Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B5/00—Optical elements other than lenses
  • G02B5/18—Diffraction gratings
  • G02B5/1866—Transmission gratings characterised by their structure, e.g. step profile, contours of substrate or grooves, pitch variations, materials

Definitions

• the present invention concerns signal amplification in fluorescence-based assays for sensitive detection and interaction analysis of chemical and biological species. It is based on plasmonic light management at the optical sensor surface by the use of multi-resonant metallic diffraction gratings.
• Optical affinity biosensors are pursued for applications in the biomolecular interaction analysis and for specific detection of chemical and biological analytes that are relevant to important areas of point-of-care medical diagnostics, food control, security, and environmental monitoring.
• Fluorescence-based readout of the affinity capture of target analytes by specific biomolecular recognition elements relies on using fluorophores that serve as labels, which (ideally) represents the most-spread method in analytical laboratories.
• Established instrumentation for the readout of fluorescence assays typically allows for the analysis of target analytes present at concentrations between pM - n M and above (1).
• optical nanostructures on a sensor chip surface has been investigated to enhance detected fluorescence signal in various readout modalities (2). These include dielectric microstructures (3) as well as nanostructures (4) that facilitate coupling to optical modes that probe the sensor surface with an evanescent profile of electromagnetic field intensity. Even stronger confinement of the
• electromagnetic field can be achieved with metallic nanostructures that support surface plasmons - collective oscillations of electron density and associated electromagnetic field (5).
• the plasmonic resonances are coupled with fluorophore emitters at their absorption A ab and emission A em wavelengths, their optical characteristics can be efficiently manipulated (6).
• a combination of locally increasing the excitation rate of fluorophores at A ab enhancing their quantum yield, and improving the collection efficiency of emitted fluorescence light at A em based on directional surface plasmon-coupled emission (SPCE) can be utilized.
• SPCE directional surface plasmon-coupled emission
• PSP surface plasmons
• fluorescence light coupled with PSPs by using optical setups compatible with epi- fluorescence microscopes and fluorescence scanner readout systems.
• optical setups compatible with epi- fluorescence microscopes and fluorescence scanner readout systems.
• These include linear diffractive gratings (12) as well as crossed gratings (13-15) with a period that allows diffraction phase matching of far-field radiation with PSPs at wavelengths in the vicinity to absorption and emission chosen emitters.
• crossed gratings 13-15
• Grating structures in the literature are most often designed to exhibit surface plasmon resonance at the absorption wavelength of the fluorescence emitter A ab in order to take advantage of plasmon- enhanced excitation and plasmon-coupled emission at longer wavelength A em that is subsequently diffracted at the same grating to angles different than the excitation beam (15, 19).
• fluorescence enhancement factors that can be achieved in applications relying on optical readout with low numerical aperture optics with low magnification (below 10 x ) and with long working distance lens such as the readout of microtiter plates or imaging of microarrays.
• optical readout with low numerical aperture optics with low magnification (below 10 x ) and with long working distance lens such as the readout of microtiter plates or imaging of microarrays.
• fluorescence sensor chips with enhanced sensitive detection and interaction analysis for chemical and biological species.
• the invention describes a method and apparatus for the amplification of fluorescence signal in biomolecular assays that rely on fluorescent labels. Strong amplification factors are achieved by a new class of multi-resonant plasmonic grating structures.
• the invention takes advantage of a confined electromagnetic field intensity of surface plasmons that is associated with locally increased field strength and local density of optical states.
• the invention concerns multi-resonant plasmonic grating structures that allow for efficient coupling of spectrally narrow surface plasmon resonances with fluorescent emitter labels at both their absorption and emission wavelengths at the same angles in the far-field.
• multi-resonant plasmonic grating structures that are tailored to simultaneously enhance the emitter excitation rate and control the angular distribution of emitted light in the far-field in the direction perpendicular to the surface.
• the multi-resonant plasmonic grating structures are implemented in the epi-fluorescence readout of assays that is suitable for rapid in situ time-resolved fluorescence measurements on arrays of sensing spots.
• the present invention thus provides a fluorescence sensor chip comprising a dielectric substrate and a metallic layer which comprises a multi-resonant grating with at least two superimposed periodic relief corrugations, wherein said multi resonant grating is plasmonically active with respect to radiation at least at two distinct wavelengths.
