This application claims the benefit of the European Patent Application EP19382521.3 filed June 20th, 2019.

Technical Field

The present invention relates to the field of carbon-based nanomaterials with optical properties. In particular, the present invention refers to fluorescent carbon quantum dots with a high quantum yield, to a process for its preparation and to its use as sensor and optical limiter.

Background Art Carbon quantum dots (CQDs) are a promising material, featuring fluorescent carbon nanoparticles of less than 10 nm in size with a high surface-to-volume ratio. Compared with other semiconductor QDs materials, such as CdS QDs, PbS QDs, etc., CQDs offer many advantages including good biocompatibility, robust chemical inertness, low toxicity, and outstanding optical properties, making them a promising carbon-based nanomaterial for applications such as biological sensing, bioimaging and photodynamic therapy, drug delivery and analysis, light-emitting diodes (LEDs), and solar cells.

To date, CQDs have been synthesized via various methods which can be generally classified into two categories,“bottom-up” approaches and“top-down” approaches. In the “bottom-up” approaches, organic or natural substances are carbonized to fabricate CQDs, and“top-down” approaches feature cutting large-sized carbon materials (e.g., graphite, activated carbon, carbon nanotubes, graphene oxide, carbon soot, etc.) into small-sized CQDs. Top-down methods which include ultrasonic synthesis, laser ablation, arch-discharge, chemical and electrochemical oxidations involve fragmentation of larger carbonaceous materials into small nanoscale sizes less than 10 nm.

In the bottom-up process, the C-dots are obtained from molecules in solutions by employing techniques such as hydrothermal treatment, microwave synthesis, plasma treatments, thermal decomposition and solvothermal synthesis.

Among the various approaches, the acidic exfoliation method, hydrothermal method, and microwave-assisted treatment method are the most common and dominant approaches. Generally, these methods are relatively complex, expensive, and environmentally hazardous in preparation processes of CQDs. Most of the obtained CQDs have only one photoluminescence (PL) emission with size-dependent and surface-passivation- dependent properties, which limit their further applications in dual-wavelength fluorescent emissions. In addition, some impurities are always produced due to the introduction of a strong acid in the preparation process of CQDs, resulting in the PL mechanism of CQDs being ambiguous.

Compared with the abovementioned methods, pulsed laser ablation (PLA) is a unique and novel approach, which is simple, cost-effective, and environmentally friendly in the fabrication process of carbon-based nanomaterials without any impurities. Anomalous reactions and growth of the fragmented species can occur under non-equilibrium conditions, such as high temperature and high pressure in liquid. Many efforts showed the possibility for the synthesis of carbonic nanomaterial by nanosecond- or femtosecond- laser ablation of highly oriented pyrolytic graphite (HOPG). At present, the study of CQDs is in a new era. However, there are still many challenging issues to be solved, including the following: (1) carbon precursors for the preparation of CQDs by pulsed laser ablation are high-cost; and (2) the fluorescence quantum yield of CQDs is relatively low in many solvents such as water and ethyl acetate oleamide (often less than 20%) using the pulsed laser ablation method, which limits their applications in biomedical sensing, imaging and optoelectronics.

On the other hand, during the last decade, there has been a great deal of progress made in the construction of boronic acid-based sensors for carbohydrates and other diol- containing compounds. Boronic acid has been widely used for the development of color sensors, fluorescent sensors, carbohydrate transporters, and for chromatographic materials. Unfortunately, majority of the carbohydrate probes based on boronic acid moiety have limited water solubility and limited fluorescence properties. These probes consume analyte and require mediators, which further restrict their use as biosensors.

Up to the filing of the present application, several publications have addressed the manufacturing of arylboronic CQD-based sensors with improved fluorescence properties. Among them, Kiran S. et al., 2015, reported the fabrication of arylboronic-functionalized CQDs by microwave and Zhang X. et al., 2013 by hydrothermal treatment. In both cases, the resulting functionalized CQDs showed a fluorescence quantum yield lower than 10%.

In spite of the efforts made, therefore, there is still the need of providing CQDs with improved optical properties. Summary of Invention

The present inventors have obtained boronic acid-functionalized CQDs with improved fluorescence properties.

As it is shown below, the present inventors found that fluorescent boronic acid- functionalized CQDs were obtained when an arylboronic acid aqueous solution (such as a 2-aminopyrimidine-5-boronic acid aqueous solution) was irradiated with femtosecond pulsed ultrafast laser beam. Surprisingly, the fluorescence quantum yield of the resulting CQDs was about at least 40%, particularly was about 60%. This is a high fluorescence quantum yield for an arylboronic-functionalized CQD, which have a remarkable impact on sensing properties: the higher quantum yield is, the better performance of the CQD as sensor is. As it has been discussed above, several attempts have been made to achieve

arylboronic-functionalized CQDs with improved fluorescence properties, such as Kiran S et al., 2015 and Zhang X. et al., 2013. In these cases, the obtained boronic-functionalized CQDs showed a fluorescence quantum yield of less than 10%. Therefore, the present invention provides a robust fluorescent boronic acid-functionalized CQD with about 6-fold higher quantum yield with respect to those of the prior art, which means a great advance in the field of fluorescent sensors.

Thus, in a first aspect the present invention provides a boronic acid functionalized carbon quantum dot which is stable against photobleaching and has a fluorescence quantum yield of at least 20%, wherein:

(a)“stable against photobleaching” means that the fluorescence intensity of the carbon dot after continuously irradiating with a laser pointer (having, for example, a wavelength of 405 nm) for a period of time of at least one hour (for example at least 10 hours, at least 15 hours, at least 20 hours), is the same or corresponds to at least a 85% (at least 86, 87,

88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %) of the fluorescence intensity of the quantum dot prior to the irradiation; and (b) the fluorescence quantum yield is determined by the formula: where m is the slope of integrated fluorescence intensity against absorbance at the wavelength from 200-450 nm (such as from 300-350, from 310-340, or 330-320 nm, or 320 nm), n is the refractive index, and subscript S and R indicates the test sample and reference quinine sulfate solution, respectively. The QY (also referred as“FU”) of the reference standard, QYR (also referred as“FUr”) is 0.59, and the refractive index of water (solvent) and 0.10 M H ₂ SO ₄ solution is 1.33.

As it is also shown below, the CQDs of the invention are also highly stable to

photobleaching, detecting a negligible loss of fluorescent when illuminated by near UV (l = 405) laser continuously for up to 15 h (see FIG. 5). This implies that repeated fluorescence measurements of the same sample could be taken without compromising the integrity of the data. These extra-data confirm the value of the CQDs of the invention as biological sensor. Photobleaching refers to the photochemical alteration of a fluorophore molecule such that it permanently is unable to fluoresce; whereas the fluorescence quantum yield is a measure of the efficiency of photon emission through fluorescence, which is the loss of energy by a substance that has absorbed light via emission of a photon. It is often defined as the ratio of the number of photons emitted to the number of photons absorbed. In other words, the fluorescence quantum yield gives the probability of the excited state being deactivated by fluorescence rather than by another, non-radiative mechanism such as internal conversion or vibrational relaxation (non-radiative loss of energy as heat to the surroundings).

The present invention also provides a facile production process of the highly fluorescent and multifunctional CQDs of the invention from a single precursor by performing one step pulsed laser irradiation. Advantageously, while the irradiation is being applied to the boronic acid compound, non-linear optical processes from the sample during laser irradiation as well as parameters such as linear fluorescence, two photon exited fluorescence and the second harmonic generation can be monitored.

