WO2020089810A1 - Technologie de détection d'acide nucléique et de protéines sans étiquette basée sur des points de carbone bruts - Google Patents

Technologie de détection d'acide nucléique et de protéines sans étiquette basée sur des points de carbone bruts Download PDF

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WO2020089810A1
WO2020089810A1 PCT/IB2019/059308 IB2019059308W WO2020089810A1 WO 2020089810 A1 WO2020089810 A1 WO 2020089810A1 IB 2019059308 W IB2019059308 W IB 2019059308W WO 2020089810 A1 WO2020089810 A1 WO 2020089810A1
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Helena Maria RODRIGUES GONÇALVES
José Ramiro AFONSO FERNANDES
Paula Filomena Martins Lopes QUINTA DE PRADOS
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Universidade De Trás-Os-Montes E Alto Douro
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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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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

Definitions

  • This application relates to label-free nucleic acid and proteins detection technology based on raw Carbon Dots.
  • Carbon Dots are carbon-based nanoparticles that ever since their serendipitous discovery in 2004 have been gathering much attention not only by their vast academic use, but also for their potential to be used as base for important scientific technologies [1].
  • the Cdots outstanding photoluminescence properties are one part of the reason why these nanoparticles are becoming the new trend in the nanotechnology area. Indeed, their photoluminescence properties are comparable to other well-known nanoparticles, quantum dots (QDs), which can be found in areas as vast as electronics, health, textiles, among others, that prove their versatility. Nonetheless Cdots go a step forward.
  • QDs quantum dots
  • Cdots are usually used upon a functionalization step, particularly if the intended application is biosensing/bio recognition [4].
  • the carbon dots surface was changed by a functionalization/activation step that rendered the nanoparticles the ability to specifically recognize a given analyte [5-7] .
  • One such example is the one described by the patent Metal-Enhanced Photoluminescence from Carbon Nanodots (PCT/US12/29609) [9-10], where the fluorescence properties of the nanoparticles are dramatically increased by the presence of metal islands, which increases their applicability in certain areas, such as, bioimaging.
  • nucleic acids sensors tend to be based on expensive and time-consuming techniques, such as, DNA sequencing and real-time polymerase chain reaction (PCR) .
  • PCR real-time polymerase chain reaction
  • gold and magnetic nanoparticles coupled with fluorescent dyes are limited by the fluorescent compound.
  • the common organic dyes used are often prone to problems like photobleaching and photoinstability, which turns the process into a complex system.
  • Cdots for DNA detection was successfully achieved in the past, however it requires Cdots functionalization [11,12] or indirect detection [13].
  • One class of nanomaterials that are now commercially available and widely used are the semiconductor Quantum Dots. These nanoparticles are traditionally composed by a heavy metal core and an organic coating. Their use as effective biosensors has already been proved [15], however they present a major drawback that limits their application in living organisms, they are intrinsically toxic. Indeed, even though there have been some attempts to eliminate/reduce its toxicity, these processes usually lead to a decrease in photostability/luminescence . Carbon dots are the non-toxic alternative to the semiconductor Quantum Dots.
  • Cdots have been used as sensors for inorganic molecules, such as, Hg 2+ sensors [5,20-21], iodine [5], Cu 2+ [5, 22-23], Fe 3+ [24-25], Pb 2+ [26] and Ag + [27], as well as, organic molecules like L-cysteine [28] and thrombin [29] .
  • a common denominator of these sensors is the functionalization of the Cdots surface with adequate molecules that renders Cdots the ability to detect/interact with a given analyte.
  • the exception of this functionalized Cdots sensor technology is the DNA sensor developed by Bay and co-workers (2011) [13] .
  • the label-free Cdots solution with the sample solution is carefully homogenized, e.g. by manually inversion, slowly and for one minute;
  • the detection method relies on a direct interaction between the Cdots and the nucleic acids and proteins.
  • the method further comprises a pre-method step consisting on the application of temperature to a double strand DNA solution.
  • the label-free Cdots have a size ranging from 1 to 100 nm.
  • the label-free Cdots have a spherical shape.
  • concentration of the label-free Cdot solution is 0.08 g/L, corresponding to 5% (w/w) .
  • the concentration of nucleic acid solution or protein solution to incubate with the label-free Cdots solutions is at least 0.07 mM.
  • the fluorescence is acquired in excitation and emission wavelengths from 400 to 850 nm.
  • the incubation time is 30 minutes.
  • the incubation time is at least 5 minutes .
  • the Cdots interaction with nucleic acids and proteins relies on label-free detection.
  • the present application relates to a sensing platform composed of raw carbon-based nanoparticles that can be used to detect and quantify the presence of nucleic acid sequences and proteins.
  • This technology emerges from the pressing need for a versatile, specific and low-cost label-free nucleic acids and proteins sensor that can not only detect but also quantify.
  • the technology here described can be applied regardless of the biological organism provided that the nucleic acids or proteins that will be analyzed can previously be extracted and sequenced.
