HK1102411B - Optically encoded particles, system and high-throughput screening - Google Patents

Description

Optically encoded particles, systems and high throughput screening

Technical Field

The field of the invention is coding. Other exemplary areas of the invention include life sciences, safety, product marking, food processing, agriculture, and chemical testing.

Background

The need for markers in society is very extensive. The markers are the basis for tracking and identification. The code may be used as a form of indicia that can be recognized by a person or machine, as in the case of bar codes. But at the micro-scale, the labeling/encoding itself becomes difficult and heavy.

Methods of encoding microscale materials have thus attracted increasing attention for high-throughput screening applications in the fields of drug discovery, genetic screening, biomedical research, and biological and chemical sensing. Research approaches to measuring increased amounts of analyte while minimizing the number of samples necessary have focused on-chip spatial discriminatory arrays or encoded beads. Large arrays have been developed for the purpose of biological and/or chemical sensing by using position coding to record the response of specific analytes. The main advantage of using an array over conventional single analyte sensors is that a large number of analytes can be processed and analyzed simultaneously. However, positional arrangements can suffer from low diffusivity and are limited in the range of concentrations of the analyte being sensed. Another approach is to use individually coded beads.

Early encoding of particles used fluorescent or infrared active molecules as binary markers. Recently, cadmium selenide quantum dots have proven to be a viable candidate for encoding particles due to their unique fluorescent properties.Compared with organic molecules, quantum dots have the advantages of improved stability to photobleaching, sharper fluorescence peak, improved solubility, large excitation frequency range and the like. With 6 colors (limited to the peak width of the visible region fluorescence) and 10 intensity levels, it is theoretically possible to match 10 colors with 10 intensity levels⁶Each particle is encoded. This number is difficult to achieve in practice due to spectral overlap and sample inhomogeneity. In addition, despite the improved photostability of quantum dots, fluorescence quenching is still possible, which leaves uncertainty in the measurement of relative intensities as a reliable encoding method.

Another encoding method is to use ultra-micro metal rods. The ultra-micro metal rod is fabricated by plating a metal onto a porous membrane in alternating bands of controlled thickness. Different reflective properties of various metals can be used as bar codes for identification purposes. The reflectance spectra do not suffer from photo-bleaching inherent in fluorophores. Furthermore, fluorescent analytes do not interfere with particle signals. Deposition of the rods is a relatively complex process and moreover may be difficult to use as an encoding method where, for example, a large number of codes are required, since each rod has to be brought into focus in an optical code reader, such as a microscope, to read out the codes.

Methods of fluorescent molecular encoding, core-shell quantum dot encoding, and photonic crystal encoding using the rabate and Bragg reflectivity theories rely on generating spectral lines as bits (bits). The number of possible encodings is limited to 2ⁿWhere n is the number of spectral lines or bits that can be discerned from the other spectral lines of the spectrum. There remains a need for methods of encoding on a microscale.

Disclosure of Invention

The invention relates to a particle having an encoding of an encoding library embedded within the physical structure of the particle according to a change in refractive index between different regions of the particle. In a desirable embodiment, the film has a porosity that varies in such a way as to produce a code that is recognizable in the reflectance spectrum. One analytical detection method uses such particles and detects the spectral shift in the presence of the analyte. Additional embodiments having other features are also disclosed.

Drawings

FIG. 1 is a schematic illustration of a multilayer encoded particle of the present invention;

FIGS. 2A and 2B illustrate Fourier transform particle decoding in accordance with a preferred embodiment;

FIG. 3A illustrates an exemplary etching waveform for a preferred embodiment of Rugate particle decoding;

FIG. 3B illustrates a preferred embodiment Rugate particle decoding;

FIG. 4 illustrates a preferred embodiment of generating encoded particles;

FIG. 5 shows optical reflectance spectra of the encoded particles of the preferred embodiment alone in experimental air (shown in solid lines) and air with a small amount of ethanol vapor (shown in dashed lines);

FIG. 6 shows the intensity of reflected laser light (632nm) from the preferred embodiment encoded porous silicon Rugate particles measured for three exposure/evacuation cycles using (from bottom to top, as shown) acetone, ethanol, formaldehyde, and water analytes at saturated vapor pressure;

FIG. 7 is an image of an exemplary preferred embodiment encoded particle formed by spatially defined, periodically varying etches in a wafer;

FIG. 8 illustrates reflectance spectra from 15 individually encoded example preferred embodiment sample particles;

FIG. 9A illustrates reflectance spectra from an exemplary preferred embodiment Rugate-encoded sample particles alone and triple-encoded Rugate sample particles;

FIG. 9B is a schematic diagram of an exemplary preferred embodiment multiple Rugate-encoded particle;

FIG. 10 illustrates the decoding results of an exemplary preferred embodiment Rugate-encoded particle alone, prepared for biological screening;

FIG. 11 illustrates an example of a code library waveform and the resulting index coding in porous silicon; and

FIG. 12 shows an example of a code library waveform.

