WO2018154078A1 - Substrat pour test analytique utilisable en tant que sonde fluorescente pour la mise en œuvre de la détection d'un analyte, dispositif portable pour la mise en œuvre d'une telle détection et système associé - Google Patents

Substrat pour test analytique utilisable en tant que sonde fluorescente pour la mise en œuvre de la détection d'un analyte, dispositif portable pour la mise en œuvre d'une telle détection et système associé Download PDF

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Publication number
WO2018154078A1
WO2018154078A1 PCT/EP2018/054583 EP2018054583W WO2018154078A1 WO 2018154078 A1 WO2018154078 A1 WO 2018154078A1 EP 2018054583 W EP2018054583 W EP 2018054583W WO 2018154078 A1 WO2018154078 A1 WO 2018154078A1
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WIPO (PCT)
Prior art keywords
gqds
test substrate
analytical test
light
fluorescence
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PCT/EP2018/054583
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English (en)
Inventor
Arben MERKOÇI
Ruslán Raulievich ÁLVAREZ DIDUK
Jahir OROZCO HOLGUÍN
Original Assignee
Fundació Institut Català De Nanociència I Nanotecnologia
Institució Catalana De Recerca I Estudis Avançats
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Publication of WO2018154078A1 publication Critical patent/WO2018154078A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7756—Sensor type
    • G01N2021/7759—Dipstick; Test strip
    • 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
    • G01N21/6489—Photoluminescence of semiconductors
    • 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
    • G01N2201/02—Mechanical
    • G01N2201/022—Casings
    • G01N2201/0221—Portable; cableless; compact; hand-held

Definitions

  • the present invention relates to the field of detection of an analyte from a sample.
  • the invention relates to an analytical test substrate comprising PL-GQDs, as fluorescent probes, suitable for performing a detection of an analyte on the basis of the Forster resonance energy transfer (FRET) effect.
  • FRET Forster resonance energy transfer
  • the present invention also relates to a portable device suitable for performing such detection, and to a system for performing such detection as well as to a kit comprising a portable device, an analytical test substrate and instructions for carrying out such detection.
  • Patent Application US2014/193839 describes the use of an image sensor of a mobile device as a spectrophotometer, with a light source to illuminate the detection platform. As a result, the mobile captures the image and the detection is made by identifying the wavelength of the spectrum being analyzed.
  • the use of a light source to excite graphene quantum dots in order to excite their own light is not described nor hinted, and neither their advantages.
  • Patent Application US2014/312247 describes the use of two filters, one for excitation at a specific wavelength of a type of fluorophore, and another to filter the emission light at a specific wavelength and this is captured by the mobile.
  • the use of a filter only to block the excitation wavelengths from being captured by the mobile is not described nor hinted, and neither their advantages.
  • the present invention was made in view of the prior art described above, and the first object of the present invention is to provide an analytical test substrate comprising PL-GQDs, as fluorescent probes, suitable for performing a detection of an analyte on the basis of the Forster resonance energy transfer (FRET) effect in a solid state.
  • FRET Forster resonance energy transfer
  • the analytical test substrate comprising PL-GQDs is characterized in that is of paper and comprises: i) PL-GQDs embedded in the paper, the PL-GQDs being deprotonated GQDs, and ii) at least one area delimited by a hydrophobic barrier to confine the embedded PL-GQDs within this area, thereby, in use, the area is suitable to receive the analyte to be detected and the PL-GQDs embedded therein emit fluorescence light when excited with UV light.
  • the analytical test substrate of the first aspect is suitable for performing a detection in solid state in a simplest, faster and easier way, which a user can be performed without requiring specific knowledges.
  • the analytical test substrate can comprise a plurality of delimited areas each one confining PL-GQDs therein.
  • sensing areas means the same as “delimited areas”.
  • the hydrophobic barrier can be made of a waxy material.
  • a waxy material is of general knowledge for a skill person in the art. It is known in the art that waxes are a diverse class of organic compounds that are lipophilic, malleable solids near ambient temperatures. They include higher alkanes and lipids, typically with melting points above about 40 °C, melting to give low viscosity liquids. Waxes are insoluble in water but soluble in organic, nonpolar solvents. Therefore, a waxy material also encompasses a liquid wax including a wax disolved in an organic, nonpolar solvent.