  • This label-free technology provides an ultrasensitive, reusable, rapid and low-cost solution for the detection and quantification of nucleic acids and/or proteins that can be applied in areas as biosafety, human and animal health diagnostics, food and beverages security, food and beverages traceability, authenticity (e. g. textiles, food), plant material control, animal pedigree, among others.
  • a method that can detect and quantify these biomolecules can be largely applied in numerous areas, namely to identify forgeries in the food industry, to detect virus, the health area, among others.
  • the Cdots used for the nucleic acids and protein detection are used in the raw state, i.e., there are no functionalization procedures required for the Cdots to perform as biosensors;
  • Cdots are photostable and non-toxic which makes them good candidates for sensing nucleic acids and proteins in vivo. Additionally, they present a remarkable improvement when compared to the existing technology.
  • the most common method for fluorescence sensing in bioimaging used organic fluorophores that are photo-unstable and are usually analyte non-specific.
  • the nanoparticle alternative are semiconductor quantum dots, that are intrinsically toxic and therefore cannot be used for in vivo studies;
  • Cdots can be produced by low cost methods and do not require a costly equipment or preparation apparatus for sensing studies. e) this method is also time efficient. The time required on the detection and quantification of the nucleic acids or proteins is only limited by the incubation time, which was optimized as 30 minutes. Upon this time, it is possible to measure each sample in only one minute, the integration time required by the fluorescence apparatus to present the emission profile of the incubated Cdots.
  • the carbon dots can be obtained using several synthetic pathways that will have an impact in shape, size and surface groups .
  • the carbon dots may be in a suspension state or immobilized into an adequate substrate before the incubation with the nucleic acids or proteins.
  • the carbon dots are incubated with the nucleic acids or proteins that need to be analysed.
  • concentration of the Cdots is 0.08 g/L, corresponding to 5% (w/w) as further described .
  • the light emission properties of the incubated carbon dots are analysed.
  • the carbon dots will the irradiated with a light ranging from 300-700 nm and their emitted wavelength will be captured and converted into an analytical signal.
  • the differences between the non-incubated and incubated carbon dots will be used to create a method for detection and quantification of the nucleic acids or proteins.
  • Figure 1 - Cdots characterization a) Transmission Electronic Microscopy image of the Cdots upon suspension in ethanol, b) Dynamic Light Scattering representation of the Cdots suspended in ultrapure water.
  • Figure 2 Fluorescence intensity emission measures of Cdots as a biosensing system after incubation with: B) Cdots; D) Cdots + ssDNA (probe specific of V. vinifera) ; C) Cdots + ssDNA + negative control (probe specific of V. vinifera and non-specific target) ; A) Cdots + ssDNA+ wine DNA (probe specific of V. vinifera and DNA extracted from wine sample) .
  • Figure 3 Portable configuration example for the biosensing system, a) layout of the chip containing Cdots in white and Cdots + specific probe in grey, placed in a microwells with at least a volume of 5 pL of solution; b) sample injection, of at least a volume of 100 pL, into the chip, that will provide all the wells with the same sample volume; c) placement of the chip in a microchamber equipped with a excitation led and a photodetector with a band pass optical filter for fluorescence acquisition, linked to a software with the required algorithms to give a report; d) the report will consist on: a positive result (when the wavelength shift/fluorescence intensity/lifetime of the Cdots + specific probe changes in relation to the control Cdots); a negative result (when the wavelength shift/fluorescence intensity/lifetime of the Cdots + specific probe does not change in relation to the control Cdots); inconclusive (when only one/
  • carbon nanoparticles were neutralized with NaOH with a concentration of 10 mol/L and the salts formed were removed by centrifugation at 5000 rpm for 1 h.
  • the produced Cdots were then dialyzed against ultrapure water using a membrane dialysis of 0.5-1.0 kD MWCO.
  • the obtained nanoparticles were lyophilized in order to obtain a fine powder.
  • the Cdots were characterized using different technologies: (1) Dynamic Light Scattering (DLS) analysis using disposable polystyrene cells from Sigma in a Zeta Sizer Nano ZS form Malvern Instruments (Malvern, UK) . In order to assure some control over the possibility of aggregation of the nanoparticles, before performing DLS analysis a Cdots diluted solution was passed two times by two continuous 200 nm Fischer Scientific RC filters.
  • DLS Dynamic Light Scattering
  • EDS Energy-dispersive X-ray spectroscopy
  • the Cdots produced have sizes ranging from 1 to 100 nm and present a spherical shape.
  • the complementary sequences to identify in the sample solutions may be part of a complex solution containing numerous biological and non-biological molecules.
  • the pre-method step comprises the application of temperature that allows the denaturation of the dsDNA, before applying this analytical sensing system based on label-free Cdots .
  • a solution of 5% (w/w) raw label-free Cdots is incubated with previously known and isolated sequences of nucleic acids or proteins and left to incubate for at least 5 min. Afterwards a sample solution that might contain a complementary sequence of DNA or complementary protein is added to the incubated solution and the fluorescence of the overall mixture is measured using a fluorescence acquisition wavelength emission and excitation range of 400 to 850 nm.