Detailed Description

The invention relates to a particle having an encoding of an encoding library embedded within the physical structure of the particle according to a change in refractive index between different regions of the particle. The refractive index is preferably changed by changing the porosity formed in the particles. The reflection from the particles produces an optical signature that uniquely corresponds to the code in the code library that is used to form the particles by computer waveform controlled etching. Reflection may occur at visible and/or invisible wavelengths. The code library provides a plurality of waveforms, each of which produces a unique optical signature in controlling the etching of the formed particles. In a preferred embodiment forming method, the multi-layered porous encoded structure is produced by an etching process in which etching conditions are varied during pore formation. The cutting may be performed to form encoded particles having a small size range (e.g., from hundreds of nanometers to hundreds of microns).

The methods and particles of the invention find use in a variety of industries, including but not limited to drug discovery, biological screening, chemical screening, biological labeling, chemical labeling, in vivo labeling, security identification, and product labeling. The various attributes of the particles and methods of the invention allow for wide application in various industries. The small size of the particles allows them to be conveniently introduced into a variety of bodies such as products, test chambers, assays, powders (e.g., explosives for identification), pastes, liquids, glasses, paper, and any other body or system that can receive fine particles. Detection in vivo is enabled by the biocompatible particles of the present invention, which can then be interrogated through tissue, for example, using near infrared and infrared wavelengths that penetrate tissue.

According to the above exemplary aspects and applications of the invention, the particles of the preferred embodiments are identified by a code that is inherent to the reflectivity spectrum of the porous structure of which they vary. In another aspect of the invention, a substance, such as a biological or chemical substance, is attached to the porous structure, and the particles become labels that identify the substance carried by the pores. In another aspect of the invention, the change in the reflectance spectrum of the encoded particle may reflect the presence, absence or amount of a substance in the particle pore.

Fig. 1 shows a cross-section of a preferred embodiment encoded particle 10. The encoded particle 10 comprises a layer or region 12 having₁-12_NThe multilayer porous film of (1). The use of multiple layers here means that there must be multiple regions with different porosities. In some embodiments, the transition between porosities may be gradual. This means that the multiple layers include both structures with a gradual transition of multiple porosities and structures with an abrupt transition of multiple porosities. Consistent with this definition, layer 12₁-12_NDefined by a varying porosity, which may vary gradually or abruptly. In addition, the use of "layers" encompasses not only individually deposited layers, for example, but also continuous structures having varying porosity. In other words, "layer" includes but is not limited to meaning a separate formation process or deposition. Layer or zone 12 of varying porosity₁-12_NThe multi-layer porous membrane structure of (a) is formed on a substrate 14, as shown in figure 1. However, embodiments of the invention include multilayer thin film particle structures, such as layer 12 that is peeled from the substrate₁-12_NThey are initially formed on or from the substrate. Porous layer 12₁-12_NThe encoding is performed by selecting from an encoding library and introducing the encoding in the layer by computer controlled etching to apply the encoding to produce an interference pattern in the reflectivity spectrum that forms an optical feature corresponding to the encoding selected from the encoding library. Porous layer 12₁-12_NThe light reflected by the interface between them interferes with the light from the interface between the other layers, creating an interference pattern in the reflectivity spectrum. The particles 10 of the present invention are specifically encoded by encoding in a library of codes that are used to control the etching conditions and layer thickness during the formation of the particles 10. Refractive index, chemical composition of layer interface and each layer 12₁-12_NThe thickness of (a) affects the optical characteristics produced by a particular particle. Thus, varying the relative porosity between layers in a particular particle (to affect the refractive index) and varying the thickness of the layers during formation of the particle 10 can tailor particular optical characteristics in the reflectance spectrum. Porosity also affects the intensity of the peaks in the reflectance spectrum, providing additional coding possibilities. Each code or group of codes selected from the library of codes may be used to replicate the same optical characteristic over and over again, producing particles with the same code. In addition, different codes may be selected from a library of codes to produce particles of different optical characteristics.

Porous layer 12₁-12_NMay be formed of any porous semiconductor or insulator. In the particles of the preferred embodiment of the present invention, porous silicon is used to form porous layer 12₁-12_N. Controlled anodic etching of crystalline silicon in hydrofluoric acid solution may simultaneously etch porous layer 12₁-12_NThe porosity and thickness of the porous material are controlled. Generally, the time of etching controls the thickness of the porous layer, while the density of the etching current controls the porosity. Furthermore, the timing of the application of the current density influences the optical characteristics to be produced. Layer 12₁-12_NMay be different from each other to produce specific optical characteristics. The codes in the library of codes differ in the duration, level and timing of the etching current density, and each code in the library produces a unique optical signature on a given material, such as silicon, which is etched to produce the coded particles.

