WO2015188215A1 - Optical biosensor - Google Patents
Optical biosensor Download PDFInfo
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- WO2015188215A1 WO2015188215A1 PCT/AU2015/000118 AU2015000118W WO2015188215A1 WO 2015188215 A1 WO2015188215 A1 WO 2015188215A1 AU 2015000118 W AU2015000118 W AU 2015000118W WO 2015188215 A1 WO2015188215 A1 WO 2015188215A1
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- mmp
- optical biosensor
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/068—Optics, miscellaneous
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
Definitions
- the present invention relates to optical biosensors for the detection of bioanalytes, such as peptides and proteins.
- the present invention relates to optical biosensors for the detection of peptides and proteins that are associated with specific diseases or pathological conditions.
- bioanalytes biological analytes
- ACS acute coronary syndromes
- CRP C- reactive protein
- Bioanalyte to be detected may be either member of the binding pair; alternatively, the bioanalyte may be a ligand analogue that competes with the ligand for binding to the complement receptor.
- a range of devices for detecting ligand/receptor interactions are known.
- chemical/enzymatic assays are used in which the presence or amount of bioanalyte is detected by measuring or quantifying a detectable reaction product, such as gold immunoparticles.
- Ligand/receptor interactions can also be detected and quantified by radiolabel assays.
- a frequently used assay method is enzyme linked immunosorbent assay (ELISA).
- ELISA enzyme linked immunosorbent assay
- Biosensors that detect bioanalytes associated with wounds would be beneficial in the management of chronic wounds such as diabetic foot ulcers, pressure ulcers and venous leg ulcers. Management of these wounds is lengthy and challenging due to the inherent complexity of the biochemical processes occurring in non-healing wounds. Typically, regular examinations and assessments of the wound bed are performed by nurses and clinicians to inform the individual subject's wound treatment plan. This assessment process consumes a significant amount of nursing time and dressing materials, which contribute to increasing medical costs in wound care.
- an optical biosensor for detecting a target bioanalyte in a sample
- the biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker capable of interacting with the target bioanalyte and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled in the absence of the target bioanalyte such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor, and interaction of the target bioanalyte with th e linker results in optical un-coupling of the luminescence donor and the luminescence acceptor to thereby result in light emission from the luminescence donor; and a plurality of light interacting pores on the surface
- an internal surface of the light interacting pores comprises an optical structure that interacts with the light emission from the luminescence donor.
- the optical structure may be an optical filter, reflector or cavity.
- the internal surface of the light interacting pores may comprise a Bragg reflector, a rugate filter, a resonant microcavity, or a combination of any of these optical features.
- the substrate is a resonant microcavity (pSiRM) substrate in which the light interacting pores comprise distributed Bragg reflectors separated by a resonant microcavity.
- the luminescence donor and the luminescence acceptor may be a fluorescence donor/acceptor pair or a phosphorescence donor/acceptor pair.
- the optical biosensor may further comprise a detector for detecting light emission from the luminescence donor and provide an output signal containing information on said light emission.
- the measurable light emission is enhanced or amplified relative to the light emission that would be measured in the absence of th e light interacting pores on the surface of the substrate. This means that higher levels of detection can be obtained using the biosensor described herein relative to a biosensor that does not include the light interacting pores.
- the biosensor further comprises a bioanalyte specific capture agent.
- the bioanalyte specific capture agent may comprise a binding agent capable of selectively binding the target bioanalyte.
- the bioanalyte specific capture agent may be deposited on or near the surface of the porous silicon or alumina substrate so that at least some of any bioanalyte captured by the capture agent is capable of interacting with the sensing domain of the detection agent.
- the bioanalyte specific capture agent may be in the form of particles comprising binding agent on the surface thereof.
- the particles may be functionalised magnetic nanoparticles (MNPs) having binding agent bound to a surface thereof.
- the functionalised MNPs may interact with and be retained on the surface of the substrate.
- the binding agent may be any agent that selectively binds the target bioanalyte.
- the binding agent may bind the target bioanalyte selectively from complex fluids comprising other components that are structurally related to the target bioanalyte.
- the biosensor of these embodiments may be used for the selective detection of a specific peptide or protein in a family of structurally related peptides or proteins.
- a method for detecting a target bioanalyte in a sample comprising: providing an optical biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker capable of interacting with the target bioanalyte and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled in the absence of the target bioanalyte such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor, and interaction of the target bioanalyte with the linker results in optical un-coupling of the luminescence donor and the luminescence acceptor to thereby result in light emission from the luminescence donor, and a plurality of light interacting pores
- a method for measuring the concentration of a target bioanalyte in a sample comprising: providing an optical biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker capable of interacting with the target bioanalyte and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled in the absence of the target bioanalyte such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor, and interaction of the target bioanalyte with the linker results in optical un-coupling of the luminescence donor and the luminescence acceptor to thereby result in light emission from the luminescence donor, and a plurality of light
- the change in light emission may be any one of a change in the wavelength of the light emitted and/or a change in the intensity of the light emitted from the optical biosensor.
- the linker is cleavable by the target bioanalyte when it contacts the linker such that cleavage of the linker results in optical un-coupling of the luminescence donor and the luminescence acceptor.
- the light interacting pores comprise a resonant microcavity and the substrate shows a resonance microcavity dip in the centre of the reflectance band in a reflectance spectrum and the wavelength of the microcavity dip is substantially the same as the emission wavelength of the luminescence donor so that the emission from the luminescence donor is enhanced by the microcavity.
- the resonance microcavity dip of the pSiRM is sensitive to refractive index changes and a relatively small refractive index change induces a relatively large shift in the optical spectrum.
- the shift in the optical spectrum is also indicative of the presence of the target bioanalyte.
- the microcavity may be formed in porous silicon or porous alumina. In certain embodiments, the microcavity is formed in porous silicon.
- the light interacting pores comprise a distributed Bragg reflector with each reflector comprising a periodic layer structure alternating between high porosity silicon and low porosity silicon.
- the substrate is a resonant microcavity (pSiRM) substrate in which the light interacting pores comprise distributed Bragg reflectors separated by a resonant microcavity.
- the optical thickness of each distributed Bragg reflector is a quarter-wavelength and the optical thickness of the microcavity is a multiple of a half-wavelength and the wavelength is the emission wavelength of the fluorescence donor.
- the target bioanalyte is a peptide or protein of interest.
- the target bioanalyte is an enzyme.
- the biosensor further comprises a bioanalyte specific capture agent.
- the bioanalyte specific capture agent may comprise a binding agent capable of selectively binding the target bioanalyte.
- the bioanalyte specific capture agent may be deposited on or near the surface of the porous silicon or alumina substrate so that at least some of any bioanalyte captured by the capture agent is capable of interacting with the sensing domain of the detection agent.
- the bioanalyte specific capture agent may be in the form of particles comprising binding agent on the surface thereof.
- the particles may be functionalised magnetic nanoparticles (MNPs) having binding agent bound to a surface thereof.
- MNPs magnetic nanoparticles
- the functionalised MNPs may interact with and be retained on the surface of the substrate.
- the binding agent may be any agent that selecti vely binds the target bioanalyte.
- the binding agent may bind the target bioanalyte selectively from complex fluids comprising other components that are structurally related to the target bioanalyte.
- the biosensor of these embodiments may be used for the selective detection of a specific peptide or protein in a family of structurally related peptides or proteins.
- the target bioanalyte is a matrix metalloproteinase (MMP).
- MMPs are clinically validated biomarkers in chronic wounds.
- a method for monitoring and/or assessing wound status in a subject comprising: providing an optical biosensor for detecting a matrix metalloproteinase in a wound fluid from said subject, the biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker that is a substrate for the matrix metalloproteinase and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor, and interaction
- a method for monitoring and/or assessing cancer status in a subject comprising: providing an optical biosensor for detecting a matrix metalloproteinase in cancer tissue or blood from said subject, the biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker that is a substrate for the matrix metalloproteinase and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor, and interaction of the matrix metalloproteinase with the linker results
- the biosensor further comprises a bioanalyte specific capture agent.
- the bioanalyte specific capture agent may comprise a binding agent capable of selectively binding a specific MMP protein selected from one of the group consisting of MMP-1 , -2, -3 and -9.
- the binding agent may be capable of selectively binding the one selected MMP in the presence of the other listed MMPs.
- the binding agent may be an antibody.
- the binding agent may be functionalised magnetic nanoparticles (MNPs) having the antibody bound to a surface thereof.
- the bioanalyte is a bacterial biomarker.
- a method for monitoring and/or assessing bacterial infection in a subject comprising: providing an optical biosensor for detecting a bacterial biomarker in a body fluid from said subject, the biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker that is a substrate for the bacterial biomarker and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor, and interaction of the bacterial biomarker with the linker results in cleavage of the linker and optical uncoupling of
- the bacterial biomarker may be used as an indicator of wound status in a subject. Accordingly, in a seventh aspect there is provided a method for monitoring and/or assessing wound status in a subject, the method comprising: providing an optical biosensor for detecting a bacterial biomarker in a wound fluid from said subject, the biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker that is a substrate for the bacterial biomarker and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor, and interaction of the bacterial biomarker with the linker results in cleavage of the linker and optical uncoupling of the lumi
- the change in light emission may be any one of a change in the wavelength of the light emitted and/or a change in the intensity of the light emitted from the optical biosensor.