• said two distinct wavelengths correspond to the absorption and emission wavelengths of a fluorophore, or to the peak wavelengths of the absorption and emission bands of a fluorophore.
• said at least two superimposed periodic relief corrugations exhibit at least two different periods between 0.2 and 1 pm.
• said superimposed periodic relief corrugations are oriented between each other with an azimuthal angle between 0 and 90 deg.
• said superimposed periodic relief corrugations have a depth of at least 5 or of at least 10 nm.
• said superimposed periodic relief corrugations exhibit a harmonic profile.
• said multi-resonant grating is prepared by using laser interference lithography, electron beam lithography or nanoimprint lithography in combination with sputtering or vacuum evaporation of thin metallic film.
• the substrate of the sensor chip described herein is of glass or plastic and the metallic layer of the sensor chip described herein is of gold, silver, aluminum, or any combination thereof.
• said metallic layer exhibits a thickness between 30 and 200 nm.
• biomolecular recognition elements are attached to said metallic layer.
• biomolecular recognition elements comprise antibodies and their fragments, aptamers, peptides, proteins, and molecularly imprinted polymers.
• the present invention further provides a fluorescence assay device comprising:
• a fluorescence sensor chip as described herein; b. an excitation source emitting a light beam incident at the multi- diffractive grating of said fluorescence sensor chip; and
• a light detector arranged to selectively detect fluorescence light in epi- illumination fluorescence geometry.
• said sensor chip may be comprised in said fluorescence assay device as an insertable element or as a disposable element.
• the assay device described herein comprises a permanent reader with a light source and a detector.
• the excitation light beam of said assay device is normally incident at the multi-diffractive grating.
• the emitted fluorescence light beam is collected at the same angle as the excitation light beam is incident at the multi-diffractive grating.
• said excitation source emits a light beam in the UV, visible or near-infrared part of the spectrum.
• a dichroic mirror is used for the separation of light at the excitation and emission wavelength in combination with a miniature metallic mirror deposited in its center to which the incident and reflected excitation beam is focused.
• the present invention further provides a method for performing a
• said fluorescence assay is a fluorescence bioassay or fluorescence biochemical assay.
• said fluorescence assay is a sandwich assay, a displacement or competitive inhibition assay with fluorophore-labeled biomolecular recognition element affinity bound to the surface of said fluorescence sensor chip, or a direct assay with fluorophore-labeled biorecognition element immobilized on the fluorescence sensor chip and undergoing conformational change due to the capture of the target analyte and inducing distance-dependent fluorescence energy transfer.
• Fig la depicts surface plasmon mode that couples with a fluorophore serving as a label in a sandwich immunoassay and that is conjugated to detection antibody (dAb).
• the capture antibody (cAb) is attached to the metal surface to capture the target analyte from the analyzed liquid sample.
• Fig lb shows a
• Fig 2a presents an interference pattern that is sequentially recorded by using UV-LIL to a photoresist layer forming a three-diffractive grating topology after the etching step.
• the AFM observation of an example of the three-diffractive grating structure is showed in Fig 2b.
• Fig 2c illustrates a specific structure with a varied composition of overlaid periodic components of corrugation profile over its surface. It shows two positions of a flow chamber in the area comprising varied corrugation profiles and a profile with all three diffractive components (left). In addition, it shows a photograph of a prepared multi-resonant grating (right).
• Fig 3a shows a schematic of the UV laser interference (UV-LI L) setup used for the recording of multi-resonant gratings by using a Lloyd’s mirror configuration.
• Figure 3b depicts the preparation of multiple copies of grating structures by UV nanoimprint lithography that is followed by the deposition of a thin layer of gold.
• UV-LI L UV laser interference
• Fig 4 presents the measured wavelength spectrum of reflectivity featuring surface plasmon resonance at two distinct wavelengths (left) that are coincident with peak absorption and emission wavelengths of the selected fluorophore (right).
• the reflectivity is measured with a normal incidence light beam at the surface of the three-diffractive grating in contact with water.
• Fig 5a shows an optical setup enabling in situ and time-dependent readout of fluorescence signal from the arrays of sensing spots.