In addition, the process of the invention is environmental-friendly, limiting wastes and enables easy in situ surface passivation with molecular functional groups. The process of the invention also facilitates straightforward up scaling of the production. Thus, in a second aspect, the present invention provides a process for obtaining a boronic acid functionalized carbon dot as defined in the first aspect of the invention, the process comprising the step of subjecting an arylboronic acid solution to a pulsed laser irradiation with a peak powder intensity which generates a filament in the liquid or breakdown of the arylboronic acid, wherein: “arylboronic acid” is understood as an aryl-(B(OH)2)n, wherein“aryl” represents a known ring system with 1-3 rings which comprises from 5 to 14 members, the rings being saturated, partially unsaturated, or aromatic, provided that at least one of the rings is an aromatic ring; and being fused, partially fused or isolated; each one of the members forming the known ring system being selected from the group consisting of: -CH-, -CH2- , -NH-, -N-, -SH-, -S-, and -0-; and the ring system being optionally substituted by one or more radicals independently selected from the group consisting of (Ci-Ce)alkyl,

(CrC ₆ )haloalkyl, (Ci-Ce)alkyl-O-, nitro, cyano, -NR1R2, and halogen; n is an integer value from 1 to 3; and

Ri and R2 are the same or different and are selected from -H, (Ci-Ce)alkyl optionally substituted by -OH, halogen, C=0, and nitro.

The requisite is the interaction of intense laser light with the sample leading to formation of plasma or filamentation. The process takes places due to the synergistic influence of the intense laser filamentation and the induced breakdown of the precursor molecules. The filamentation appears due to the dynamic counterbalance between self-focusing and defocusing of the intense laser beam by the optical Kerr effects and the self-generated plasma, respectively. This equilibrium also clamps the laser intensity inside the filament core to a constant value and leads to the formation of uniform CQDs.

The inventors have surprisingly found that an arylboronic acid solution could be efficiently used as the single precursor in the one-step pulsed laser irradiation process. In fact, emissions of intense blue light were detected when the aqueous 2-aminopyrimidine-5- boronic acid (2-APBA) solution was irradiated with a femtosecond pulsed laser. The inventors attributed it to the second harmonic generation (SHG), which occurred at an intermediate stage before the precursor material is fully converted to CQDs. This could be due to the formation of dimmers, trimers, or some supramolecular structures which elongate the charge transfer conjugation length, hence leading to maximization of the SHG. Without being bound to the theory, it is believed that such maximization of the SHG could explain the remarkable improvement in the fluorescence quantum yield of the CQDs of the invention. This is because SHG involve inter/intramolecular charge transfer (ICT) in the sample which also increases fluorescence quantum yield.

In a third aspect the present invention provides the boronic acid-functionalized CQDs obtainable by the process of the second aspect of the invention. For the purposes of the invention the expressions "obtainable", "obtained" and equivalent expressions are used interchangeably, and in any case, the expression "obtainable" encompasses the expression "obtained".

In a fourth aspect the present invention provides an article comprising the CQD as defined in the first or third aspect of the invention.

As it has been mentioned above, the CQDs of the invention are functionalized with boronic acid groups (B(OH)2). As it is shown below, the CQDs resulting from the process of the second aspect of the invention were characterized by Fourier Transform Infra-red (FTIR) spectroscopy and X-ray Photoelectron Spectroscopy (XPS) to distinguish the chemical functional groups in the obtained CQDs. FTIR vibrational bands shown in FIG. 2 indicated the presence of hydroxyl (OH), amino groups (NH2) C=N, C=C, B-C, B-0 and B-OH functional groups. The presence of B-OH moieties on the surface of the CQDs is very important for sensing applications because this group forms highly selective covalent complexes specifically with diols (such as saccharides or catechols) but also with strong Lewis bases, such as fluoride or cyanide anions. The attachment of the diol or Lewis base to the CQDs via the B-OH groups changes CQD’s fluorescence. In the particular case of diols, the boronic acid reacts and transforms from the neutral trigonal form into the anionic tetrahedral form, leading to the formation of tetrahedral boronate esters. The formation of a cyclic boronate ester via interaction with a diol intensifies the electrophilicity of the boronic acid group and thereby reduces the value of acid dissociation constant (pKa). Thus, at pH in the range of 6.5-8.5, the boronic acid exists in trigonal form, but in the presence of the target molecule, it transforms to tetrahedral anionic form. This may induce changes in intensity, decay time, and polarization. The magnitude of this change depends on the analyte concentration: the greater the amount of analyte, the greater the fluorescence intensity.

Thus, in a fifth aspect the present invention provides the use of the boronic acid- functionalized CQD as defined in the first or third aspect of the invention or of the kit as defined in the fourth aspect of the invention, as sensor. The sensing applications can be homogeneous assays or heterogeneous detection. Detection can be at the interface of the sensing material or within the bulk sample. Furthermore, the key interaction of boronic acids with the target analyte allows utilisation in various areas ranging from biological labelling, protein manipulation and modification, separation and the development of therapeutics. QDC-based sensors have distinct advantages over other fluorophores, including their compact size, high brightness, continuous excitation by any wavelength shorter than the emission wavelength, high extinction coefficients, resistance to photobleaching, and narrow emission that enables multiplexing with QDCs emitting at different wavelengths.

In a sixth aspect, the present invention also provides a method for the determining the presence or concentration of a target analyte in a test sample, the method comprising (a) contacting the sample with the carbon quantum dot of the first aspect of the invention, (b) exposing the sample and the carbon quantum dots to light energy that causes the carbon quantum dots to emit photons; and (c) detecting a change in fluorescence intensity, whereby said change indicates the presence or amount of the target analyte.

The present inventors have also found that the CQDs of the invention have optical limiting properties since peak absorption increases with increasing laser intensity (see FIG. 7).

Optical limiting occurs when the power of high intense laser beam (for instance pulsed lasers) decreases upon interaction with a material that has non-linear optical properties such as multiphoton absorption, non-linear scattering and reflection. Optical limiting materials are in high demand because of their use in protecting human eyes and optical sensors from damages that can be incurred when exposure to high intense laser beams.

Thus, in a seventh aspect the present invention provides the use of the CQD as defined in the first aspect of the invention or the kit as defined in the fourth aspect of the invention as optical limiter.

Nonlinear optical limiting materials and devices that passively filter out damaging laser radiation at any incoming wavelength from an optical system. Nonlinear materials and devices are transparent to the optical system until a laser threat is present, then turn-on, blocking the threat laser, but still allowing the viewing system to function normally in the presence of the threat.

Brief Description of Drawings

FIG. 1 : (a) Transmission electron microscopy (TEM) image of 1000 fold diluted carbon quantum dots (CQDs); inset: High resolution TEM image of a representative single particle (b) TEM image of 100 fold diluted CQDs.

FIG. 2: Characterization of the obtained carbon quantum dots by: Fourier Transform Infrared (FTIR) spectroscopy (a) and X-ray Photoelectron Spectroscopy (XPS) (b-f). The XPS survey spectrum of the sample indicating the presence of Oxygen, Nitrogen, Carbon and Boron species is shown in (b). The corresponding high-resolution XPS spectra are depicted in (c), (d), (f) and (f), respectively. FIG.3: UVA/is absorption spectrum (labeled Abs 1) of CQDs dispersed in water. The absorption spectrum (dotted curve) labeled Abs 2 is from the diluted sample

photoluminescence excitation spectrum (open circular symbols) shows two excitation peaks at wavelengths 233 and 333 nm. The Fluorescence emission spectra at the two excitation wavelengths of 233 and 333 nm are represented by filled circles and open star symbols, respectively.

