WO2025069117A1

WO2025069117A1 - Method for highly sensitive analytes detection

Info

Publication number: WO2025069117A1
Application number: PCT/IT2023/000030
Authority: WO
Inventors: Rudy IPPODRINO; Bruna MARINI
Original assignee: Ulisse Biomed SpA
Current assignee: Ulisse Biomed SpA
Priority date: 2023-09-26
Filing date: 2023-09-26
Publication date: 2025-04-03
Anticipated expiration: 2026-03-26

Abstract

A method for the highly sensitive detection of analytes, e.g. antigens, small molecules, proteins, and antibodies, in samples, uses a combination of local concentration increase and PCR-based amplification to achieve sensitivity and quantification in a one-step reaction. The method utilizes polymerase enzymes, nucleic acid (e.g. DNA) arms, and binding moieties for analyte detection, enabling rapid and highly sensitive detection in complex matrices.

Description

“METHOD FOR HIGHLY SENSITIVE ANALYTES DETECTION”

FIELD OF THE INVENTION

Embodiments described herein concern a novel technology for the highly sensitive detection of analytes, e.g. antigens, proteins, small molecules and antibodies, in purified and crude samples. The technology leverages a combination of local concentration increase and Polymerase Chain Reaction (PCR) amplification to achieve a highly sensitive detection and quantification in a one- step reaction. The methods described herein utilize polymerase enzymes, nucleic acid (e.g. DNA) arms, and binding moieties for analyte detection, enabling rapid and highly sensitive detection in samples, such as biological samples, for instance in complex biological matrices or purified samples, and possibly also inorganic samples, drugs or others.

Embodiments described herein address the challenge of highly sensitive antigen, protein, and antibody detection. The technology is designed to detect even fewer than 10 molecules of proteins, antigens, small molecules or antibodies per reaction within minutes. This approach allows for target detection using PCR in a streamlined one-step reaction that may take less than 90 minutes

BACKGROUND OF THE INVENTION

The state of the art of the detection of analytes such as proteins, antigens, small molecules, antibodies is represented by traditional immunoassays, such as lateral flow-based systems, Enzyme-Linked Immunosorbent Assays (ELISA) and Chemiluminescent Immunoassays (CLIA) that have limitations in sensitivity, especially when detecting low-abundance analytes lower than picomolar range. Polymerase Chain Reaction (PCR) is a sensitive technique that can generate copies of very small amounts of DNA sequences, which are amplified in a series of cycles of temperature changes. PCR has been exploited also for the detection of not- nucleic acid related targets through PCR-based immunoassays, such as Polymerase Chain Reaction-Enzyme Linked Immunosorbent Assay (PCR-ELISA, Sue, M. J. et al. https://doi.org/10.1155/2014/653014), Proximity Ligation Assay (PLA, Soderberg, O. et al. https://doi.org/10.1038/nmeth94, Lof, L. et al. https://doi.org/10.1002/cpcy.22), Agglutination-PCR (ADAP, Tsai, C. et al. https://doi.org/10.1021/acscentsci.5b00340), and Proximity Extension Assay (PEA, Petrera, A. et al._https://doi.org/10.1021/acs.jproteome.0c00641). Such PCR based immunoassays have shown higher sensitivity’ compared to traditional immunoassays like lateral flow-based systems, enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA). However, these techniques are often complex, time-consuming, requiring multiple experimental steps (multiple incubation and washing steps) which would require complex automation systems, especially in high-throughput settings.

In fact, PLA, PEA, ADAP and ELISA PCR are technologies that require from 2 to 5 experimental steps to get results, are complex and time-consuming processes ranging from 3 to 24 hours per analysis run. Moreover, it is important to underline that multiple and complex experimental steps dramatically increase the probability of amplicons contamination thus affecting test usability.

In processes that involve multiple steps, including e.g. binding of detection molecules, ligation, and subsequent PCR amplification, each of these steps requires time for completion, and the need of transitioning from one step to another can add to the overall time required to obtain results. This multi-step nature of the known methods leads to a longer overall analysis time. In multi-step methods, moreover, different reagents and buffers might need to be added at different stages, and intermediate steps such as washing, dilution or incubation might be needed to achieve the desired binding. These additional steps and reagent additions can contribute to a longer overall analysis time. Moreover, multiple steps can increase errors incidence and therefore negatively affect the performance of the assay.

Additionally, due to their complexity, these technologies often require the use of a complex automation (De Jesus Cortez, F. et al. https://doi.Org/10.1016/j.slast.2021.10.001) that helps limiting human intervention and consequently potential related errors, but it is expensive and takes up laboratory⁷ space.

