U.S. patent application number 14/291532 was filed with the patent office on 2015-03-19 for manufactured product with optically encoded particle tag and id method.
This patent application is currently assigned to The Regents of the University of California. The applicant listed for this patent is The Regents of the University of California. Invention is credited to Frederique Cunin, Jamie Link, Michael J. Sailor, Thomas Schmedake.
Application Number | 20150076412 14/291532 |
Document ID | / |
Family ID | 27734488 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150076412 |
Kind Code |
A1 |
Sailor; Michael J. ; et
al. |
March 19, 2015 |
MANUFACTURED PRODUCT WITH OPTICALLY ENCODED PARTICLE TAG AND ID
METHOD
Abstract
The invention concerns an article of manufacture that is a
manufactured product marked with a microparticle and a method of
marking manufactured produces. The microparticle tag includes a
predetermined code embedded in its physical structure by refractive
index changes between different regions of the particle. The
particle has a diameter of a few hundred microns or less and has a
plurality of layers.
Inventors: |
Sailor; Michael J.; (La
Jolla, CA) ; Schmedake; Thomas; (Charlotte, NC)
; Cunin; Frederique; (Cardiff, CA) ; Link;
Jamie; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
27734488 |
Appl. No.: |
14/291532 |
Filed: |
May 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10503217 |
Aug 2, 2004 |
8765484 |
|
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PCT/US03/03040 |
Jan 31, 2003 |
|
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14291532 |
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60355234 |
Feb 7, 2002 |
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Current U.S.
Class: |
252/408.1 ;
427/212 |
Current CPC
Class: |
Y10T 436/143333
20150115; B05D 2451/00 20130101; G01N 21/31 20130101; G06K 19/0614
20130101; G06K 19/06084 20130101; G09F 3/00 20130101; B05D 1/00
20130101; Y10T 436/13 20150115; Y10T 436/144444 20150115; G01N
21/45 20130101 |
Class at
Publication: |
252/408.1 ;
427/212 |
International
Class: |
G06K 19/06 20060101
G06K019/06; B05D 1/00 20060101 B05D001/00 |
Claims
1. An article of manufacture, comprising: a manufactured product
marked with a microparticle, wherein the microparticle is
incorporated into the manufactured product; and the microparticle
has a diameter of a few hundred microns or less and comprises a
first porous layer having a predetermined first porosity and
thickness; and a plurality of n additional porous layers having
predetermined thicknesses and porosities, at least one of which has
a predetermined porosity differing from said first porosity;
wherein said predetermined porosities and thicknesses of said first
porous layer and said n additional porous layers are configured to
produce an interference pattern in the reflectivity spectrum having
a distinct series of peaks such that the interference pattern forms
an optical signature having a particular predetermined code
determined by the porosities and thicknesses of said first porous
layer and said n additional porous layers.
2. The article of claim 1, wherein the manufactured product
comprises a powder.
3. The article of claim 1, wherein the manufactured product
comprises a paste.
4. The article of claim 1, wherein the manufactured product
comprises a liquid.
5. The article of claim 1, wherein the manufactured product
comprises a biological product.
6. The article of claim 1, wherein the manufactured product
comprises a chemical product.
7. The article of claim 1, wherein said first porous layer and said
n additional porous layers are formed from a semiconductor.
8. The article of claim 7, wherein said semiconductor comprises
silicon.
9. The article of claim 1, wherein said first porous layer and said
n additional porous layers are formed from an insulator.
10. The article of claim 1, wherein the microparticle has a
diameter in the range from a few hundred nanometers to a few
hundred microns.
11. An article of manufacture, comprising a manufactured product
marked with a microparticle, wherein the microparticle is
incorporated into the manufactured product and the microparticle
comprises an encoded micron-sized porous particle having a
particular predetermined code embedded in its physical porous
structure by refractive index changes between different porous
regions of the particle, wherein the physical porous structure has
a plurality of layers some of which have separate predetermined
mismatched optical thicknesses and the microparticle has a diameter
of a few hundred microns or less and is freestanding separated from
any substrate other than the manufactured product itself.
12. The article of claim 11, wherein the porosity varies
periodically.
13. The article of claim 12, wherein the particle comprises sets of
porosities each varying to produce a code detectable in the
reflectivity spectrum.
14. The article of claim 11, wherein the manufactured product
comprises a powder.