The variation in porosity and thickness follows a pattern established in accordance with a code selected from a library of codes. In some embodiments, the porosity varies gradually between layers, while in other cases the porosity may be abruptAnd (6) changing. Porous silicon is layer 12₁-12_NAre preferred. Porous silicon has a number of proven advantages. For example, porous silicon has proven to be biocompatible. In addition, the surface chemistry of oxidized porous silicon is as effective as silicon oxide. Thus, the surface chemistry is well understood due to biochemical derivatization and ligand immobilization.

In a preferred embodiment, layer 12₁-12_NFormed to include the acceptor material in the porous structure. The purpose of the receptor is to bind to a particular analyte of interest. Exemplary receptors (also referred to as binders) are disclosed, for example, in U.S. patent No.6,248,539 entitled "port Semiconductor B derived Optical interferrometric sensor". By binding receptor molecules to porous layer 12₁-12_NBy any means of (1), the receptor molecules are allowed to contact the porous silicon layer 12₁-12_NAdsorption or association. This includes, but is not limited to, covalent binding of the receptor molecule to the semiconductor, ionic association of the receptor molecule with the layer, adsorption of the receptor molecule to the surface of the layer, or other similar techniques. Association may also include covalently linking the receptor molecule to another moiety which, in turn, is covalently linked to porous layer 12₁-12_NOr by hybridization or other biological association mechanism to link the target molecule to another moiety that is linked to porous layer 12₁-12_N. Other specific examples include receptor ligands that have been attached to a porous silicon layer to form a biosensor. The analyte bound to the particles 10 of the present invention becomes identifiable and traceable by the code provided by the particles 10.

Multilayer film 12₁-12_NMay be encoded, both in intensity and wavelength properties. The preferred embodiment is a multilayer film 12 with mismatched optical thicknesses₁-12_NThe particles 10 of (a). Optical thickness is defined as the refractive index of a layer multiplied by its metric thickness. Referring to fig. 2A and 2B, particles 10 encoded in this manner exhibit optical characteristics in the fourier transform of the generated reflectivity interference spectrum. An exemplary generated interference spectrum is shown in fig. 2A.The fourier transform shown in fig. 2B shows an optical signature with well-resolved peaks. The particles 10 can be set to have a series of distinct peaks (a, b, c).

The intensity of the peaks in the reflection spectrum passes through the layer 12₁-12_NThe refractive index of the interface between them, which is determined by the change in porosity between adjacent layers. This change may be gradual or abrupt. The position of the peaks can be controlled by adjusting the thickness of the layer. By varying the relative intensity of each reflectivity peak, additional encoding may be performed, which may be designed into the inventive particle 10 by adjusting the electrochemical etch parameters to control the layer 12₁-12_NThe porosity of (a). Accordingly, N layers of particles 10 having A resolvable positions and B resolvable intensities per peak can be encoded (A × B)^NParticles. Furthermore, a particle 10 having any combination of N peaks with A resolvable positions for each peak and relative intensity order can encode N! (A)^NOne of them.

Embodiments of the invention include complex encoded particles and particle systems. Wherein the particles are encoded by electrostatic anodic etching of crystalline P + (-1 mOhm/cm) silicon wafers. The thickness and porosity of the porous layer are controlled by the current density over time and the composition of the etching solution. Computer generated waveforms allow complex encoding strategies to be implemented. The duration of the etch cycles is controlled using computer generated waveforms so that each is unique to generate porosity so that the effective refractive index varies in direct correspondence with the applied current waveform. After the encoded portion of the current waveform has been conventionally run, a short high-magnitude current pulse may be applied to remove the porous matrix created in the wafer. The free porous matrix includes photonic crystal particles. Masking of the wafer prior to etching can generate particles of different shapes. These shapes provide additional opportunities for recognition.

Repeating the above process using carefully controlled computer waveforms can form a large library of unique particle types. These libraries may be used to form test chambers. The library and the specific particle type form the basis for high throughput screening and bioassay methods.

The method of data extraction and analysis may embody all the complexities of the spectrum that result from the reflectivity properties of the photonic crystal. Unlike traditional bioassays that combine fluorescence encoding methods with fluorescence analysis, our technique does not have the problem of spectral coincidence with the encoding methods read analytically. Spectral lines are not used as bits in the method of the invention. For example, analytical detection methods are based on reflectance and detect spectral shifts rather than the presence, concentration or absence of spectral peaks. Possibilities for spectral identification include multivariate analysis and relative and ratiometric multimodal analysis.

