WO2023007298A1

WO2023007298A1 - Fluorescence-based biosensor for sars-cov-2 detection and quantification

Info

Publication number: WO2023007298A1
Application number: PCT/IB2022/056509
Authority: WO
Inventors: Abel José ASSUNÇÃO DUARTE; Helena Maria RODRIGUES GONÇALVES; Paula Filomena Martins Lopes
Original assignee: Universidade De Trás-Os-Montes E Alto Douro; Requimte – Rede De Química E Tecnologia; Instituto Superior Engenharia Do Porto
Priority date: 2021-07-30
Filing date: 2022-07-14
Publication date: 2023-02-02

Abstract

The present disclosure relates to a method for detection of biomolecules comprising the steps of collecting a biological sample comprising an analyte at a concentration of at least 0.01 pM; exposing the collected sample to a temperature ranging from 70-100 °C; incubating the sample with Cdots previously incubated with the probe, therefore obtaining a solution; carefully homogenising the resulting solution; subjecting the solution to an excitation source for acquisition of fluorescence; determining the fluorescence shift between a negative control and the sample; wherein the Cdots are in a suspension state and the proportion of Cdots for analyte in the sample are in a ratio of at least 1:0.5; and wherein a shift of fluorescence from the negative control to the sample indicates the presence of the biomolecule. The present disclosure also relates to an apparatus for carrying out the process according to the present invention comprising a sensing unit (A), connected by an optical fiber (B) to a sample holder (C), and a LED (D) and a kit for detection of biomolecules, in particular for diagnosis of SARS-CoV-2.

Description

DESCRIPTION

FLUORESCENCE-BASED BIOSENSOR FOR SARS-CoV-2 DETECTION AND

QUANTIFICATION

Technical Field

[0001] The present disclosure relates to the field of Molecular Biology, in particular to a fluorescence-based technology for detection and quantification of SARS-CoV-2 virus using carbon-based nanoparticles commonly known as carbon dots (Cdots) in the suspension, non-functionalized state.

Background

[0002] The use of nanoparticles in the development of highly sensitive and specific biosensors has been evolving over the years. These appealing biosensors found applications in numerous areas, namely environment, food safety, bioimaging, health, among others. The use of nanoparticles in fluorescence biosensing is particularly attractive due to their superior stability over the traditional organic fluorophores. The use of the Quantum Dots family in the development of new biosensing systems has been gathering much attention. The main problem found in the use of these nanoparticles is the fact that they are cytotoxic and even when their surface is masked with biocompatible molecules they still induce high rates of immunologic responses. An alternative has been found in 2004 with the discovery of Carbon Dots (Cdots). This class of nanoparticles is carbon-based, presenting all the good optoelectronic properties of the traditional metal-based Quantum Dots, with the main advantage of the inherent non-toxicity. The versatility that these nanoparticles offer, particularly the fact that their surface can be adjusted in order to develop a highly specific sensor has been the ground- base that has boosted the use of Cdots. In the past they have been successfully applied for the detection of chemical species, namely mercury (II) ion, iodine, Reactive Oxygen Species, among others. Additionally, they have been proved useful for pH sensing and bioimaging applications. The interesting work developed around these nanoparticles have gave rise not only to excellent academic publications but also interesting patents. The Cdots application in a raw state for bio-detection is a recent line of investigation that has already gave rise to a patent request.

[0003] 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. 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.

[0004] Nowadays there are several synthetic pathways for producing these nanoparticles. 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.

[0005] In the end of 2019, the first human case of SARS-CoV-2 infection was reported that led to the development of the new COVID19 disease. Ever since its appearance it has been widely spread worldwide with devastating effects. The high infection rate and the fact that not all people infected have visible symptoms lead to world chaos. In order to attempt to control the dissemination of SARS-CoV-2, the methodology has been to test, detect and isolate people. However, the only test available in the market for this virus was quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). This technique requires specialized personnel and technical knowledges that are not widely available for all, leading to a limited testing capacity which is more pronounced in countries with fragile economies. In this sense there was a pressing need to develop innovative methods for SARS-CoV-2 detection. Currently these methods can be grouped in (i) nucleic acid testing and (ii) antibody testing.