15. The article of claim 11, wherein the manufactured product
comprises a paste.
16. The article of claim 11, wherein the manufactured product
comprises a liquid.
17. The article of claim 11, wherein the manufactured product
comprises a biological product.
18. The article of claim 11, wherein the manufactured product
comprises a chemical product.
19. A method of marking a manufactured product, the method
comprising incorporated an encoded microparticle in the
manufactured product during manufacture, wherein the encoded
microparticle comprises an encoded micron-sized porous particle
having a particular predetermined code embedded in its physical
porous structure by refractive index changes between different
porous regions of the particle, wherein the physical porous
structure has a plurality of layers some of which have separate
predetermined mismatched optical thicknesses and the microparticle
has a diameter of a few hundred microns or less and is freestanding
separated from any substrate other than the manufactured product
itself.
Description
PRIORITY CLAIM AND REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of and claims priority
under 35 U.S.C. .sctn.120 from prior co-pending application Ser.
No. 10/503,217 filed Aug. 2, 2004, which was a 35 U.S.C. .sctn.371
application from PCT application PCT US 03/03040, filed Jan. 31,
2003, which claimed priority under 35 U.S.C. .sctn.119 from prior
provisional application Ser. No. 60/335,234, which was filed Feb.
7, 2002.
TECHNICAL FIELD
[0002] A field of the invention is encoding. Additional exemplary
fields of the invention include the life sciences, security,
product marking, food processing, agriculture, and chemical
detection.
BACKGROUND ART
[0003] A well-appreciated need for labeling exists in society.
Labeling is a fundamental basis for tracking and identifying.
Encoding can be used as a form of labeling understood by persons or
equipment, as in the case of bar coding. At the microscale,
however, labeling/encoding itself becomes difficult.
[0004] Strategies to encode microscale materials have accordingly
received increased attention for such uses as high-throughput
screening in the fields of drug discovery, genetics screening,
biomedical research, and biological and chemical sensing.
Concurrent research strategies for measuring an increased number of
analytes while minimizing the necessary sample volume have focused
on either on-chip spatially differentiated arrays or encoded beads.
Large arrays have been developed for biological and/or chemical
sensing purposes by making use of positional encoding to register
specific analyte responses. The main advantage of using an array
over a conventional single analyte sensor is the ability to process
and analyze a large number of analytes simultaneously. Positional
arrays, however, can suffer from slow diffusion rates and limits on
the concentration ranges of analytes being sensed. An alternative
approach is to use individually encoded beads.
[0005] Early attempts to encode particles used fluorescent or
infrared-active molecules as binary markers. More recently, cadmium
selenide quantum dots have been demonstrated as viable candidates
for particle encoding based on their unique fluorescent properties.
Quantum dots have the advantage over organic molecules of increased
stability towards photobleaching, sharper fluorescence peaks,
improved solubility characteristics, and large excitation frequency
ranges. With six colors (limited to the peak width of the
fluorescence in the visible range) and ten intensity levels,
10.sup.6 particles could theoretically be encoded. In practice,
this number is difficult to obtain because of spectral overlap and
sample inhomogeneities. Also, despite the increased photostability
of quantum dots, fluorescence quenching is still possible, casting
uncertainty on using relative intensity measurements as a reliable
encoding method.
[0006] Another encoding strategy has used sub-micron metallic rods.
The sub-micron metallic rods are prepared by electrodeposition of
metals on a porous membrane in alternating strips of controlled
thickness. Different reflection characteristics of the various
metals are used as a barcode for identification purposes.
Reflection spectroscopy does not have the disadvantage of
photobleaching inherent with fluorophores. Additionally,
fluorescent analytes do not interfere with the particle signal.
Deposition of rods is a relatively complex process, however, and
may be difficult to apply as an encoding strategy where, for
example, a large number of codes is desirable because each rod must
be brought into focus in an optical reader (such as a microscope)
in order to read out the codes. There remains a need for encoding
strategies at the microscale.