Another encoding strategy includes a periodic structure. An exemplary periodic structure includes a layer 12 having₁-12_NThe layer 12 of particles 10₁-12_NThe layers are configured via porosity and thickness to form a Bragg stack or Rugate filter. For example, a Bragg stack may be created by alternating layers having matching optical thicknesses. By varying the layer 12 in the particles 10 of the invention₁-12_NThe porosity-defined Bragg stack will produce peaks in the reflectance spectrum with full width half maximum peaks (peak) well resolved in the reflectance spectrum (e.g., -10 nm). Through the multilayer structure 12₁-12_NThe Rugate filter generated by the refractive index change at the interface of the layers also generates similar narrow peaks in the reflection filter spectrum and also suppresses sidebands and higher order (higher order) reflections.

Fig. 3A and 3B illustrate a preferred embodiment Rugate particle encoding strategy. Rugate encoded particles may be produced by etching a semiconductor or insulator with periodic changes in etching conditions such that the refractive index in the material varies as a sinusoidal (or diffracted) sinusoidal) function. The structure may be produced by etching a semiconductor wafer with a pseudo-sinusoidal current waveform. Fig. 3A shows that the period of an exemplary sinusoidal variation of the etch current density (n) used to produce the etch of the exemplary embodiment is 18 seconds. As can be seen in fig. 3B, the encoding produces clearly discernable narrow peaks. The intensity and position of the peaks may vary with layer thickness and refractive index.

A preferred method of constructing the encoded porous particles 10 is illustrated in fig. 4. A suitable semiconductor or insulator, such as a silicon wafer, is selected for processing (step 14). For example, a silicon wafer may be cut and masked to have portions exposed for etching. One exemplary suitable semiconductor material is a single crystal silicon wafer. After which the spatial coding is defined (step 16). The spatial encoding defines a range of encodings on the material to be etched. A spatially resolved (resolved) etch is performed so that the code is encoded in the grain-sized portion of the silicon wafer. An exemplary space dissolution etching process is disclosed in U.S. Pat. No.5,318,676 (published on 6/7 of 1994) entitled "Photolithographical interface of luminescence on silicon structures". In an alternative process, the step of spatially defining (step 16) is omitted. For example, individual silicon wafers or regions of silicon wafers may be etched to include particles having individual codes. In this way, other silicon wafers may be etched to contain particles with different codes. Anodic etching is then started, for example in an aqueous solution of hydrofluoric acid and ethanol (step 18). Thereafter, etching is performed with etching conditions that vary according to the defined coding strategy (step 20). One or more of the inventive codes are etched into a silicon wafer. The laterally (longitudinally in fig. 1) encoded but still associated particles can be removed from the wafer (step 22), such as by a high level of electropolishing current. The regions between the spatially defined etched portions may be cut to separate different coded silicon wafer portions. The individual particles can thereafter be separated in a cut, for example by mechanical agitation or ultrasonic disruption (step 24). Particle separation (step 24) preferentially produces micron-sized particles, such as particles in the range from hundreds of nanometers to hundreds of microns. The particle separation (step 24) or the particle assignment step (step 26) can be carried out after step 20 or step 22. Particle assignment may include, for example, porous multilayer structure 12 for a particular biological, biomedical, electronic, or environmental application₁-12_NChemical modification of (2). For example, the particles may be used as receptors for an analyte or modified with a targeting moiety, such as a sugar or polypeptide.In addition, binding can be indicated (signal), for example, by fluorescent labeling of the analyte or analyte autofluorescence. When using particles 10, the particles can be identified based on their optical characteristics when bound to a given target analyte. This designation step may also be omitted in embodiments of the invention.

In other embodiments of the invention, the encoded particles may be placed in a suitable body, i.e. any liquid, powder, fine dust or other material that will carry the inventive micron-sized encoded particles. Particles disposed in the body may be used, for example, to identify the source of an artificial powder, such as an explosive. Another potential subject is an animal. The biocompatible particles of the present invention may be implanted in vivo in an animal subject. The reflectance spectrum of the porous silicon particles 10 of the preferred embodiment of the present invention includes, for example, visible, near infrared and infrared spectra. This shows the possibility of perceiving the encoding of the particles of the present invention through obstacles such as living tissue.

Examples of embodiments and Experimental data

Examples of embodiments of the invention will now be discussed. The experimental data set forth herein are intended to demonstrate potential applications of the invention to the skilled artisan. The devices listed here are only for the skilled person to understand the data reported here. For example, commercial embodiments of the invention the inventive apparatus may take a substantially different form so as to enable low cost mass production.

The first embodiment is exemplified by remote detection, which is a chemical detection technique for identifying analytes from a distance. The particles 10 of the present invention include receptors for sensing specific analytes. The encoding of the particles and the indication of binding to the analyte may be detected in the reflectance spectrum, for example using a low power laser. The receptor may for example be specific for sensing a biomolecule or attaching the encoded particle to a cell, spore or pollen particle.