• the system relies on epi- fluorescence microscope optical configuration and it images a fluorescence signal originating from the sensing spots at a detector.
• the detector is a CCD camera, CMOS-based camera, photomultiplier or avalanche photodiode.
• Fig 5b presents an example of a sensor chip with arrays of spots that affinity capture fluorophore- labeled biomolecules and respective fluorescence image acquired by the optical setup with a CCD camera.
• Fig 6b shows the fluorescence intensity images acquired with a CCD camera from these areas that carry affinity bound
• the polarization of the excitation beam is controlled so the resonant excitation of PSPs by the A 1 component of the multi-resonant grating was switched“on” (horizontal) or“off” (vertical).
• Fig 6c provides an example of a time-dependent fluorescence signal measured from area (A) and reference area (B) upon flow of series of liquid samples with concentrations of target anti-mouse IgG molecules labeled with Alexa Fluor 790 between 100 fM and 10 nM.
• Fig 7 shows a calibration curve for a model immunoassay
• the present invention concerns a method and implementation of combined surface plasmon-enhanced fluorescence excitation and surface plasmon-coupled fluorescence emission for sensitive detection of molecular and biological analytes (see Fig 1). It allows for the development of fluorescence sensor chips with enhanced sensitivity of fluorescence assays that are compatible with optical readers for the rapid parallel analysis of arrays for sensing spots relying on epi - fluorescence geometry.
• the whole fluorescence sensor chip can be fabricated by mass production- compatible technologies such as nanoimprint lithography, hot embossing or injection molding to inexpensive polymer materials.
• the master structure that comprises the superimposed periodic relief corrugations can be prepared by using laser interference lithography, electron beam lithography or nanoimprint
• the present invention thus provides a fluorescence sensor chip comprising a dielectric substrate and a metallic layer which comprises a multi-resonant grating with at least two superimposed periodic relief corrugations, wherein said multi resonant grating is plasmonically active with respect to radiation at least at two distinct wavelengths.
• plasmonically active refers to the resonant excitation of surface plasmons traveling along the surface of the metallic layer at two distinct wavelengths. Specifically, said wavelengths are engineered to overlap with absorption and emission bands of fluorophores that are used as biomolecular labels.
• the term“multi-resonant” encompasses the terms“multi- diffractive” and“multi-period”.
• the two or more periodic corrugations exhibit at least two different periods between 0.2 and 1 pm.
• the superimposed periodic relief corrugations are oriented with respect to each other with azimuthal angle ( ⁇ ) between 0 and 90 deg, or between 0 and 45 deg.
• said relief corrugations are coated with a conformal or non-conformal metallic layer with a thickness between 30 and 200 nm that supports surface plasmons on its top.
• a substrate comprising relief corrugation coated with a metallic layer with components comprising spatial frequencies enabling diffraction coupling of an optical wave in the far-field to surface plasmons at two distinct wavelengths.
• each periodic component of the relief corrugation is between 10 and 100 nm assuring optimum diffraction coupling strength between surface plasmons and far-field optical waves.
• the fluorescence sensor chip described herein comprises a transparent glass or plastic substrate and on the outer surface, a polymer layer with a relief corrugation profile comprising at least two superimposed periodic relief corrugations, which are coated with a metallic layer as described herein.
• said metallic layer is of gold, silver or aluminum, or any combination thereof.
• a fluorescence sensor chip wherein said excitation of surface plasmons occurs in the ultraviolet, visible or near-infrared part of the spectrum.
• a fluorescence sensor chip wherein biomolecular recognition elements for selected chemical or biological compounds are attached to the metallic layer on the corrugated area.
• the biomolecular recognition element is able to capture target analytes.
• Various types of biomolecular recognition elements are known in the art, e.g.
• biomolecular element specific to the target analyte is immobilized in the sensing area on the biochip. It needs to be assured that the biological activity of the immobilized biomolecular recognition elements is conserved and the biochip surface exhibits non-fouling properties.
• regenerate the biomolecular recognition elements i.e., break their complex with the analyte molecules and make them available for another use
• n-a I kylthiols or disulfides may spontaneously self-assemble on gold into well-ordered arrays.
• SAMs have been employed in many immobilization methods for spatially controlled attachment of biomolecular recognition elements to surfaces of sensors.