SUMMARY OF THE INVENTION
[0007] The invention concerns an article of manufacture having a
manufactured product marked with a microparticle and a method of
marking a manufactured product. The microparticle tag includes a
predetermined code embedded in its physical structure by refractive
index changes between different regions of the particle. The
particle has a diameter of a few hundred microns or less and has a
plurality of layers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a schematic diagram of a multi-layer encoded
particle of the invention;
[0009] FIGS. 2A and 2B illustrate a preferred embodiment Fourier
transform particle decoding;
[0010] FIG. 3A illustrates an exemplary etching waveform for a
preferred embodiment method of Rugate particle encoding;
[0011] FIG. 3B illustrates a preferred embodiment Rugate particle
decoding;
[0012] FIG. 4 illustrates a preferred embodiment method of
fabricating encoded particles;
[0013] FIG. 5 shows the optical reflectivity spectrum of a single
preferred embodiment encoded particle in laboratory air (solid
line) and in air containing a small amount of ethanol vapor (dashed
line);
[0014] FIG. 6 shows the intensity of reflected laser light (632 nm)
from a preferred embodiment encoded porous silicon Rugate particle
measured for three exposure/evacuation cycles using (from bottom to
top as indicated) acetone, ethanol, toluene and water analytes at
their saturation vapor pressures;
[0015] FIG. 7 is an image of exemplary preferred embodiment encoded
particles formed in a wafer by a spatially defined, periodically
varying etch;
[0016] FIG. 8 plots the reflectivity spectra from 15 separately
coded exemplary preferred embodiment sample particles;
[0017] FIG. 9A plots the reflectivity spectra from exemplary
preferred embodiment single Rugate encoded sample particles and
triply encoded Rugate sample particles;
[0018] FIG. 9B is a schematic diagram of an exemplary preferred
embodiment multiple Rugate encoded particle; and
[0019] FIG. 10 plots decoding results for exemplary preferred
embodiment single Rugate encoded particles prepared for biological
screening.
BEST MODE OF CARRYING OUT THE INVENTION
[0020] The invention concerns a particle having a code embedded in
its physical structure by refractive index changes between
different regions of the particle. A change in the refractive index
is preferably obtained by varying porosity formed in the particle.
Reflections taken from the particles produce an optical signature,
in the visible and/or non-visible wavelengths. In preferred
embodiments, the number of peaks, their locations, and intensities
can be used to produce a high number of unique optical signatures.
In preferred embodiment formation methods, a multi-layer porous
encoded structure is produced by an etching process during which
the etching conditions are varied during pore formation. A dicing
may be conducted to form individual encoded particles having a
range of small sizes, e.g., from hundreds of nanometers to hundreds
of microns.
[0021] Methods and particles of the invention are applicable to 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 marking. Various attributes of the particles and methods of
the invention enable a wide range of applications in various
industries. The small size of the particles facilitates ready
incorporation into various hosts, e.g., products, test kits,
assays, powders (such as explosives for identification), pastes,
liquids, glass, paper, and any other host or system that can accept
small particles. In vivo detection is enabled by biocompatible
particles of the invention, which may then be queried, for example,
through tissues using near infrared and infrared wavelengths that
penetrate tissues.
[0022] In accordance with the aforementioned exemplary aspects and
applications of the inventions, preferred embodiment particles are
identified by the code inherent to the reflectivity spectrum of
their varying porous structure. In another aspect of the invention,
matter, e.g., biological or chemical matter, is hosted by the
porous structure and the particle becomes a tag identifying the
matter hosted by the pores. In another aspect of the invention, a
variance in the reflectivity spectrum of an encoded particle can
indicate the presence, absence or quantity of matter within the
particle's pores.
[0023] Referring to FIG. 1, a preferred embodiment encoded particle
10 is shown in cross-section. The encoded particle 10 includes a
multi-layer porous thin film having layers or regions
12.sub.1-12.sub.N. Multi-layer, as used herein, means that there
must be a plurality of regions having distinct porosity.
Transitions between porosity in some embodiments may be gradual.
This means that multi-layer encompasses both structures having
multiple gradual transitions of porosity and structures having
multiple abrupt transitions of porosity. Consistent with this
definition, the layers 12.sub.1-12.sub.N are defined by varying
porosity, which may change gradually or abruptly. Also, the use of
"layer" encompasses separate deposits, for example, but also
encompasses continuous structures having the varying porosity. In
other words, "layer" includes but does not solely imply separate
formation processes or depositions. The multi-layer porous thin
film structure of layers or regions 12.sub.1-12.sub.N having
varying porosity is shown in FIG. 1 as being formed on a substrate
14. However, embodiments of the invention include particle
structures of multi-layer thin films such as the layers
12.sub.1-12.sub.N released from a substrate upon or from which they
were initially formed. The porous layers 12.sub.1-12.sub.N are
encoded to produce an interference pattern in the reflectivity
spectrum that forms an optical signature. Light reflected at the
interfaces between the porous layers 12.sub.1-12.sub.N interferes
with light from interfaces between other ones of the layers to
generate an interference pattern in the reflectivity spectrum.