Remote probing techniques were tested using an exemplary encoded multilayer porous silicon film. By mixing in a 1: 3 ethanol: 48% aqueous HF solutionMedium electrochemical etching (100) directionally polished silicon wafer (p)⁺⁺-type, B doping, < 1m Ω -cm resistivity) to prepare a multilayer porous silicon film. The etching current density is periodically changed from quasi-sine wave (at 11.5-34.6 mA/cm)²In between) to produce a sinusoidally varying porosity gradient. By applying a current density of 600mA/cm for 30 seconds²The electropolishing pulse of (a) separates the thin film from the substrate. The free-standing thin film is thereafter formed into particles by mechanical grinding or ultrasonic fracturing to produce particles ranging in size from a few hundred nanometers to a few hundred micrometers. The optical reflectance spectrum in fig. 5 approximates a Rugate filter, showing sharp reflection maxima at wavelengths and source-sample-detector angles that satisfy the Bragg equation and appropriate matching conditions.

The particles were fixed on a glass plate and placed in a gas formulation chamber equipped with an optical window and a Baratron manometer. The particles were irradiated with 10MW He/Ne laser. As shown in fig. 5, the formed particles strongly reflect light of the He/Ne laser at a wavelength of 632nm in air. The spectral position of the laser used to acquire the data of fig. 5 is shown for comparison (vertical arrows). These data were obtained using an Ocean Optics CCD visual spectrometer at the focal plane of an optical microscope. When exposed to an analyte gas, capillary condensation causes the reflectance spectrum of the particles to shift to longer wavelengths due to the increase in the refractive index of the porous medium, and the particles are observed to darken.

The relative change in light intensity reflected simultaneously from many particles can be quantified at a fixed wavelength (632nm) for a series of condensable analyte vapors, as shown in fig. 6. The vapor pressure of each analyte was 222, 60, 28, and 24 torr at 25 c, respectively. The relative reflected light intensity was measured as the photocurrent from a magnified silicon photodiode mounted on the objective lens of an 8 inch Schmidt-Cassegrain collection optic. The sample was 20 meters from the laser and detection optics. The spectra are shifted in the Y-axis direction for clarity. These vapors are introduced into the exposure chamber under saturated vapor pressure. The intensity of the reflected light was measured at a distance of 20 meters using an interrupt lamp and phase sensitive probe (Stanford Instruments SR-510 lock-in amplifier) in the presence of normal fluorescent room lighting. No other optical or electronic filtering was used. The nature of adsorption and/or microcapillary condensation at the surface of the porous silicon is largely dependent on the surface chemistry, and for hydrophobic analytes the hydrogen-terminated, hydrophobically-formed materials used in the experiments have much greater affinity relative to hydrophilic analytes. Thus, the particles are relatively insensitive to water vapor when the partial pressure is comparable to that used for more hydrophobic organic analytes. No attempt was made to provide acoustic or vibrational isolation of the sample or optical device, and most of the noise observed in the data could be attributed to laboratory shaking. With a near infrared laser light source, the sensitivity should be further improved, with less significant background radiation and atmospheric adsorption and scattering.

Another preferred exemplary application of the invention is the screening of biomolecules by the encoded particles 10 of the invention. Millions of codes may be generated for a small number of layers. Simple antibody-based biological assays using fluorescently labeled proteins have been tested. For the exemplary chemical sensing embodiment, periodic Rugate format encoding as previously described is used. By masking the wafer prior to etching, a well-defined particle plate is created, as shown in fig. 7.

The particles of FIG. 7 were used to show the peak of the photon (photonic) spectrum at 632 nm. The proportion of the insert (reproduced above the figure for clarity) corresponds to 2 μm per cell. The multilayer encoded particles generated in this way show very sharp lines in the optical reflectance spectrum. This line can occur anywhere in the visible to near infrared spectral range, depending on the waveform used in the programmed etch.

Exemplary waveforms for the 15 independent codes are presented in fig. 8. Fig. 8 presents reflectance spectra of 15 porous silicon multilayer samples prepared using sinusoidal etching (Rugate code structure). Each sample contains a separate Rugate frequency code. The spectra were obtained using a Cambridge Instruments microscope with a 70 x objective lens. The sample was illuminated with a tungsten lamp and the reflectance spectrum was measured by an Ocean Optics SD2000 CCD spectrometer. Sample particles pass through at 4Anodic etching p in 8% aqueous HF: ethanol (3: 1, volume ratio) solution⁺⁺Type, B-doped, (100) oriented silicon (resistivity < 1m omega-cm)²) And (4) preparation. Typical etch parameters for Rugate structures used in pseudo-sinusoidal current waveforms are oscillation range 11.5 to 19.2mA cm^-250 cycles with a period of 18 seconds. 460 mA cm with use time of 40 seconds^-2The current pulse separates the film from the substrate. Lithographically defined particles were prepared by applying S-1813 photoresist (Shipley) on silicon wafers prior to electroetching (spin coating at 4000 r.p.m. for 60 seconds before development (development), soft bake at 90 ℃ for 2 minutes, uv exposure using a contact mask aligner, hard bake at 120 ℃ for 30 minutes). The spectral features presented in fig. 8 may be much narrower than the fluorescence spectra obtained from molecular or core-shell quantum dots.