  • the scope of the present invention is not restricted to a determined analytical test paper, it is preferable that the analytical test paper be of nitrocellulose for properties reasons.
  • the detection assays carried out in a nitrocellulose paper have shown higher capabilities of PL-GQDs confinement.
  • the PL-GQDs are embedded in the nitrocellulose paper.
  • the substrate or the paper preferably, has a strip shape.
  • the present invention refers to the use of the analytical test substrate comprising PL-GQDs of the first aspect for performing a detection of an analyte.
  • analytes can be detected by using the analytical test substrate of the first aspect like flavonols such as quercetin, miricetin, kaemferol, morin; anthocyanidins such as cyanidin, delfinidine and aurantadine, pollutants such as nitrophenols; or PL-GQDs.
  • flavonols such as quercetin, miricetin, kaemferol, morin
  • anthocyanidins such as cyanidin, delfinidine and aurantadine, pollutants such as nitrophenols
  • PL-GQDs PL-GQDs
  • the analytical test substrate works as a fluorescent probe, that is as a sensor, due to the PL-GQDs in a particular arrangement and configuration in the paper.
  • a fluorescence quenching, a fluorescence light emitted by PL-GQDs, an intensity of the fluorescence or even a brightness of an image can be sensed and quantified.
  • the term "sensor” means everything that has a property sensitive to a magnitude of the medium, and by varying this magnitude also varies with some intensity the property, that is, the presence of that magnitude, and also its measurement.
  • a sensor in the industry is an object capable of varying a property before physical or chemical magnitudes, called instrumentation variables, and transforming them with a transducer into electrical variables.
  • the invention provides a method for manufacturing the analytical test substrate of the first aspect.
  • the method comprises the following steps: a. imprinting hydrophobic barrier patterns delimiting at least one area in the paper; b. preparing an aqueous solution including PL-GQDs, wherein the aqueous solution is buffered at a pH ranging from 8 to 13 by using a base, thereby the PL-GQDs in the basic solution result in deprotonated PL-GQDs, which show their fluorescence when they are irradiated with UV light; and
  • step a) a waxy material is employed to imprint the hydrophobic barrier pattern.
  • the barrier pattern is circular.
  • a thermal treatment is performed to melt the wax printed in the paper to form the hydrophobic barrier and, thus, to delimit a sensing area in which the PL-GQDs are adsorbed.
  • the PL-GQDs are deposited onto or into the analytical test substrate after a wax-printing method as defined in step a) above is performed.
  • the aqueous solution includes PL-GQDs in a concentration between 0.5 mg/ml and 500 mg/ml. More preferable, the aqueous solution includes PL-GQDs in a concentration higher than 12.5 mg/ml.
  • the minimum concentration of PL-GQDs can vary depending on factors such as the analyte to detect, the type of paper.
  • the solution including PL-GQDs are buffered with an inorganic base, preferably at a concentration from 8 mg/ml to 10 mg/ml, in order the solution has a pH ranging from 8 to 13.
  • step c) is performed.
  • step c) the prepared solution is deposited into the delimited area in order to wet the paper in such area.
  • the solution can be pipetted on the paper substrate.
  • the solution can be buffered to a pH ranging from 8 to 10, specially for detecting anthocianidins, flavonols, as analyte.
  • the solution can be buffered to a pH ranging from 10 to 13, specially for detecting nitrophenol pollutants, as analyte.
  • the amount of solution deposited on the paper sensing area can vary depending on the area size to be wetted.
  • the PL-GQDs are pipetted on the substrate at a concentration which ranges from 1 ⁇ of a solution at 6.25 mg/ml of PL-GQDs to 4 ⁇ of a solution at 50mg/ml of PL-GQDs.
  • a solution containing PL-GQDs with a concentration from 0.5 mg/ml to 500 mg/ml is equivalent to a concentration of PL-GQDs in the paper from 5 to 20 mg/mm 3 .
  • the invention provides a portable device suitable for performing a detection of an analyte from a sample received in an analytical test substrate on the basis of the Forster resonance energy transfer (FRET) effect by means of an analytical test substrate.
