"LABEL-FREE NUCLEIC ACID AND PROTEINS DETECTION TECHNOLOGY

BASED ON RAW CDOTS"

Technical field

This application relates to label-free nucleic acid and proteins detection technology based on raw Carbon Dots.

Background art

Carbon Dots (Cdots) 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. These carbon-based nanoparticles are equally versatile but with the main advantage of a non-toxic nature.

Nowadays there are several synthetic pathways for producing these nanoparticles [2-3] . In fact, one of the advantages of using Cdots is their ability to be produced using a wide variety of raw materials, including organic wastes from several industries. The high diversity of starting materials and production methods has helped researchers to better understand the fluorescence mechanism that lies beneath these nanoparticles. Indeed, their appealing fluorescence properties are due to two concerning factors: the surface defects and the surface groups. These elements are both introduced on the nanoparticles upon their production, as such, the starting materials and the synthetic pathway represent a core issue that need to be considered according to their future application.

Regardless of their production method Cdots are usually used upon a functionalization step, particularly if the intended application is biosensing/bio recognition [4]. There are some examples in the literature, where 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] . Additionally, it is also possible to alter their fluorescence properties by placing the carbon dots near a metal [8] . 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.

The fluorescence properties of these nanoparticles can truly be used for several applications, in the present application their use is intended for specific detection and quantification of nucleic acids and proteins. Indeed, the specific detection and quantification of these biomolecules in a given sample is an area of tremendous interest. The known interest in these biomolecules is not constrained to the scientific/academic area, but there are also several economically relevant areas that can greatly benefit with the use of a cost effective, practical and reproducible sensor, namely the food industry and the health diagnostics area. In this framework the detection of a specific nucleic acid's sequence and/or protein (s), with high reproducibility and low cost, has been a major concern in the biosensing technology field.

Nowadays there are some nucleic acids sensors available, however they tend to be based on expensive and time-consuming techniques, such as, DNA sequencing and real-time polymerase chain reaction (PCR) . Alternatively, to these traditional methods are the use of gold and magnetic nanoparticles coupled with fluorescent dyes. These techniques 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. The use of Cdots for DNA detection was successfully achieved in the past, however it requires Cdots functionalization [11,12] or indirect detection [13]. Indeed, and even though there have been some efforts into the development of a highly sensitive nucleic acid sensor, a common trend can be found between almost all these sensors: the low reproducibility and reusability, which makes the process rather expensive. It is easy to see the tremendous breakthrough associated with the development of a specific, cheap, reproducible and reusable nucleic acid or protein sensor that could provide a fast analysis and is based on non-toxic, and easy to produce nanoparticles.

The biosensing technology has been growing exponentially over these past few years. With the growing demand for more effective and specific sensing devices, along with the increased knowledge and interest in nanoparticles, it is now possible to see a wide variety of nanosensors for metal ions [14], biomolecules [15], nucleic acids [16], proteins [17], among others. The use of nanomaterials can be truthfully considered widely spread. Their high surface-to-volume ratio and their tuneable surface turns these nanomaterials into efficient starting materials for the development of new sensing platforms.

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. Indeed, their non-toxic nature [19], photostability and biocompatibility, as well as, tuneable optical properties makes them the optimal candidates for detecting and quantifying nucleic acids and proteins in biological complex samples using optical based techniques, such as, fluorescence, bioimaging, UV detection, among others.

In the present literature 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] . It is important to highlight the fact that in this work the Cdots DNA-based sensor relies on indirect detection. Indeed, these investigators report a DNA sensor where the Cdots fluorescence is initially quenched by methylene blue and then restored in the presence of calf thymus DNA.

Summary

The present application relates to a label-free method of detection of nucleic acids and proteins using non- functionalized Cdots comprising the following steps:

- Fluorescence acquisition of one of the incubated label- free Cdots solutions, considered the control;

- Incubation of at least two separate solutions of label- free Cdots with a nucleic acid solution or protein solution, in a 1:1 ratio ;

- Addition of at least 0.07 mM of a sample solution to the other incubated label-free Cdots solution;

- The label-free Cdots solution with the sample solution is carefully homogenized, e.g. by manually inversion, slowly and for one minute;

- Fluorescence acquisition of the overall solution of label- free Cdots with the sample solution.

- The detection method relies on a direct interaction between the Cdots and the nucleic acids and proteins.