Fig. 9A shows the reflectance spectra of porous silicon Rugate encoded particles etched with a single period (bottom) and 3 independent periods (top). FIG. 9B diagrammatically shows a multiple-coded particle 10a of the preferred embodiment, which contains three sets of coding layers 12, 16 and 18. The multiple Rugate codes can be spatially isolated, or can be etched at the same physical location, such as multiple packets formed at different depths, each forming an independent Rugate code. Each set of coding layers 12, 16 and 18 has a porosity that varies periodically to form an independent Rugate or Bragg code.

The exemplary particles exhibit peaks in the reflectance spectrum that represent their multilayer structure. These exemplary particles shown on the bottom spectrum are used at 11.5 to 19.2mA cm^-2A varying sinusoidal current (cycle 50, period 18 seconds) was etched. Triple-encoded particles (triple Rugate) represented by the top spectrum were shaken at 11.5 to 34.6mA cm^-2Is etched (cycle 20, cycle 10 seconds (520 nm); cycle 45, cycle 12 seconds (610 nm); and cycle 90, cycle 14 seconds (700 nm)). The thickness of the exemplary particles is approximately 15 μm. The spectra are shifted in the y-axis direction for clarity.

To test the reliability of coding methods in biological screening applications, we aimed toTwo different batches of encoded particles were prepared as separate Rugate structures. Both batches of particles were treated by ozone oxidation to increase their stability in aqueous media and to provide them with a hydrophilic surface layer. O diluted with compressed air₃The flow oxidizes the particles. Control particles encoded with 750-nm spectral features were treated with concentrated BSA (Sigma, 5g in 100 ml of double distilled water) and air at 37 ℃ at 5% CO₂The cells were incubated for 3 hours. 540-nm encoded test particles were exposed to coating buffer (2.93 g NaHCO in 1000 mL of double distilled water)₃1.61 g of Na₂CO₃) 50 μ g ml of^-1Murine Albumin, and 5% CO at 37 ℃₂The incubation was continued for 2 hours. The test particles were then tested at 37 ℃ in 5% CO₂The rabbit primary anti-murine albumin antibody was exposed to a 1: 100 dilution of BSA in concentrated solution for 1 hour. The two batches of particles were then mixed together and incubated for 1 hour in the presence of FITC (fluorescein isothiocyanate) conjugated goat anti-rabbit immunoglobulin-G in BSA solution. Analytes bound to the encoded particles are subsequently detected using fluorescence and spectral emission coefficient microscopy.

The result of the decoding is presented in fig. 10. Decoding 16 particles yields the following results: of the 8 green fluorescent particles, 8 particles were definitively decoded, belonging to the functionalized murine albumin batch (curve a of fig. 10). Of the 8 non-fluorescent particles, 6 particles were correctly decoded (curve B of fig. 10), one particle showed the wrong code, and the other particle was not decipherable. It may be shown that the incorrectly encoded particles belong to the first batch of particles, but are not sufficiently functionalized with murine albumin, so that no fluorescence is produced in the antibody detection. This is understandable because murine albumin was not covalently attached to silica-coated particles in the experiment. A number of stable chemical modification chemistries have been developed for porous silicon, either oxidized or non-oxidized, and some of these have been demonstrated with specific antibodies or receptors. The problem of immobilizing biochemical or chemical components is therefore easily solved. In addition, the chemical modification may prevent erosion in aqueous media that may lead to undesired movement and/or unreadable particles in the optical encoding. In experiments that have been carried out, without passivation chemical treatment, except for the ozone oxidation to produce a silicon oxide layer, a spectral coding shift between 0 and 50nm according to incubation time was observed when immersed in an alkaline aqueous medium.

The multilayer porous silicon coding structure has many advantages over the current coding methods. Porous silicon coding structures can be constructed that display spectra spanning the visible, near infrared and infrared regions of the spectrum. In addition, the reflectivity spectrum of the Rugate filter may show much sharper spectral features than the spectrum obtained from the gaussian set of quantum dots. In other embodiments, spectral shifts may be used for detection, thus avoiding the encoding method from coinciding with the spectrum of the detection readout. The invention comprises different coding particle libraries and also comprises particles with different shapes.