FIG.4: (a) and (b) Photoluminescence (PL) emission spectra with maxima at 407, 430 and 510 nm for different excitation wavelengths as indicated. The voltage of the spectrometer was set to 430 mV. (c) PL emission at the voltage of 580 mV and excitation wavelength of 380 nm. (d) PL emission at around 510 nm for different excitation wavelength at a voltage of 580 mV. (e) and (f) PL emission spectra with maxima at around 750 and 800 nm for different excitation wavelengths as indicated. The spectra in (e) and (f) were recorded when the voltage was adjusted to 700 mV. The CQDs have overall large stoke shift of 500-600 nm. In this case the stoke shift is defined as the difference between excitation wavelength and the wavelength at maximum emission.

FIG. 5: Fluorescence intensity as function of time for the CQDs illuminated with a laser pointer (l=405 nm) continuously for 15 h. FIG. 6: Solid state fluorescence spectra of carbon quantum dots-doped porous silica sheets (CQDs-pSi0 ₂ ) with different excitation wavelengths.

FIG. 7: Normalize power transmission as a function z-position of the CQDs-pSi0 ₂ sample. The data was obtained from the open z-scam measurement done at different input power which is given in terms of fluence in the legend.

FIG. 8: Represents the change of fluorescence of the prepared CQDs-pSi0 ₂ against glucose concentration in the range 1-100 mg/dL. The inset depicts the fluorescence spectra of the CQDs-pSi0 ₂ before and after adding different concentrations of glucose as indicated. The fluorescence of CQDs-pSi0 ₂ is quenched depending on the concentration of the added glucose. Detailed description of the invention

All the terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition. In addition, for the purposes of the present invention, any ranges given include both the lower and the upper end-points of the range. Ranges given, such as temperatures, times, weights, and the like, should be considered approximate, unless specifically stated.

In a first aspect the present invention provides a boronic acid functionalized carbon quantum dot which has a fluorescence quantum yield of at least 15%. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the CQD is a nitrogen-doped CQD. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the CQD is functionalized with boronic acid and -NH2 groups.

In the present invention by“boronic acid”, when referred to the first aspect of the invention, means *-B(OH)2, wherein * denotes the attachment of the boron atom to the CQD’s surface. In the present invention, the term“functionalized” means that the boronic acid is bound to the QDC surface.

In the present invention the term“carbon quantum dot” is understood as small carbon nanoparticles, with a size lower than 10 nm and higher than 1 nm as measured by transmission electron microscope (TEM). In one embodiment of the invention, the CQD has a size of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nm when measured by TEM.

In the present invention the term“fluorescence quantum yield” (0F) is to be understood as the ratio of photons absorbed to photons emitted through fluorescence at a particular wavelength. In other words, the quantum yield gives the probability of the excited state being deactivated by fluorescence rather than by other, non-radiative mechanisms. The most reliable method for recording 0F is the comparative method of Williams et al., which involves the use of well characterised standard samples with known 0F values. Essentially, solutions of the standard and test samples with identical absorbance at the same excitation wavelength can be assumed to be absorbing the same number of photons. Hence, a simple ratio of the integrated fluorescence intensities of the two solutions (recorded under identical conditions) will yield the ratio of the quantum yield values. Since 0F for the standard sample is known, it is trivial to calculate the 0F for the test sample. An alternative way is provided in examples below. Briefly, absorption and fluorescence spectra of both reference standard and test sample are measured under identical conditions. The 0F is calculated from the gradients of the plots of integrated fluorescence versus absorbance of both the test sample and the standard reference sample using the formula: where m is the slope of integrated fluorescence intensity against absorbance, n is the refractive index, and subscript S and R indicates the test sample and reference quinine sulfate solution, respectively. The QY (also referred as“FU”) of the reference standard, QYR (also referred as“FUr”) is 0.59, and the refractive index of water (solvent) and 0.10 M H ₂ SO ₄ solution is 1.33.

In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the fluorescence quantum yield is at least of 30%, at least of 40%, at least of 50% or at least of 60%. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the fluorescence quantum yield is comprised from 40 to 80%, from 50 to 70% or from 55 to 65%. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the fluorescence quantum yield is selected from 15, 16, 17, 18, 19, 20, 21 , 22, ,23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,

42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65,

66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89,

90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 and 100%. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the intensity after continuously irradiating it with the laser after 15 hours corresponds to at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the fluorescence intensity of the quantum dot prior to irradiation.

In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the QDC further has UV/vis absorption peaks at 220, 250, 294, and 343 nm when measured between 200-700 nm. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the QDC further has four fluorescence emission windows at around 400 nm, 430 nm, 510 nm and at about 750-800 nm, when excited at wavelength between 200-330, 340-380, 390-480, and 200-370 nm, respectively.

The four emission windows allow for imaging and sensing at different wavelengths especially the near IR region which is very attractive for imaging biological samples due to deep tissue penetration and reduced scattering of the light within this region. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the QDC further has (a) UV/vis absorption peaks at 220, 250, 294, and 343 nm; and (b) four fluorescence emission windows at around 400 nm, 430 nm, 510 nm and at about 750-800 nm (see FIG. 4). In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the QDC further has one or more excitation/emission stoke shifts of up to 200 nm for emission at about 400 nm, excitation/emission stoke shifts of up to 180 nm for emission at about 430 nm, excitation/emission stoke shifts of up to 250 nm for emission at about 510 nm, excitation/emission stoke shifts of up to 540 nm for emission at about 760 nm, and excitation/emission stoke shifts of up to 550 nm for emission at about 800 nm.

The emission maximum at 760 nm with possible excitation wavelength of 220 nm represent the largest stokes shift (540 nm) ever reported between the emission and excitation wavelengths. In this case the stoke shift is defined as the difference between excitation wavelength and the wavelength at maximum emission. Fluorescence materials with large stoke shifts are very important for many applications because of the possibility to achieve high signal to noise ratio. For example, such materials are applied in super resolution microscopy imaging utilizing stimulated emission depletion (STED) technique to overcome the diffraction limit.

In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the QDC further has the

excitation/emission stoke shifts of up to 200 nm for emission at about 400 nm, excitation/emission stoke shifts of up to 180 nm for emission at about 430 nm,

excitation/emission stoke shifts of up to 250 n for emission at about 510 nm,

excitation/emission stoke shifts of up to 560 nm for emission at about 760 nm, and excitation/emission stoke shifts of up to 550 nm for emission at about 800 nm. (FIG. 4).

Furthermore, the broad emission with excitation at 380 nm implies that the obtained CQDs can find applications in white light emitting diodes.

In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the CQD is optical limiting.

In one embodiment, the CQD of the invention is attached to a solid support. The presence of amino groups on the surface of the obtained CQDs (as confirmed by FTIR in the above discussion) facilitates their anchoring on solid surface through H-bonding with the silanol groups. It is therefore inferred that the CQDs can be located inside the nanoporous and/or on the surface of exemplified silica sheets.

The support may be, among others; glass, plastic, cellulose, nitrocellulose or paper.

Attachment of the probe to the solid support may succeed by methods well known in the art (Angenendt P, Drug Discovery Today, 2005, vol. 10(7), p. 503). For example, the CQD of the invention may be attached to two-dimensional plain glass slides, which are activated with a variety of coupling chemistries such as aldehyde, epoxy or carboxylic esters. Slides with these surfaces bind biomolecules (for example peptides or nucleic acids) either by electrostatic interactions or through the formation of covalent bonds. Alternatively, the CQD of the invention can be attached to three-dimensional gel or membrane-coated surfaces, such as polyacrylamide, agarose and nitrocellulose. These surfaces bind biomolecules mainly by physical adsorption. Alternatively, the CQD of the invention can be attached to surface coatings, such as dendrimer or avidin slides, which mix both concepts mentioned above. In one embodiment the solid support is amino, epoxy or carboxy-functionalised. In another embodiment the support comprises aminosilane glass surface.