• the mapping of biomolecular recognition elements to different sensing areas the mapping of biomolecular recognition elements to different sensing areas
• immobilization chemistry needs to be spatially controlled.
• Most of the current technologies of protein arrays are based on the surfaces and formats that were earlier developed for DNA arrays.
• Most DNA array production techniques were developed for glass supports, but they can be tailored to noble metal surfaces with appropriate immobilization chemistries.
• a combination of SAMs with the covalent coupling of biomolecular recognition elements or non-covalent streptavidin-biotin system as a linker for attachment of biotinylated biomolecular recognition elements are most frequently used approaches for the development of protein assays on metal.
• a fluorescence sensor chip wherein said resonant coupling of light to surface plasmons occurs at wavelengths overlapping with the absorption and emission bands of a fluorophore that serves as a label in an assay.
• a fluorescence sensor chip wherein fluorophores are employed as labels and after their capture, they are excited by resonantly coupled surface plasmons at the emitter excitation wavelength and simultaneously emit via surface plasmons at the emission wavelength that are subsequently out-coupled to the direction
• the characteristic surface plasmon- driven excitation and surface plasmon-coupled fluorescence emission is tuned to allow increased excitation rate and highly efficient collection of emitted
• a fluorescence sensor chip wherein the area with multi-resonant relief corrugation comprises arrays of sensing spots and the enhanced fluorescence intensity of each spot is imaged to a detector such as CCD or CMOS camera.
• a fluorescence sensor chip wherein the fluorophore labels at the sensing area are organic chromophores or quantum dots emitting fluorescence light in UV, visible or near-infrared part of the spectrum.
• a fluorescence sensor chip wherein said fluorophores are excited with a light beam at lower wavelengths than their emission wavelength.
• a fluorescence sensor chip wherein the said excitation beam is collimated and hits the sensing area at a normal angle of incidence.
• a fluorescence assay device for detecting fluorescence in biochemical assays by combined surface plasmon-enhanced excitation and surface plasmon-coupled emission methods, comprising:
• a light detector arranged to selectively detect fluorescence light in epi - illumination fluorescence geometry.
• said assay device is used for performing a fluorescence bioassay or fluorescence biochemical assay.
• the fluorescence assay device described herein comprises said fluorescence sensor chip as an insertable or disposable element.
• the fluorescence assay device described herein comprises a permanent reader with a light source and a detector.
• a fluorescence assay device where a dichroic mirror is used for the separation of light at the excitation and emission wavelength in combination with a miniature metallic mirror deposited in its center to which the incident and reflected excitation beam is focused.
• the excitation light beam of said assay device is normally incident at the multi-diffractive grating. Specifically, the excitation light beam is normally incident at the multi-diffractive grating and the emitted fluorescence light beam is collected from the same side.
• the emitted fluorescence light beam is collected at the same angle as the excitation light beam is incident at the multi-diffractive grating.
• said excitation source emits a light beam in the UV, visible or near-infrared part of the spectrum.
• concentration of an analyte in a sample by combined surface plasmon-enhanced fluorescence and surface plasmon-coupled fluorescence emission comprising the steps of:
• said fluorescence assay is a bioassay or biochemicall assay. Specifically, it is a sandwich assay, a displacement or competitive inhibition assay with fluorophore-labeled biomolecular recognition element affinity bound to the surface of said fluorescence sensor chip, or a direct assay with fluorophore-labeled biorecognition element immobilized on the fluorescence sensor chip and
• Positive photoresist Microposit S1805 was purchased from Shipley and its developer AZ 303 was acquired from MicroChemicals.
• Polydimethylsiloxane elastomer (PDMS) Sylgard 184 was obtained from Dow Corning and the UV- curable polymer Amonil M MS 10 was from AMO GmbH.
• N-(3- dimethylaminopropyl)-N’- ethylcarbodiimide hydrochloride (EDC), N- hydroxysuccinimide (N HS), ethanolamine, as well as acetic acid and sodium acetate for the preparation of acetate buffer (ACT) were bought from Sigma- Aldrich.
• Phosphate buffered saline (PBS) at a pH of 7.4 was from Calbiochem.