[0006] The nucleic acid testing differs in the approach and the nucleic acid targets used. According to the literature there are: the traditional qRT-PCR that extracts RNA from a respiratory swab. A simplification of this method suggests heating the swab transport media before RNA extraction. Another interesting alternative that is both cost-effective and presents a simplification of the traditional PCR methods is the SalivaDirect^®. This method differs from the rest by the use of saliva, instead of the invasive respiratory swabs; it does not require nucleic acids preservatives as it replaces them by the addition of proteinase K and a heat pre-treatment step. Nonetheless they still use a dualplex quantitative reverse transcription PCR (RT-qPCR) assay for the final determination of SARS-COV2. An alternative method has been proposed by Lamb et al., 2020, that requires the Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) technique.

[0007] The antibody tests available use the IgM and /or IgG antibodies for SARS-CoV-2 quantitative detection in a given serum, plasma (EDTA, citrate) or venipuncture whole blood specimen. There are some currently available: the lateral flow test strip (LFTS) or lateral flow immunoassay (LFIA) has been widely used for this purpose. A chemiluminescence-immunoassay for the detection of SARS-CoV-2 infections and surveillance of changing antibody patterns based on the recombinant nucleocapsid antigen and magnetic beads; a colloidal gold-based immunochromatographic (ICG) strip test detecting viral IgM or IgG. In these antibodies' tests, there have been descriptions of methods that rely on the use of: colloidal gold nanoparticles, due to their colorimetric visualization, latex nanoparticles, fluorophores, among others. These tests are not available for home testing yet; however, they can be performed by laboratories and healthcare workers at a point-of-care. Nonetheless they are useful tools for intermediate or late stages of infection. Our technology relies on the use of carbon dots and nucleic acids for SARS-CoV-2 detection and quantification using a fluorescence- based method. [0008] Nowadays there are some nucleic acids sensors available; nevertheless, 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 it is possible to use gold and magnetic nanoparticles coupled with fluorescent dyes for nucleic acid sensing. 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 or indirect detection. 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.

[0009] 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, biomolecules, nucleic acids, proteins, among others. The use of nanomaterials can be truthfully considered widely spread. Their high surface-to-volume ratio and their tuneable surface makes nanoparticles efficient starting materials for the development of new sensing platforms.

[0010] 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; 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. Certainly, their non-toxic nature, 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.

[0011] Document US10962529 discloses a method for detecting a SARS-CoV-2 protease in a biological sample is provided. The method includes contacting the biological sample with a fluorescent probe-based sensor, wherein the sensor comprises an L-Histidine-D- aspartic acid peptide substrate, a fluorophore, and a quencher molecule; and detecting the SARS-CoV-2 protease when an increase in fluorescence is observed. The present invention differs from this disclosure by providing a simpler and faster technology, wherein there is no need of a quencher nor a fluorophore.

[0012] Document US2016168615 discloses compositions and methods for detecting pathogens. In one aspect, a method for detecting a pathogen on or within an object comprises: providing a detection agent configured to generate a visible indication when exposed to the pathogen; contacting the detection agent with the object; and visually detecting the presence or absence of the visible indication, wherein the presence of the visible indication indicates that the pathogen is present on or within the object.

[0013] W02020/089810 (WO^'810) discloses 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 of the present invention is performed upon a direct interaction between the Cdots and the nucleic acids and proteins. [0014] The present invention differs from WO^'810 essentially because a sample pre heating step is used, comprising the use of an inactivation virus solution, allowing the virus RNA exposure that is directly used in the detection process.

[0015] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

General Description

[0016] The present disclosure presents a method for identifying biomolecules, namely SARS-CoV-2 biomolecules.

[0017] The present method relates to a fluorescence detection technology for nucleic acids, proteins and other relevant biomolecules for direct virus detection using non- functionalized Cdots.

[0018] The expression "direct virus detection" shall be interpreted as a virus detection which is possible without the need of performing a DNA/RNA extraction from a sample step before the detection method. It shall also be understood that the present method is not performed directly on the human or animal body.

[0019] In an embodiment, the method comprises the following steps:

The analyte concentration is of at least 0.01 fM.

The solution containing the analyte passes through a series of pre-steps, including the following:

(i) exposition to temperature ranging from 70-lOO^C;

(ii) optionally, the temperature exposition is performed using a single temperature or a ramp;

(iii) optionally, the temperature exposition is performed over time at least for 5s. Upon these pre-steps the analyte solution is placed in contact with the raw Cdots. The raw Cdots are on a suspension state and the proportion of Cdots for analyte is at least 1:0.5 ratio.