  • the label-free Cdots solution with the sample solution is carefully homogenized, e.g. by manually inversion, slowly and for one minute;
  • the method comprises a pre-method step comprising the application of temperature, depending on the melting temperature of the DNA strain and base content, to a double strand DNA solution to allow the denaturation of the dsDNA. This step occurs prior to the incubation with the label-free Cdots solution.
  • the concentration of label-free Cdots is 0.08 g/L, corresponding to 5% (w/w) .
  • the concentration of nucleic acid solution or protein solution to incubate with the label-free Cdots solutions is at least 0.07 mM.
  • the fluorescence is acquired in excitation and emission wavelengths from 400 to 850 nm.
  • the incubation time is at least 5 minutes. In a preferred embodiment, the incubation time is 30 minutes.
  • Fluorescence emission measurements were made by a portable CCD-USB-spectrometer (Ocean Optics USB-4000 FL, USA) using a quartz cuvette of 1 cm using an integration time of 50 s. Emission spectra were recorded, at room temperature, in the 400-850 nm range using an excitation wavelength of 405 nm generated from semi-conductor laser with 100 mW of emission power. All the UV-visible measurements were acquired on a Perkin Elmer double beam Lambda 25 UV/VIS Spectrometer using a 200-700 nm range.
  • a Cdots solution of 5% (w/w) was prepared and used as blank sample. Afterwards, the analyzing solutions were prepared and were composed by Cdots and ssDNA. These solutions were prepared as follows: 750 pL of a ssDNA 0.25 mM stock solution, was added to 750 pL Cdots solution with a concentration of 5% (w/w) to obtain the most concentrated ssDNA/Cdots solution used in this example. This 1:1 ratio was adjusted in order to obtain ssDNA concentrations ranging from 0.07 to 0.50 pM, while maintaining the overall volume. These analysing solutions were left to incubate for different times, and it was found that it is necessary a minimum incubation period of at least 5 minutes.
  • the method was also tested with different DNA strands differing in its sequence presenting one or more single nucleotide polymorphisms - SNPs (these DNA sequences are presented in Table 2) .
  • the solutions were carefully homogenized by inverting slowly the tube and the fluorescence was measured.
  • Table 2 List of oligonucleotides, probes and targets, used in the fluorescence Cdots assay. In the sequence, marked in bold, are the SNPs positions.
  • This example demonstrates the applicability of the Cdots sensing system using a complex DNA sample extracted from wine using specific Vitis vinifera L. probes.
  • the Cdots as a biosensing system of the present application were applied to the identification of a specific DNA sequence (shown in Table 2) from Vitis vinifera
  • a profile of the emission spectrum of the Cdots is presented, representing the experimental control (Spectrum B) .
  • the fluorescence intensity increased when the Cdots were incubated with a ssDNA probe specific to V. vinifera for 30 minutes (Spectrum D) , demonstrating that the probe linked to the Cdots.
  • the fluorescence signal was equal to the Cdots + ssDNA probe (Spectrum C) , proving that the non- complementary strand did not change the fluorescence profile of the sensing system.
  • Example 3 This example demonstrates on one of the possible portable configurations for a biosensing system based on Cdots direct detection.
  • Figure 3 a layout of a portable configuration for the biosensing system and the protocol flow is presented.
  • the chip based on micro wells with at least a volume of 5 pL will be carved on a glass slide ( Figure 3) .
  • the simplest layout, in Figure 3a, may consist on 6 wells, three with
  • Cdots negative control, in white
  • Cdots + specific probe or protein positive control, in grey
  • the sample will be feed into the system through a unique pore, using a microfluidic layout ( Figure 3b, indicated with ->) .
  • the chip will be placed in a microchamber equipped with an excitation led and a photodetector for fluorescence acquisition, linked to a software with the required algorithms to give the report based on the data acquired on the 6 wells ( Figure 3c) .
  • the report will be presented on the top of the microchamber (Figure 3D) that will consist on the following symbols: + - positive (when the wavelength shift/fluorescence intensity/lifetime of the Cdots + specific probe or protein changes in relation to the control Cdots); - - negative (when the wavelength shift/fluorescence intensity/lifetime of the Cdots + specific probe or protein does not change in relation to the control Cdots); ? - inconclusive (when only one/two wells does not have wavelength shift/fluorescence intensity/lifetime change) .

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Abstract

La présente invention concerne une technologie sans étiquette pour la détection et la quantification d'acides nucléiques et de protéines à l'aide de nanoparticules à base de carbone non fonctionnalisées communément appelées points de carbone (Cdots). Ces nanoparticules de carbone peuvent être utilisées à l'état brut.
PCT/IB2019/059308 2018-10-31 2019-10-30 Technologie de détection d'acide nucléique et de protéines sans étiquette basée sur des points de carbone bruts WO2020089810A1 (fr)

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WO2023007298A1 (fr) 2021-07-30 2023-02-02 Universidade De Trás-Os-Montes E Alto Douro Biocapteur à base de fluorescence pour la détection et la quantification du sars-cov-2

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023007298A1 (fr) 2021-07-30 2023-02-02 Universidade De Trás-Os-Montes E Alto Douro Biocapteur à base de fluorescence pour la détection et la quantification du sars-cov-2

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