• PBS with addition of 0.05 % Tween 20 (Sigma-Aldrich) and 1 % bovine serum albumin (Thermo Fisher Scientific) (PBST) was used as running buffer in all detection experiments.
• the thiols for self-assembled monolayer formation were purchased from SensoPath Technologies (Bozeman, Montana, US).
• Mouse IgG (mlgG) was obtained from Sigma-Aldrich and anti-mouse IgG labeled with Alexa Fluor 790 (A790) was acquired from Thermo Fisher Scientific.
• Laser interference lithography is adopted for the preparation of master gratings as showed in Fig 3a.
• the exposure is performed with an interference field formed by a coherent beam emitted from HeCd laser (model I K 3031 R-C from Kimmon) at a wavelength of 325 nm.
• the beam is expanded and split to two collimated beams that are made spatially overlapping at controlled angle Q by using a Lloyd’s mirror configuration.
• the interference field is recorded to a photoresist layer (Shipley Microposit S1805) prepared on a glass substrate with a thickness of about 100 nm.
• the exposures are sequentially repeated for different interfering angles Q .
• the azimuthal angle f between different periodic components is controlled by using a dedicated holder.
• the relief corrugation profile of multi-resonant grating is etched with a developer AZ-303, rinsed with water and dried.
• the master grating is cast to PDMS (serving as a working stamp) and transferred to a UV curable polymer followed by the gold deposition as shown in Fig 3b.
• Cleaned BK7 glass substrates are coated with the UV-curable polymer Amonil M MS 10 by spin-coating at 3000 rpm for 120 s.
• the PDMS working stamp is placed on top of the fluid Amonil layer and the structure is cast into the layer is irradiated by UV light (UV lamp Bio-Link 365, Vilber Lourmat). Finally, the PDMS stamp is detached from the cured Amonil M MS 10 leaving a copy of the master structure on the BK7 glass substrate. The grating copies are subsequently coated with 4 nm of chromium and 100 nm of gold by vacuum thermal evaporation (H HV AUTO 306 from H HV LTD) in vacuum better than 10 -6 mBar.
• a self-assembled monolayer is formed by immersion for at least 12 hours in a 1 mM ethanolic solution of PEG and COOH thiols at a 1:9 ratio. Afterwards, the gold grating surface is rinsed with ethanol, dried, and stored under inert
• the chip surface with gold multi-resonant grating is further modified either in-situ or ex-situ with mouse IgG (cAb) through amine coupling to the COOH groups.
• cAb mouse IgG
• it is incubated for 15 minutes in an aqueous solution containing NHS and EDO, 21 mg/mL and 75 mg/mL, respectively, to activate the carboxy-terminal groups on the thiol SAM.
• the sensor chip is already mounted in the reader system (see section 1.6 below) and, similar to the ex-situ immobilization, an aqueous solution with 21 mg/mL NHS and 75 mg/mL EDO is flowed over the surface for 15 min, followed by rinsing with ACT buffer and incubation for around 90 min with the cAb dissolved in ACT at a concentration of 50 jU g/mL.
• any unreacted active ester groups are passivated by a solution of ethanolamine (1M, pH 8.5) for 15 minutes.
• the sensor chip Prior to the experiment, either before or after immobilization, the sensor chip is mounted into the optical system, and a microfluidic cell made from fused silica glass is clamped on top.
• the microfluidic flow cell is sealed using a 100 pm thick gasket with a volume of only several microliters and it comprises in- and outlets which allow connecting a peristaltic pump with tubing.
• FIG. 1 A schematic of the used optical system is presented in Fig 5.
• a fiber- coupled laser diode LM L-785.0CB-10 (PD-LD Inc.) served as a light source and a FC-multimode fiber (100 m m diameter) is used to deliver the excitation light beam from the laser diode to the reader through an FC/APC adapter.
• the beam was collimated by an achromatic lens 1 (AC254-035-B, Thorlabs) and spectrally filtered by using a bandpass (BP) filter for 785 nm (LL01-785-25, Shamrock).
• the beam polarization is controlled by a linear polarizer (POL, LPVIS100, Thorlabs).
• the excitation beam is then focused on a dichroic mirror (69-905, Edmund Optics) with a miniature mirror made of a gold layer (thickness of 100 nm, elliptical shape with dimensions 1 and 1.4 mm) in its center, which serves as a spatial filter.