Moreover, all the products based on proximity principle and PCR amplification require hours to produce results and are mainly used for research use only in the field of biomarkers discovery⁷ and protein-protein interaction. Complex protocols are often required for the discrimination of multiple targets in the same reaction such as post analysis amplicons sequencing step. To date, no one-step PCR-based immunoassay is available on the market mainly because: a) often, multiple polymerase enzymes are not able to properly and effectively work in the same reaction, in the same chemical and thermal conditions thus implying the use of multiple buffers which traduces in multiple analysis steps; b) in the cases where multiple enzymes can work together the chemical and thermal condition of the reaction do not allow to the binding moieties to properly bind the target. This implies long initial incubations to favor target binding or a separate buffer which allow target binding; c) long process of DNA purification and sequencing are required to discriminate among different targets in the same reaction; d) PCR reactions lack often of specificity due to the high basal signal background, meaning the reaction can take place even in the absence of the target of interest.

Moreover, another problem of the known PCR-based protein assays is that they require dilution of the sample since using the undiluted sample would not provide a usable outcome.

There is therefore a need to provide a novel technology and related methods for highly sensitive one-step detection of analytes.

In particular, one purpose of the present invention is to provide a one-step technology and related methods that combine the specificity of binding moieties (e.g., antibodies, antigens, small molecules or aptamers) with the signal amplification capabilities of nucleic acid arms to achieve highly sensitive and specific detection of analytes, while providing results quickly (e.g. in less than 90 minutes) and being usable by the majority of the real-time PCR machines available on the market.

The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

SUMMARY OF THE INVENTION

The present invention is set forth and characterized in the independent claims, while the dependent claims describe other characteristics of the present invention or variants to the main inventive idea.

The present invention provides methods for the qualitative and/or quantitative detection and discrimination of one or more analytes, such as proteins, antigens, small molecules and antibodies, in a one-step reaction format from crude and purified samples. The methods exploit target-induced nucleic acid (e.g. DNA) local concentration increase and PCR to achieve rapid and highly sensitive detection and quantification. A nucleic acid-based nanosensor, comprising arms with specific template sequences and anchor sequences, is coupled with binding moieties capable of binding to the target analytes of interest. The interaction of the analytes with the binding moieties leads to an increase in the proximity' of the nucleic acid arms, allowing polymerase-mediated isothermal nucleic acid (e.g. DNA) synthesis and subsequent PCR amplification.

The presence, the quantification and the discrimination of specific analytes may be determined by post-PCR melting curve analysis (which correlates specific amplicon melting temperatures to the presence of individual analytes) and/or probes (correlating different fluorescence wavelengths to the presence of multiple targets) enabling the discrimination of multiple targets in the same reaction thus allowing the design of assays with deep multiplexing capability⁷.

According to embodiments, a method for one-step highly sensitive detection of one or more analytes in samples is provided. The method occurring in a one-pot PCR machine system. In one embodiment, the method comprises:

- providing a reaction solution comprising: a buffer solution, single stranded nucleic acid arms, at least one polymerase enzyme, nucleic acid (e.g. DNA) primers complementary to reverse complement of arm template sequence portions, possibly fluorescent intercalating dyes and/or fluorescent probes, possibly additives and stabilizers; each single stranded nucleic acid arm containing a binding portion for binding moieties, capable of simultaneously binding nucleic acid sensor structures and target analytes of interest, a template sequence, a partially complementary anchor sequence, and a 3’ priming portion;

- adding an amount of a sample to said reaction solution;

- implementing a PCR thermal cycle allowing binding moieties target binding, isothermal polymerase polymerization and PCR amplification.

In embodiments, said reaction solution can be provided in a liquid format or, alternatively, in a freeze-dried or lyophilized format.

Embodiments of the present disclosure overcomes the mentioned above limits of the prior art. In particular, the composition of the buffer solution and specific thermal protocol in the PCR thermal cycling combined with specific polymerase enzyme(s) may allow the polymerase(s) to perform isothermal nucleic acid synthesis and PCR amplification in the same reaction, thus generating the conditions to use one buffer only.

Moreover, the buffer solution according to the embodiments described herein may allow a quick target binding to the binding moieties. This implies that each reaction passage occurs in a single buffer solution in a single step directly in the PCR machine.