The raw Cdots solution and analyte solution is carefully homogenized, e.g. by manually inversion, slowly and left in contact fora period of time of at least one min before fluorescence acquisition. Fluorescence acquisition of the overall solution of label-free Cdots with the sample analyte.

Determination of the fluorescence shift between the control and sample.

[0020] In one embodiment, the method further comprises a pre-method step consisting on the application of temperature to the analyte solution before contact with the Cdots.

[0021] For the purposes of the present invention an analyte is to be considered as a substance whose chemical constituents are being identified and measured, particularly biomolecules, preferably nucleic acids.

[0022] In one embodiment the Cdots are in a suspension state with a concentration of at least 0.01 g/L.

[0023] In an embodiment, the size of the Cdots is from 1 to 100 nm and its shape is spherical.

[0024] In yet another embodiment, the fluorescence is acquired using an excitation and emission wavelengths ranging from 400 to 850 nm, and an integration time of at least 1 ms.

[0025] In another embodiment, the analyte solution and the Cdots solution are left to interact for at least one minute.

[0026] In another embodiment the present invention also discloses a sensor to carryout the method of the present invention. This sensor comprises means for time and/or temperature control, a heating chamber, optical fibers, LEDs and a spectrometer.

[0027] In a preferred embodiment the present disclosure relates to a method for detection of biomolecules comprising the following steps: collecting a biological sample comprising an analyte at a concentration of at least 0.01 pM; exposing the collected sample to a temperature ranging from 70-100 °C; incubating the sample with Cdots previously incubated with the probe, therefore obtaining a solution; carefully homogenising the resulting solution; subjecting the solution to an excitation source for acquisition of fluorescence; determining the fluorescence shift between a negative control and the sample; wherein the Cdots are in a suspension state and the proportion of Cdots for analyte in the sample are in a ratio of at least 1:0.5; and wherein a shift of fluorescence from the negative control to the sample indicates the presence of the biomolecule.

[0028] In a further embodiment the present disclosure relates to a method for detection of biomolecules, wherein the Cdots are in a suspension state at a concentration of at least 0.01 g/L.

[0029] In a further embodiment the present disclosure relates to a method for detection of biomolecules, wherein the biomolecules are nucleic acids or proteins.

[0030] In a further embodiment the present disclosure relates to a method for detection of biomolecules, wherein the biomolecule is a fragment of a gene or protein of SARS- CoV-2.

[0031] In a further embodiment the present disclosure relates to a method for detection of biomolecules, wherein the temperature exposition is performed using a single temperature or a temperature ramp, either one for at least five seconds.

[0032] In a further embodiment the present disclosure relates to a method for detection of biomolecules, wherein homogenization is carried out and left for at least one minute.

[0033] In a further embodiment the present disclosure relates to a method for detection of biomolecules, wherein the excitation source is a LED and a photodetector with a band pass optical filter.

[0034] In a further embodiment the present disclosure relates to a method for detection of biomolecules, wherein acquisition of fluorescence is carried out by excitation and emission wavelengths ranging from 400 to 850 nm, and an integration time of at least 1 ms.

[0035] In a particular embodiment, the present invention relates to an apparatus for carrying out the process according to the present invention comprising a sensing unit (A), connected by an optical fiber (B) to a sample holder (C), and a LED (D). [0036] In a further embodiment, the present invention relates to an apparatus for carrying out the process according to the present invention comprising a heating unit with means for controlling both temperature and time.

[0037] In a further embodiment, the present disclosure relates to a kit for detection of biomolecules according to the present invention, preferably for detecting viruses.

[0038] In a further embodiment, the present disclosure relates to a kit for detection of SARS-CoV-2 according to the present invention.

[0039] In a further embodiment, the present disclosure relates to a kit for detection of SARS-CoV-2 comprising Cdots previously incubated with at least one SARS-CoV-2 probe.