  • FRET Forster resonance energy transfer
  • the portable device comprises an analytical test substrate, a body, light means, energy connection means, light-readout means, and electronic processor means, and is characterized in that
  • the analytical test substrate is as defined in the first aspect of the invention, and is configured to receive, in use, the sample containing the analyte to be detected, and to emit fluorescence light when excited with UV light, and
  • the light means are UV means, wherein o the body is configured and adapted to house therein at least part of the analytical test substrate,
  • the UV means operates on the basis of ultraviolet wavelength and is configured and arranged in the body
  • the energy connection means are configured and adapted to be coupled to an energy source
  • o the electronic processor means are operatively connected to the energy connection means to supply energy to the UV means; and o the light-readout means are associated to the analytical test substrate for taking of an optical readout of the fluorescence- indicative signal (S21 1 , S212) generated in the analytical test substrate, and are adapted to provide the optical readout to a user of a quenching of the fluorescence light emitted by the PL-GQDs.
  • the portable device provides a fast and reliable data of the presence or absence of the analyte without requiring the use of expensive equipment, nor requiring the data resulting from the detection to be subsequently analyzed in a computer.
  • the portable device is capable to detect several chemical compounds as analytes, without requiring adaptations of the portable device to each specific analyte in each detection. It is preferable the use of this variant for performing a detection of an analyte selected from an anthocianidin, a flavonol, and a nitrophenol pollutant.
  • the portable device of this variant provides the optical readout of a fluorescence quenching of the fluorescence light emitted by the PL-GQDs to an user in a more versatile way, in situ-application, and whose operations of detection are easier to be practiced by a user.
  • the fluorescence-indicative signal indicates a signal Yes/Not (positive/negative).
  • the invention provides a second variant of the portable device suitable for performing a detection of an analyte from a sample received in an analytical test substrate on the basis of the Forster resonance energy transfer (FRET) effect by means of an analytical test substrate.
  • FRET Forster resonance energy transfer
  • the portable device of this variant is capable to detect directly the presence of PL-GQDs in a sample with a high level of sensibility.
  • the analyte to be detected are the PL- GQDs directly by fluorescence.
  • the portable device is particularly suitable for copy detection of products in a solid state, that is to say a suitable device for the analysis counterfeiting of a solid product.
  • the second variant of the portable device is suitable for performing a detection of an analyte received in an analytical test substrate on the basis of the Forster resonance energy transfer (FRET) effect by means of an analytical test substrate, the portable device comprising an analytical test substrate, a body, light means, energy connection means, light-readout means, and electronic processor means, and is characterized in that:
  • the analytical test substrate is as defined in the first aspect of the invention, and is configured to receive, in use, the analyte from a sample to be detected, and to emit fluorescence light when excited with UV light, wherein the analyte from a sample to be detected are PL-GQDs embedded in a sample, and
  • the light means are UV means, wherein o the body is configured and adapted to house therein at least part of the analytical test substrate,
  • the UV means operates on the basis of ultraviolet wavelength and is configured and arranged in the body
  • the energy connection means are configured and adapted to be coupled to an energy source
  • o the electronic processor means are operatively connected to the energy connection means to supply energy to the UV means; and o the light-readout means are associated to the analytical test substrate for taking of an optical readout of the fluorescence- indicative signal (S21 1 , S212) generated in the analytical test substrate, and are adapted to provide the optical readout to a user of a fluorescence of the fluorescent PL-GQDs;
  • the device further comprises
  • a UV-blocking filter arranged between the light-readout means and the analitycal test substrate and is configured to block, in use, the UV light of the UV means (21 ), thereby enhancing the detection level of PL-GQDs.
  • the enhancing of detection level can be two or three orders of magnitude.
  • the analyte to be detected are the PL-GQDs.
  • the sample can be, for example, a picture.
  • the portable device is suitable for performing a detection of PL-GQDs embedded in the picture, for example, for preventing a copy thereof.
  • the PL-GQDs can be detected in a concentration of 0.1 mg/mm 3 (0.5mg/ml) even in a concentration as lower as 0.0001 mg/mm 3 (0.0005mg/ml).
  • the portable device provides an optical readout of the fluorescence-indicative signal generated in the analytical test substrate in a reliable, easy, inexpensive and secure way for carrying out in situ by a user, who does not need technical acknowledgment.