Particles 10 of the invention may be specifically encoded by
controlling etching conditions and layer thicknesses during
formation of the particle 10. The refractive index at layer
interfaces, chemical composition, and thickness of each layer
12.sub.1-12.sub.N affects the optical signature generated by a
particular particle. Thus, varying the relative porosity between
layers in an individual particle (to affect the refractive index)
and varying the layer thickness during formation of the particle 10
permits the tailoring of particular optical signatures in the
reflectivity spectrum. Porosity also affects the intensity of peaks
in the reflectivity spectrum, providing additional encoding
potential.
[0024] The porous layers 12.sub.1-12.sub.N may be formed of any
porous semiconductor or insulator. In preferred embodiment
particles of the invention, porous silicon is used to form the
layers 12.sub.1-12.sub.N. Controlled anodic etching of crystal
silicon in hydrofluoric acid solution permits control of both the
porosity and thickness of porous layers 12.sub.1-12.sub.N. The time
of etching controls the thickness of a porous layer, while the
etching current density controls the porosity. The thicknesses and
porosities of layers 12.sub.1-12.sub.N are varied with respect to
each other to produce a particular optical signature.
[0025] Variance in the porosity and thicknesses may in some
embodiments be arbitrary, and in other embodiments follow a
periodic pattern. In some embodiments, the porosity may vary
gradually and in others the porosity may change abruptly from layer
to layer. Porous silicon is a preferred material for the layers
12.sub.1-12.sub.N. Porous silicon has a number of demonstrated
advantages. For example, porous silicon has been demonstrated to be
biocompatible. In addition, the surface chemistry of oxidized
porous silicon is effectively that of silica. Accordingly, the
surface chemistry is well understood for biochemical derivatization
and ligand immobilization.
[0026] In preferred embodiments, the layers 12.sub.1-12.sub.N are
formed to include a receptor material within the porous structure.
The purpose of the receptor is to bind a particular analyte of
interest. Exemplary receptors (also referred to as binders) are
disclosed, for example, in U.S. Pat. No. 6,248,539 entitled "Porous
Semicoductor Based Optical Interferometric Sensor". Receptor
molecules may be adsorbed or otherwise associated with the porous
silicon layers 12.sub.1-12.sub.N by any approach that leads to the
tethering of the receptor molecules to the porous layers
12.sub.1-12.sub.N. This includes, without limitation, covalently
bonding the receptor molecules to the semiconductor, ionically
associating the receptor molecules to the layers, adsorbing the
receptor molecules onto the surface of the layers, or other similar
techniques. Association can also include covalently attaching the
receptor molecules to another moiety, which is in turn covalently
bonded to the porous layers 12.sub.1-12.sub.N, or binding the
target molecule via hybridization or another biological association
mechanism to another moiety which is coupled to the porous layers
12.sub.1-12.sub.N. Specific additional examples include receptor
ligands that have been attached to porous silicon layers to produce
biosensors. An analyte bound to a particle 10 of the invention
becomes identifiable and traceable by the encoding provided by the
particle 10.
[0027] Encoding is possible for both intensity and wavelength
properties of multi-layer films 12.sub.1-12.sub.N. A preferred
embodiment is a particle 10 having multi-layer films
12.sub.1-12.sub.N that have mismatched optical thicknesses. Optical
thickness is defined as the refractive index of a layer multiplied
by its metric thickness. Referring to FIGS. 2A and 2B, a particle
10 encoded in such a manner reveals an optical signature in a
Fourier transform of the resulting reflectivity interference
spectrum. An exemplary resulting interference spectrum is shown in
FIG. 2A. The Fourier transform shown in FIG. 2B reveals an optical
signature with well-resolved peaks. Particles 10 may be set to have
a distinct series of peaks (a, b, c).