- the biosensor further comprises a bioanalyte specific capture agent.
- the bioanalyte specific capture agent may comprise a binding agent capable of selectively binding the bacterial biomarker.
- the binding agent may be an antibody.
- the binding agent may be functionalised magnetic nanoparticles (MNPs) having the antibody bound to a surface thereof.
- the bacterial biomarker of the fifth and seventh aspects may be a peptide, protein or other molecule that is indicative of infection by a bacterial species such as Bacillus anthracis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Borrelia burgdorferi, Treponema pallidum, Chlamydia trachomatis, Chlamydophila psittaci, Corynebacterium diphtherias, Mycobacterium tuberculosis, and Mycobacterium avium, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Anaplasma phagocytophilum
- pseudomallei Neisseria gonorrhoeae, Neisseria meningitides, Campylobacter jejuni, Helicobacter pylori, Legionella pneumophila, Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa, Aeromonas sp., Vibrio cholerae, Vibrio parahaemolyticus, Thiotrichales sp., Haemophilus influenzae, Klebsiella pneumoniae, Proteus mirabilis, Yersinia pestis, Yersinia enterocolitica, Shigella flexneri, Salmonella enterica or Escherichia coli.
- the optical biosensor is part of a detection device.
- the detection device may be a point-of-care (POC) device.
- the detection device comprises a fluid inlet through which the sample can be introduced, the optical biosensor described herein, and an optical output for outputting information on the emission intensity of the luminescence donor.
- the detection device may also comprise a means for directly collecting and transferring a test sample from a subject to the detection device. Specifically, the detection device may utilise a microneedle or one or more microneedle arrays designed to transfer a bodily fluid, such as a wound fluid, from the subject to the device via capillary action and/or surface tension.
- the detection device may be used for detection of multiple target bioanalytes.
- the housing may comprise a plurality of spatially arranged optical biosensors with each biosensor comprising detection agents specific to different bioanalytes so that each biosensor is capable of selectively detecting a different bioanalyte relative to an adjacent biosensor.
- each biosensor may be capable of detecting a bioanalyte that is indicative of a specific bacterial species and the detection device can thereby be used in the detection of a plurality of bacterial infections in a single step.
- each biosensor may have the same luminescence donor and acceptor pair but different linkers.
- a single or multiple biosensors may comprise detection agents having the same linker but each having different luminescence donor and acceptor pairs.
- the optical biosensor may be part of a wound dressing or bandage.
- the optical biosensor may be fixed or otherwise attached to a wound dressing or bandage material and may provide information to a practitioner regarding the status of a wound.
- a theranostic device for the diagnosis and/or treatment of a disease or pathological condition in a subject, the device comprising: providing an optical biosensor for detecting and/or determining the concentration of a bioanalyte that is a biomarker of said disease or pathological condition in a sample of bodily fluid obtained from said subject, the optical biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker capable of interacting with the target bioanalyte and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled such that emission of light from the luminescence donor is substantially quenched by the lumin
- the biosensor further comprises a bioanalyte specific capture agent.
- the bioanalyte specific capture agent may comprise a binding agent capable of selectively binding the bioanalyte.
- the binding agent may be an antibody.
- the binding agent may be functionalised magnetic nanoparticles (MNPs) having the antibody bound to a surface thereof.
- the measurable light emission is advantageously enhanced or amplified relative to the light emission that would be measured in the absence of the light interacting pores on the surface of the substrate.
- Figure 1 shows: (a) a schematic representation of surface functionalisation reactions of a hydride-terminated porous silicon resonance microcavity (pSiRM) surface involving hydrosilylation with undecylenic acid, NHS ester formation and reaction with fluorogenic matrix metalloproteinase (MMP) substrate; and (b) baseline-corrected FTIR-ATR spectra of the pSiRM surface (i) after hydrosilylation with undecylenic acid, (ii) activation with EDC/NHS and (iii) immobilisation of the fluorogenic MMP peptide substrate;
- pSiRM hydride-terminated porous silicon resonance microcavity
- Figure 2 shows: top view (a-e) and cross-sectional (f-j) SEM images of the single layer pSi etched at five different current densities as listed at Table 1.
- (a) and (f) are for current density 25 mA/cm”
- (b) and (g) are for current density 30 mA/cm 2
- (c) and (h) are for current density 40 mA/cm'
- (d) and (i) are for current density 50 mA/cm
- (e) and (j) are for current density 60 mA/cm 2 .
- the scale bars presented in figure (a-e) and (f-j) are 300 nm and 2 ⁇ , respectively;
- Figure 3 shows: (a) a simulated reflectance spectrum of pSiRM (grey trace) and reflectance spectrum obtained using IRS from a freshly etched pSiRM (black trace); and (b) top view and (c) cross- sectional SEM images of a freshly etched pSiRM;
- Figure 4 shows EDANS emission spectra immobilised (a) in the pSiRM matrix; (b) in buffer solution from 3 different MMP-1 concentrations: 1.2 x 10 "7 M (full line); 1.2 x 10 12 M (dashed line); 1.2 x 10 1 ' M (dotted line) and 0 M (dot and dashed line); and (c) a plot of emission intensity at 446.5 nm for each concentration of MMP-1 in the buffer solution (solid) and on the pSiRM surface (pattern), with the error bars calculated from three separate experiments;
- Figure 5 shows fluorescence spectra of different pSi architectures (a): single layer pSi with low porosity (pSi-LP, grey dashed line), single layer pSi with high porosity (pSi-HP, grey full line), multilayer pSi with alternating HP and LP layers (pSiML with resonance at 446.5 nm, black dashed line) and pSiRM (black full line).
- the dotted line represents the reflectance spectrum of pSiRM; and
- figure 6 shows: (a) comparison of fluorescence emission spectra observed at 446.5 nm from the pSiRMs with cavity dip at 446.5 nm (full line) and at 506.7 nm (dashed line) after incubation with MMP- 1.
- the dotted line corresponds the control sample, which was not incubated with MMP- 1 ; and
- Figure 7 shows plots of EDANS emission from (a) the pSiRM with (HP/LP) 3 (HP) 4 (LP/HP) 3 and a 16.4% porosity contrast (full line) compared to a pSiRM with (HP/LP) 3 (HP) 4 (LP/HP) 3 and 19.3% porosity contrast (dotted line) and (b) the pSiRM with (HP/LP) 3 (HP) 4 (LP/HP) 3 and a 16.4% (full line) compared to a pSiRM with (HP/LP) 4 (HP) 4 (LP/HP) and 16.4% porosity contrast.
- the concentration of MMP-1 added to the surface was 1.2 x 10 7 M;
- Figure 8 is a plot showing the simulation of electric field distribution at excitation wavelength of EDANS (340 nm) throughout the porous layers of the same pSiRMs.
- the black trace represents the pSiRM with a configuration of (HP/LP) 3 (HP) 4 (LP/HP) 3 and the grey trace represents the pSiRM with a configuration of (HP/LP) 4 (HP) (LP/I1P) .
- the grey square shows the position of the defect layer in the layer stack of the pSiRM structure;
- Figure 9 shows a plot of fluorescence emission intensities for different incubation times. The error bars were calculated from three separate experiments;
- Figure 10 shows a plot of fluorescence emission intensity of peptide-functionalised pSiRM after incubation with MMP-1 at different concentrations for 15 min. The error bars were calculated from the three separate experiments;
- Figure 11 shows a Western Blotting analysis for wound fluid sample and MMP-1 as a positive control
- Figure 12 shows: (a) a plot of fluorescence emission spectra of peptide-functionalised pSiRM after immersion in wound fluid (WF) (full line). The dotted line corresponds to the control pSiRM not incubated with wound fluid; and (b) a plot of average emission intensity of different concentration of MMP-1 in buffer solution and spiked to wound fluid sample with error bars calculated from three separate experiments;
- Figure 13 shows a plot of the fluorescence emission intensity of neat dye in the presence and absence of wound fluid at four different concentrations with error bars calculated from three separate experiments;
- Figure 14 shows a plot of the fluorescence emission intensity of peptide-functionalised pSiRM after incubation with tissue extract (left) and two different sources of wound fluid (middle and right);
- Figure 15 shows FTIR spectra of MNP surface functionalization.