  • the portable device can be coupled to a handheld computing device, that wears almost all the users in their pocket, in an easy way and low cost for a rapid detection of an analyte in situ, making this technology accessible to the whole public.
  • the handheld computing device can be selected from the group consisting of a handheld digital camera device, a cellular phone, a smart phone, and a tablet computer.
  • the energy connection means can be adapted to be coupled to an energy source of a handheld computing device, so as to provide the energy source to the energy connection means from the handheld computing device.
  • the energy connection means is a USB connector.
  • the light-readout means can also independently be adapted to be coupled to an optical readout, i.e. a camera, of a handheld computing device, so as to provide the optical readout from the handheld computing device.
  • the light-readout means is the structural part of the body that allows take a direct readout of the fluorescence-indicative signal generated in the analytical test substrate upon UV irradiation.
  • the UV means is a LED.
  • the electronic processor means is an electric circuit of the UV means: (365nm) UV LED, connected to the energy connection means at the male USB port.
  • the authors of the present invention has found that the intensity of the fluorescence of PL-GQDs is directly dependent upon the concentration of the analyte in the sample. Therefore, the concentration of the analyte in the sample can be quantified.
  • the portable device is suitable for determining the concentration of the analyte once detected their presence in the sample.
  • the electronic processor means are configured to receive data relating to the fluorescence-indicative signal (S21 1 , S212), at an intensity indicative of the concentration of the analyte in the sample, and are adapted to perform by means of processing means a calculation of said intensity, based on the received data, to generate a concentration-indicative output signal (S3).
  • the portable device is sized to be suitable stored in a pocket of a user.
  • the portable device can be sized to a size as small as a matchbox.
  • the body is configured to isolate the analytical test substrate housed therein of the external light of the body, so to protect the analytical test substrate from external light fluctuations.
  • the body is a black chamber.
  • highly reproducible data is obtained in a portable device embodiment having this configuration of the body.
  • PL-GQDs can be detected in a very low concentration in the sample.
  • the presence of the UV-blocking filter permits to barr the ultraviolet wavelength through thereof.
  • the ultraviolet light emitted by UV means LED
  • the fluorescence indicative signal S21 1 S212
  • the UV-blocking filter can be also used in the first variant of the portable device in order to enhance the level of detection.
  • UV-blocking filter means a filter that at least barr the pass of ultraviolet wavelengths through thereof.
  • a filter that exclusively prevent the pass of the UV wavelengths is also contemplated in the present invention.
  • a "longpass filter” from which wavelenghts higher that 400 nm can through thereof is also contemplated as a UV-blocking filter according to the invention.
  • a system suitable for performing a detection of an analyte from a sample received in an analytical test substrate on the basis of the Forster resonance energy transfer (FRET) effect by means of an analytical test substrate comprising an analytical test substrate, and a portable device having a body, light means, energy connection means, light-readout means, and electronic processor means, and is characterized in that
  • an analytical test substrate is as define in the first aspect, and is configured to receive, in use, the analyte from a sample to be detected, and to emit fluorescence light when excited with UV light, and
  • - - a portable device comprises:
  • the body is configured and adapted to house therein at least part of the analytical test substrate
  • the UV means operates on the basis of ultraviolet wavelength and is configured and arranged in the body
  • a fluorescence-indicative signal (S21 1 , S212) in the analytical test substrate which is indicative of the quenching of the fluorescence light emitted by the excited PL-GQDs and reveals the presence of an analyte by quenching the fluorescence light emitted of the excited PL- GQDs in case an analyte is received in the analytical test substrate and the analyte has an absorption wavelength range that at least in part overlaps the fluorescence light emitting wavelength range of the excited PL-GQDs; then o the energy connection means are configured and adapted to be coupled to an energy source;
  • o the electronic processor means are operatively connected to the energy connection means to supply energy to the UV means; and o the light-readout means are associated to the analytical test substrate for taking of an optical readout of the fluorescence- indicative signal (S21 1 , S212) generated in the analytical test substrate, and are adapted to provide the optical readout to a user of a quenching of the fluorescence light emitted by the PL-GQDs; or alternatively,
  • - a portable device which comprises:
  • the body is configured and adapted to house therein at least part of the analytical test substrate
  • the UV means operates on the basis of ultraviolet wavelength and is configured and arranged in the body
  • ⁇ to generate a fluorescence-indicative signal (S21 1 , S212) in the analytical test substrate, which is indicative of the fluorescence light emitted by the excited PL-GQDs and reveals the presence of PL-GQDs by fluorescence; then o the light-readout means are associated to the analytical test substrate for taking of an optical readout of the fluorescence- indicative signal (S21 1 , S212) generated in the analytical test substrate, and are adapted to provide the optical readout to a user of a fluorescence of the fluorescent PL-GQDs;
  • a UV-blocking filter arranged between the light-readout means and the analitycal test substrate and is configured to block, in use, the UV light of the UV means (21 ), thereby enhancing the detection level of PL-GQDs.
