# US 2025/0154585 A1 · Illumination of integrated analytical systems

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- Number: US 2025/0154585 A1
- Document ID: US20250154585A1
- Status: Published application
- Kind: A1
- Application number: 19/020,171
- Filed: 2025-01-14
- Published: 2025-05-15
- Earliest claimed: 2010-02-19
- Assignees: Pacific Biosciences of California, Inc.
- Applicants: Pacific Biosciences of California, Inc.
- Inventors: Adrian FEHR; Nathaniel Joseph McCAFFREY; Stephen TURNER
- Web page: https://patentgrep.com/patent/US20250154585A1

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- B01L 3/502707: {characterised by the manufacture of the container or its components}
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B82Y 20/00: Nanooptics, e.g. quantum optics or photonic crystals
- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/6869: Methods for sequencing
- G01N 21/03: Cuvette constructions
- G01N 21/0303: {Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment (G01N21/031 - G01N21/15 take precedence)}
- G01N 21/05: Flow-through cuvettes (G01N21/09 takes precedence; handling fluid samples G01N1/10)
- G01N 21/64: Fluorescence; Phosphorescence
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/645: {Specially adapted constructive features of fluorimeters}
- G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates}
- G01N 21/6454: {using an integrated detector array}
- G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging}
- G01N 21/648: {using evanescent coupling or surface plasmon coupling for the excitation of fluorescence}
- G01N 21/75: Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated (systems in which material is burnt in a flame or plasma G01N21/72, G01N21/73)
- G01N 21/77: by observing the effect on a chemical indicator
- G01N 33/54373: {involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings}
- G02B 6/1226: {involving surface plasmon interaction}
- B01L 2300/0654: Lenses; Optical fibres
- B01L 2300/0663: Whole sensors
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/168: Specific optical properties, e.g. reflective coatings
- G01N 2021/0346: {Capillary cells; Microcells}
- G01N 2021/6434: {Optrodes}
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- G01N 2021/6441: {with two or more labels}
- G01N 2021/6463: {Optics}
- G01N 2021/757: {using immobilised reagents}
- G01N 2021/7786: {Fluorescence}
- G01N 2201/067: Electro-optic, magneto-optic, acousto-optic elements
- G01N 2201/068: Optics, miscellaneous
- G01N 2201/08: Optical fibres; light guides
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

An analytical device including an optically opaque cladding, a sequencing layer including a substrate disposed below the cladding, and a waveguide assembly for receiving optical illumination and introducing illumination into the device. The illumination may be received from a top, a side edge, and a bottom of the device. The waveguide assembly may include a nanoscale aperture disposed in the substrate and extending through the cladding. The aperture defines a reaction cell for receiving a set of reactants. In various aspects, the device includes a sensor element and the illumination pathway is through the sensor element. Waveguides and illumination devices, such as plasmonic illumination devices, are also disclosed. Methods for forming and operating the devices are also disclosed.

## Claims

### Claim 1

An analytical device, comprising:
an optically opaque cladding;
a sequencing layer comprising:
a substrate disposed below the cladding; and
at least one nanoscale aperture disposed in the substrate and extending through the cladding, the at least one nanoscale aperture separated by regions of the substrate from any other nanoscale aperture, wherein the at least one nanoscale aperture defines a reaction cell for receiving a set of reactants; and
a waveguide assembly for receiving optical illumination from one of a top of the sequencing layer, a side edge of the sequencing layer, and a combination of the same and for directing the illumination to the at least one reaction cell.

### Claim 2 (depends on 1)

The device of claim 1, further comprising a sensor element disposed in optical communication with the at least one reaction cell, wherein the sensor element is positioned below the waveguide assembly.

### Claim 3 (depends on 2)

The device of claim 2, wherein the illumination is directed to the sensor element with evanescent irradiation.

### Claim 4 (depends on 1, 2, 3)

The device of any one of claims 1 to 3, further comprising a fluidics layer including a fluidic conduit on top of the sequencing layer and in fluid communication with the at least one reaction cell.