- Figure 16 shows fluorescence emission spectra of the EDANS from MMP fluorogenic peptide substrate detected in the solution (a) and the pSi surface after incubated with MMP- 1 immobilised in MNP-MMP-lAb;
- Figure 17 shows a plot of fluorescence intensity of the EDANS from the MMP fluorogenic peptide substrate after cleavage by MMP-1 bound with MNP-MMP-lAb at different concentration of MMP-1 Ab;
- Figure 18 shows a plot of fluorescence intensity of the EDANS from the MMP fluorogenic peptide substrate after cleavage by MMP-1 bound with MNP-MMP-lAb at different interaction time between MMP-1 Ab and MMP- 1 ;
- Figure 19 shows a plot showing the selectivity of MNP -MMP Ab in buffer solution containing MMP-1 , MMP-9 and mixed MMP-1 MMP-9;
- Figure 20 shows a plot showing the selectivity test of MNP-MMPAb in wound fluid sample, wound fluid spiked with MMP-1 , MMP-9 and mixed MMP-1 MMP-9;
- Figure 21 shows a confocal microscope image of a pSiRM surface functionalised using a microcontact printing technique to immobilise the MMP peptide substrate and then incubated with MMP- 1 solution;
- Figure 22 shows the fluorescence emission (top trace) of FITC detected on solution (a) and pSiRM surface (b) after cleaved by Sortase A, while the lower trace is the blank which is the Sortase A substrate solution (a) and functionalised pSiRM surface (b) before contact with Sortase A enzyme;
- Figure 23 shows the fluorescence intensity of the FITC from the Sortase A peptide substrate after cleavage by Sortase A at different contact times with the error bars calculated from three different experiments;
- Figure 24 shows the fluorescence intensity of the FITC from the Sortase A peptide substrate after cleavage by Sortase A at different concentrations of Sortase A enzyme with the error bars calculated from three separate experiments;
- Figure 25 shows the fluorescence intensity of the FITC from the Sortase A peptide substrate from a wound fluid experiment
- Figure 26 shows the fluorescence intensity of the FITC from the Sortase A peptide substrate after cleaved by bacteria supernatant sample
- Figure 27 shows the fluorescence intensity of the FITC from the Sortase A peptide substrate after cleavage by bacteria supernatant sample with different inoculation times (0, 0.5, 1 , 3, 5 and 24 h) and wound fluid sample inoculated with bacteria for 0 and 24 h with the error bars calculated from three separate experiments;
- Figure 28 shows a comparison of emission intensity of different concentration of Sortase A in buffer solution (full circle), added in wound fluid sample (open circle) and added in bacterial supernatant sample (triangle). The error bars were calculated from three separate experiments.
- the biosensor comprises a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface.
- the detection agent comprises a sensing domain and a signaling domain.
- the sensing domain comprises a linker capable of interacting with the target bioanalyte and the signal ing domain comprises a luminescence donor and a luminescence acceptor.
- the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled in the absence of the target bioanalyte such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor.
- the surface of the substrate further comprises a plurality of light interacting pores, wherein the pores are configured to interact with the light emission from the luminescence donor to provide a measurable light emission which is indicative of the presence of the target bioanalyte.
- the light emission from the luminescence donor is enhanced by interaction with the light interacting pores of the substrate.
- An internal surface of the light interacting pores may comprise an optical structure that interacts with the light emission from the luminescence donor.
- the optical structure may be an optical filter, reflector or cavity.
- the internal surface of the light interacting pores may comprise a Bragg reflector, a rugate filter, a resonant microcavity, or a combination of any of these optical features.
- a substrate that is a porous silicon resonant microcavity (pSiRM) substrate in which the light interacting pores comprise distributed Bragg reflectors separated by a resonant microcavity.
- pSiRM porous silicon resonant microcavity
- the light interacting pores comprise another type of optical feature, such as a rugate filter, a resonant microcavity or a Bragg reflector.
- luminescence donors and the luminescence acceptors that are fluorescence donors and fluorescence acceptors. It will be appreciated that the luminescence donors and the luminescence acceptors could be phosphorescence donors and
- porous silicon resonant microcavity provide an optical biosensor to monitor the presence of specific biomarkers found in wound exudate, such as matrix metalloproteinases (MMPs) and bacterial enzyme biomarkers.
- MMPs matrix metalloproteinases
- the pSiRM is functionalised using a fluoiOgenic MMP peptide substrate featuring both a fluorophore and a quencher. The peptide -functionalised pSiRM is then used as a fluorescence-based optical biosensor for MMPs.
- Active MMPs interact with and cleave the linker, producing an immobilised peptide fragment carrying the fluorophore.
- the fluorescence intensity of the fluorophore embedded within the pSiRM matrix is enhanced by the photonic structure of the pSiRM compared to other pSi photonic structures. This fluorescence enhancement translates into high sensitivity, enabling detection of MMP-1 at a limit of detection as low as 7.5 x 1 0 19 M after only 15 min incubation time.
- the biosensor comprises a porous silicon resonant microcavity (pSiRM) substrate.
- Porous silicon (pSi) has been used previously in optical biosensors.
- Gao et /. [20] achieved MMP-2 detection as low as 1.5 x 10 ⁇ I2 M using a biosensor based on a pSi rugate filter coated with gelatin, which can be digested by MMP-2. The digestion products then entered in to the pSi matrix and induced color changes that could be observed by the naked eye.
- Martin et al A ⁇ designed a biosensor to detect MMP-8 based on antibody-functionalised pSiRM and monitored the presence of MMP-8 by observing a shift in the resonance cavity dip of the pSiRM. This device was able to detect MMP-8 down to 1.5 x 10 9 M. However, neither of these biosensors was used with complex biological fluids. Kilian et developed a label-free biosensor to detect MMPs secreted by human macrophages as an example of a biological fluid. The biosensor was based on photonic crystals of anodized silicon loaded with a biopolymer. It successfully detected MMP-9 down to a level of 1.2 x 10 M.
- the substrate may be a porous alumina substrate.
- the pSiRM is a photonic structure comprising two distributed Bragg reflectors (DBR) separated by a microcavity layer, producing a reflectance spectrum with a sharp resonance cavity dip in the center of the reflectance band.
- DBR distributed Bragg reflectors
- Each DBR consists of periodic layers of alternating low porosity (LP) and high porosity (HP) pSi, with high and low refractive index, respectively, but the same optical thickness.
- the optical thickness for each DB R is 1/4, where 1 is the central wavelength of the photonic resonance band with near 100% reflectance.
- the resonance microcavity has an optical thickness of an integer multiple of 1/2.
- the position of the central wavelength of the resonance cavity dip can be tuned by changing the electrochemical etching parameters. ⁇
- the pSiRM substrate can be formed by electrochemical etching.
- pSiRM substrates can be fabricated by anodically etching a Si wafer using a current density alternating between 50 mA/cm' for 2288 ms and 25 mA/cm' for 1820 ms to form HP and LP layers, respectively.
- the resonant microcavity can be etched at a current density of 50 mA/cm 2 for 9152 ms.
- the resulting pSiRM had the configuration (HP/LP) 3 (HP) 4 (LP/HP) 3 .
- the Si wafer may be pre-treated in order to remove the parasitic layer from the substrate prior to the electrochemical etching.
- pSi substrates have many advantages when used in label-free optical biosensors. Specifically, pSi substrates have a very large surface area (up to 600 m 2 /g), tunable morphological and optical properties, they are biocompatible and have a range of surface chemistries. Pore size can be tuned to allow ingress of even large biomolecules such as antibodies.
- the morphological properties such as pore size, porosity and thickness can be tuned to fabricate pSi substrates suitable for ingress of the targeted bioanalyte while excluding others.
- Biocompatibility of the substrate material is also essential when direct contact of the biosensor occurs with the human body, such as through a smart dressing.
- pSi is well tolerated in vitro and in vivo and degrades into orthosilicic acid, the natural form of silicon in humans.
- the pSiRM substrates described herein have two interesting optical features for biosensing applications. Firstly, the resonance cavity dip of the pSiRM is sensitive to the refractive index changes. Specifically, small refractive index changes induce large shifts in the optical spectrum. This optical feature lends itself to biosensor design and has been previously explored in biosensing applications, such as glucose detection ' 51 bacteria detection/' 3 ' 411 viruses and DNA detection. 4 " 1
- the second optical feature is a confinement effect of light inside the microcavity to a specific wavelength contributing to the enhancement of fluorescence emission of the fluorescence donor immobilised on the pSiRM. The optimum enhancement of the fluorescence emission is obtained if the wavelength of the microcavity dip is aligned with the emission wavelength of the fluorescence donor.
- the surface of freshly etched pSiRM substrates may be unstable and prone to oxidation in the presence of oxygen or to hydrolysis in the presence of water leading to uncontrollable optical properties which is undesirable for biosensor applications.
- the surface of the freshly etched pSiRM substrate may be functionalised.
- the surface may be functionalised with an alkylating agent to produce a functionalised alkyl monolayer on the surface of the substrate, or by surface modification with alkenes, yielding organic monolayers covalently attached to the surface' 661 or by grafting of alkynes to the hydride-terminated silicon surface through a direct Si-C bond via nanoscale cathodic electrografting reaction through the use of conducting atomic force microscope (ATM).
- ATM conducting atomic force microscope
- the surface of the freshly etched substrate is functionalised with an alkylating agent.