In another embodiment, the solid support has microarray format, meaning that CQD of the invention is deposited or immobilized onto the solid support following a microarray design, i.e. , the CQDs are densely spotted onto the support on a few square microns such that large number of samples may be analyzed simultaneously. Different immobilization strategies known in the art may be employed to this end, including, among others;

immobilization of antibodies by DNA-directed immobilization (DDI), direct spotting, and streptavidin-biotin attachment (Wacker, R. et al.“Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study”. Anal. Biochem. 2004, vol. 330, p. 281-287). Usually, such microarray format comprises a glass surface. In another embodiment the solid support has lateral flow format. Lateral flow tests, also known as lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in a sample without the need for specialized and costly equipment, though many lab based applications exist that are supported by reading equipment. These lateral flow tests are well known in the art and appropriate for point of use applications. Often they comprise a series of capillary beds, each of which has the capacity to transport fluid. The sample thus flows through the different capillary beds contacting the reagents so that a detection result is finally observed. In another embodiment the solid support with lateral flow format comprises a nitrocellulose membrane to which the QD-binding molecule label is attached.

In other embodiments the solid support has microliter plate format, such as those used for ELISA assays.

In a second aspect the present invention provides a process for obtaining the QDCs of the invention.

The term (Ci-Ce)alkyl refers to a saturated straight or branched alkyl chain having from 1 to 6 carbon atoms. Illustrative non-limitative examples are: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neo-pentyl and n-hexyl.

The term“halogen” refers to the group in the periodic table consisting of five chemically related elements: fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At).

The term (Ci-Ce)haloalkyl refers to a group resulting from the replacement of one or more hydrogen atoms from a (Ci-Ce)alkyl group with one or more, preferably from 1 to 6, halogen atoms, which can be the same or different. Examples include, among others, trifluoromethyl, fluoromethyl, 1-chloroethyl, 2-chloroethyl, 1-fluoroethyl, 2-fluoroethyl, 2- bromoethyl, 2-iodoethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 3-fluoropropyl, 3- chloropropyl, 2,2,3,3-tetrafluoropropyl, 2,2,3,3,3-pentafluoropropyl, heptafluoropropyl, and 4-fluorobutyl, and nonafluorobutyl.

In the present invention, the term“arylboronic acid” is represented by aryl-(B(OH)2) _n , wherein the boronic acid radical(s) (-B(OH)2) can be located in any position or the aryl moiety and, in case that the ring system comprises more than one ring, these radicals can be either in the aromatic or non-aromatic ring. The“aryl” moiety is as defined above under the second aspect of the invention. In one embodiment the one, two or three boronic acid radicals are bound to the aromatic ring. In an embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, n is 1

In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic acid

(aryl-(B(OH)2) _n ) is a ring system consisting of one aromatic ring having 5 or 6 members from the group consisting of: -CH-, -NH-, and -N-, one of the members having a boronic acid radical (-B(OH)2) as substituent, and the others being optionally substituted with one or more radicals independently selected from the group consisting of (Ci-Ce)alkyl, (CrC ₆ )haloalkyl, (Ci-Ce)alkyl-O-, nitro, cyano, -NR1R2, and halogen. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic acid (aryl-(B(OH)2) _n ) consists of an aromatic ring having 6 members selected from -CH-, - -NH-, and -N-, wherein one of the members is substituted by a boronic acid radical and the others are optionally substituted with one or more radicals independently selected from the group consisting of (CrC ₆ )alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyl-O-, nitro, cyano, -NRiR2 and halogen. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic acid (aryl-(B(OH)2) _n ) consists of an aromatic ring selected from the group consisting of: phenyl, pyrimidine, pyrazine, pyridine, pyridazine, and 1 ,2,3-triazine, wherein from 1 to 3 members of the aromatic ring are substituted by a -B(OH)2 radical and the remaining members are optionally substituted by one or more radicals independently selected from the group consisting of (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyl-O-, nitro, cyano, -NRiR2 and halogen. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic acid (aryl-(B(OH)2) _n ) consists of an aromatic ring selected from the group consisting of: phenyl, pyrimidine, pyrazine, pyridine, pyridazine, and 1 ,2,3-triazine, wherein 1 of the members of the aromatic ring is substituted by a -B(OH)2 radical and the remaining members are optionally substituted by one or more radicals independently selected from the group consisting of (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyl-O-, nitro, cyano, -NRiR2 and halogen. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic acid (aryl-(B(OH)2) _n ) consists of an aromatic ring selected from the group consisting of: phenyl, pyrimidine, pyrazine, pyridine, pyridazine, and 1 ,2,3-triazine, wherein 1 of the members of the aromatic ring is substituted by a -B(OH)2 radical and the remaining members are optionally substituted by one or more radicals independently selected from the group consisting of (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyl-O-, nitro, cyano, -Nhh and halogen. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic acid (aryl-(B(OH)2) _n ) consists of an aromatic ring selected from the group consisting of: phenyl, pyrimidine, pyrazine, pyridine, pyridazine, and 1 ,2,3-triazine, wherein 1 of the members of the aromatic ring is substituted by a -B(OH)2 radical and the remaining members are optionally substituted by -IMH2. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic acid (aryl-(B(OH)2) _n ) is 2-aminopyrimidine-5-boronic acid (2-APBA).

In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic acid (aryl-(B(OH)2)) solution is prepared by mixing the arylboronic acid (aryl-(B(OH)2) _n ) with a protic solvent. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, 2-APBA solution is prepared by mixing the 2-APBA with a protic solvent.

In the present invention, a“protic solvent” means a solvent that has a hydrogen atom bound to an oxygen (as in a hydroxyl group), a nitrogen (as in an amine group) or a fluorine (as in hydrogen fluoride). In general terms, any solvent that contains a labile H+ is called a protic solvent. The molecules of such solvents readily donate protons (H+) to reagents. In one embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the solvent is selected from water; a (Ci-Cio)alkyl substituted by one or more -OH, -COOH, and -NO2; (C2-Cio)alkenyl substituted by one or more -OH, -COOH, and -NO2; and (C2-Cio)alkynyl substituted by one or more -OH, -COOH, and -NO2. In one embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the solvent is selected from water, ethanol, butanol, isopropanol, methanol, nitromethane. In one embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the solvent is water (and, hence, the arylboronic acid (aryl-(B(OH)2) _n ) solution is also referred as“aqueous solution”). In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic (aryl-(B(OH)2) _n ) solution is transparent. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylboronic (aryl-(B(OH)2) _n ) solution shows an optical transmission of at least 50% at the wavelength of the laser used in the irradiation step. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylbroronic acid (aryl-(B(OH)2) _n ) solution is prepared at a

concentration comprised from 0.0005 to 1 M, from 0.0001 to 0.5 or from 0.005 to 0.1 M. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the arylbroronic acid (aryl-(B(OH)2) _n ) solution is prepared at a concentration of 0.002 or 0.05 M.

The process of the second aspect of the invention comprises irradiating the arylboronic acid (aryl-(B(OH)2) _n ) solution with a pulsed laser.

In one embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pulsed laser irradiation is a nano-, pico- or femtosecond pulse laser irradiation. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pulsed laser irradiation is a femtosecond laser irradiation. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pulse irradiation corresponds to pulses from 10 to 150 femtoseconds (pulse duration) full width at half maximum (FWHM) with a central wavelength from 700-900 nm. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pulse irradiation corresponds to pulses from 20 to 40 femtoseconds intensity full width at half maximum (FWHM) with a central wavelength from 750-850 nm. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pulse irradiation corresponds to pulses from 30 femtoseconds (pulse duration) full width at half maximum (FWHM) with a central wavelength of 800 nm.