Due to its porous coding structure, more codes can be placed in a narrower spectral window. Unlike encoding methods based on layered metal nanorods, fluorescence or vibrational features, the inventive encoded particles can be detected using light diffraction techniques, and thus do not require the use of imaging optics to read the code. The encoded particles can be detected using conventional fluorescent labeling techniques, and sensitive chemical and biochemical detection techniques can also be introduced into the optical structure of the encoded particles, eliminating the need for fluorescent probes and focusing optics. Furthermore, since the silica porous silicon encoded particles of the preferred embodiments present a silica-like surface to the environment, they do not readily quench fluorescence from organic chromophores, and can be treated and modified using chemistries developed for glass bead bioassays. Silicon-based encoded particles are easy to combine with existing chip technology.

The use of the encoded silicon particles of the present invention is advantageous over organic dyes or quantum dots in medical diagnostic applications. In vivo studies have shown biocompatibility of porous silicon and long-term stability of reflectance data of multilayer structures. Furthermore, the possibility of optically addressing particles at near-infrared, tissue-penetrating wavelengths without the losses associated with low fluorescence quantum yields makes these materials amenable to in vivo diagnostic assays. Finally, since the porous code is an integral ordered part of the porous structure, it is not possible for the coded part to be missing, confused or photobleached, as may occur with quantum dots or fluorescent molecules.

We discuss specific codes below, and by way of example, illustrate complex codes that can be used to construct a library of codes. FIG. 11 shows example encodings and resultant refractive indices in porous silicon. The code is a waveform with a specific etch current density applied as a function of time, as shown by the current versus time curve in fig. 11, and produces a code of a specific index of refraction versus depth map in the etched deformed porous material, as shown on the right side of fig. 11.

Through experimental verification, the code library can be constructed experimentally for different etch currents over time, based on the information in the following table.

Table I

Encoding	tdow (minutes)	taller (minutes)
			1	0	8
2	2	6
			3	4	4
4	6	2
			5	8	0

Table II

Sample (I)

Encoding

Start time of etching

W3_19	3	10:50
			W3_15	1	11:05
W3_11	5	11:18
			W3_1	2	11:33
W3_7	4	11:48
			W3_10	2	12:08
W3_12	5	12:20
			W3_9	3	12:36

W3_21	4	12:51
			W3_6	1	1:06

Table I above describes the timing of the waveforms shown in fig. 12. The waveform includes an initial low current of 15 milliamps and a subsequent high current of 45 milliamps at a particular time. A piece of silicon wafer type approximately 1 milliohm T was energized with current at 2000 updates per second through a platinum mesh electrode using a PCI-6042E DAQ card in a Princeton instruments model 363 potentiostat/galvanostat. The etching solution is prepared by 48% hydrofluoric acid and ethanol in a ratio of 3: 1. The sample was etched, rinsed with ethanol and placed in a vacuum mixing dish (descretor) until a spectrum was obtained. Spectra were obtained using an ocean optical (ocean optical) SD2000 CCD spectrometer with 0.1 second delay, no averaging, at 22 milliseconds during the ten minute cycle. Table II shows different samples and the codes applied to the different samples. Table I gives five encodings. Results were obtained for each of the two samples and the codes given for each of the two samples showed that the resulting optical features had uniquely identifiable characteristics. Slight drift between repeated experiments is caused by inaccuracies in the experimental manufacturing process. Commercial manufacturing processes can reduce this offset. In any case, subtle differences between crystals etched with the same code are permissible, as will be appreciated by those skilled in the art. There are many techniques available today for comparing slightly different optical characteristics and determining whether to substantially match a code. For example, the total waveform envelope (envelope), and the number of stripes, frequency, etc., associated with an internal reference (i.e., first, middle, and last stripes) within the code may be used to distinguish between copies and other codes that employ methods such as primary (principal) component analysis or other multivariate analysis methods. For example, patterns in an optical feature created by etching according to the present invention form a recognizable code, and thus, sufficient information to identify the feature pattern can be stored in a pattern code library for identification purposes.

While particular embodiments of the present invention have been shown and described, other modifications, substitutions and alternatives will be apparent to those skilled in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims.

Various features of the invention are set forth in the following claims.

Claims (44)

1. A method of generating a library of optically encoded particles (10, 10a), comprising:

selecting one of a set of computer control waveforms; and

applying this waveform in a set of computer controlled waveforms during etching of a material to produce particles of a particle library from the material, the particles of the particle library comprising,

a porous layer having a refractive index versus depth map that uniquely corresponds to the waveform of a set of computer controlled waveforms, the refractive index versus depth map uniquely defining an interference pattern in the reflectance spectrum that forms an optical signature corresponding to the waveform of the set of computer controlled waveforms.

2. The method for generating a library of particles according to claim 1, wherein the particles have a diameter of several hundred micrometers or less.

3. The method for particle library generation as in claim 1, wherein said interference pattern in the reflectance spectrum extends beyond the visible spectrum.

4. The particle library generation method of claim 1, performed to form a first porous layer and an n-layer additional porous layer, wherein the first porous layer and the n-layer additional porous layer periodically alternate and form a Bragg stack.