[0028] The intensity of peaks in the reflectance spectrum is
controlled by the refractive index at interfaces between layers
12.sub.1-12.sub.N, determined by a change in porosity between
adjacent layers. Such change may be gradual or sharp. The position
of peaks is controlled by adjusting layer thicknesses. Additional
encoding is possible by variation of the relative intensities of
each reflectivity peak, which can be engineered into particles 10
of the invention by adjustment of the electro chemical etch
parameters to control porosity of the layers 12.sub.1-12.sub.N.
Accordingly, an N-layer particle 10 having A resolvable positions
for each peak and B resolvable intensities can encode (A*B).sup.N
particles. Additionally, a particle 10 having N peaks with A
resolvable positions for each peak with any combination of order of
relative intensities can encode one of N!(A).sup.N.
[0029] Another encoding strategy involves periodic structures.
Exemplary periodic structures include particles 10 having layers
12.sub.1-12.sub.N configured by porosity and thickness to form a
Bragg stack or a Rugate filter. Bragg stacks, for example, may be
generated by alternating layers having matched optical thicknesses.
A Bragg stack defined by varying porosity layers 12.sub.1-12.sub.N
in a particle 10 of the invention will produce peaks in the
reflectivity spectrum with full width half maximum peaks in the
reflectivity spectrum that are very well resolved, e.g., .about.10
nm. Rugate filters produced by variation of the refractive index of
the interfaces through multi-layer structures 12.sub.1-12.sub.N
also generate similarly narrow peaks in the reflectivity spectrum
while also suppressing side bands and higher order reflections.
[0030] FIGS. 3A and 3B illustrate a preferred embodiment Rugate
particle encoding strategy. A Rugate encoded particle may be
created by etching a semiconductor or insulator with a periodic
variance of etching conditions, such that the refractive index in
the material varies in a sinusoidal (or apodised sinusoidal)
function. The structures can be generated by etching the silicon
wafer with a pseudo-sine current waveform. FIG. 3A indicates that a
period for an exemplary sine wave variation of etching current
density (n) in an etch used to produce the exemplary embodiment was
18 seconds. As seen in FIG. 3B, a well-resolved narrow peak results
from the encoding. The intensity and location of the peak can be
varied with layer thickness and refractive index.
[0031] Referring now to FIG. 4, a preferred method for forming an
encoded porous particle 10 is shown. A suitable semiconductor or
insulator, e.g., a silicon wafer, is selected for processing (step
14). For example, silicon wafers may be cut to size and be masked
to have portions exposed for etching. An exemplary suitable silicon
material is a single crystalline silicon wafer. Spatial encoding is
then defined (step 16). The spatial encoding defines a range of
codes over the material to be etched. Conducting a spatially
resolved etch allows codes to be programmed in particle-sized
sections of the wafer. An exemplary spatially resolved etch is
disclosed in U.S. Pat. No. 5,318,676, entitled "Photolithographic
fabrication of luminescent images on porous silicon structures",
published Jun. 7, 1994. In an alternative process, the step of
spatial defining (step 16) is omitted. For example, a single wafer
or an area of wafer could be etched to include particles having a
single code. In that case, other wafers could be etched to have
particles having a different code. Anodic etching is then
commenced, for example, in an aqueous solution of hydrofluoric acid
and ethanol (step 18). Etching is then conducted with etching
conditions varying according to the defined encoding strategy (step
20). A code or codes of the invention are etched into the wafer.
The traverse (vertical direction in FIG. 1) encoded but still
connected particles may be lifted off from the silicon wafer (step
22), for example by a high level of electropolishing current. Areas
between spatially defined etch sections may be cut to separate
differently encoded wafer sections. Individual particles are then
separated (step 24) in a dicing that may be conducted, for example,
by mechanical agitation or ultrasonic fracturing. The particle
separation (step 24) preferably produces micron-sized particles,
e.g., particles in a range from a few hundred nanometers to a few
hundred micrometers. A step of particle designation (step 26) may
be conducted after the particle separation (step 24) or subsequent
to step 20 or step 22. Particle designation may comprise, for
example, chemical modification of the porous multi-layer structure
12.sub.1-12.sub.N for specific biological, biomedical, electronic,
or environmental applications. As an example, the particles can be
modified with a receptor for a desired analyte or with a targeting
moiety (such as a sugar or a polypeptide). Additionally, binding
can be signaled, for example, by fluorescence labeling of analytes
or analyte autofluoresence. In use of particle 10, the particle can
be identified by its optical signature upon binding of the
designated targeted analyte. This step of designation may also be
omitted in embodiments of the invention.