In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pulses have a repetition rate from 0.5 to 1.5 kHz. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pulses have a repetition rate of 1 kHz. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pulse duration corresponds to time from 10 to 50 femtoseconds full width at half maximum (FWHM) with a central wavelength from 700-900 nm, at a repetition rate from 0.5 to 1.5 kHz. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pulse duration corresponds to time from 30 femtoseconds full width at half maximum (FWHM) with a central wavelength of 800 nm at a 1 kHz repetition rate. In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the peak power density of the laser is in the range from 10 ¹⁰ to 10 ²² W/cm ² . In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the peak power density of the laser is about 10 ¹² W/cm ² .

In the present invention, the term“peak powder density” also referred to as irradiance of the pulsed laser beam, can be determined by formula 2: where pulse energy is given by average laser power/repetition rate and focus area is given by TT(d/2) ² with d being the diameter (in cm) of laser beam at the focal spot. A power meter can measure average laser power while the pulsed width and repetition rate are the characteristics of the pulsed laser in use.

In a fourth aspect, the present invention provides an article comprising the CQD of the invention. All the embodiments provided above under the first aspect of the invention, are also embodiments of the CQD referred in the fourth aspect of the invention. In one embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the article is a kit further comprising a solid support. In another embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the solid support is a transparent material made of either glass, ceramic or polymeric material in the bulk state or when it contains pores, open or closed channels. The solid support facilitates easy handling and avoids possible contamination of CQDs in the solution. There are

commercially available transparent polymeric matrix useful as solid supports. Illustrative non-limitative examples of transparent porous materials are: porous silica, glass, transparent porous ceramics, and other porous polymeric matrices comprising of

Poly(methyl methacrylate) (PMMA), polyvinyl alcohol (PVA), Polymethylphynlysiloxane (PDMS).

The CQD of the invention can be applied by routine means on the solid support. In one embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the CQDs of the invention are entrapped in the silica matrix. In another embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the CQDs are entrapped in transparent porous silica matrix by soaking an already-formed transparent matrix film in a solution comprising the CQDs of the invention. In another embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the CQDs are entrapped in transparent porous silica matrix by soaking an already-formed transparent silica sheet in an aqueous solution comprising the CQDs of the invention.

In another embodiment of the fourth aspect of the invention, the article can be a sensor, an optical limiter device, a light-emitting device (LED), a biomedicine delivery system, dye- sensitized solar cells (DSCs), organic solar cells (OSCs), supercapacitor, or

photocatalyzer. The skilled person, making use of the general knowledge, can

manufacture articles for their commercialization. The article will take one or other form depending the final use.

In a fifth aspect the present invention provides the use of the CQD as defined in the first or third aspect of the kit as defined in the fourth aspect as sensor. All the embodiments provided under the first and fourth aspects are also embodiments of the fifth aspect of the invention.

In one embodiment of the fifth aspect of the invention, the CQD of the invention or kit is used as a sensor of monosaccharides. In another embodiment of the fourth aspect of the invention, the CQD as defined in the first aspect of the invention is used as a sensor of glucose. As it is shown in FIG. 8, there is a linear relationship between the change in fluorescence and glucose concentration in the range 1-100 mg/dL. This means that the CQDs of the invention are able to appropriately detect glucose concentration as low as 0.055 mM, which is indicative of the high sensitivity of the CDQ of the invention. This so low detectable level characteristic of the CQDs of the invention make them useful as very sensitive sensors, providing a robust and reliable method for detecting/determining the amount of a particular analyte.

Thus, in a sixth aspect the present invention provides a method for determining the presence or amount of a target metabolite in a test sample. All the embodiments provided under the first aspect of the invention are also embodiments of the sixth aspect of the invention. In one embodiment of the sixth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the target analyte is selected from a monosaccharide, catechol, halogen and cyanide. In another embodiment of the sixth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the target analyte is a monosaccharide, preferably glucose.

In another embodiment of the sixth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the test sample is an isolated sample from a body fluid or tissue from an animal, particularly a human. In another embodiment of the sixth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the test sample is a body fluid selected from blood, serum, plasma and tears.

In another embodiment of the sixth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the test sample is blood, serum, plasma or tears; and the target analyte is glucose.

In a seventh aspect the present invention provided the use of the CQD as defined in the first or third aspect of the invention or the kit as defined in the fourth aspect of the invention as optical limiter, particularly an optical laser limiter.

The CQDs of the invention can be formulated in suitable macroscopic systems such as porous silica sheets or polymer films with a glassy morphology. Additionally, a number of porous sol-gel host matrices containing CQDs can be cast into thin films. CQDs can also be incorporated into elastic polymer and viscoelastic gels and elastomers as guest-host materials for optical-limiting functions. For use as eye protection, the CQDs can be coated onto the optical eyeglass wares using the common glass tinting procedures. Alternatively, the mixture of CQDs with transparent polymeric gels and elastomers can be cast or moulded to form eyeglasses for protection against intense laser radiations.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps.

Furthermore, the word“comprise” encompasses the case of“consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim, and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred

embodiments described herein.

Examples

1. Obtaining of boronic acid-functionalized CQD of the invention

2-aminopyrimidine-5-boronic acid (2-APBA) sample with purity of 95% was purchased from Sigma Aldrich. Ultrapure water (resistivity 18 MW cm at 25°, Milli-Q system) was used as a solvent. Two different concentrations; 0.002 M, and 0.05 M of 2-APBA were prepare by dissolving appropriate amount of 2-APBA in pure water. The mixture was ultrasonicated at 60 °C using ultrasonic probe UP400s (Hielscher Ultrasonics Gmbh, Germany) for 4 hours. To increase solubility, two drops equivalent to about 20 pl_ of ammonia solution at 25% was added.

In the fabrication of CQDs, 5 ml or 10 ml of solution of 2-APBA was irradiated with a Ti:sapphire laser (Femtopower Compact Pro, Femto Lasers) using 30 fs full width at half maximum (FWHM) pulses at the central wavelength of 800 nm, and a repetition rate of 1 kHz. To achieve more precise pulse compression at the sample, a programmable acousto-optic filter (DAZZLER, Faslite) was used. A laser beam with a diameter of 6 mm, and a mean power of 170 mW was focused onto the liquid with a spherical convex 75 mm lens to achieve a focal spot size of about 15 pm and a peak power density of about 10 ¹⁵ W/cm ² . The position of the sample or the lens was adjusted so that the laser focal spot was approximately 2 cm below the surface of the liquid.

In order to monitor the fabrication process, a laser pointer l = 405 nm placed orthogonally to the direction of propagation of fs laser beam was used to illuminate the sample and the emitted fluorescence was collected by an optical fiber (with core is 1000 pm) to the spectrometer. BLK-CXR-SR-50, StellarNet Inc. spectrometer was used and the spectrum was displayed and stored on a computer. The optical fiber was place on the side of the cuvette perpendicular to both the laser pointer and fs excitation light beams. Its position was manually adjusted for maximum coupling of the emitted light.