In one embodiment, the method further comprises a pre-method step consisting on the application of temperature to a double strand DNA solution.

In another embodiment, the label-free Cdots have a size ranging from 1 to 100 nm.

In yet another embodiment, the label-free Cdots have a spherical shape. In one embodiment the concentration of the label-free Cdot solution is 0.08 g/L, corresponding to 5% (w/w) .

In another embodiment, the concentration of nucleic acid solution or protein solution to incubate with the label-free Cdots solutions is at least 0.07 mM.

In yet another embodiment, the fluorescence is acquired in excitation and emission wavelengths from 400 to 850 nm.

In one embodiment, the incubation time is 30 minutes.

In another embodiment, the incubation time is at least 5 minutes .

In yet another embodiment the Cdots interaction with nucleic acids and proteins relies on label-free detection.

General Description

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 technology described herein is significantly different from what is observed nowadays in the sense that:

a) 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;

b) it is not necessary to use any type of fluorescence labels, since Cdots are fluorescent it is possible to follow in real-time the sensing reaction;

c) 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;

d) 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.

Based on the previously exposed the present technology is sufficiently different and present several main advantages over the existing technologies.

Thus, herein is disclosed a new detection and quantification system for nucleic acids and proteins based on carbon dots. The carbon dots can be obtained using several synthetic pathways that will have an impact in shape, size and surface groups .

The presently described sensing method comprises the following characteristics:

a) the light emission properties of the produced carbon dots are extensively analysed, including light exposure stability, lifetime, among others. Some of these properties are summarized in Table 1.

b) The carbon dots may be in a suspension state or immobilized into an adequate substrate before the incubation with the nucleic acids or proteins.

c) the carbon dots are incubated with the nucleic acids or proteins that need to be analysed. The concentration of the Cdots is 0.08 g/L, corresponding to 5% (w/w) as further described .

d) upon this stage the light emission properties of the incubated carbon dots are analysed. In this stage 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.

e) This method can and will be applied into simple and complex matrixes, i.e., in simple and synthetic nucleic acids and proteins, as well as, complex biological samples where these biomolecules can be present.

It is also intended the description of a portable, easy-to- use technology of immobilized carbon dots.

Table 1. Optical characteristics of the Cdots

⁺ This measurement presents the lifetime calculated using a decay profile described by A+Bl ^a .exp (—i/T1 ) + ... + B3 ^a .exp (—i/T3 ) , with a X ² of 0.491.

*These measurements refer to three independent assays where a solution with the concentration used in the biosensing assay was left under the excitation source for 0, 60 and 120 min, respectively.

Brief description of drawings

For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

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/two of three wells does not have wavelength shift/fluorescence intensity/lifetime change) . Description of the embodiments

Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.

The Cdots of the present technology were synthetized using a carbohydrate thermal method described as follows:

Ten grams of biomass, e.g. chestnut shells, not previously treated, were dissolved in 50 mL of deionized water to form a clear solution. Then, 50 mL of a 95-97 % H ₂ SO ₄ solution was added to the previous solution, and the mixed solution was ultrasonically treated for 4 hours, and oven-dried at 80 °C for 24 h. The resulting black solution was mixed with 50 mL of deionized water, centrifuged at 5000 rpm for 1 h, and filtered to remove the solid supernatant. A dark brown solution was obtained along with the formation of the carbon nanoparticles. Afterwards 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.

(2) Surface characterization using X-ray photoelectron spectroscopy (XPS) with a Kratos AXIS Ultra HSA, with VISION software for data acquisition and CasaXPS software for data analysis. A monochromatic A1 Ka X-ray source (1486.7 eV) , operating at 15 kV (90 W) , in fixed analyser transmission

(FAT) mode, with a pass energy of 40 eV for regions ROI and 80 eV for survey. All data acquisition was performed with a pressure lower than 1.0 ^c 10 ⁶ Pa, and a charge neutralization system was used. The effect of the electric charge was corrected by the reference of the carbon peak (285 eV) . The spectra deconvolution was carried out using the XPSPEAK41, in which a peak fitting is performed using Gaussian- Lorentzian peak shape and Shirley type background subtraction .