The buffer solution may also help create the appropriate conditions for the interaction of binding moieties with target analytes, as well as the subsequent isothermal nucleic acid synthesis and PCR amplification. The buffer components, such as 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), MgCh. and nucleoside triphosphate (dNTPs), may contribute to maintaining the necessary pH, ionic strength, and cofactors required for the enzymatic activities involved in the assay. Moreover, it may contain component helping in the stabilization of enzyme and other cofactors, and additives needed to work in complex matrices. In the context of the one-step approach and rapidity of analysis, the buffer solution contributes to the efficiency of the process. By having the necessary components readily available in the reaction solution, it is ensured that the binding of binding moieties to the target analytes, the isothermal polymerase polymerization, and the PCR amplification can all occur smoothly within a single reaction without the need for additional steps or external reagent additions. The read-out is ensured by means of probes or dye that generate one or more target-specific fluorescent signal that is read by the thermocycler machine. This streamlined approach reduces the time required for the assay since all required components are present and active from the beginning of the reaction. Therefore, the presence of the appropriate chemicals in the buffer solution coupled with the appropriate polymerases may be a significant factor for the one-step approach and the rapidity of analysis in the present invention.

Moreover, utilizing a one-pot PCR machine system the method avoids any external transfers of reaction mixtures thus minimizing the need of manual intervention and further reducing contamination risk. The embodiments according to the present disclosure contributes also to the rapidity of the analysis by reducing handling steps and potential sources of error. The above mentioned features allow to perform the analysis in simple manual settings or by using a basic and simple PCR-setup liquid handler.

Further, nucleic acid arms and template sequence used in the embodiments described herein may be designed to have a peculiar and distinctive melting profile thus allowing to couple each analyte target with a peculiar melting peak allowing to discriminate multiple targets in the same reaction without sequencing, i.e. allowing the correlation of a specific analyte to a particular amplicon melting temperature without the need of post-PCR sequencing. In other embodiments nucleic acid-based probes with fluorescent reporter and quencher are added allowing multiplex target detection by means of different fluorescence wavelengths.

Still further, the chemical modifications of the arms, including e.g. SAMRS (Self-Avoiding Molecular Recognition Systems) modification, may allow to reduce the signal background increasing assay specificity. This specificity may help in achieving accurate and reliable results in a shorter time frame.

Moreover, multiple binding moieties may be employed for each target, increasing test sensitivity and assay specificity.

In the method according to the present disclosure, the one-step approach means that all the steps or phases required for the detection of one or more analytes occur within a single reaction, especially in a one-pot PCR machine system, without the need for intermediate steps such as binding, ligation, and multiple washing steps, nor pouring off, decanting or buffer changes where each reaction occurs separately. This streamlining of the process reduces the time required to perform the assay, as it eliminates the need for external transfers and associated contamination risk and waiting time. As a result, the entire assay can be completed much more rapidly compared to methods that involve multiple sequential steps as known in the prior art. The elimination of intermediate steps and the concurrent execution of various assay stages result in a quicker overall analysis time when compared to methods involving multiple sequential steps.

In some embodiments, the reaction solution may include PCR additive(s), stabilizer(s), possibly fluorescent probe(s) and/or fluorescent intercalating dye(s). In some embodiments, a part of said reaction solution can be mechanically separated from the other by means of physically layers or jellifying agents that can be disrupted by temperature steps allowing complementation of the reaction solution.

In some embodiments, isothermal nucleic acid (e.g. DNA) synthesis and PCR amplification may be performed by a specific polymerase enzyme, having low activity during the isothermal phase and high activity over a higher temperature, enabling both reactions to occur in the same buffer. Alternatively, at least two polymerase enzymes may be used, i.e. an isothermal nucleic acid (e.g. DNA) polymerase for the isothermal nucleic acid synthesis and a hot-start nucleic acid (e.g. DNA) polymerase for the PCR amplification.

In some embodiments, the nucleic acid probes may be labeled with fluorophores such as CY5.5 or Alexa 680, enhancing detectability in blood serum samples. Alternatively, the fluorescence read-out of the real time PCR of both amplification and melting curve can be provided by using intercalating dyes, in particular including Syto 9. In some particular embodiments, nucleic acid probes can be used in combination with intercalating dyes aiming to increase assay multiplexing capacity, since different target analytes with overlapping melting profile can be differentiated by a different probe wavelength. In addition, the use of serum albumin blockers such as caffeine, theophylline, theobromine, chlorpromazine, with the aim to increase the signal from Syto 9 may also be contemplated in some embodiments.