### Claim 5 (depends on 1, 2, 3, 4)

The device of any one of claims 1 to 4, wherein the waveguide assembly is integrated into the substrate.

### Claim 6 (depends on 1, 2, 3, 4)

The device of any one of claims 1 to 4, wherein the waveguide assembly comprises a channel waveguide for directing the illumination to an underside of the reaction cell.

### Claim 7 (depends on 4)

The device of claim 4, the device comprising a plurality of reaction cells, wherein the waveguide assembly comprises a planar waveguide for directing the illumination to an underside of one or more the plurality of reaction cells.

### Claim 8 (depends on 6, 7)

The device of claim 6 or 7, the waveguide assembly further comprising an illumination inlet port for receiving the illumination from above the sequencing layer and directing the illumination into the waveguide assembly.

### Claim 9 (depends on 8)

The device of claim 8, wherein the illumination inlet port is an optical pillar having opaque walls, the pillar being positioned adjacent to the at least one reaction cell and extending above the cladding for optically shielding a respective one reaction cell from direct contact with incident illumination.

### Claim 10 (depends on 1)

The device of claim 1, wherein the device comprises a plurality of reaction cells, each reaction cell dimensioned and configured to receive a single molecule.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The device of any one of claims 1 to 10, wherein the device comprises a number of reaction cells selected from the group consisting of less than 1,000, between about 1000 and about 1,000,000, between about 1,000 and about 100,000, between about 100,000 and about 1,000,000, between about 1,000,000 and 10,000,000, and greater than 10,000,000.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The device of any one of claims 1 to 10, wherein the illumination is received in flood fashion from above the sequencing layer.

### Claim 13 (depends on 1)

The device of claim 1, further comprising:
a sensor element disposed in optical communication with the at least one respective reaction cell along a detection pathway;
wherein at least part of the illumination pathway is through the sensor element and along a common pathway as the detection pathway.

### Claim 14 (depends on 1)

The device of claim 1, further comprising an optical element for receiving incident illumination near a zero angle relative to the sequencing layer and redirecting the incident illumination to the at least one reaction cell at a sufficiently high angle for excitation of the set of reactants.

### Claim 15 (depends on 14)

The device of claim 14, wherein the optical element is a prism.

### Claim 16

An analytical device, comprising:
an optically opaque cladding;
a sequencing layer comprising:
a transparent substrate disposed below the cladding; and
at least one nanoscale aperture extending through the cladding to a top of the substrate, each aperture separated from any other aperture by regions of the transparent substrate;
a sensor element disposed in optical communication with the at least one nanoscale aperture along a detection pathway;
a waveguide for receiving illumination and directing the illumination along an illumination pathway through the sensor element to the at least one nanoscale aperture for exciting a material of interest therein;
wherein part of the illumination pathway is essentially coextensive with the detection pathway.

### Claim 17 (depends on 16)

The device of claim 16, wherein the cladding is composed of metal, a metal oxide, and/or a composite of metal and metal oxide.

### Claim 18 (depends on 16, 17)

A system for monitoring analytical reactions, the system comprising:
the device of claim 16 or 17, wherein the at least one nanoscale aperture is a reaction cell;
a set of reactants contained in the at least one reaction cell, the set of reactants including an upconverting phosphor for receiving two or more photons of a first energy level and emitting fewer photons of a second energy level in response, the second energy level being greater than the first energy level.

### Claim 19 (depends on 18)

The system of claim 18, wherein the received two or more photons have a longer wavelength than the emitted photons.

### Claim 20 (depends on 18)

The system of claim 18, wherein the phosphor is selected to have an emission wavelength near the detection edge of the sensor element and substrate.

### Claim 21 (depends on 18)

The system of claim 18, wherein the sensor element is selected to be transmissive to photons of the first energy level and absorbable to photons of the second energy level.

### Claim 22 (depends on 21)

The system of claim 21, wherein the waveguide includes an optical lens for directing the illumination from below the system through the sensor element.