- the alkylating agent may be an alkyl carboxylic acid, such as a C5-C20 carboxylic acid, ester, suphonate, alkyne, azide, alkene, or combination of any of the aforementioned.
- the surface is hydrosilylated using undecylenic acid. This produces a dense alkyl monolayer with stable Si-C bonds protecting the pSiRM surface from oxidative hydrolysis.
- the functional group on the alkylating agent can be used to covalently attach the detection agent.
- a carboxylic acid can be used to covalently attach the detection agent using known peptide synthesis methods.
- a carboxylic acid-terminated surface can be activated to form an NHS ester- terminated surface by reacting the pSiRM samples with N-hydroxysuccinimide (NHS) in the presence of a coupling agent, such as 1 -(3 -dimethyl aminopropyl)-3-ethylcarbodiimide (EDC).
- NHS N-hydroxysuccinimide
- EDC 1 -(3 -dimethyl aminopropyl)-3-ethylcarbodiimide
- the detection agent can then be coupled to the NHS ester-terminated surface by reacting the functionalised pSiRM surface with the detection agent to provide a modified pSiRM surface that is ready for use in biosensing.
- the detection agent may be a fusion peptide or protein comprising a signaling domain and a sensing domain.
- the signaling domain comprises a fluorescence donor and a fluorescence acceptor.
- the sensing domain comprises a bioanalyte -binding peptide.
- the fluorescence donor and the fluorescence acceptor are fused to both termini of the bioanalyte -binding peptide.
- the fluorescence donor and/or the fluorescence acceptor may be fused to the bioanalyte-binding peptide or protein via a linker.
- FRET fluorescence resonance energy transfer
- fluorescence donor means a fluorophore acting as a donor in the FRET mechanism
- fluorescence acceptor refers to a fluorophore acting as an acceptor in the FRET mechanism.
- the fluorescence donor can be any dye molecule that absorbs light which places the dye in an excited state and then returns to the ground state by emitting light (fluorescence).
- the fluorescence acceptor can be any dye molecule with no native fluorescence which nonradiatively accepts energy from the fluorescence donor to generate an acceptor excited state. The fluorescence acceptor then preferably returns to the ground state nonradiatively by giving off energy as heat.
- the energy transfer efficiency of FRET varies depending on the range in which the emission spectrum of the fluorescence donor and the absorption spectrum of the fluorescence acceptor overlap with each other, the quantum efficiency of the fluorescence donor, the relative orientation of transition dipoles of the fluorescence donor and the fluorescence acceptor, and the distance between the fluorescence donor and the fluorescence acceptor.
- the energy transfer efficiency of FRET vari es depending on the distance between the fluorescence donor and the fluorescence acceptor and the relative orientation thereof.
- any fluorescence donor and fluorescence acceptor pair for which the emission spectrum of the donor and the absorption spectrum of the acceptor can overlap with each other to cause FRET may be used.
- fluorescence donors that may be used include fluorescent proteins, fluorescent dyes, bioluminescent proteins, and quantum dots, which have various wavelengths.
- fluorescence acceptors that may be used include fluorescent proteins, fluorescent dyes, and quantum dots, which have wavelengths different from those of the fluorescence donor.
- the fluorescence acceptor may consist of quenchers or gold nanoparticles, which reduce the fluorescence intensity of the fluorescence donor.
- EDANS 5-[(2 -Aminoethyl)amino]naphthalene-l -sulfonic acid
- EDANS can be paired with the fluorescence acceptors 4-((4- (dimethylamino)phenyl)azo)benzoic acid (Dabcyl) or 4-((4-(dimethylamino)phenyl)azo)sulfonic acid (Dabsyl).
- FRET pairs that may be used include: ECFP (enhanced cyan fluorescent protein) and EYFP (enhanced yellow fluorescent protein), which are fluorescent proteins acting as a fluorescence donor and a fluorescence acceptor, respectively; fluorescein and Dabcyl acting as a fluorescence donor and a fluorescence acceptor, respectively; fluorescein and Cy5 acting as a fluorescence donor and a fluorescence acceptor, respectively; gold nanoparticles and Cy3 acting as a fluorescence donor and a fluorescence acceptor, respectively; or gold nanoparticles and Cy5 acting as a fluorescence donor and a fluorescence acceptor, respectively.
- ECFP enhanced cyan fluorescent protein
- EYFP enhanced yellow fluorescent protein
- the fluorescence donor and fluorescence acceptor pair may be colloidal semiconductor nanocrystals (ie. quantum dots).
- quantum dots The broad absorption spectra of quantum dots allow flexibility in choosing the desired excitation wavelength where direct excitation of the acceptor molecules can be substantially reduced.
- luminescent CdSe-ZnS core-shell quantum dots QDs
- QDs luminescent CdSe-ZnS core-shell quantum dots
- the detection agent can be prepared by conjugating the fluorescence acceptor with the bioanalyte -binding peptide and then allowing the product to self-assemble on appropriately functionalised quantum dots (eg. quantum dots functionalised with a dithiol-alkyl-COOII ligands).
- the methods described in Clapp et al. can be used to prepare detection agents based on quantum dots 0]
- the sensing domain links the fluorescence donor and the fluorescence acceptor and comprises a bioanalyte -binding domain.
- the bioanalyte-binding domain may be a peptide, protein, sugar, amino acid, lipid or other agent that selectively binds the bioanalyte and undergoes a conformational or compositional change as a result of that binding.
- a conformational change in the bioanalyte-binding domain may result from the bioanalyte competitively binding to a domain that is otherwise intermolecularly bound to another portion of the domain in the absence of the bioanalyte (i.e. unfolding of the bioanalyte-binding domain).
- the intramolecular binding is reduced, thereby resulting in the fluorescence donor and the fluorescence acceptor spatially separating from one another to give a measureable fluorescence emission.
- the bioanalyte-binding domain undergoes a compositional change when it interacts with the bioanalyte.
- the bioanalyte may be a protease enzyme and the bioanalyte-binding domain may be a peptide or protein that is a substrate for the enzyme.
- the biosensor may further comprise a bioanalyte specific capture agent.
- the bioanalyte specific capture agent may comprise a binding agent capable of selectively binding the target bioanalyte.
- the bioanalyte specific capture agent may be deposited on or near the surface of the porous silicon or alumina substrate so that at least some of any bioanalyte captured by the capture agent is capable of interacting with the sensing domain of the detection agent.
- the bioanalyte specific capture agent may be in the form of particles comprising a binding agent on the surface thereof.
- the particles may be functionalised nanoparticles (NPs) having the binding agent bound to a surface thereof.
- the functionalised NPs may interact with and be retained on the surface of the substrate.
- the functionalised NPs may be functionalised magnetic nanoparticles (MNPs).
- the binding agent may be any agent that selecti vely binds the target bioanalyte.
- the binding agent may bind the target bioanalyte selectively from complex fluids comprising other components that are structurally related to the target bioanalyte.
- the biding agent may be an antibody.
- the biosensor of these embodiments may be used for the selective detection of a specific peptide or protein in a family of structurally related peptides or proteins.
- the bioanalyte is a matrix metalloproteinase (MMP) (described in detail later) and the bioanalyte-binding domain of the detection agent is a substrate for the MMP.
- MMP matrix metalloproteinase
- the bioanalyte-binding domain may comprise the MMP substrate Gaba-Pro-Gln-Gly-Leu- Glu-Ala-Lys-NIL in which case the detection agent may be Dabcyl-Gaba-Pro-Gln-Gly-Leu- Glu(EDANS)-Ala-Lys-NH 2 .
- the distance between Dabcyl and EDANS is about 5- 6 nm, at which FRET can occur.
- the bioanalyte is a biological molecule of interest in a sample that is to be detected, analysed, and/or quantified.
- bioanalytes include, but are not limited to, amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty acids, lipids, hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, metabolites, cofactors, inhibitors, drugs, pharmaceuticals, nutrients, prions, toxins, poisons, explosives, pesticides, biohazardous agents, carcinogens, mutagens, narcotics, amphetamines, barbiturates, and hallucinogens.
- monitoring of glucose levels in diabetic subjects For example, monitoring of glucose levels in diabetic subjects.
- the bioanalyte is a peptide or protein associated with wounds.
- a clinically validated biomarker in chronic wounds is in the group of matrix
- MMPs metalloproteinases
- collagenases such as MMP-1 and MMP-8
- gelatinases including gelatinase-A or MMP-2
- stromelysins such as stromelysin-1 or MMP -3
- membrane-type MMPs such as MTl -MMP or MMP- 14
- MMP -7 heterogeneous subgroup containing matrilysin
- MMP-20 macrophage metalloelastase
- MMP-12 macrophage metalloelastase
- MMPs The activity of MMPs can be inhibited by tissue inhibitors of metalloproteinases
- TIMPs 112 ' 14'17
- synthetic inhibitors 112 ' 16
- the inhibition involves forming a chelate complex between TIMPs and a zinc ion at the active site of MMP.