To be able to also monitor the emissions due the interactions of the femtosecond (fs) laser pulses with the sample, two shutters with digital controllers were used to alternatively block the laser pointer and fs pulsed laser beam. During the capture of the linear fluorescence signal, the fs laser beam was blocked for 1.05 seconds using SHB1T - 01" Low-Reflectance Diaphragm Optical Beam Shutter with Controller obtained from Thorlabs. This shutter then opens for 5 minutes during which the laser (l = 405 nm) pointer is blocked by SH05 - Optical Beam Shutter, Thorlabs. This cycle constantly repeats during entire course of the CDs fabrications. Meanwhile, the StellarNet spectrometer was set to continuously record the spectra at integration time of 600 ms. When using the initial sample volume of 10 ml at concentration of 0.002M, the evolution of the monitored spectra of the laser light of wavelength 405 nm and the femtosecond pulsed light showed progressive increase in linear fluorescence (LFL) emitted at around 520 nm, two photon exited fluorescence (2PEF) which also appeared at 520 nm and second harmonic generation (SHG) with a peak at 400 nm. The intensity of each of the peaks after every 10 minutes for the data collected over a period of 18 h was analyzed and plotted as a function irradiation time. Notably, the LFL and SHG increased to maxima values after about 10 h of irradiation before the intensity of SHG sharply decreased. This was in contrast to the 2PEF which remained relatively constant toward the end of the irradiation process. The sharp decrease in the intensity of the SHG signal after attaining a maximum was attributed to the sensitivity of this process to structural changes which imply the formation of CQDs because the color of the solution also changed to pale yellow and green fluorescence intensity under excitation at 405 nm was at its maximum. We noted that continued irradiation of the sample after this optimum time lead to a decrease in fluorescence. The irradiation process was therefore stopped at this point and the time was noted as the optimum condition for the formation of CQDs for this sample. Similar approach was followed for different starting volume and different concentration of the sample. For instance, by reducing the volume from 10 ml to 5 ml and maintaining the same concentration and laser parameters resulted to optimum time of production of CQDs of 5 h instead of 10 h noted before. These conditions were used as reference for the subsequent sample preparation. For high concentrated sample (0.05M), the color change of the sample was very remarkable which was used together with the changes in the intensities of the monitored light signals to indicate when to stop the irradiation process as discussed above. The fabricated CDs were first purified by filtration using polycarbonate membrane filters (from Filter-Lab) having pore size of 0.1 pm. The sample was then centrifuged at 4000 rpm for 1 h. In order to remove small organic molecules, the sample was finally dialyzed for 24 h using 1 kDa molecular weight cut-off (MWCO) Pur-A-Lyzer mega tubes (from Sigma Aldrich). 2. Characterization of the CQDs of the invention

2.1. Confirmation that the obtained particles were quantum dots Thermionic transmission electron microscope (TEM) equipped with W JEOL JEM 1010 emitter with 0.4 nm resolutions was used to probe the sizes and intrinsic lattice spacing of the obtained particles. Prior to the TEM measurement, the sample was dispersed onto a carbon-coated copper-based TEM grid. In the isolated state, the average size of the CQDs as measured by TEM was 5-6 nm. High resolution TEM image in the inset of FIG. 1 (a) revealed lattice fringes with a spacing of 0.21 nm, attributed to the (100) lattice distance of the graphitic carbon.

2.2. Analysis of the composition of the CQDs of the invention Fourier Transform Infrared (FTIR) measurements were carried out at room temperature (about 22 °C) using a Bio-Rad FTS 6000 spectrometer from 700 cm ^-1 to 5000 ¹ cm with a resolution of 2 cm ^-1 .

The samples were prepared on Calcium Floride (CaF2) windows. First, about 10 mg of Barium Floride (BaF2) powder was placed on a clean CaF2 surface and mixed with about 200 pl_ of colloidal suspension of CQDs in water. The sample was then heated on a hot plate at 80 °C for five minutes to remove excess water. To completely remove the water content, the sample was further annealed at 100 °C in high vacuum (10 ^~6 mbar) for 24 h. The vacuum consisted of a custom-made vacuum annealing chamber with two-step pumping system that employs a turbo molecular pump (Pfeiffer Vacuum GmbH) and an ME2C NT oil-free diaphragm vacuum Pump (Thomas fisher). A wide range vacuum gauge was connected via four angle valves. The sample was placed in a chamber made of fused silica that was heated by an oven equipped with a temperature controller. After 24 h of annealing, the sample was taken for FTIR measurement. The results are shown in FIG. 2. (a). The IR peaks at around 3345 and 3193 cm ^-1 are attributed to the stretching vibrations of the H-bonded OH and NH moieties. The absorption peaks appearing at 1677, 1602, and the shoulder at 1176 cm ^-1 were due to C=N, C=C and C-N stretching vibrations, respectively. The boron containing species were detected at 1376, 1061 , and 950 cm ^-1 representing the IR vibrations of B-C, B-0 and B-OH groups, respectively.

From this, it could be concluded that the obtained CQDs contain p-conjugated bonds in the core structure and primary amide and the boronic acid groups of the precursor molecule at the surface of the carbogenic cores. The surface sensitive X-ray photoelectron spectroscopy (XPS) (K-ALPHA, Thermo Scientific) technique was used to probe the surface states of CQDs. All spectra were collected using Al-K- radiation (1486.6 eV), monochromatized by a twin crystal monochromator, yielding a focused X-ray spot (elliptical in shape with a major axis length of 400pm) at 3 mA ^c 12 kV. The alpha hemispherical analyser was operated in the constant energy mode with survey scan pass energies of 200 eV to measure the whole energy band and 50 eV in a narrow scan to selectively measure the particular elements. XPS data were analysed with Avantage software. A smart background function was used to approximate the experimental backgrounds and surface elemental composition were calculated from background-subtracted peak areas. Charge compensation was achieved with the system flood gun that provides low energy electrons and low energy argon ions from a single source. The colloidal suspension of CQDs was dropped cast and dried on to TEM grids for five times to ensure complete surface coverage. The XPS survey spectra (FIG. 2 (b)) indicate the presence of Oxygen (01s), Nitrogen (N1s), Carbon (C1s), and Boron (B1s), species at 531 , 398, 284, and 291 eV binding energies, respectively. More details about the chemical bonds of these species are shown by the high resolution XPS spectra (FIG. 2 (c-f)) where 01s is convoluted into two peaks at 533.5 and 531.8 eV representing O-B and O-C bonds, respectively. Similarly, N1s spectrum was convoluted to three peaks at 401.3, 398.8 and 299.9 eV which are assigned to Graphitic-N, Pyrolic-N and Pyridinic-N, with relative percentages of 0.05 %, 38 % and 56 % respectively.

Convoluted C1s spectrum (FIG. 2 (e)) show the presence of C-C, C=C and C-B bonds at around 284.6 eV, C-N bond at 286 eV and C-O-B bond at 288.6 eV. Lastly, FIG. 2 (f) shows the presence of B-C and B-0 bonds at 190.0 and 192.8 eV, respectively.

These XPS results corroborate the observation from the FTIR discussed above and confirm the CQDs are functionalized with boronic acid species that are responsible for glucose sensing. In addition, it could also be concluded that most of the Nitrogen species are located at the outer edge of the CQDs. This could explain the high fluorescence quantum yields characterizing the CQDs of the invention.

2.3. Fluorescent properties of the CQDs of the invention

2.3.1. Measurement of quantum yield (FU)

The FU measurement was done using a comparative method. This method involves measurement of absorption and fluorescence of the sample and a reference material under the same conditions. Thus, freshly prepared stock solution containing 8 mg/ml of reference quinine sulfate in a 0.10 M H ₂ SO ₄ solution was used as reference material. The following steps were followed:

(i) The concentration of quinine sulphate in 0.10 M H ₂ SO ₄ (QS) stock solution was dilute to 4 mg/ml.

(ii) Cuvettes having optical path length of 10 mm were cleaned using acetone and rinsed with Milli-Q water in ultrasonic bath and dried with a jet of dry nitrogen gas.

(iii) Absorbance and fluorescence of the clean empty cuvette were measured and used as baseline.

(iv) Cuvette was then filled with QS and its absorption spectrum as a function wavelength was obtained. When necessary, the QS reference sample was further diluted so that the absorption intensity at a wavelength of 320 nm was about 0.01. After waiting for a bout 5- 10 min, the absorption spectrum was re-measured to be sure that it remained constant over time.

(v) The fluorescence spectrum of the same QS sample in the same cuvette was taken at excitation wavelength of 320 nm.

(vi) A small drop of the QS solution in (i) above was then added and mixed thoroughly before the next absorption and fluorescence spectra were measured.