  • the electronic processor means are connected to a handheld computing device, the handheld computing device being selected from the group consisting of a handheld digital camera device, a cellular phone, a smart phone, and a tablet computer.
  • the electronic processor means are connected to the handheld computing device wirelessly.
  • the energy connection means are coupled to an energy source of the handheld computing device, and/or the light-readout means are coupled to an optical readout of the handheld computing device, so as to supply the energy to the UV means and/or to provide an optical readout by means of the handheld computing device.
  • the portable device further comprises remote means associated to the light-readout means for wireless communication with the handheld computing device.
  • the handheld computing device coupled to the portable device it is allowed to record data, process data, and display readings at any time after finalized the detection of the analyte, which makes the portable device more versatile, of simplified design, reduced size and low cost in the detection.
  • the portable device further comprises a lens configured to couple to the optical readout, i.e. the camera, of the handled computing device.
  • the lens is arranged in the optical readout of a handheld computing device, making such lens possible that the size of the portable device be reduced to a smaller size.
  • the invention provides a kit comprising a portable device, an analytical test substrate and instructions for carrying out such detection.
  • kits for detecting an analyte from a sample comprising a portable device according any one of variants described above, an analytical test substrate of the first aspect of the invention and instructions for carrying out the detection by using a handheld computing device, the instructions comprising:
  • the optical readout of the analytical test substrate can be read directly by the eyes of the user.
  • Figure 1 A depicts a schematic view of an embodiment of the portable device 2 of this invention showing the different parts thereof, and showing the portable device coupled with a smart phone and the analytical test substrate 1 housed therein.
  • Figure 1 B depicts a schematic view of another embodiment of the portable device 2 of this invention showing the different parts thereof, without a smart phone 3 coupled thereon, and with the analytical test substrate 1 outside the portable device.
  • Figure 1 C depicts a schematic view of another embodiment of the portable device 2 of this invention showing the different parts thereof, with a smart phone 3, the analytical test substrate 1 and the UV-blocking filter 4.
  • Figure 2 depicts a graph showing the absorption spectrums of two analytes (Quencher 1 and Quencher 2) and the emission spectrum of the PL-GQDs depending on the normalized absorbance and the normalized fluorescence.
  • Figure 3A depicts how fluorescence decreased as the concentration of 4-nitrophenol (D) and paraoxon (E) increases.
  • Figure 3B depicts two graphs of the corresponding Stern-Volmer relationship for Paraoxon and 4-nitrophenol.
  • Figure 4 depicts two bar diagram that show the fluorescence quenching of the portable device according to the second aspect of the invention by using the analytical test paper as a response to A) wine samples of different vintage and B) seawater samples spiked with 4-nitrophenol (NP) and Paraoxon (PO).
  • NP 4-nitrophenol
  • PO Paraoxon
  • Figure 5A depicts both the UV-visible absorbance spectrum (left), and the Photoluminescence spectra (right) when PL-GQDs are excited at 365 nm.
  • Figure 5B is a HRTEM image depicted showing the morphology of synthesized PL- GQDs.
  • the PL-GQDs were placed on top of the nitrocellulose substrate 1 .
  • the substrate resulted ideal for the assay where the PL-GQDs as fluorescent probes were bound to the paper interstices and reagents (whether needed) and samples placed on its surface.
  • Some wax- printed hydrophobic circular patterns 10 were previously delineated on the nitrocellulose matrix 1 by a wax printer to confine the sensing probes, thus defining the sensing area.