(3) Transmission Electron Microscopy (TEM) analysis was performed by placing 10 pL of samples mounted on Formvar/carbon film-coated mesh nickel grids (Electron Microscopy Sciences, Hatfield, PA, USA) and left to dry for 2 min. The excess liquid was carefully removed with filter paper. The visualization was carried out on a JEOL JEM 1400 TEM at 120kV (Tokyo, Japan) . Images were digitally recorded using a CCD digital camera Orious 1100W (Tokyo, Japan) .

(4) Energy-dispersive X-ray spectroscopy (EDS) analysis, was carried out by mounting the section on nickel grids and a beryllium holder (EM-21150, Jeol Ltd.) was used. An X-Max 80 mm ² (Oxford Instruments, Bucks, England) operated at 120 kV was coupled to the microscope.

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.

In one embodiment, if the Cdots are incubated with ssDNA or protein, the signal shift relies on the interaction process between the nanoparticles and the added molecules, as such, it may be necessary to pre-process the samples. In another embodiment 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.

When hybridization or chemical connection/recognition occurs, there is a shift in the Cdots fluorescence. This is an indication that the solution added to the incubated Cdots contains a complementary sequence of the one initially incubated with the Cdots. If it is not possible to observe a fluorescence shift of the Cdots solution, it is possible to say that the complementary sequence was not present on the mixture added or it is present in a concentration lower than the method detection limit. A practical example applied in the laboratory using synthetic strands (present on Table 2) can be seen below. When a positive chemical interaction occurs, e.g. protein- antibody, a shift in the Cdots fluorescence intensity/ wavelength/ lifetime takes place, indicating the presence of a specific protein.

Method of detection of nucleic acids and proteins using label free Cdots comprising the following steps:

- Fluorescence acquisition of one of the label-free Cdots solutions, considered the control;

- Incubation of at least two separate solutions of label- free Cdots with a nucleic acid solution or protein solution, in a 1:1 ratio ;

- Addition of at least 0.07 mM of a sample solution to the other label-free Cdots solution;

- The label-free Cdots solution with the sample solution is carefully homogenized, e.g. by manually inversion, slowly and for one minute;

- Fluorescence acquisition of the overall solution of Cdots with the sample solution.

In one embodiment, 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.

In one embodiment, the concentration of label-free Cdots is 0.08 g/L, corresponding to 5% (w/w) .

In one embodiment, the concentration of nucleic acid solution or protein solution to incubate with the label-free Cdots solutions is at least 0.07 mM. In one embodiment, the fluorescence is acquired in excitation and emission wavelengths from 400 to 850 nm.

These measurements were performed immediately after the homogenization process.

In an embodiment 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.

Example 1 :

This example demonstrates the applicability of the Cdots sensing system using synthetic DNA strands that can be found in Table 2.

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. In this particular example we used synthetized targets that were in a pure state. This was necessary, because it was the only way to discriminate what happens in each process, e.g, upon addition of ssDNA solution, upon the addition of complementary DNA and upon the addition of non-complementary DNA. Moreover, by using this procedure it was also possible to determine the minimum concentration of Cdots/ssDNA needed for the technique to be successful, a solution of 5% pure Cdots with 0.07 mM of ssDNA. However, further work allowed us to determine that the targets do not need to be in a pure state, nor the ssDNA. The fact that there was no observable change in the fluorescence upon the addition of the non- complementary DNA was quite relevant, since it means that the presence of other DNA sequences on the solution rather than the complementary will not interfere with the method. Upon incubation the fluorescence of the samples was measured and the signal shift was determined, (functionalization signal: 71 Ua of light intensity; hybridization signal: 1415 Ua of light intensity) , was consistent throughout the different tubes. Consequently, and in order to test the specificity of the biosensor, complementary DNA was added to one tube, 750 L of 0.25 mM stock solution and, total non- complementary DNA (with the same concentration) was added to another tube. 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.

Example 2 :

This example demonstrates the applicability of the Cdots sensing system using a complex DNA sample extracted from wine using specific Vitis vinifera L. probes.

In one example, 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

L.

In Figure 2, 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. When the Cdots + ssDNA probe were incubated with a non-complementary DNA sequence, the negative control, 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.

Whereas, when Cdots + ssDNA probe were incubated with wine DNA there was an increase in the fluorescence signal due to the hybridization process (Spectrum A) .

Example 3: This example demonstrates on one of the possible portable configurations for a biosensing system based on Cdots direct detection.

In 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) and three containing 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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