(vii) Step (vi) above was repeated to obtain seven different measurement of absorption and fluorescence while ensuring the absorption intensity at the wavelength of 320 nm remained below 0.1.

(vii) Steps (ii) to (vii) were repeated for our CQDs samples.

In order to determine the FU, integrated fluorescence intensities of the standard QS sample and CQDs were plotted against their corresponding absorbance. The FU is calculated from the slopes of these plots. The equation used for calculation is as follows:

where m is the slope of integrated fluorescence intensity against absorbance, n is the refractive index, and subscript S and R indicates the sample and reference quinine sulfate solution. The FU of the reference standard (FUr) is 0.59, and the refractive index of water (solvent) and 0.10 M H2SO4 solution is 1.33. From this analysis, the values of ms and A7?R were 1.0 x 10 ⁶ and 9.38 x 10 ⁵ respectively, which resulted to FU of 63 % for the CQDs of the invention. This high FU especially for boronic acid base CQDs is very useful for sensing

applications. This is because of improved sensitivity due to high signal to noise ratio and the fact the CQDs are stable against photoblinking and photobleaching effects. 2.3.2. Measurement of the other optical properties of the CQDs of the invention

The optical properties of the CQDs in an aqueous dispersion were investigated by UV-vis absorption spectroscopy (Cary (Varian) 500 Scan UV-Vis Spectrometer) at wavelength between 200-700 nm. The spectrum labeled Abs1 in Fig 3 is obtained from undiluted sample and Abs 2 is from 1000 fold diluted sample. Fluorescence spectroscopy was done by (Cary (Varian) Eclipse Fluorescence spectrometer.

The absorption spectra (as function wavelength) of the CQDs dispersed in water is shown in FIG. 3. For a higher concentrated sample, the spectra labeled (Abs 1) in Fig 3 displayed peaks at around 220, 250, 295, and 340 nm and a shoulder at 400 nm. After dilution of the sample the peaks at 220 and 250 merged into a single strong absorption peak at around 235 nm (dotted line, spectrum labeled Abs 2). The low wavelength peaks (220-295 nm) could be attributed to the p-p* transition of the conjugated C=C, and C=N bonds of the carbon core and the peak at 340 nm including a shoulder at 400 nm could be due to the h-p* transition of the C-N/C-0 functional groups on the carbon dots.

The Fluorescence excitation spectrum (open circular symbols labeled Em) was recorded within a wavelength of 200-500 nm and showed two excitation peaks at 235 and 333 nm which merged with the absorption peaks within the experimental uncertainty. The fluorescence emission spectra for the two excitation wavelengths show strong emission centered at around 430 nm and a shoulder at 500 nm.

Detailed fluorescence spectra (FIG. 4) under different excitation wavelengths revealed that the obtained CQDs have four distinct emission windows at around 400, 430, 510 and 760 where the fluorescence maxima were independent to the excitation wavelength. The emissions with the highest quantum yield was in the blue region, i.e. 400 and 430 nm. These measurements were taken at the voltage of 430 mV. The intensity of the peaks at higher wavelengths are weak. Nevertheless, strong emissions even in the near infra-red region were achieved by simply adjusting the voltage of the excitation light. For instance, by changing the voltage to 580 mV, emissions at around 510 nm reached the same level of intensities as the blue emissions at around 400. Similar intensities of the fluorescence spectra in the near infra-red (IR) region was recorded at voltage of 700 mV. In terms of the excitation wavelength, the near IR emission at 760 nm was a complete parallel of the emission spectra at around 400 nm (where excitation is in the deep UV region) implying that they were of the same origin. However, to have a single main peak around 510 required excitation wavelengths of 400 nm and above. The red shift of the emission peaks was attributed to the excited state inter/intramolecular charge transfer between the emissive species.

Abroad emission spectrum at excitation wavelength of 380 nm at voltage of 580 mV was due to double emission at nearly equal intensities at the blue and green region.

In summary, the absorption and fluorescence results showed that the obtained CQDs had useful unique optical properties. On one hand, four emission windows allow for imaging and sensing at different wavelengths especially the near IR region which is very attractive for imaging biological samples due to deep tissue penetration and reduced scattering of the light within this region. The emission maximum at 760 nm with possible excitation wavelength of 220 nm represent the largest stokes shift (540 nm) ever reported between the emission and excitation wavelengths. Fluorescence materials with large stoke shifts are very important for many applications because of the possibility to achieve high signal to noise ratio. For example such materials are applied in super-resolution microscopy imaging utilizing stimulated emission depletion (STED) technique to overcome the diffraction limit. Furthermore, the broad emission with excitation at 380 nm implies that the obtained CQDs can find applications in white light emitting diodes.

2.3.3. Photostability

The stability of the prepared CQDs against photobleaching was investigated by continuous illumination of the sample by a laser pointer having a power of 5 mW (l = 405 nm) for over 15 h while collecting fluorescence signal by use of an optical fiber placed perpendicular to direction of excitation light and connected to StellarNet Inc. spectrometer. The spectra were collected at interval of 5 min and stored on a computer. The intensity of each spectra was then analyzed and plotted as a function of time. The results are shown in FIG. 5 which shows negligible loss of fluorescence intensity of merely 10 % during the entire period of laser (l = 405 nm) illumination. This confers the obtained CQDs with high stability for imaging and sensing applications. 3. Formulation of the CQDs of the invention in silica sheets

The nanoporous silica (pSi) membranes were prepared by the electrochemical etching of highly doped (0.005 W cm) p-type <100> oriented mono-crystalline silicon wafers in a home built anodization cell. The electrolyte contained hydrofluoric acid (HF-48%) and ethanol (C ₂ H ₅ 0H-99%) purchased from Sigma-Aldrich and mixed in the ratio of 1 :1.

Current densities (j) in the range of 20-120 mA cm ^-2 were applied to obtain pore diameters between 4 and 10 nm with porosity varying from 9% to 23%. In each case, the etching time was adjusted to maintain the thickness of the pSi at around 50 pm. As the final electro-polishing step (j=700 mA cm ^-2 ) was applied for 3-4 s to lift the pSi from the Si substrate.

The obtained pSi membranes were subsequently oxidized thermally in an oven (Vulcan 3- 550, Neytech) at 1100 K (heating rate 3 K/min) for 6 h to form completely transparent and insulating nanoporous silica (pSiC>2) with unidirectional pores.

The CQDs could be incorporated into porous silica sheets in two ways: First during in situ fabrication and secondly, by soaking the pSiC>2 in solution containing already prepared CQDs for at least 12 h.

In the first case, the porous silica sheet was supported by a glass slide and placed at the top of the surface of the precursor liquid such that the focus of the laser spot was in the liquid just below the lower surface of the porous silica sheet. In this way, the coulomb explosion from the pulsed laser causes infiltration of the formed CQDs directly into the porous silica sheets. The whole sample setup could be placed on a translational table or magnetic stirrer for gentle shaking.

Alternatively, the silica sheets could be placed at the bottom of a cuvette/ container filled with the precursor solution for the last 2-3 h of the laser irradiation. This approach enabled immediate infiltration of the CQDs in the nanopores before any aggregation took place.

The presence of amino groups on the surface of the obtained CQDs (as confirmed by FTIR in the above discussion) also facilitates anchoring of the CQDs on silica surface through H-bonding with the silanol groups. It is therefore inferred that the CQDs can be located inside the nanoporous and/ or on the surface of silica sheets. This composite material in this invention is referred to as carbon quantum dots doped porous silica (CQDs-pSi0 ₂ ).

To confirm the presence of CQDs fluorescence measurement CQDs-pSi0 ₂ was carried out using the Cary (Varian) Eclipse Fluorescence spectrometer in the reflection mode.