5. The particle library generation method of claim 1, performed to form a first porous layer and an n-layer other porous layer, wherein said first porous layer and said n-layer other porous layer form a Rugate reflector.

6. The method for generating a library of particles of claim 1, wherein the material comprises a semiconductor.

7. The method of particle library generation of claim 6, wherein said semiconductor comprises silicon.

8. The method for generating a library of particles of claim 1, wherein the material comprises an insulator.

9. The method of generating a library of particles of claim 1, further comprising a receptor that binds a predetermined analyte.

10. Optically encoded particles (10, 10a) comprising a film wherein the porosity is varied in accordance with a waveform in a library of computer controlled waveforms in a manner to produce an encoding in the library identifiable in the reflectance spectrum.

11. The particle of claim 10 wherein the analytical detection method comprises the step of detecting a spectral shift.

12. The particle of claim 10, further comprising a receptor.

13. The particle of claim 12, wherein said receptor is a receptor for a biological analyte.

14. The particle of claim 12, wherein said receptor is a receptor for a chemical analyte.

15. The particle of claim 12, wherein said receptor is a receptor for a gaseous analyte.

16. The particle of claim 10, further comprising a fluorescent label for detecting the particle.

17. The particle of claim 10 wherein the film comprises porous silicon.

18. The particles of claim 10 which are micron-sized.

19. A method of encoding a film, comprising the steps of:

etching the semiconductor or insulator substrate to form a thin film containing holes;

the etching conditions are varied according to one of a set of computer controlled waveforms to produce a map of refractive index versus depth that produces a pattern that will produce an identifiable code in the reflectance spectrum in response to illumination.

20. The method of claim 19, further comprising the step of separating said thin film from said semiconductor or insulator substrate.

21. The method of claim 20, further comprising the step of separating the film into particles.

22. The method of claim 21, further comprising the preliminary step of masking the semiconductor or insulator substrate to define a pattern to define morphology in the particles as they separate from the thin film.

23. The method of claim 19, further comprising the step of:

generating an interference pattern in the reflectance spectrum by illuminating one or more particles;

a particle code is determined from the interference pattern.

24. The method of claim 19 further comprising the step of spatially defining the semiconductor or insulator substrate to perform said etching step at a spatially defined location or locations.

25. The method of claim 24, wherein said varying step further varies the etching conditions in different spatially defined locations to perform multiple encodings in the thin film.

26. The method of claim 25, further comprising the step of separating said thin film from said semiconductor or insulator substrate.

27. The method of claim 26, further comprising the step of separating the film into particles.

28. A method of identifying an analyte attached to the encoded particle of claim 10, or identifying a subject containing the encoded particle of claim 10, the method comprising the steps of:

associating the encoded particles with an analyte or a subject;

generating an interference pattern in the reflectance spectrum by illuminating the particle;

determining a particle code from the interference pattern;

identifying the analyte or subject according to the determining step described above.

29. The method of claim 28, further comprising the step of assigning the particles to the analyte by modifying the particles with specific receptors or targeting moieties.

30. The method of claim 29, wherein the targeting moiety is a sugar or a polypeptide.

31. The method of claim 30, further comprising the step of indicating binding of the analyte by fluorescent labeling or autofluorescence of the analyte.

32. A method of encoding micron-sized particles, the method comprising the steps of:

etching the wafer to form a thin film having an altered porosity that will produce a detectable optical feature in response to the illumination, the optical feature selected from a library of optical features;

subjecting the wafer to an electropolishing current to remove the porous membrane from the wafer;

the film is cut into micron-sized particles, each of which retains the optical features resulting from the etching step described above.

33. The method of claim 32, further comprising the step of modifying the particles with specific receptor or targeting moieties.

34. Encoded micron-sized particles (10, 10a) having an encoding library, the encoding being embedded in their physical structure by refractive index variations between different regions of the particles.

35. The encoded micron-sized particle of claim 34 wherein the refractive index change is caused by a varying porosity.

36. The encoded micron-sized particle of claim 34 wherein different regions of the particle have different thicknesses.

37. The particle of claim 34, further comprising a receptor.

38. The particle of claim 37, wherein said receptor is a receptor for a biological analyte.

39. The particle of claim 37, wherein said receptor is a receptor for a chemical analyte.

40. The particle of claim 37, wherein said receptor is a receptor for a gaseous analyte.

41. The particle of claim 37, further comprising a fluorescent label for analyzing the particle.

42. The particle of claim 34 wherein the film comprises porous silicon.

43. Optically encoded particles (10, 10a) having a porosity whose optical reflectance spectrum can be identified as a significant interference pattern from one of the patterns in the library of patterns in order to positively identify the particles, and to identify spectral shifts in the presence of an analyte.

44. A method of identifying particles as claimed in claim 43, the method comprising performing a principal component analysis to match the optical reflectance spectrum with the one pattern in the library of patterns.