Brief Description of the Drawings

[0040] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0041] Figure 1 illustrates the fluorescence emission measures of Cdots as a biosensing system after incubation with: A) Cdots prior to the incubation with a concentration of 0.01 g/L; B) Cdots + SARS-CoV-2 ssDNA sequence Orfab (probe specific of Spike gene); C) Cdots + SARS-CoV-2 ssDNA sequence Orfab + negative control (PCR-proved negative test used here as negative control); D) Cdots + SARS-CoV-2 ssDNA sequence Orfab + positive control (PCR-proved positive test used here as positive control). The fluorescence emission profile of the Cdots previous to the incubation with a specific SARS-CoV-2 probe (spectrum A). When the Cdots are incubated with a specific SARS- CoV-2 probe, in this particular case Orfab (a sequence that is present in the spike protein of the virus), the fluorescence intensity increases (Spectrum B). The Cdots that already have been incubated with a SARS-CoV-2 probe when in contact with a sequence that does not match the probe is followed by a decrease in the fluorescence intensity to a value that is closer to the Cdots emission profile before the incubation step. This is considered a negative result (Spectrum C). On the other hand, when in the presence of the complementary sequence of the probe, the emission profile of the incubated Cdots decreases to a value higher than the control. This corresponds to a positive control (Spectrum D). [0042] Figure 2 illustrates the portable configuration example for the biosensing system. This innovative sensing solution relies on the fluorescence measurements. In this sense it was necessary to adjust the configuration of the biosensor to the specific particularities of the SARS-CoV-2 biosensing, by introducing a pre-heating chamber. In Figure 2 is described a possible configuration of the biosensor that does not intent to be exhaustive or restrictive. Figure 2 shows two schematics, the top view of the biosensor (1) and the inside view (2) where it is possible to observe the components. The biosensor is composed by sensing unit (A), connected by an optical fiber (B) to a sample holder (C), that is irradiated by an LED (D) with an adequate wavelength that can be adjusted according to specific excitation needs. The biosensor also comprises a heating unit I that has a temperature and time control. The positions of the different components are here presented in one of the possible configurations. The roman letters i-vi, represent typical distances which are only here indicated as suggestions, it shall not be regarded as restricting the scope of the invention (i - 4 cm; ii - 6 cm; iii - 7 cm; iv - B cm; v- 20 cm; vi - 10 cm).

[0043] Figure 3 - The sensitivity of the biosensor to detect the sequences Orfab, E and N was tested. The Cdots solution was incubated with each probe sequence for a period ranging from 10 min to 1 h. Upon this time the incubated solution was placed in contact with a complementary synthetic sequence for each probe. Additionally, real samples were also tested, provided by the UTAD COVID testing center. The samples were previously tested by RT-PCR and the result was known to us before using them in the biosensor platform. In this sense a negative and a positive sample was selected for testing. The data obtained are depicted in this figure and prove that using the biosensor allows the achievement of a positive or negative result that is in agreement with the one obtained using RT-PCR that is the standard method for these samples.

[0044] Figure 4 - In order to test the sensitivity of the biosensor, a sample was chosen based on the high CT (Cycle Threshold) value obtained in RT-PCR (Reverse Transcriptase Real Time PCR). This is an indication of low virus content. This sample was tested with the biosensor and a positive result was obtained, which is depicted in the figure, as expected by comparison with the RT-PCR data (not shown). Detailed Description

[0045] 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 SARS-CoV- 2 nucleic acids 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 SARS-CoV-2.

[0046] This label-free technology provides an ultrasensitive, reusable, rapid and low- cost solution for the detection and quantification of SARS-CoV-2 nucleic acids and/or proteins that can be applied in all biological samples, regardless of the specie.

[0047] The technology described herein is significantly different from what is observed nowadays in the sense that:

It does not require PCR for the virus detection;

It does not require specialized personnel for performing the test (considering extraction to analysis, it can be performed by a non-specialized individual);

It does not require RNA extraction steps;

It does not require RNA reverse transcription amplification steps;

It does not require PCR amplification steps;

It uses raw Cdots which are incubated with SARS-CoV-2 probes specific for SARS- CoV-2 and can be adjusted as mutations occur;

It can be used for the infection detection in early stages;

It can be used to detect and quantify the viral charge in one analysis;

Cdots can be produced by low-cost methods and do not require a costly equipment or preparation apparatus for sensing studies;

[0048] The method of the present invention presents the surprising technical effect of being extremely time efficient when compared with the state-of-the-art. The time required on the detection and quantification of the nucleic acids or proteins is only limited by the incubation time of the raw Cdots solution with the SARS-CoV-2 probes, of at least 5 minutes. To overcome this limitation the present invention also discloses a kit, where the raw Cdots where previously incubated with SARS-CoV-2 probes therefore achieving a diagnostic in less than one minute. The time from collecting the sample from the individual until obtaining a result, considering that the test is performed simultaneously using three different SARS-CoV-2 specific probes (e.g. Orfab, N and E genes) is less than 10 min.