In the context of the present disc losure, an amplicon refers to a nucleic acid (e.g. DNA) fragment that is generated through the process of amplification using PCR. PCR is a technique used to create multiple copies of a specific nucleic acid sequence, and the amplicon is the result of this amplification process. It is a region of nucleic acid that has been replicated multiple times, leading to an increase in the amount of that particular nucleic acid sequence. In the present disclosure, the amplicon is generated during the isothermal phase of the detection process. The nucleic acid arms, which are initially designed to anneal to each other in the presence of the target antigen, serve as templates for the synthesis of the second nucleic acid strand by the nucleic acid polymerase enzyme. This synthesis leads to the creation of amplicons that contain the target nucleic acid sequence. The amplicons are then further amplified through PCR thermal cycling, resulting in a significant increase in their quantity. The detection of these amplicons using e.g. fluorescent dyes/probes provides a quantifiable signal indicating the presence of the target antigen, small molecule, protein, or antibody.

Further embodiments relate to a kit for one-step highly sensitive detection of one or more analytes in samples, comprising:

- a buffer solution, possibly containing also additives and stabilizers;

- single stranded nucleic acid arms, each single stranded nucleic acid arm containing a binding portion for binding moieties capable of simultaneously binding nucleic acid sensor structures and target analytes of interest, a template sequence, a partially complementary anchor sequence, and a 3^! priming portion;

- at least one polymerase enzyme;

- nucleic acid primers complementary to reverse complement of arm template sequence portions;

- fluorescent dyes and/or probes; and

- possibly a PCR machine.

In embodiments of the kit, said a buffer solution, single stranded nucleic acid arms, at least one polymerase enzyme, nucleic acid primers, fluorescent dyes and/or probes, can be provided in a freeze-dried or lyophilized format.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments. The accompanying drawings relate to embodiments of the disclosure and are described in the following:

- Fig. 1 shows a nucleic acid arm structure that may be used in embodiments of the present disclosure;

- Fig. 2 shows a nanosensor scheme that may be used in embodiments of the present disclosure;

- Fig. 3 shows a nanosensor scheme that may be used in further embodiments of the present disclosure;

- Fig. 4 is a diagram showing phases A, B, C, D of a method according to embodiments of the present disclosure; - Fig. 5 schematically shows phase E of a method according to embodiments of the present disclosure;

- Fig. 6 schematically shows a phase of a method according to further embodiments of the present disclosure;

- Fig. 7 schematically shows a phase of a method according to further embodiments of the present disclosure;

- Fig. 8 is a graph showing Trastuzumab quantification;

- Fig. 9 is a graph showing anti-Digoxigen antibody quantification;

- Fig. 10 is a graph showing TNF-alpha quantification.

DESCRIPTION OF EMBODIMENTS

We shall now refer in detail to the various embodiments of the invention, of which one or more examples are shown in the attached drawings. Each example is supplied by way of illustration of the invention and shall not be understood as a limitation thereof. For example, the characteristics shown or described insomuch as they are part of one embodiment can be adopted on, or in association with, other embodiments to produce another embodiment. It is understood that the present invention shall include all such modifications and variants.

Before describing these embodiments, we must also clarify that the present description is not limited in its application to details of the construction and disposition of the components as described in the following description using the attached drawings. The present description can provide other embodiments and can be obtained or executed in various other ways. We must also clarify that the phraseology and terminology used here is for the purposes of description only and cannot be considered as limitative.

All the percentages and ratios indicated refer to the weight of the total composition (for example indicated as % w/w), unless otherwise indicated. All the measurements are made, unless otherwise indicated, at 25°C and atmospheric pressure. All the temperatures, unless otherwise indicated, are expressed in degrees Centigrade.

All the ranges reported here shall be understood to include the extremes, including those that report an interval "between" two values. Furthermore, all the ranges reported here shall be understood to include and describe the punctual values included therein and all the sub-intervals. The present invention provides methods to perform target analytes, e.g. proteins, antigens, small molecules and antibodies, one-step detection in crude and also purified samples (e.g. blood serum, blood, gut, urine, sputum, saliva, vaginal discharges, anal discharges, sweat and bodily fluids). These methods provide a quick and simple assay in a homogeneous phase, without any washing steps which exploits target-induced nucleic acid (e.g. DNA) local concentration increase and PCR.

The embodiments described herein can be used for nucleic acid and in the following reference may be made to nucleic acids in general and sometimes to DNA as possible example. Reference to DNA may not be considered as limiting the scope of the present invention since it might be possible that other nucleic acids other than DNA are used in the embodiments described herein.