### Claim 23

A method of monitoring analytical reactions, the method comprising:
providing an analytical chip comprising a sequencing layer comprising at least one reaction cell in an optically opaque cladding, the at least one reaction cell for receiving a set of reactants, and a sensor element in a substrate in optical communication with the at least one reaction cell along a detection pathway;
delivering a set of reactants to the at least one reaction cell, the set of reactants including an upconverting phosphor for receiving illumination light having two or more photons at a first energy level and emitting a detection signal having fewer photons at a second energy level in response, the second energy level being greater than the first energy level; and
directing the illumination light to the at least one reaction cell along an illumination pathway under sufficient conditions to cause the phosphor to emit the detection signal at the second energy level.

### Claim 24 (depends on 23)

The method of claim 23, wherein the sensor element is positioned in the illumination pathway.

### Claim 25 (depends on 24)

The method of claim 24, further comprising selecting the upconverting phosphor and sensor element such that the illumination light at the first energy level is transmissive to the sensor element and the detection signal at the second energy level is absorbable by the sensor element.

### Claim 26 (depends on 23, 24, 25)

The method of any one of claims 23 to 25, further comprising directing the detection signal along a common but opposite path of the illumination pathway to the sensor element for detection.

### Claim 27

A device for exciting a fluorophore, said device comprising:
a first region having a reaction cell configured to receive a fluorophore;
a second region laterally adjacent the first region, the second region including a metal-insulator-metal (MIM) structure for providing plasmonic energy to the reaction cell;
a fourth region including an optical waveguide and inlet for receiving optical energy; and
a third region operationally positioned between the second region and the fourth region, the third region including a transition portion at one end of the waveguide adjacent the MIM structure, the transition portion dimensioned and configured to direct the received optical energy to the MIM structure.

### Claim 28 (depends on 27)

The device of claim 27 wherein the inlet is positioned along a side edge of the device.

### Claim 29 (depends on 27)

The device of claim 27 wherein the waveguide is a planar waveguide.

### Claim 30 (depends on 27)

The device of claim 27 wherein the MIM structure and waveguide are essentially concentric rings and the reaction cell is positioned in a center of the ring.

### Claim 31 (depends on 27)

The device of claim 27 wherein the waveguide extends along one surface of the insulator and the insulator is configured for plasmonic confinement of the waveguide.

### Claim 32 (depends on 27)

The device of claim 27 wherein the transition portion is tapered.

### Claim 33 (depends on 27)

The device of claim 27 wherein the transition portion has a height decreasing in direction towards the reaction cell.

### Claim 34 (depends on 27)

The device of claim 27 wherein the transition region is dimensioned and configured to focus the energy into the MIM structure.

### Claim 35 (depends on 27)

The device of claim 27 wherein the waveguide has a tunnel portion extending from an end of the taper to the reaction cell.

### Claim 36

A device for exciting a fluorophore, said device comprising:
a metal layer for generating plasmons based on plasmon modes in response to optical energy;
a dielectric slab extending between the metal layer;
a waveguide disposed between the metal layer and dielectric slab, the waveguide configured to receive optical energy and transmit the optical energy to metal-dielectric interface to generate the plasmon modes; and
a reaction cell disposed adjacent ends of the dielectric slab and metal layer, the reaction cell configured to receive a fluorophore;
wherein the metal layer and slab are in communication with the reaction cell whereby plasmons generated in the metal are transmitted to the reaction cell.

### Claim 37 (depends on 36)

The device of claim 36, wherein the reaction cell is defined by an aperture extending upwardly through the metal layer and dielectric slab.

### Claim 38 (depends on 37)

The device of claim 37, wherein the waveguide extends around at least a portion of a circumference of the reaction cell, an inner diameter of the waveguide extending inward toward the reaction cell.

### Claim 39 (depends on 38)

The device of claim 38, wherein the waveguide and metal layer are ring-shaped and extend entirely around the reaction cell.

### Claim 40 (depends on 37)

The device of claim 37, wherein the aperture is a nanoscale aperture.