Examples

Example 1

[0049] In a first example the sensitivity of the biosensor to detect the sequences Orfab, E and N was tested. The Cdots solution was incubated with each probe sequence for a period ranging from 10 min to 1 h. Upon this time the incubated solution was placed in contact with a complementary synthetic sequence for each probe. Additionally, real samples were also tested, provided by the UTAD COVID testing center. The samples were previously tested by RT-PCR and the result was known to us before using them in the biosensor platform. In this sense a negative and a positive sample was selected for testing. The data obtained (Figure 3) prove that using the biosensor allows the achievement of a positive or negative result that is in agreement with the one obtained using RT-PCR that is the standard method for these samples.

Example 2

[0050] In another example, in order to test the sensitivity of the biosensor, a sample was chosen based on the high CT (Cycle Threshold) value obtained in RT-PCR (Reverse Transcriptase Real Time PCR). This is an indication of low virus content. This sample was tested with the biosensor and a positive result (Figure 4) was obtained, as expected by comparison with the RT-PCR data.

[0051] Based on the previously exposed, the present technology is sufficiently different and presents several main advantages over the existing technologies, namely the fact that it does not need previous steps of extraction, reverse transcription nor amplification of the biologic sample, it is a direct detection method, not using intermediate entities such as quenchers or fluorophores, it is a very quick detection method, it only takes 5-10 minutes to detect the three sequences used in the RT-PCR from collecting the sample until identifying the result and can be applied in situ, due to the fact that the sensor is, in an embodiment, portable and can be operated by non health professionals. Example 3

Lot-to-Lot Reproducibility studies of SARS-CoV-2 detection kit

To demonstrate lot-to-lot reproducibility of: the SARS-CoV-2 detection kit, three lots were tested using three or four positive samples and one negative sample. For each lot the samples were tested in 12 replicates in one assay run.

Table 1. Mean and CV (Coefficient of variation) for each positive and negative sample for each of the three lots used.

IB 2. Analytical limits

The Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantitation (LoQ) for SARS-CoV-2 detection kit was calculated. In order to do so, two lots of each component were tested with 5 negative samples and 5 low positive samples with 5 replicates in one run each day during 3 days.

Table 2. LoB, LoD and LoQ determinations

Table 3. Estimated CV(%)(Classical approx.) and Full precision (Precision Profile)

Table 4. Values of Min, max and quotient for the positive samples considering the three lots used.

[0052] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0053] The above-described embodiments are combinable.

[0054] The following claims further set out particular embodiments of the disclosure.

Claims

1. Method for direct detection of biomolecules comprising the following steps: collecting a biological sample comprising an analyte at a concentration of at least 0.01 pM; exposing the collected sample to a temperature ranging from 70-100 °C; incubating the sample with Cdots previously incubated with the probe, therefore obtaining a solution; carefully homogenising the resulting solution; subjecting the solution to an excitation source for acquisition of fluorescence; determining the fluorescence shift between a negative control and the sample; wherein the Cdots are in a suspension state and the proportion of Cdots for analyte in the sample are in a ratio of at least 1:0.5, respectively; and wherein a shift of fluorescence from the negative control to the sample indicates the presence of the biomolecule.

2. Method according to the previous claim, wherein the Cdots are in a suspension state at a concentration of at least 0.01 g/L.

3. Method according to any of the previous claims, wherein the biomolecules are nucleic acids or proteins.

4. Method according to any of the previous claims, wherein the biomolecule is a fragment of a gene or protein of SARS-CoV-2.

5. Method according to any of the previous claims, wherein the temperature exposition is performed using a single temperature or a temperature ramp, either one for at least five seconds.

6. Method according to any of the previous claims, wherein homogenization is carried out and left for at least one minute.

7. Method according to any of the previous claims, wherein the excitation source is a LED and a photodetector with a band pass optical filter.

8. Method according to any of the previous claims, wherein acquisition of fluorescence is carried out by excitation and emission wavelengths ranging from 400 to 850 nm, and an integration time of at least 1 ms.

9. Apparatus for carrying out the process according to any of the previous claims comprising a sensing unit (A), connected by an optical fiber (B) to a sample holder (C), and a LED (D).

10. Apparatus according to the previous claim further comprising a heating unit with means for controlling both temperature and time.

11. Kit for detection of biomolecules according to claim 1, preferably for detecting viruses.

12. Kit according to the previous claim for detection of SARS-CoV-2.

13. Kit according to the previous claim for detection of SARS-CoV-2 comprising Cdots previously incubated with at least one SARS-CoV-2 probe.