[0032] In other embodiments of the invention, encoded particles can
be placed into a suitable hosts, namely any liquid, powder, dust,
or other material that will hold encoded micron sized particles of
the invention. Particles placed in hosts, for example, could be
used to identify the source of a manufactured powder such as an
explosive. Another potential host is an animal. Particles of the
invention being biocompatible may be implanted in vivo into an
animal host. The reflectivity spectrum of preferred embodiment
porous silicon particles 10 of the invention, for example,
encompasses the visible, near infrared, and infrared spectra. This
presents the opportunity to sense the code of a particle of the
invention through barriers such as living tissue.
Example Embodiments and Experimental Data
[0033] Example embodiments of the invention will now be discussed.
Experimental data is included for the purpose of illustrating to
artisans the potential of the invention. Where given, equipment is
specified only to allow artisans to understand experimental data
reported herein. Commercial embodiment devices of the invention may
take substantially different form, permitting low cost mass
manufacturing, for example.
[0034] A first example embodiment is stand-off detection. This is a
chemical detection technique to identify an analyte from a
distance. A particle 10 of the invention includes a receptor to
sense a particular analyte. Both the code of the particle and an
indication of binding of the analyte can be detected in the
reflectivity spectrum, for example, with use of a low power laser.
The receptor, for example, can be specific to sense biomolecules or
to attach the encoded particle to a cell, spore, or pollen
particle.
[0035] A test of stand-off detection was conducted with exemplary
encoded multi-layer porous silicon films. The multi-layered porous
silicon films were prepared by an electrochemical etch of a (100)
oriented polished Si wafer (p.sup.++-type, B doped, <1
m.OMEGA.-cm resistivity) in a 1:3 ethanol:48% aqueous HF solution.
The etching current density was modulated periodically with a
pseudo-sine wave (between 11.5 and 34.6 mA/cm.sup.2) to generate a
sinusoidally varying porosity gradient. The films were removed from
the substrate by applying a 30 second electropolishing pulse of
current density of 600 mA/cm.sup.2. The freestanding films were
then made into particles by mechanical grinding or by ultrasonic
fracture to produce particles of sizes ranging from several hundred
nanometers to a few hundred microns. The optical reflectivity
spectrum in FIG. 5 approximates a Rugate filter, displaying a sharp
reflection maximum at a wavelength and source-sample-detector angle
that satisfies the Bragg equation and appropriate phase matching
condition.
[0036] The particles were immobilized on a glass plate and mounted
in a gas dosing chamber fitted with an optical window and Baratron
pressure gauge. The particles were illuminated with a 10 MW He/Ne
laser. The as-formed particles strongly reflect the 632 nm light of
the He/Ne laser at a wavelength in air, as seen in FIG. 5. The
spectral position of the laser used to acquire the data presented
in FIG. 5 is shown for comparison (vertical arrow). The data were
taken using an Ocean Optics CCD visible spectrometer at the focal
plane of an optical microscope. When exposed to analyte vapors,
capillary condensation causes the reflectivity spectrum of the
particles to shift to longer wavelengths due to an increase in the
refractive index of the porous medium and the particles are
observed to go dark.
[0037] The relative change in light intensity simultaneously
reflected from many of the particles was quantified at a fixed
wavelength (632 nm) for a series of condensable analyte vapors, as
seen in FIG. 6. The vapor pressure at 25.degree. C. for each of
these analytes is 222, 60, 28, and 24 Torr, respectively. Relative
reflected light intensity was measured as the photocurrent from an
amplified Si photodiode mounted at the objective of the 8-inch
Schmidt-Cassegrain collection optics. The sample was 20 m from the
laser and detection optics. Spectra are offset along the y-axis for
clarity. The vapors were all introduced to the exposure chamber at
their saturation vapor pressures. The intensity of reflected light
was measured at a distance of 20 m in the presence of normal
fluorescent room lighting using chopped light and phase-sensitive
detection (Stanford Instruments SR-510 lock-in amplifier). No other
optical or electronic filtering was used. The specificity of
adsorption and/or microcapillary condensation at porous Si surfaces
depends dramatically on the surface chemistry, and the
hydrogen-terminated, hydrophobic as-formed material used in the
experiments has a much greater affinity for hydrophobic versus
hydrophilic analytes. Thus, the particles are relatively
insensitive to water vapor at a partial pressure comparable to that
used for the more hydrophobic organic analytes. No attempt was made
to provide acoustic or vibrational isolation of the sample or
optics, and most of the noise observed in the data is attributed to
laboratory vibrations. Sensitivity should be further enhanced using
a near infrared laser light source, where background radiation and
atmospheric adsorption and scattering are less significant.