• the excitation beam is focused (lens 2) and re-collimated again (lens 3) by using two achromatic lenses (AC254-040-B, Thorlabs) and made incident at the biochip surface through its transparent flow-cell.
• the excitation beam is partially coupled to PSP modes and partially its intensity is reflected.
• the back-reflected excitation beam is blocked by its directing towards the light-source arm of the reader with the use of the miniature gold mirror and the intensity of the scattered excitation beam at A ex travelling towards the detector arm is suppressed by the dichroic mirror.
• Fluorescence light emitted from the biochip surface at A em passes through the dichroic mirror and is imaged at the detector by an achromatic lens 4 (AC254-080-B, Thorlabs). Between this lens 4 and the detector, the remaining excitation light intensity at A ex is blocked by a notch filter (NF-785-33, Thorlabs) and a bandpass filter for the fluorescence wavelength A em (BP-810-10, Thorlabs).
• a detector either a Peltier cooled CCD camera (EM-CCD iXon + 885 from Andor Technology) or a photomultiplier (PMT, H6240-01, Plamamatsu), that is connected to a counter (53131A from Agilent), is used.
• E-CCD iXon + 885 from Andor Technology or a photomultiplier (PMT, H6240-01, Plamamatsu), that is connected to a counter (53131A from Agilent)
• the overall reader with a CCD detector is controlled by an in-house developed LabVIEW-based software. It allows real-time recording of the
• the CCD detector iXon is operated at the temperature -70 degC with a gain setting of 100 and acquisition time 0.3 sec.
• the acquired images were accumulated 10 times and the intensity of the excitation beam was adjusted to 30 mW/cm 2 in order to suppress the effect of bleaching.
• the operating software allows for averaging of fluorescence signal F on pre-defined areas representing sensing spots and for the recording and visualization of their kinetics A(t).
• the CCD detector was replaced by PMT and the output from the connected counter was recorded in counts per second (ops) by using software Wasplas developed at Max Planck Institute for Polymer Research in Mainz (Germany).
• the excitation beam is made to pass through a spatial filter with a pin-hole diameter of 10 pm.
• the optical fiber was mounted to a custom-made mode mixer where the fiber coil was mechanically actuated with a frequency of several Hz.
• the sensor surface Prior to each assay experiment, the sensor surface is rinsed with PBST for a minimum of 30 min or until the baseline is stable. Afterwards, the fluorescent labelled dAb is flowed through the sensor in varying concentrations for 15 min each, followed by 5 min rinsing with PBST. The difference in the sensor signal before and after each concentration of dAb is determined.
• the coupling of fluorescent emitters with the confined field of surface plasmons can be utilized for enhancing the emitted fluorescence light intensity that is associated with the capture of the target analyte and its subsequent reaction with fluorophore-labeled affinity binding partner at the sensor surface.
• a multi-resonant plasmonic grating that allows to simultaneously couple light to surface plasmons at absorption A ab and emission A em wavelengths of fluorescent labels.
• the structure is designed in order to provide strong
• microfluidic device and for systems that rely on imaging of arrays of sensing areas on two-dimensional arrays of detectors (such as CCD or CMOS) for rapid and parallelized measurements of fluorescence signal kinetics. Then the large numerical objective lens optics (that are mostly used in e.g. fluorescence scanners) cannot be used and only a small fraction of emitted light intensity can be detected.
• detectors such as CCD or CMOS
• the target structure was made by the use of UV-LI L with a sequential recording of corrugations and A 1 and A 2 .
• Fig 2a shows the simulated interference field that was used to generate the multi-resonant grating corrugation. In order to test the contribution of each component of the grating ( A l and A 2 ) to the
• the recordings were made in a way that different sub-areas of the sensor chip carried all or only part of them, see Fig 2c.
• Fig 3a illustrates, the recording of the grating structure was performed with laser interference lithography in Lloyd’s mirror configuration.
• the structure was etched by using a developer and cast to a working stamp.
• the working stamp was used in the replication step when multiple copies of the structure were made by using UV-nanoimprint lithography.
• the copies made into a UV-curable resin were subsequently coated with a 100 nm thick gold film (see example in Fig 2c).
