one host cell according to the invention. The preparation of medicaments comprising proteins or host cells, preferably attenuated or killed host cells, and the preparation of medicaments comprising nucleic acids and/or vectors for gene therapy are well known in the art. The preparation scheme for the final pharmaceutical composition and the mode and details of its administration will depend on the protein, the host cell, the nucleic acid and/or the vector employed.
In a preferred embodiment, the pharmaceutical composition of the invention comprises a pharmaceutically acceptable excipient, diluent and/or adjuvant.
The present invention provides for a pharmaceutical composition comprising at least one of the following, (i) a recombinant protein, a host cell, a nucleic acid and/or a recombinant vector being/encoding/expressing a recombinant protein according to the present invention, and (ii) a pharmaceutically acceptable excipient, diluent and/or adjuvant.
Suitable excipients, diluents and/or adjuvants are well-known in the art. An excipient or diluent may be a solid, semi-solid or liquid material which may serve as a vehicle or medium for the active ingredient. One of ordinary skill in the art in the field of preparing compositions can readily select the proper form and mode of administration depending upon the particular characteristics of the product selected, the disease or condition to be treated, the stage of the disease or condition, and other relevant circumstances (Remington's Pharmaceutical Sciences, Mack Publishing Co. (1990)). The proportion and nature of the pharmaceutically acceptable diluent or excipient are determined by the solubility and chemical properties of the pharmaceutically active compound selected, the chosen route of administration, and standard pharmaceutical practice. The pharmaceutical preparation may be adapted for oral, parenteral or topical use and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions, or the like. The pharmaceutically active compounds of the present invention, while effective themselves, can be formulated and administered in the form of their pharmaceutically acceptable salts, such as acid addition salts or base addition salts, for purposes of stability, convenience of crystallization, increased solubility, and the like.
A further aspect of the present invention is directed to a method for producing N-linked glycosylated proteins, comprising the steps of:
a) providing a recombinant organism, preferably a prokaryotic organism, comprising nucleic acids coding for                i) a functional pgl operon from Campylobacter spp., preferably C. jejuni, and        ii) at least one recombinant target protein comprising one or more of the following N-glycosylated optimized amino acid consensus sequence(s):                    D/E-X-N-Z-S/T (SEQ ID NO:7),                        wherein X and Z may be any natural amino acid except Pro, and wherein at least one of said N-glycosylated optimized amino acid consensus sequence(s) is introduced, and
b) culturing the recombinant organism in a manner suitable for the production and N-glycosylation of the target protein(s).
Preferably, the target protein is one of the above described recombinant proteins according to the invention.
In a preferred method of the invention, the functional pgl operon from Campylobacter spp., preferably C. jejuni, comprises nucleic acids coding for                i) recombinant OTase from Campylobacter spp., preferably C. jejuni, and        ii) recombinant and/or natural specific glycosyltransferases from Campylobacter spp., preferably C. jejuni, and/or        iii) recombinant and/or natural specific glycosyltransferases from species other than Campylobacter spp.,
for the assembly of an oligosaccharide on a lipid carrier to be transferred to the target protein by the OTase.
Moreover, in a preferred embodiment the present invention relates to a method for preparing a host cell according to the invention comprising the steps of:
i) providing a gram-negative bacterium,
ii) introducing into said bacterium at least one nucleotide sequence encoding for                a) at least one recombinant specific glycosyltransferase for the assembly of an oligosaccharide on a lipid carrier, and/or        b) at least one recombinant oligosaccharyl transferase (OTase) from Campylobacter spp., preferably C. jejuni, and/or        c) at least one recombinant protein comprising one or more of the following N-glycosylated optimized amino acid consensus sequence(s):                    D/E-X-N-Z-S/T (SEQ ID NO:7),                        wherein X and Z may be any natural amino acid except Pro, and wherein at least one of        said N-glycosylated optimized amino acid consensus sequence(s) is introduced, and iii) culturing said bacterium until at least one recombinant N-glycosylated protein coded by the nucleotide sequence of c) is located in and/or on the outer membrane of the gram-negative bacterium.
For practicing the preferred methods above, the recombinant procaryotic organism or host cell is preferably selected from the group of bacteria consisting of Escherichia ssp., Campylobacter ssp., Salmonella ssp., Shigella ssp., Helicobacter ssp., Pseudomonas ssp., Bacillus ssp., preferably Escherichia coli, preferably E. coli strains Top10, W3110, CLM24, BL21, SCM6 and SCM7, and S. enterica strains SL3261, SL3749 and SL3261ΔwaaL.
Another preferred method for producing, isolating and/or purifying a recombinant protein according to the invention comprises the steps of:
a) culturing a host cell according to claims 15 or 16,
b) removing the outer membrane of said recombinant gram-negative bacterium and
c) recovering said recombinant protein.
Exemplary methods for removing the outer membrane of a cell, preferably a prokaryotic cell, more preferably a gram-negative bacterial cell, are suitable enzymatic treatment methods, osmotic shock detergent solubilisation and the French press method.
Most preferred, the present invention relates to a method, wherein recombinant or natural specific glycosyltransferases from species other than Campylobacter spp., preferably C. jejuni, are selected from the group of glycosyltransferases and epimerases originating from bacteria, archea, and/or eukaryota that can be functionally expressed in said host cell.
The following examples serve to illustrate further the present invention and are not intended to limits its scope in any way.

Recombinant N-glycosylated proteins from procaryotic cells


A foldable tricycle, including a front wheel assembly; a mainframe, latched to the front wheel assembly via a front mount; a pair of rear-wheel assemblies; a pair of folding ring assemblies coupling the rear-wheel assemblies to the mainframe; and a mechanism for simultaneously unlocking the pair of ring assemblies and unlatching the mainframe from the front wheel assembly so that a single pulling motion on the mechanism unlocks and unlatches the foldable tricycle from an assembled state thereof so as to be converted to a folded state thereof.

The present invention relates to a tricycle and, more particularly, to an improved multi-stage tricycle with a single-motion release mechanism.
Multi-stage tricycles are known in the art such as multi-stage tricycles including steering handles which are intended to be used by an adult to propel and steer the tricycle when the child is too young to successfully do so himself and to be removed later as the child ages. All of the known steering handles suffer similar drawbacks. Namely, the steering handles are coupled to the steering column of the tricycle via a complex set of wires and mechanisms. Furthermore, the single-column configuration of the steering handle makes the steering process difficult.
Collapsible polycycles are also known in the art. The known processes of collapsing polycycles, include numerous separate steps which are necessary to perform in order to convert the assembled polycycle into the folded state.
It would be highly advantageous to have a multi-stage tricycle with a simplified steering handle providing improved control and also having an improved free-wheeling feature as well as a free steering feature. It would furthermore be advantageous to have single-motion release mechanism for folding the tricycle.

Free-wheeling, free-steering, foldable multi-stage tricycle with a single-motion release mechanism