[0038] Another preferred exemplary application of the invention is
for biomolecular screening via the encoded particle 10 of the
invention. Millions of codes are possible with a small number of
layers. A simple antibody-based bioassay using fluorescently tagged
proteins has been tested. Periodic Rugate style encoding was used
as described above with respect to the exemplary chemical sensing
embodiments. By masking the wafer before etching, well-defined
slabs of particles were generated, as seen in FIG. 7.
[0039] The FIG. 7 particles were prepared to display a photonic
spectral maximum at 632 nm. The scale in the inset (reproduced
above the figure for clarity) corresponds to 2 .mu.m per small
division. The multi-layered encoded particles generated in this
fashion display a very sharp line in the optical reflectivity
spectrum. This line can appear anywhere in the visible to
near-infrared spectral range, depending on the waveform used in the
programmed etch.
[0040] Exemplary waveforms for 15 separate codes are shown in FIG.
8. FIG. 8 shows the reflectivity spectra of 15 porous-silicon
multi-layered samples prepared using a sinusoidal etch (Rugate
encoded structure). Each of the samples contains a single Rugate
frequency code. Spectra were obtained using a Cambridge Instruments
microscope with a 70.times. objective. The sample was illuminated
using a tungsten lamp, and the reflected light spectrum was
measured with an Ocean Optics SD2000 CCD (charge-coupled device)
spectrometer. The sample particles were prepared by anodically
etching p.sup.++ type, B-doped, (100)-oriented silicon
(resistivity<1 m.OMEGA.-cm.sup.2) in a solution of 48% aqueous
HF:ethanol (3:1 by volume). Typical etch parameters for a Rugate
structure used in a pseudosinusoidal current waveform oscillating
between 11.5 and 19.2 mA cm.sup.-2 with 50 repeats and a
periodicity of 18 s. Films were removed from the substrate using a
current pulse of 460 mA cm.sup.-2 for 40 s. Lithographically
defined particles were prepared by applying an S-1813 photoresist
(Shipley) to the wafer before the electrochemical etch (spin coated
at 4,000 r.p.m. for 60 s, soft-baked at 90.degree. C. for 2 min.,
ultraviolet-exposed using a contact mask aligner, hard-baked at
120.degree. C. for 30 min. before development). The spectral
features exemplified by FIG. 8 can be much narrower than the
fluorescence spectrum obtained from a molecule or core-shell
quantum dot.
[0041] FIG. 9A shows the reflectivity spectra of porous silicon
Rugate encoded particles etched with a single periodicity (bottom)
and with three separate periodicities (top). FIG. 9B schematically
illustrates a preferred embodiment multiple encoded particle 10a,
wherein there are three sets of encoded layers 12, 16, and 18.
Multiple Rugate codes may be separated spatially, but also may be
etched in the same physical location, as sets of multi-layers
formed at different depths, each forming a separate Rugate
encoding. Each of the layer sets 12, 16, and 18 includes a
periodically varying porosity to produce a separate Rugate, or
alternatively, Bragg, code.
[0042] The example particles display peaks in the reflectivity
spectrum characteristic of their multi-layered structures. The
sample represented in the bottom spectrum was etched using a
sinusoidal current variation between 11.5 and 19.2 mA cm.sup.-2
with 50 repeats and a periodicity of 18 s. The triply encoded
particle (triple Rugate) represented by the top spectrum was
prepared using a sinusoidal current variation oscillating between
11.5 and 34.6 mA cm.sup.-2 with a periodicity of 10 s for 20
periods (520 nm), 12 s for 45 periods (610 nm), and 14 s for 90
periods (700 nm). The approximate thickness of this sample is 15
.mu.m. Spectra are offset along the y axis for clarity.
[0043] To test the reliability of the encoding approach in a
biological screening application, we prepared two different batches
of encoded particles as single Rugate structures. Both batches of
particles were ozone-oxidized to improve their stability in aqueous
media and to provide a hydrophilic surface. The particles were
oxidized in a stream of O.sub.3 diluted with compressed air.