- concentration of MMPs needs to be known to correctly dose the inhibitor since over-inhibition is also deleterious.
- fl l Therefore, chronic wound management would benefit from a POC biosensor that is able to rapidly establish MMP levels in wound fluid.
- a method for monitoring and/or assessing wound status in a subject comprising: providing an optical biosensor for detecting a matrix metalloproteinase in a wound fluid from said subject, the biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker that is a substrate for the matrix metalloproteinase and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor, and interaction of the matrix metalloproteinase with the linker results in cleavage of the linker and optical un-coupling of the luminescence donor and the luminescence acceptor to thereby result in
- the substrate is a porous silicon resonant microcavity (pSiRM) substrate and there is provided a method for monitoring and/or assessing wound status in a subject, the method comprising: providing an optical biosensor for detecting a matrix metalloproteinase in a wound fluid from said subject, the biosensor comprising: a porous silicon resonant microcavity (pSiRM) substrate comprising a surface comprising a plurality of light interacting pores, each pore comprising distributed Bragg reflectors separated by a microcavity; and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker that is a substrate for the matrix metalloproteinase and the signaling domain comprising a fluorescence donor and a fluorescence acceptor wherein the fluorescence donor and the fluorescence acceptor are connected by the linker and are optically coupled such that emission of light from the
- MMPs assays already exist, but they have not been developed and demonstrated for chronic wounds.
- EDANS 5-((2-aminoethyl)amino)naphthalene- 1 -sulfonic acid
- the selectivity of the biosensor toward specific MMPS may be achieved using a bioanalyte specific capture agent.
- the bioanalyte specific capture agent may comprise a binding agent capable of selectively binding a specific MMP protein, such as one selected from one of the group consisting of MMP- 1, -2, -3 and -9.
- the binding agent may be capable of selectively binding the one selected MMP in the presence of other MMPs.
- the binding agent may be an antibody.
- the binding agent may be functionalised nanoparticles (NPs), such as functionalised magnetic nanoparticles (MNPs), having the antibody bound to a surface thereof.
- NPs are functionalised with the MMP antibody (MMPAb) to harvest the target MMP from buffer solution or from wound fluid samples.
- MMP antibody MMP antibody
- the NPs can be modified with any type of MMPAb depending on the targeted MMP.
- the MNPs are immobilised with MMP-lAb in order to target MMP- 1.
- a method for monitoring and/or assessing cancer status in a subject comprising: providing an optical biosensor for detecting a matrix metalloproteinase in cancer tissue or blood from said subject, the biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker that is a substrate for the matrix metalloproteinase and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled such that
- the cancer tissue or blood can be obtained from a subject using known techniques, such as biopsy.
- the bioanalyte is a bacterial biomarker. Accordingly, there is provided a method for monitoring and/or assessing bacterial infection in a subject, the method comprising: providing an optical biosensor for detecting a bacterial biomarker in a body fluid from said subject, the biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker that is a substrate for the bacterial biomarker and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled such that emission of light from the luminescence donor is substantially quenched by the luminescence acceptor, and interaction of the bacterial biomarker with the linker results in cleavage of the linker and optical uncoupling of the luminescence donor
- the body fluid can be any fluid or tissue suspected of containing the bacterial biomarker of interest including, but not limited to, blood, wound fluid, sweat, saliva, excreta, body tissue and tissue fluids.
- the body fluid can be collected using known techniques.
- the substrate is a porous silicon resonant microcavity (pSiRM) substrate and there is provided a method for monitoring and/or assessing wound status in a subject, the method comprising: providing an optical biosensor for detecting a bacterial biomarker in a wound fluid from said subject, the biosensor comprising: a porous silicon resonant microcavity (pSiRM) substrate comprising a surface comprising a plurality of light interacting pores, each pore comprising distributed Bragg reflectors separated by a microcavity; and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker that is a substrate for the bacterial biomarker and the signaling domain comprising a fluorescence donor and a fluorescence acceptor wherein the fluorescence donor and the fluorescence acceptor are connected by the linker and are optically coupled such that emission of light from the fluorescence donor is substantially
- the bacterial biomarker may be a peptide, protein or other molecule that is indicative of infection by a bacterial species such as Bacillus anthracis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Borrelia burgdorferi, Treponema pallidum, Chlamydia trachomatis, Chlamydophila psittaci, Coryne bacterium diphtherias, Mycobacterium tuberculosis, and Mycobacterium avium, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Anaplasma phagocytophilum, Ehrlichia
- Burkholderia mallei B. pseudomallei, Neisseria gonorrhoeae, Neisseria meningitides, Campylobacter jejuni, Helicobacter pylori, Legionella pneumophila, Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa, Aeromonas sp., Vibrio cholerae, Vibrio parahaemolyticus,
- Thiotrichales sp. Haemophilus influenzae, Klebsiella pneumoniae, Proteus mirabilis, Yersinia pestis, Yersinia enterocolitica, Shigella flexneri, Salmonella enterica or Escherichia co i.
- Detection agents that are able to selectively detect any one of the aforementioned bacteria can be prepared using a suitable bioanalyte-binding peptide or protein and FRET pair, as described previously.
- the bioanalyte-binding peptide or protein may be MBP (maltose-binding protein), ALBP (allose-binding protein), ARBP (arabinose-binding protein) or GGBP (galactose/glucose-binding protein).
- MBP maltose-binding protein
- ALBP allose-binding protein
- ARBP arabinose-binding protein
- GGBP galactose/glucose-binding protein
- proteinase K is secreted by Pseudomonas aeruginosa and can digest poly-lysine.
- a bioanalyte-binding peptide or protein comprising a poly-lysine motif can be used to detect P. aeruginosa.
- Hyaluronidase is secreted by Staphylococcus aureus and can digest hyaluronic acid. Therefore, a bioanalyte-binding peptide or protein comprising a hyaluronic acid motif can be used to detect S. aureus.
- any bioanalyte-binding peptide or protein may be used in the method and biosensor so long as it can undergo a conformational or compositional change as a result of binding of the bioanalyte thereto.
- sample refers to a composition that is suspected to contain the bioanalyte of interest and is to be analysed.
- the sample may comprise or be derived from a biological source such as a bodily fluid, including for example, wound fluid or exudate, blood, saliva, milk, mucous, urine, etc.
- a biological source such as a bodily fluid, including for example, wound fluid or exudate, blood, saliva, milk, mucous, urine, etc.
- other samples that may be tested include water samples and food and beverages products that may be monitored for toxins and/or contaminating pathogenic microorganisms.
- the sample may be collected from one or more of cells, water, soil, air, foods, waste, and animal and plant organs and tissues.
- Detection of the target bioanalyte in a sample is performed by measuring emissions from the fluorescence donor and the fluorescence acceptor using a fluorescence analysis system.
- Suitable fluorescence analysis systems include filter-type and monochrome -type fluorescence spectrophotometers. If a sample contains the target bioanalyte, changes in the emissions from the fluorescence donor and the fluorescence acceptor are sensed, whereby the target bioanalyte can be detected. Furthermore, if a change in the concentration of the target bioanalyte occurs, a change in the emissions from the fluorescence donor and the fluorescence acceptors occurs. Thus, the biosensor can be also be used to measure a change in the bioanalyte concentration.
- Emissions from the fluorescence donor and the fluorescence acceptor can also be measured using a confocal microscope.
- the detection agent can be spatially arranged on the substrate surface by microcontact printing and the emissions observed using confocal microscopy. This method can be used to form a multi-analyte biosensor.
- the optical biosensor is part of a detection device.
- the detection device may be a point-of-care (POC) device.
- POC point-of-care
- the detection device comprises a fluid inlet through which the sample can be introduced, a housing for the optical biosensor described herein, and an optical output for outputting information on the emission intensity of the fluorescence donor.
- the detection device may also comprise a means for directly collecting and transferring a test sample from a subject to the detection device.
- the detection device may utilise a microneedle or one or more microneedle arrays designed to transfer a bodily fluid, such as a wound exudate, from the subject to the device via capillary action and/or surface tension.
- the optical biosensor may be part of a wound dressing or bandage.
- the optical biosensor may be fixed or otherwise attached to a wound dressing or bandage material and may provide information to a practitioner regarding the status of a wound.
- the bioassays described herein are designed to detect one or more bioanalytes of interest in a sample, the presence of which is correlated to a specific disease or predisposition to a disease. The presence of the bioanalytes of interest can function as a warning to a subject, or a healthcare professional, that a disease is present or may develop in the future.
- the detection device may be used for detection of multiple target bioanalytes.
- the housing may comprise a plurality of spatially arranged optical biosensors with each biosensor capable of selectively detecting a different bioanalyte relative to an adjacent biosensor.
- each biosensor may be capable of detecting a bioanalyte that is indicative of a specific bacterial species and the detection device can thereby be used in the detection of a plurality of bacterial infections in a single step.
- compositions of the invention may also have veterinary uses for diagnosing diseases in animals.