### Claim 41 (depends on 36)

The device of claim 36, wherein the dielectric slab extends entirely through the metal layer in a transverse direction to the reaction cell.

### Claim 42 (depends on 36)

The device of claim 36, wherein the dielectric slab is disposed below the waveguide, the slab configured for plasmonic confinement of the waveguide.

### Claim 43 (depends on 36)

The device of claim 36, wherein a portion of a top of the waveguide at an outlet end is optically open to the metal layer.

### Claim 44 (depends on 43)

The device of claim 43, further comprising a cladding layer disposed between another portion of the top of the waveguide at an inlet end and the metal layer for optically confining the waveguide.

### Claim 45 (depends on 36)

The device of claim 36, wherein the waveguide is a channel waveguide.

### Claim 46 (depends on 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)

The device of any one of claims 36 to 45, wherein the waveguide includes a projection extending adjacent the reaction cell, the projection for adjusting the optical energy at an interface with the metal layer.

### Claim 47 (depends on 46)

The device of claim 46, wherein the projection has a taper shape, a dimension of the taper being based on a wavelength of the introduced optical energy and a desired resulting plasmonic wavelength in the metal layer.

### Claim 48 (depends on 36)

The device of claim 36, further comprising a sensor element disposed in a substrate below the dielectric slab and metal layer, the sensor element in optical communication with the reaction cell along a detection pathway.

### Claim 49 (depends on 48)

The device of claim 48, wherein the sensor element is positioned essentially below the reaction cell.

### Claim 50 (depends on 48)

The device of claim 48, further comprising an optically opaque top layer for shielding the reaction cell and the detection pathway.

### Claim 51 (depends on 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)

The device of any one of claims 36 to 50, wherein the introduced optical energy is light having a wavelength of about 0.5 micrometer.

### Claim 52 (depends on 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)

The device of any one of claims 36 to 50, wherein the optical energy is light having a wavelength of about 650 nm, and preferably about 647 nm.

### Claim 53 (depends on 37, 52)

The device of claim 37 or 52, wherein the aperture has a diameter of less than or equal to about 0.6 micrometer.

### Claim 54 (depends on 37)

The device of claim 37, wherein the aperture has a diameter of less than 200 nm.

### Claim 55 (depends on 37)

The device of claim 37, wherein the aperture has a diameter of about 50 nm to about 180 nm.

### Claim 56 (depends on 37)

The device of claim 37, wherein the waveguide has a thickness of about 1 micrometer in a region adjacent the reaction cell.

### Claim 57 (depends on 37)

The device of claim 37, wherein the dielectric slab has a thickness of about 10 nm to about 20 nm.

### Claim 58 (depends on 46)

The device of claim 46, wherein a portion of the dielectric slab extending below the waveguide projection and adjacent to the reaction cell has a length of about 1 micrometer.

### Claim 59 (depends on 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The device of any one of claims 36 to 58, wherein insertion loss in the reaction cell is less than or equal to about 10 dB.

### Claim 60 (depends on 37)

A system comprising:
a planar waveguide layer for receiving light from a light source;
a plurality of analytical devices according to claim 37 disposed within the planar waveguide, the apertures being reaction cells; and
a set of reactants labeled with a fluorophore for excitation by plasmons in at least one of the reaction cells.

### Claim 61 (depends on 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)

A method of monitoring analytical reactions, comprising:
providing the device of any one of claims 36 to 59;
introducing optical energy to the waveguide thereby generating plasmon modes in the metal layer;
applying the plasmons to the reaction cell through the dielectric slab; and
delivering a set of reactants labeled with a fluorophore to the reaction cell under sufficient conditions to cause a reaction to occur.

### Claim 62 (depends on 61)

The method of claim 61, further comprising detecting the reaction.

### Claim 63 (depends on 61)

The method of claim 61, wherein the plasmons are applied to the reaction cell essentially continuously.

### Claim 64

A system comprising a combination of features as described in the foregoing disclosure.