Control particles coded with a 750-nm spectral feature were treated
with concentrated BSA (Sigma, 5 g in 100 ml of double-distilled
water) and incubated at 37.degree. C. under 5% CO.sub.2 in air for
three hours. The 540-nm-encoded test particles were exposed to 50
.mu.g ml.sup.-1 rat albumin in coating buffer (2.93 g NaHCO.sub.3,
1.61 g Na.sub.2CO.sub.3 in 1,000 ml double-distilled water), and
incubated at 37.degree. C. under 5% CO.sub.2 for two hours. The
test particles were then exposed to a 1:100 dilution of primary
rabbit anti-rat-albumin antibody in a concentrated solution of BSA
at 37.degree. C. under 5% CO.sub.2, for one hour. Both batches of
particles were then mixed together and incubated for one hour in
the presence of FITC-(fluorescein isothiocyanate) conjugated goat
anti-rabbit immunoglobulin-G in a BSA solution. Detection of
analyte binding to the encoded particles was then performed by
fluorescence and spectral reflectance microscopy.
[0044] Decoding results are shown in FIG. 10. Decoding, performed
on 16 particles, yielded the following results: among eight green
fluorescent particles, eight particles were positively decoded as
belonging to the functionalized rat albumin batch (plot A in FIG.
10). Among the eight non-luminescent particles, six particles were
correctly decoded (plot B in FIG. 10), one particle displayed the
incorrect code and one particle was unreadable. Presumably, the
particle that displayed the incorrect code belonged to the first
batch but was not sufficiently functionalized with rat albumin to
generate fluorescence in the antibody assay. This is understandable
because in the experiment the rat albumin was not covalently
attached to the silica-coated particles. A variety of stable
chemical modification chemistries have been developed for oxidized
and non-oxidized porous silicon, and some of these have been
demonstrated with specific antibodies or receptors. Thus, the issue
of immobilizing biochemical or chemical components is easily
addressable. Additionally, chemical modification can prevent
corrosion in aqueous media, which may otherwise lead to undesirable
shifts in the optical code and/or unreadable particles. In the
conducted experiments, no passivating chemical treatments, other
than ozone oxidation to generate a layer of silica, were performed,
and upon immersion in basic aqueous media the spectral codes were
observed to shift between 0 and 50 nm depending on the incubation
times.
[0045] The layered porous-silicon encoded structures offer several
advantages over existing encoding methodologies. Porous-silicon
encoded structures can be constructed that display features
spanning the visible, near-infrared and infrared regions of the
spectrum. In addition, the reflectivity spectra of Rugate filters
can exhibit much sharper spectral features than can be obtained
from a gaussian ensemble of quantum dots. Thus, more codes can be
placed in a narrower spectral window with the porous encoded
structures. Unlike encoding schemes based on stratified metallic
nanorods, fluorescence or vibrational signatures, encoded particles
of the invention can be probed using light diffraction techniques;
thus it is not necessary to use imaging optics in order to read the
codes. Encoded particles may be assayed using a conventional
fluorescence tagging technique, and sensitive chemical and
biochemical detection can also be built into the optical structure
of the encoded particles, eliminating the need for fluorescent
probes and focusing optics. In addition, because preferred
embodiment oxidized porous-silicon encoded particles present a
silica-like surface to the environment, they do not readily quench
luminescence from organic chromophores, and they can be handled and
modified using the chemistries developed for glass bead bioassays.
Silicon-based encoded particles may be readily integrated with
existing chip technologies.
[0046] The use of encoded silicon particles of the invention in
medical diagnostic applications has advantages over organic dyes or
quantum dots. In vivo studies have shown the biocompatibility of
porous silicon, as well as the long-term stability of reflectance
data from multilayer structures. Additionally, 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
diagnostics. Finally, because the porous codes are an integral and
orderly part of the porous structure, it is not possible for part
of the code to be lost, scrambled or photobleached, as can occur
with quantum dots or fluorescent molecules.
[0047] While specific embodiments of the present invention have
been shown and described, it should be understood that other
modifications, substitutions and alternatives are apparent to one
of ordinary skill 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.
[0048] Various features of the invention are set forth in the
appended claims.
* * * * *