- Appropriate bioassays can be designed to selectively detect the intended target bioanalyte.
- the optical biosensor described herein may be part of a theranostic device.
- the term “theranostic” refers to a delivery system, which may be used to at least one of treating, preventing, monitoring or diagnosing a disease or pathological condition.
- a theranostic device for the diagnosis and/or treatment of a disease or pathological condition in a subject, the device comprising: providing an optical biosensor for detecting and/or determining the concentration of a bioanalyte that is a biomarker of said disease or pathological condition in a sample of bodily fluid obtained from said subject, the optical biosensor comprising: a porous silicon or alumina substrate comprising a surface and a detection agent immobilised on the surface, the detection agent comprising a sensing domain and a signaling domain, the sensing domain comprising a linker capable of interacting with the target bioanalyte and the signaling domain comprising a luminescence donor and a luminescence acceptor wherein the luminescence donor and the luminescence acceptor are connected by the linker and are optically coupled such that emission of li ght from the luminescence donor is substantially quenched by the luminescence acceptor, and i nteraction of the target bioanalyte
- pathological condition means an abnormal anatomical or physiological condition and objective or subjective manifestations of disease, not classified as disease or syndrome.
- the delivery system may comprise a microparticle or a nanoparticle that is loaded with the therapeutic agent and is activated to release the therapeutic agent to treat the disease or pathological condition by the controller.
- the microparticle or nanoparticle can be a multistage particle, a porous particle, a porous silicon particle, a porous silica particle, a non-porous particle, a fabricated particle, a polymeric particle, a synthetic particle, a semiconducting particle, a virus, a gold particle, a silver particle, a quantum dot, an indium phosphate particle, an iron oxide particle, a micelle, a lipid particle, a liposome, a silica particle, a mesoporous silica particle, a PLGA-based particle, a gelatin-based particle, a carbon nanotube or a fullerene.
- the delivery system could also be a pump for delivering a liquid therapeutic agent to the subject.
- the therapeutic agent may be a physiologically or pharmacologically active substance that can produce a desired biological effect in a targeted site in an animal, such as a mammal or a human.
- the therapeutic agent may be any inorganic or organic compound. Examples include, without limitation, peptides, proteins, nucleic acids (including siRNA, miRNA and DNA), polymers, and small molecules.
- Non-limiting examples of therapeutic agents include wound repair agents, tissue repair agents, thermal therapy agents, anti-bacterial agents, anti-inflammatory agents, anti-cancer agents, antiproliferative agents, anti-vascularisation agents, and combinations thereof.
- therapeutic agents include anti-infective agents; antibiotics, such as penicillins, cephalosporins, macrolids, tetracyclines, aminglycosides, and antituberculosis agents; antifungal/antimycotic agents; antiviral agents, such as acyclovir, gancyclovir, ribavirin, anti-HIV agents, and anti-hepatitis agents; anti -inflammatory agents, such as NSAlDs, steroidal agents, cannabinoids; anti-allergic agents, such as antihistamines, (e.g., fexofenadine); vaccines or immunogenic agents, such as tetanus toxoid, reduced diphtheria toxoid, acellular pertussis vaccine, mumps vaccine, smallpox vaccine, anti-HIV vaccines, hepatitis vaccines, pneumonia vaccines and influenza vaccines; anesthetics, including local ane
- MMP- 1 as one of collagenases because this enzyme is one of key enzymes responsible for cleaving interstitial fibrillar collagen' 441 which is crucial during wound healing. 1451
- the pSiRM structure afforded enhanced emission in comparison to other pSi structures and allowed detection of MMP-1 down to the attomolar level in buffer. This pSi optical biosensor was also successfully applied to detect MMPs in human wound fluid.
- High purity solvents (methanol, ethanol, acetone and dichloromethane) were purchased from Chem Supply. All pSi samples were prepared from highly doped, ( 100)-oriented, phosphorus doped n-type Si wafer (0.008 - 0.02 ⁇ cm, Siltronix). The Si wafer was diced using a diamond cutter into pieces of 3-4 cnf .
- Example 1 Fabrication and characterisation of porous silicon resonance microcavity substrates
- pSi substrates were prepared in a Teflon-based electrochemical etching cell using aluminium tape as a contact for the silicon piece as anode and a platinum mesh as cathode.
- the electrochemical etching solution contained 25:200: 1 volume ratio of aqueous hydrofluoric acid (48%, Scharlau)/water/surfactant (NCW1001, Wako Pure Chemical Industries).
- the Si wafer was pre-treated in order to remove the parasitic layer from the substrate by anodically etching the Si wafer at a current density of 40 mA/cm 2 for 30 s, followed by a current density of 250 mA/cm 2 for 6 s which led to electropolishing. Following this step, the surface was exposed to MilliQ water for 1 min to remove the sacrificial layer, then rinsed with methanol, acetone, dichloromethane and dried under a stream of nitrogen gas.
- pSiRM porous silicon resonance microcavity
- the pSiRM substrate used in the optical biosensors was then designed via the SCOUT program based on the obtained porosity and thickness values of the single layer pSi substrate.
- the contrast of porosity and thickness was chosen to obtain an appropriate refractive-index profi le of the pSiRM substrate with the position of the resonance cavity dip at the desired wavelength.
- the required current densities and etching time were also obtained via this method.
- the pSiRM substrates were fabricated by anodically etching a Si wafer using a current density alternating between 50 mA/cnT for 2288 ms and 25 mA/cnf for 1820 ms corresponding to HP and LP layers, respectively.
- the defect layer was etched at a current density of 50 mA/cm' for 9152 ms.
- the resulting pSiRM had the configuration (HP/LP) 3 (HP) 4 (LP/HP) 3 .
- the pSiRM substrate was also characterised using IRS to ensure that the reflectance spectrum matched that of the simulation.
- the single layer pSi and pSiRM substrates were analysed using SEM.
- a Quanta 450 field emission gun (FEG) Environmental SEM fitted with a Solid- State Detector (SSD) and an accelerating voltage of 30 kV was used.
- FIG. 1 A schematic schematic representation of surface functionalisation reactions of a hydride- terminated pSiRM surface is shown in Figure 1.
- Freshly etched pSiRM substrates from Example 1 were functionalised by thermal hydrosilylation of neat undecylenic acid in a glass reaction flask. Before performing the reaction, the undecylenic acid was purged with argon for 15 min to remove any oxygen. The pSiRM substrates were then immersed in the undecylenic acid and purged for an additional 30 min. Afterwards, the reaction flask was immersed in an oil bath at 120 °C and the reaction proceeded for 3 h under an argon flow.
- the hydrosilylated pSiRM substrates were removed from the flask, rinsed with ethanol and dried gently under a stream of nitrogen gas.
- the hydrosilylated pSiRM substrates with a carboxylic acid- terminated surface was activated to form an NHS ester-terminated surface by reacting the pSiRM substrates with N-hydroxysuccinimide (NHS) (5 mM) in water in the presence of l-(3- dimethylaminopropyl)-3-ethylcarbodiimide (EDC, Fluka) (5 mM) for 20 min at room temperature.
- NHS N-hydroxysuccinimide
- EDC l-(3- dimethylaminopropyl)-3-ethylcarbodiimide
- FTIR spectra were obtained using a Vertex 70 Hyperion microscope (Bruker) in the ATR mode.
- the freshly etched pSiRM substrate features a hydride-terminated surface. This surface is unstable and tends to oxidise in the presence of oxygen or to hydrolyse in the presence of water leading to uncontrollable optical properties which is undesirable for biosensor applications. 129, 31 ' 49 ' We
- the pSiRM substrates were characterised by Fourier Transform Infrared (FTIR) spectroscopy in the attenuated total reflectance (ATR) mode after every surface functionalization step ( Figure 1(b)). Hydrosilylation of the freshly etched pSiRM surface using neat undecylenic acid (Spectrum (i)) replaced the Si-H bonds on the surface with Si-C bonds. This was confirmed by the appearance of characteristic bands at 1459 cm 4 , 2865 cm 1 and 2935 cm 1 which were assigned to the 5cn tet. deformation mode of methylenes and the stretching vibrational of aliphatic C-H bonds, respectively.
- FTIR Fourier Transform Infrared
- the characteristic band of v ( 0 stretching mode of a carboxylic acid was observed at 1714 cm 4 .
- the presence of very faint bands at 904 cm 1 and 2100 cm assigned to Si -IT scissor vibrational mode and Si-H x stretching vibrational mode of the freshly etched pSi, respectively, indicate that there is a small amount of unreacted silicon hydride groups left on the surface.
- the band at 1033 cm 1 attributed to the Si-0 stretching vibrational indicates the presence of silicon dioxide at the surface of the pSiRM substrate. Residual silicon hydrides and a small amount of surface oxidation are commonly observed in the hydrosilylation of p Si/ 49 5 "'
- the 5 nm red shift after hydrosilylation can be explained by an increase in effective refractive index due to the monolayer formation of undecylenic acid within the porous layer.