  • a hydrophobic barrier spread all around the hydrophilic nitrocellulose matrix was ready and rapidly delineated and the PL-GQDs were physically adsorbed into, while the barrier contained the fluorescent probes in the as-defined area.
  • PL-GQDs pH value showed to be the most crucial parameter to be controlled. While at pH 4 and 6 protonated GQDs showed to dramatically quench the fluorescence, the deprotonated GQDs at pH 8 and 13 were absolutely fluorescent.
  • the portable device 2 has been used to detect two groups of phenolic compounds from a sample with no instrumentation required other than the analytical test paper 1 and a simple mobile phone 3.
  • PL-GQDs as sensing probes were embedded into a nitrocellulose matrix 1 .
  • the sensing probes were synthesized from citric acid by a pyrolysis procedure, and with no modifications further physisorbed and confined into small wax-traced spots 10 at the nitrocellulose substrate 1 .
  • a UV LED 21 that excites the PL-GQDs was placed into a dark chamber 20 and fed by a mobile phone 3, used for both as the energy source 22 and digital color imaging capture 23.
  • the dark chamber 20 was isolated the analytical test paper 1 comprising embedded PL-GQDs from external light fluctuations.
  • the nitrocellulose-based device 2 was able to detect the antioxidant capacity related with flavonoid content in wine samples and 4-nitrophenol and paraoxon pollutants in seawater samples.
  • the sensing mechanism is based on fluorescence quenching of the sensing probes because of their interfacing with some phenolic compounds through the polar-polar resonance of acceptor-donor interaction.
  • the portable device is able to detect the presence of a compound (Quencher 1 and Quencher 2, see Fig 2) which absorption spectrum is overlapped with the emission spectrum of the phonoluminiscent GQDs.
  • the overlapped area, see Fig 2 is proportional to the percentage of quenching.
  • the quantum dots play a donor role transferring the light to the compound that absorbs it in the overlapped region (acceptor).
  • the dark chamber 20 contains a strip hole where the paper strip 1 passes through (see Fig 1 B). Each spot 10 is passed manually one by one to reach the UV LED area, where the confined PL-GQDs are excited by the LED 21 at 365 nm.
  • the LED 21 is fed by a smartphone through a USB port 22, utilizing the simple circuit.
  • the smartphone 3 captures the light that the PL-GQDs emit at 460 nm.
  • White paper provides a proper background for optical readouts. The amount of captured fluorescent light is further related to the concentration of the analyte, which can be quantified after processing by the image J program.
  • the UV-blocking filter 4 would prevent the light coming from the UV LED 21 from interfering with the light captured by the camera, thus increasing the exposure time. In other words, the camera cannot see the light coming from the LED 21 and only see the light of the excitation.
  • An increase in the detection limit is obtained by increasing the exposure time to capture more light when the concentration of the GQDs is very low. It is also possible to detect a minimum difference of quenching. As described above in detail, this invention has at least one of the following advantages:
  • the portable devices provide a simplest, faster, and easier way for detecting an analyte in- situ, which further is reliable, inexpensive, with reproducible data and which can be performed by a user without requiring specific knowledges.
  • Photoluminiscent Graphene oxide quantum dots can be synthesized according to the procedure reported by Dong, Y. et al. "Blue luminescent graphene quantum dots and graphene oxide prepared by tuning the carbonization degree of citric acid. Carbon 50, 4738-4743 (2012)”. Briefly, 1 g of citric acid was pyrolyzed at 200 °C until the color changed from transparent to orange in approximately 30 min, then the orange liquid was slowly transferred to a 50 ml 10 mg/ml NaOH solution, under vigorous stirring. The stock suspension of PL-GQDs was adjusted with 1 M HCI to get 50 mg/ml PL-GQDs solution with a pH value down to 9 and kept in the fridge at 4 °C.
  • the citric acid was pyrolyzed in a microwave oven for 2 min and then was added to the NaOH solution (10 mg/ml) under vigorous stirring. This methodology produces similar graphene quantum dots with the same emission wavelength range.
  • the GQDs were further characterized by spectrophotometry, fluorimetry, Fourier transform infrared (FT-IR) spectroscopy and high-resolution transmission electron microscopy (HRTEM) imaging.