This measurement was done using excitation wavelength of 320 nm. It resulted to fluorescence emission peaked at around 400 nm (FIG. 6) which is similar to emission from the colloidal CQDs. Embodying the CQDs in or onto solid support while preserving their fluorescence properties is more advantageous than the colloidal suspension due to their ease of handling and the possibility to reduce contaminations. 4.1. CQDs-pSi02 of the invention as optical limiting material

The optical limiting property of the obtained CQDs-pSiC>2 was investigated using the open Z-scan technique. This involves recording the power that is transmitted through the sample as it is moved across the focal spot along the direction of the beam propagation. This measurement was done using femtosecond pulsed laser beam with wavelength in the near infra-red region (l = 800 nm) and repetition rate of 1 kHz. A converging lens of focal length 75 mm was used to focus the light beam. Another converging lens of focal length 200 mm was placed in front a power meter (Gentec-eo, Tuner) to ensure that all the light that is transmitted through the sample strike the sensor of the power meter.

The CQDs-pSiC>2 sample was mount on a support and fixed on to XR25C/M-25 mm travel linear translation stage (Thorlabs). The position of the sample was initially adjusted to about 5 mm from the focal spot of the laser beam and power transmitted through the sample was recorded. The sample was then manually moved across the focal sport in varying steps of 0.2, 0.1 and 0.05 mm with the aid of the translational stage. Small steps were used closer to the focus. The transmitted power was recorded at each step and the normalize transmission was plotted as a function of position Z of the sample from the focal spot. This was repeated for different input power, given in term of fluence (pJ/crrr ² ) in Fig 7. The resulting data is shown in Fig.7 indicate that the obtained CQDs-pSiC>2 had optical limiting property since peak absorption increases with increasing laser intensity.

Optical limiting occurs when the power of high intense laser beam (for instance pulsed lasers) decreases upon interaction with a material that has non-linear optical properties such as multiphoton absorption, non-linear scattering and reflection. Optical limiting materials are in high demand because of their use in protecting human eyes and optical sensors from damages that can be incurred when exposed to high intense laser beams.

4.2. Use as sensor

4.2.1. CQDs of the invention as glucose sensor

The prepared CQDs in the solid state (CQDs-PSi0 ₂ ) were used for glucose sensing. First the fluorescence of CQDs-PSi0 ₂ was recorded using the Cary Eclipse spectrometer in order to obtain the baseline. The excitation wavelength was 320 nm. Thereafter, 10 pL of specific glucose concentration in water was dropped onto the CDS-PS1O2 without dismounting it from the spectrometer sample holder. Fluorescence quenching was then monitored until there was no further change. The final fluorescence intensity was subtracted from the baseline.

This was done for difference concentration of glucose to obtain the calibration line shown in FIG. 8. It was found low a glucose detection limit of 1.0 mg/dl with wide linear range of up to 100 mg/dl.

Citation List

Non Patent Literature

Xianfeng Zhang et al. ,“Facile synthesis of boronic acid-decorated carbon nanodots as optical nanoprobes for glycoprotein sensing”, Analyst, 2019,144, 1975-1981 ;

Kiran S. et al.,“Mechanism of intracellular detection of glucose through nonenzymatic and boronic acid functionalized carbon dots”, J Biomed Mater Res A., 2015, 103(9), 2888- 2897;

Angenendt P, Drug Discovery Today, 2005, vol. 10(7), p. 503; and Wacker, R. et al.“Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidinbiotin attachment: a comparative study”.

Anal. Biochem. 2004, vol. 330, p. 281-287.

For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

1. A boronic acid functionalized carbon quantum dot which is stable against

photobleaching radiation and has a fluorescence quantum yield of at least 15%, wherein: (a)“stable against photobleaching” means that the fluorescence intensity of the carbon dot after continuously irradiating it with laser for a period of time of at least one hour, is the same or corresponds to at least 85% of the fluorescence intensity of the quantum dot prior to irradiation; and (b) the fluorescence quantum yield is determined by the formula: wherein:

m is the slope of integrated fluorescence intensity against absorbance at a wavelength from 200-450 nm;

n is the refractive index;

subscript S and R indicates the test sample and reference quinine sulfate solution, respectively;

QYR is 0.59; and

ns/n _R is equal to 1.

2. The boronic acid functionalized carbon quantum dot of clause 1 , which has a fluorescence quantum yield comprised from 40 to 80% or from 55 to 65%.

3. The boronic acid functionalized carbon dot of any one of the clauses 1-2, which is an optical limiter.

4. The boronic acid functionalized carbon dot of any one of the clauses 1-3, which has UV/vis absorption peaks at 220, 250, 294, and 343 nm at 200-700 nm; and, alternatively, one or more of the following features: (a) four fluorescence emission peaks at around 400 nm, 430 nm, 510 nm and at about 750-800 nm when excited between 200-330, 340-380, 390-480, and 200-370, respectively; and (b) a stoke shift from 500-600 nm, this parameter being calculated as the difference between the maximum fluorescence emission wavelength and the minimum excitation wavelength. 5. A process for obtaining a boronic acid functionalized carbon dot as defined in any one of the clauses 1 to 4, the process comprising the step of subjecting an arylboronic acid solution to a pulsed laser radiation which generates a filamentation in the liquid or breakdown the arylboronic acid, wherein: “arylboronic acid” is understood as an aryl-(B(OH)2)n,

“aryl” represents a known ring system with 1-3 rings which comprises from 5 to 14 members, the rings being saturated, partially unsaturated, or aromatic, provided that at least one of the rings is an aromatic ring; and being fused, partially fused or isolated; each one of the members forming the known ring system being selected from the group consisting of: -CH-, -CH2-, -NH-, -N-, -SH-, -S-, and -0-; and the ring system being optionally substituted by one or more radicals independently selected from the group consisting of (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-C ₆ )alkyl-0-, nitro, cyano, -NR1R2, and halogen; n is an integer value from 1 to 3; and

Ri and R2 are the same or different and are selected from -H, (Ci-Ce)alkyl optionally substituted by -OH, halogen, C=0, and nitro.

6. The process of clause 5, wherein the arylboronic acid is a ring system consisting of one aromatic ring having 5 or 6 members. 7. The process of any one of the clauses 5-6, wherein the arylboronic acid consists of one aromatic ring having 6 members selected from -CH-, -CH2-, -N-, and -NH-, wherein one of the members is substituted by one boronic acid, such as is a 2-aminopyrimidine-5-boronic acid. 8. The process of any one of the clauses 5-7, wherein the pulse laser radiation is a femtosecond pulsed laser radiation.

9. The process of any one of the clauses 5-8, wherein the pulse laser radiation corresponds to pulses from 10 to 150 femtoseconds full width at half maximum (FWHM) with a central wavelength from 700-900 nm, at a repetition rate from 0.5 to 1.5 kHz.

10. An article comprising the boronic acid carbon dot as defined in any one of the clauses 1-4. 11. The use of the boronic acid carbon quantum dot as defined in any one of the clauses 1-4 or the article as defined in clause 10 as sensor.

12. The use of the boronic acid carbon quantum dot as defined in any one of the clauses 1-4 or the article as defined in clause 10 as optical limiter.

13. A method for determining the presence or concentration of a target analyte in a test sample, the method comprising (a) contacting the sample with the carbon quantum dot of any one of the clauses 1 to 4; (b) exposing the sample and the carbon quantum dots to light energy that causes the carbon quantum dots to emit photons; and (c) detecting a change in fluorescence intensity, whereby said change indicates the presence or amount of the target analyte.

14. The method of clause 13, wherein the target analyte is a monosaccharide, preferably glucose. 15. The method of any one of the clauses 13-14, wherein the test sample is a biological sample.