- Ouyang et al. observed the shift of microcavity dip after binding of thin layer molecules with different thickness considering some parameters, such as pore diameter and the refractive index changes before and after binding. They reported that a 10 nm red shift of the resonance cavity dip in the macroporous microcavity was produced by a 3 nm thick coating, which means, in our case, that for a 5 nm red shift, the monolayer thickness should be 1.5 nmJ 25] This thickness is in reasonable agreement with what Bocking et al. observed for an undecylenic acid monolayer using X-ray reflectometry (0.9 - 1.1 nm). [53]
- Example 3 Use as an optical biosensor
- the peptide functionalised pSiRM substrates were incubated in activated MMP-1 at varying concentrations at 37 °C for a few min and then rinsed with water, 2: 1 water/ethanol, 1 :2 water/ethanol and ethanol to remove unbound analytes. Afterwards, the substrates were dried gently under a stream of nitrogen gas. The dried pSiRM surface was placed in a cuvette with a special holder to support the pSiRM substrate. The cuvette was then placed in a fluorometer with the posi tion of pSiRM surface facing the light source at a 36° angle.
- the fluorescence intensity of the fluorophore was measured using a fluorometer (Parkin Elmer LS 55 Luminescence Spectrometer). The emission was measured over a wavelength range of 360 - 540 nm at a fixed excitation wavelength of 340 nm, excitation and emission slit widths of 5 nm each, and a scan speed of 200 nm/min.
- the angle formed by the light source of the fluorometer and the defect layer of the pSiRM substrate in respect to the surface normal was set to 36° since we obtained highest fluorescence signals at this angle.
- Human wound fluid sample was collected from Women's and Children's Hospital (Adelaide, South Australia).
- the study protocol which conformed to the ethical guidelines of the 1975 Declaration of tlelsinki, was approved by the Health Service Human Research Ethics Committee and Central Northern Sydney Health Service Ethics of Human Research Committee.
- the wound fluid was diluted 10-fold in buffer solution.
- the sensing platform was incubated in the wound fluid sample at 37 °C and then treated in the same ways as described above.
- the optical biosensor investigated in this study was based on a photonic pSiRM substrate which consisted of two DBR and one resonance cavity layer. Each DBR had a periodic layer structure alternating between different porosities (HP and LP) with a quarter-wavelength ( ⁇ /4) optical thickness while the defect layer had a HP layer with an optical thickness of a multiple of half-wavelength ( ⁇ /2).
- the first task was to design a pSiRM substrate with appropriate porosity contrast between HP and LP layers. We therefore prepared five single layer pSi substrates etched at different current densities for 120 s. The samples were characterised morphologically and optically to determine pore size, porosity and thickness (see Table 1).
- m is the fringe order
- ⁇ is the wavelength of the incident light for maximum constructive interferences
- n is the refractive index of the porous film
- L is the film thickness
- the factor of 2 is derived from the factor of 90° backscatter configuration of the light source and detector.
- Each DBR featured three periodic bilayers with a porosity of 83.4% for HP and a porosity of 67.0% for LP starting with HP for the first DBR and LP for the second DBR.
- Those porosity values produce pore diameters ranging from 40 - 60 nm for LP layer and 1 10 - 140 nm for IIP layer, respectively.
- Those pore sizes were large enough to allow ingress of the desired target molecules while retaining the sensitivity of the biosensor.
- the pore size is an important parameter because it affects the internal surface area of the pSiRM substrate where the biorecognition molecules are attached and the target bioanalyte is captured.
- the pore size By decreasing the pore size, the internal surface area and the density of available binding sites for target bioanalytes are increased, which translates into higher sensitivity.' 40 '
- too small pore sizes prevent infiltration of large biomolecules into the entire porous layers.' 25, 36] The chosen parameters therefore represent a compromise between these two requirements.
- the pSiRM substrate was designed with a center wavelength ( ⁇ ) of the resonance cavity dip at 440 nm measured at a light incidence angle of 36° or at 478 nm at 0° (a 38 nm blue shift from the angle of 0° to 36°).
- ⁇ center wavelength
- the experimental result obtained by IRS also showed that the pSiRM substrate designed at 440 nm produced the resonance cavity dip at 448 nm (at an angle of 36°) corresponding to an 8 nm red shift different between design and the IRS experiment (Figure 3(a)).
- the pSiRM substrate was re-designed at 432 nm.
- the refractive indices (n) were 1.3 and 1.8 for the HP and LP layer, respectively, calculated using Bruggeman effective medium approximation.
- the value of n and ⁇ were used to determine the thickness of each periodic layer considering the ⁇ /4 for each DBR and ⁇ /2 for the defect layer.
- the HP layer formed an 83 nm thick layer, while the LP layer formed a 60 nm thickness.
- the cross-sectional SEM image in Figure 3(c) reveals the periodic layers forming the pSiRM substrate, with the top and bottom DBR each featuring 3 periodic layers of HP/LP separated by an HP resonance cavity layer.
- the thickness of the periodic layer of the pSiRM substrate was 1.19 ⁇ .
- the peptide-functionalised pSiRM substrate was then used to detect MMP- 1 in buffer solution.
- MMP-1 was chosen since this MMP is prominent in wound fluid 154 ' ⁇ and is known to cleave the fluorogenic peptide sequence.
- the sensing was performed by incubating the peptide-functionalised pSiRM substrate in the MMP- 1 then rinsed and dried for measurement.
- this small shift would limit the sensitivity of the device if the sensing was only done by IRS.
- Figure 5(a-b) shows that the EDANS emission intensity of the fluorophore embedded in the pSiRM substrate was about three times and two times higher compared to the single pSi layers (both HP and LP) and the pSi multilayer, respectively. This result confirms that the microcavity architecture is indeed able to enhance fluorescence emission' 4 ' 1 and that this platform may serve as a sensitive transducer for the presence of MMP- 1 in solution.
- Figure 6 shows that after incubation with MMP-1 (1.2 x 10 ⁇ 7 M), the emission intensity of the tuned pSiRM substrate was about four times higher compared to the untuned one. This shows that optimal fluorescence is obtained when the wavelength of resonance cavity dip is tuned to the emission wavelength of fluorophore 14 ' 1 and underscores that the resonance cavity layer is the sensitive part of the pSiRM substrate.
- the Q factor can be increased by increasing the porosity contrast between LP and HP layers and also the number of periods in each DBR.
- Figure 9 presents the EDANS fluorescence intensity at different incubation times. After 5 min of incubation, a significant fluorescence emission indicating the presence of MMP-1 was already detectable. The fluorescence intensity increased with increasing incubation time (due to increasing amounts of peptide cleavage) and then plateaued at 15 min incubation time (when apparently all fluorogenic peptide was cleaved). Therefore, 5 min of incubation time and a single incubation and washing step sufficed to generate a strong optical signal in response to MMP- 1 solution, which is encouraging for a POC biosensor.
- MMP-1 (logarithmic scale) is shown in Figure 10.
- y 10.345x + 153.37
- R ⁇ 0.99535
- the fluorescence intensity increased only gradually with increasing MMP-1 concentration. This effect was attributed to the diffusion of the small amount MMP-1 inside the cavity layer generated a pre -concentration effect.
- the lowest concentration of MMP- 1 we attempted to detect was 2.4 x 10 "18 M.
- LOD limit of detection
- Std b is the standard deviation of blank. From this equation, the calculated LOD was 7.5 X 10 "19 M.
- MMP was detected in real biological samples, including wound fluid samples and a tissue extract sample using the method described in Example 3.
- the samples were obtained from the patients attending wound clinic, but the identity and also the type of wound were concealed, thus the samples were labelled based on the label on the sample vials as received.
- the MMP specific fluorogenic peptide substrate immobilised on the pSiRM substrate is not selectively cleaved by any one type of MMP. Indeed, the fluorogenic peptide substrate can be cleaved by MMP- 1 , -2, -3 and -9 with different catalytic activity. t69] As a consequence, during sensing in complex biological media such as wound fluid or other body fluids, the specific MMP detected by the pSiRM sensing platform cannot be identified.
- MNPs magnetic nanoparticles
- the MNPs are functionalised with MMP antibody (MMPAb) to harvest the MMP from buffer solution or from wound fluid samples.
- MMP antibody MMPAb
- the MNPs can be modified with any type of MMPAb depending on the targeted MMP.
- the MNPs are immobilised with MMP- lAb in order to target MMP-1.
- the MNP-MMP-1 Ab binding MMP- 1 (MNP-MMP-lAb-MMP-1) is then incubated with pSiRM functionalised with the fluorogenic peptide substrate.
- the fluorescence signal after cleaving, observed fluorimetrically, can be used to confirm the presence of MMP- 1 without any interference from other MMPs. Therefore, the biosensor can be used for the selective detection of a specific peptide or protein in a family of structurally related peptides or proteins.
- the MNPs used for this experiment have a particle size of 10 nm with carboxylic acid terminated groups enabling immobilisation of an MMP antibody via amide coupling.