  • Figure 5A shows both the UV-visible absorbance spectrum (left), characterized by a wide pick between 300 and 430 nm; and the Photoluminescence spectra with a strong fluorescent emission peak at 460 nm (right) when is excited at 365 nm.
  • the top inset depicts a picture of the PL-GQDs under UV (right) and visible (left) light, respectively.
  • HRTEM image depicted in Figure 5B shows the morphology of fresh synthesized GQDs. The image depicts well-dispersed GQDs with a circular shape and an average size of 2.33 ⁇ 0.12 nm.
  • Analytical test paper preparation and set up Wax circumferences of 4 or 5 mm of diameter and 0.5 mm of width were printed on top of a nitrocellulose paper strip of (9 mm x 20 mm) with a Xerox ColorQube 8570 solid ink printer. Afterward, the strip was put on a hot plate at 84 °C for 2 minutes to melt the wax and create an internal structure confining spot. Then 4-8 ⁇ _ of the as-prepared PL-GQDs was added to the spots; and let it dry at room temperature, for approximately 45 min. The paper strip was placed either under the UV lamp or into the dark chamber.
  • nitrocellulose-embedded PL-GQDs strip was tested first with quercetin as a model phenolic compound of double bonds conjugated within multiple aromatic rings. Its molecular structure contains five -OH groups conjugated with the double bonds of its three aromatic rings.
  • Fluorescence quenching at different concentrations of quercetin was collected: 0 (0.01 M PBS buffer), 1 .66 x 10 5 , 4.98 x 10 5 , 8.30 x 10 5 , 1 .33 x 10 4 , 1 .99 x 10 4 , 2.51 x 10 4 , 5.01 x 10 4 , 7.52 x 10 "4 , 1 .25 x 10 "3 , 1 .50 x 10 "3 M.
  • the PL-GQDs-modified paper platform under UV light showed that the fluorescence quenching was higher as concentrations of quercetin buffered solutions increasing in a range of two orders of magnitude. Quenching of the fluorescence was negligible in the first spot with only PBS buffer.
  • the deprotonated quercetin and PL-GQDs have shown to produce an efficient FRET with the concomitant quenching of the PL-GQDs.
  • Stabilization of delocalized electrons resonating within the quercetin structure by electron transference may be accounting for such PL-GQDs intermolecular deactivation processes (quenching).
  • quercetin structurally analogous compounds with antioxidant properties (e.g. morin, myricetin, and kaempferol), also demonstrated to quench the fluorescence of QDs.
  • Stability of the analytical test paper comprising embedded PL-GQDs was evaluated upon time based on the ⁇ 3 ⁇ criterion. For this purpose, a control chart was plotted with the average intensity of the sensing area the first day of the study as central value. The estimated fluorescence was still within the control chart even after 15 days of storage at 4 5 C, thus demonstrating the stability of the analytical test paper comprising embedded PL- GQDs.
  • the analytical information was extracted from pictures taken with a smartphone camera after illuminating the sensing areas with a simple UV light lamp.
  • nitrocellulose-embedded PL-GQDs were tested for sensing another group of phenolic compounds of environmental interest.
  • 4-nitrophenol has shown to quench PL-GQDs in aqueous solutions.
  • deprotonated 4-nitrophenolate molecules work as acceptors of electrons while PL-GQDs are the donors, being the FRET susceptible of taking place.
  • basic pH given by a 1 M NaOH solution, hydrolyzes of the paraoxon pesticide produces 4-nitrophenol as bi-product.
  • Figure 3A shows how fluorescence decreased as the concentration of 4-nitrophenol (D) and paraoxon (E) increased from 2.5 x 10 ⁇ 5 to 5 x 10 ⁇ 4 M (in the direction of the purple arrow in the figure).
  • the versatile sensing portable device was used for testing in food and environmental samples, see Figure 4.
  • Sensing capabilities of the analytical test paper strip were also tested in environmental samples.
  • a seawater sample was spiked with 50 and 100 mM 4-nitrophenol (NP), which produced a quenching of the PL-GQDs of 17.1 ⁇ 1 .2 and 35.7 ⁇ 2.6 %, respectively.