• the topology of the prepared multi-resonant corrugation profile was observed with AFM, as can be seen in Fig 2b.
• the modulation amplitude of each periodic modulation was set of about 15 nm resulting in a structure with a rather shallow profile and overall modulation depth below 100 nm.
• the reflectivity of the plasmonic multi-resonant grating was measured for the normally incident beam and aqueous sample brought in contact with its surface. As can be seen in Fig 4a and Fig 4b, the reflectivity spectra show two narrow dips at wavelengths that are coincident with targeted A ex and A em for the polarization of the beam
• the gold surface of the multi-resonant plasmonic grating was modified with a thiol SAM linker layer that carried PEG moieties with antifouling properties and functional carboxyl groups that were used for amine coupling of ligands.
• the sensor chip with already immobilized ligands was interfaced with a microfluidic device in order to transport liquid samples to be analyzed over its surface.
• a flow cell with a PDMS gasket was placed on top of the gold grating and was pressed against a transparent quartz glass with drilled input and output ports by using a dedicated holder. Liquid samples were pumped through these ports by the use of a peristaltic pump connected via tubing.
• the optical system for the measurement of fluorescence signal on the biochip was designed to image fluorescence light emitted from the array of sensing spots to a detector of low intensity of light.
• the optical configuration was designed to provide magnification by a factor of 2 and thus imaging arrays of sensing spots on the footprint of 4 x 4 mm (object plane) to a detector with the area of about 8 x 8 mm (image plane area corresponding to the size of the sensor chip in used camera iXon +885, Andor, Ireland).
• the numerical aperture of the lens that collects the fluorescence light emitted from the sensor chip is about 0.2. Spectral filtering of light imaged to a detector is performed hence the excitation wavelength
• the schematic of the system is presented in Fig 5a and it was further used for the imaging of fluorescence signal from the sensor chip areas carrying different plasmonic gratings and for time-dependent measurement of fluorescence response due to the affinity binding of fluorophore-labeled molecules (Fig 5b). It is worth of noting that characteristics of filters used for the spectral filtering are highly angular
• a grating with four areas schematically showed in Fig 6a is used. It comprises a wedge zone where all three corrugations [(A), A 1 and orthogonally oriented A 2 ] are
• the area below that comprises a zone with a flat profile (B), an area that carries only one corrugation with /l 2 (C), and an area that carries superimposed corrugation with the period A 1 and single A 2 (D). All these areas were coated with the same 100 nm thick gold film and IgG biomolecules labelled with Alexa Fluor 790 emitter were affinity bound to its surface by using a
• the gold surface was modified with a thiol SAM linker that exhibits a thickness of about 1-2 nm.
• the average distance between the emitter attached to target biomolecule (antibody against mouse IgG, cAb) that is affinity bound to a ligand (mouse IgG, dAb) attached by amine coupling to the linker layer can be estimated of about f ⁇ 15 nm based on the size of IgG molecules (6 x 6 x 12 nm). At this distance f, the quenching of fluorescence signal due to the energy transfer to metal is weak and the interaction with PSPs is dominant (15).
• Fig 6c shows a fluorescence signal kinetics F[t) measured on areas with the complete corrugation (A) and reference flat area (B) upon the sequential flow of series of samples (PBST) that were spiked with the concentration of target analyte (a-mlgG-A790, dAb) at concentrations of 1 pM, 10 pM, 0.1 nM, 1 n M, and 10 nM.
• PBST target analyte
• the obtained time-dependent signal F[t) in Fig 6c shows, that on the area with the developed plasmonic multi-resonant grating the fluorescence signal is measurable from the concentration 1 pM and that it gradually increases with the concentration of the target analyte.
• samples only generate sufficiently strong fluorescence intensity at a concentration of 1 nM.
• the comparison of the fluorescence intensity change D Fupon the binding of analyte at the highest concentration of 10 nM shows that the plasmonic grating provides enhanced response by a factor of 300 which is similar to that determined in section 2.5.
• the CCD detector was replaced by a PMT and the titration experiment was performed in triplicate. As seen in the obtained calibration curve in Fig 7, the measured data follows Langmuir isotherm dependence and the limit of detection of 6 fM is determined for the incubation time of analyzed sample of 15 min.
• SPCE plasmon-coupled emission

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