- the particle size of MNP is small enough to facilitate an easy infiltration of nanoparticles throughout the porous layer of pSiRM.
- the surface chemistry used to immobilise the MMPAb on the MNP surface is similar to that used to immobilise MMP fluorogenic peptide substrate on the pSiRM surface.
- FTIR Fourier transform infrared spectroscopy
- MNP-MMP-1 Ab The MNPs immobilised with 500 ⁇ g/mL MMP-1 Ab (MNP-MMP-1 Ab) were then interacted with 1.2 x 10 12 M MMP-1 at 37 °C for 5 min.
- the functionalised nanoparticle (MNP-MMP- lAb-MMP-1) was then separated from the MMP-1 solution using a magnetic column and diluted in 100 ⁇ buffer solution. This 100 ⁇ buffer solution containing MNP-MMP- l Ab-MMP-1 was then incubated in the solution containing MMP fluorogenic MMP peptide substrate for 15 min and then the fluorescence intensity of ED AN S after cleavage was measured (Figure 16(a)).
- the sensing test was also conducted in the pSiRM sensing platform (pSiRM functionalised with fluorogenic MMP peptide substrate) for 15 min and then the fluorescence intensity was measured ( Figure 16(b)).
- MMP-1 Ab The concentration of MMP-1 Ab used was 25 ⁇ g/mL as the lowest concentration tested in the previous experiment but this already provided an obvious signal. Six different time was tested; 0, 5, 10, 15, 30 and 60 min, as shown in Figure 18. From the figure, it can be seen that within 5 min most of MMP-1 was captured by MMP-lAb and there was no significant difference when the time was increased. This shows that the utilisation of MNP-MMP- l Ab is an effective and fast method to harvest MMP in buffer solution.
- the first two bar charts in Figure 19 show the fluorescence intensity of the EDANS after cleavage by MMP-1 captured by MNP-MMP-lAb (left bar (dotted)) and MNP-MMP9Ab (right bar). These charts show that the fluorescence intensity was higher in the sample incubated with MNP-MMP- lAb binding MMP-1 (about 17.3 ⁇ 1. 1) than the sample incubated with MNP-MMP9Ab binding MMP- 1 (about 1.0 ⁇ 0.1).
- the small amount of MMP-1 detected in MNP-MMP9Ab may due to the small amount of MMP-1 trapped in the MNP during the washing steps after MNP-MMP9Ab interacted with MMP- 1 solution thus interfere the measurement.
- MNP-MMP-lAb and MNP-MMP9Ab The MNP functionalised MMPAb was added to a wound fluid sample, in separate vials for each MMPAb, to harvest the MMP-1 and MMP-9.
- the MNP-MMPAb-MMP was then incubated with the functionalised pSiRM for 15 min before fluorescence intensity was observed by means of a fluorimeter.
- the fluorescence intensity of EDANS emitted from the surface incubated with MNP-MMP9Ab (7.7 ⁇ 0.8) is higher than the intensity of the EDANS emitted from the surface incubated with MNP-MMP-lAb (4.1 ⁇ 0.2) indicating the wound fluid sample contains more MMP-9 than MMP-1.
- the MNP-MMP-lAb and MNP-MMP9Ab were also contacted with wound fluid sample spiked with 1.2 x 10 12 M of MMP-1 , 1.2 x 10 12 M of MMP-9 and mixed MMP-1 MMP-9 with each concentration of 1.2 x 10 12 M (WF MMP- 1 , WF MMP9 and WF mixed MMP in Figure 20, respectively).
- the fluorescence intensity of EDANS observed in the functionalised pSi surface after incubation with MNP-MMP-lAb was higher compared to the surface incubated with MNP- MMP9Ab (right bar, 6.5 ⁇ 0.6).
- the value of the fluorescence intensities were contributed to by the MMP-1 present in wound fluid and MMP-1 spiked in the wound fluid sample and if they are reduced from the fluorescence signal observed only in the wound fluid sample (signal in the first two bar charts), the fluorescence intensity was similar to the fluorescence signal observed after MNP-MMPAb binding the MMP-1 in buffer solution (the first two bar charts in Figure 19).
- the fluorescence signal detected on the pSiRM functionalised surface after incubation with MNP-MMP-l Ab and MNP-MMP9Ab was 21.9 ⁇ 1.6 and 28.9 ⁇ 1.1 , respectively. These intensity values were also in agreement with the fluorescence intensity of mixed MMP in buffer solution if they were reduced from their fluorescence intensity in the wound fluid sample. This confirmed that selective binding using MNP-MMPAb can be employed in complex biological samples.
- Example 6 - MMP detection by means of a confocal microscope [00200] Besides using a fluorimeter, the fluorescence detection of EDANS emissions after cleavage by MMP were investigated using a confocal microscope.
- the pSiRM surface was functionalised using a microcontact printing technique to immobilise the MMP peptide substrate.
- the functionalised surface was then incubated with MMP-1 solution and viewed under microscope ( Figure 21).
- the lighter circle is the surface where the MMP peptide substrate was immobilised while the darker surrounding surface is the area where there was no immobilised peptide.
- the lighter colour (blue) is the colour of EDANS emission confirming the cleavage of the MMP substrate. This result confirms that it is also possible to detect the fluorescence emission under microscope.
- the biosensor described can also be used to detect other analytes for example bacterial enzymes, such as the bacterial Sortase A enzyme.
- This enzyme is used by Gram-positive bacteria Staphylococcus aureus to anchor surface protein to the cell wall by cleaving LPXTG at the amide bond between threonine and glycine.
- the LPXTG is a general tag where X is any amino acid, however LPETG (where X is E) is the optimal isoform of the tag.' 72]
- LPETG-(K-F1TC)-NH 2 where 2,4-dinitrophenol (Dnp) is the fluorescence acceptor, FITC (Fluorescein isothiocyanate) is the fluorescence donor and T-G (Threonine-Glycine) as the linker.
- Dnp 2,4-dinitrophenol
- FITC Fluorescein isothiocyanate
- T-G Threonine-Glycine
- the immobilisation of the Sortase A substrate was conducted in a similar way to the MMP peptide substrate, as described in Example 2.
- the pSiRM as sensing platform was re-designed to have a microcavity dip aligned with FITC emission of the FRET substrate after cleavage, which is about 514 nm.
- Figure 22 shows the emission of FITC after the Sortase A substrate cleaved by Sortase A enzyme and the fluorescence emission of the FITC is 4.3 times higher in pSiRM surface ( Figure 22(b)) compared to the emission detected in solution ( Figure 22(a)). It confirms the fluorescence enhancement effect of the pSiRM sensing platform.
- the next step was to optimise the concentration of Sortase A that can be detected.
- the concentration of the Sortase A substrate was kept constant at 1 mM and then we tested six different concentrations from 1 ⁇ ig/mL down to 1 x 10 ⁇ 10 ng/mL as shown in Figure 24.
- the figure shows that the fluorescence signal decreased along with the lower concentration of Sortase A enzyme and this sensing platform still had an obvious signal down to fg/mL of Sortase A concentration.
- Sortase A was also detected in wound fluid (Figure 25).
- the Sortase A substrate immobilised on pSiRM sensing platform was contacted with wound fluid sample (lower trace) and wound fluid spiked with 1 x l O "4 ⁇ g/mL Sortase A (upper trace) for 30 min and then the fluorescence intensity of FITC was measured.
- Figure25 shows that the wound fluid sample did not give any fluorescence signal (lower trace) indicating the wound fluid did not contain any Sortase A enzyme. Therefore, we tried to add the wound fluid sample with 1 x 10 4 ⁇ g/mL Sortase A enzyme and measured the emission (upper trace). The wound fluid sample spiked with Sortase A emitted the FITC fluorescence with the intensity about 82.34 a.u., which is close to the emission of that concentration in buffer solution as presented in Figure 24 (100.2 ⁇ 22.7 a.u.). This result indicates the enzyme was still active and able to cleave the Sortase A substrate even in a complex sample.
- Figure 27 shows that fluorescence emission generated from the surface incubated with bacterial supernatant from bacteria culture media increased along with the increasing inoculation time. It confirms the longer the inoculation time, the more Sortase A enzyme produced. In the supernatant sample from bacterial culture media and wound fluid sample at 0 h inoculation time had very small amount of Sortase A enzyme as indicated by the very low emission fluorescence detected. However, after 24 h inoculation time, the Sortase A enzyme produced in the inoculated bacterial culture sample was higher compared to the enzyme produced in the inoculated wound fluid sample indicating the faster Sortase A enzyme in bacterial culture media.
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WO2019079860A1 (en) * | 2017-10-26 | 2019-05-02 | The University Of Queensland | Detection method |
CN108645832B (en) * | 2018-03-22 | 2021-07-27 | 苏州英菲尼纳米科技有限公司 | SERS chip and preparation method and application thereof |
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CN112763423A (en) * | 2020-12-08 | 2021-05-07 | 武汉纺织大学 | Self-assembly photonic crystal bacteria detection film and preparation method thereof |
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