  • 50 and 100 mM hydrolyzed paraoxon (PO) led to similar quenching extents (16.9 ⁇ 1 .5 and 35.3 ⁇ 1 .7, respectively) as expected from the 4-nitrophenol, stoichiometrically produced as the byproduct of the hydrolysis of PO.
  • the camera's sensor captures the light coming from the ultraviolet light source, which can be more intense than the emission light of the GQDs.
  • a filter is added that allows the light captured by the camera sensor to come only from the emission of the GQDs.
  • the shutter speed of the chamber should be increased to values between 1 ⁇ 4 sec to 10 sec. This causes the camera to capture the LED's light signal, producing a saturation in the image. This translates to RGB values close to 255, 255, 255 respectively (white image) the GQDs are not different from anything because the image is completely white.
  • the use of the filter allows detection of GQDs even at concentrations lower than 0.5mg, even two or three orders of magnitude, that is about 0.0005 mg/ml. Because only the signal of the GQDs is seen and when increasing the shutter speed the light of GQDs accumulates in the time until completing the photo.
  • the filter is placed exclusively between the camera and the paper.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention concerne un substrat pour test analytique (1) comprenant des PL-GQD (points quantiques de graphène photoluminescents), en tant que sondes fluorescentes, convenant à la mise en œuvre de la détection d'un analyte sur la base de l'effet de transfert d'énergie par résonance de Förster (FRET). La présente invention concerne également un dispositif portable (2) comportant un corps (20), un moyen d'éclairage (21), un moyen de raccordement électrique (22), un moyen de détection de lumière (23) et un moyen formant processeur électronique (24), convenant à la mise en œuvre de la détection d'un analyte. La présente invention concerne également un système pour la mise en œuvre de la détection d'un analyte comprenant le substrat pour test analytique (1) et le dispositif portable (2), et qui est apte à détecter l'analyte sur la base de l'effet de transfert d'énergie par résonance de Förster (FRET). La présente invention concerne en outre un kit comprenant le dispositif portable (2), le substrat pour test analytique (1) et des instructions pour la mise en œuvre d'une telle détection.
PCT/EP2018/054583 2017-02-24 2018-02-23 Substrat pour test analytique utilisable en tant que sonde fluorescente pour la mise en œuvre de la détection d'un analyte, dispositif portable pour la mise en œuvre d'une telle détection et système associé WO2018154078A1 (fr)

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TWI682172B (zh) * 2019-01-10 2020-01-11 國立臺灣大學 檢測套組及檢測毒品的方法
US11360082B2 (en) 2019-01-10 2022-06-14 National Taiwan University Detection kit and method for detecting abused drugs
WO2020150403A1 (fr) * 2019-01-15 2020-07-23 Quidel Corporation Système et procédés d'évaluation à distance d'un dosage d'échantillon pour un diagnostic de maladie
CN113424051A (zh) * 2019-01-15 2021-09-21 奎多公司 用于疾病诊断的样本分析的远程评估的系统和方法
WO2021126650A1 (fr) 2019-12-17 2021-06-24 Lantha Inc. Dispositifs mobiles pour l'analyse chimique et méthodes associés
EP4077712A4 (fr) * 2019-12-17 2024-01-24 Lantha Inc. Dispositifs mobiles pour l'analyse chimique et méthodes associés
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WO2022243842A1 (fr) * 2021-05-20 2022-11-24 Council For Scientific And Industrial Research Détecteur de fluorescence portable
CN113588607B (zh) * 2021-07-05 2022-11-11 山西大学 基于比率荧光和比色双模式的纳米探针及其制备方法和在检测桑色素中的应用
CN113588607A (zh) * 2021-07-05 2021-11-02 山西大学 基于比率荧光和比色双模式的纳米探针及其制备方法和在检测桑色素中的应用
US11760928B2 (en) * 2021-07-06 2023-09-19 Jiangnan University Enhanced yellow-emitting carbon dots and its preparation method and application
CN114518358A (zh) * 2022-02-16 2022-05-20 四川农业大学 农药残留检测用三色传感探针及其制备方法、应用和深度学习的视觉智能监测装置及方法
CN114518358B (zh) * 2022-02-16 2023-10-27 四川农业大学 农药残留检测用三色传感探针及其制备方法、应用和深度学习的视觉智能监测装置及方法
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