Embodiments described herein relate to a novel technology and methods offering an innovative approach to highly sensitive analyte detection. They involve the use of a nanosensor 10 with nucleic acid (e.g. DNA) arms 11 (Fig. 1, 2 and 3), which comprise different portions, including a binding moiety binding portion 16, a template sequence 19, an arm anchor sequence 20, and a 3’ priming portion 21. The binding moiety binding portion 16 can be directly conjugated to binding moieties 17 or be annealed, directly or indirectly, to them through hydrogen bonds. In case of indirect annealing, a nucleic acid based or PNA based adapter contiguous with the binding moiety may be used.

Upon binding of the target analyte 18 to the binding moieties 17 (Fig. 4, phase A), the proximity of nucleic acid arms 11 increases, leading to the annealing of arm anchor sequences 20. This allows the nucleic acid (e.g. DNA) polymerase enzyme 12, 13 to bind to the 3’ priming portion (Fig. 4, phase B) and initiate the synthesis of the second strand 23 of nucleic acid (e.g. DNA) using the template sequence as a guide (Fig. 4, phase C).

The newly synthesized nucleic acid (e.g. DNA) undergoes amplification through PCR cycling through polymerase 12, 14 and primers 15 (Fig. 4, phase D and Fig. 6), resulting in exponential PCR amplification of the nucleic acid amplicons (Fig. 5, phase E and Fig. 7), thereby enabling the highly sensitive detection of the analyte.

Thus, the methods described herein enable highly sensitive detection of analytes, also at femtomolar concentration levels.

The same nucleic acid (e.g. DNA) polymerase enzyme 12 may perform the isothermal nucleic acid synthesis and PCR amplification phases are performed. In this case such polymerase enzyme 12 may have differential activities at defined temperatures.

Alternatively, an isothermal nucleic acid polymerase 13 that works at room temperature, or generally at a low temperature ranging from about 15 to about 50 °C, to synthesize DNA may be used for the isothermal nucleic acid synthesis and a hot-start nucleic acid polymerase 14 that is activated after heat treatment may be used for the PCR amplification.

Aspects of the present disclosure involve a one-step reaction format detection method. The method utilizes a one-step reaction format, which means that all the steps required for the detection of multiple antigens, proteins, small molecules and antibodies occur in a single reaction, in particular the method occurs in a one-pot PCR machine system. This eliminates the need for multiple experimental steps, reducing the assay time significantly.

Aspects of the present disclosure involve a nucleic acid-based nanosensor 10 (Fig. 2 and 3). The design of the nucleic acid-based nanosensor 10 is one aspect of the disclosure. The nanosensor 10 comprises binding moieties 17 capable of binding to the target analytes 18, arms 11 with anchor sequences 20, 3’ priming portions 21 and template sequences 19. The specific interaction of these components and the target analytes 18 enables accurate and specific detection.

Aspects of the present disclosure involve proximity-induced PCRThe use of proximity-induced PCR amplification allows for highly sensitive and specific detection of the target analytes 18. The binding of the target analytes 18 to the nanosensor 10 leads to increased proximity of nucleic acid arms 11, triggering PCR amplification only when the target analyte 18 of interest is present. This approach enhances the sensitivity of the assay.

Aspects of the present disclosure involve a fluorescent readout. The use of fluorescent dyes and/or nucleic acid-based probes for real-time PCR amplification readout allows for quantification of the detected analytes. The fluorescence signal increases after each PCR cycle and is correlated, e.g. directly proportional, to the initial amount of the target analyte 18 in the sample, providing a reliable and quantitative measure.

Aspects of the present disclosure involve post-PCR melting curve analysis. The invention may employ post-PCR melting curve analysis, which correlates specific amplicon melting temperatures to the presence of specific target analytes 18. Melting curve can be used alone or coupled to specific fluorescent probes to discriminate different targets in the same reaction. This capability enables the discrimination of multiple analytes in the same reaction, further enhancing the assay’s multiplexing capability.

The combination of a one-step reaction format, proximity-induced PCR, and real-time PCR with fluorescent readout may allow for rapid results. The entire assay can be completed in less than 90 minutes, even less than 60 minutes, making it much faster compared to existing techniques that require several hours to produce results.

The compatibility of the assay with PCR machine available on the market ensures its widespread applicability and accessibility to laboratories with standard PCR equipment.

The capability of method according to the present disclosure to detect one or more analytes in crude and also purified samples, such as blood serum, blood, urine, and saliva, makes it practical for various diagnostic applications without the need for extensive sample preparation.

The combination of these aspects may result in a highly sensitive, rapid, and easy-to-use assay method. Its ability to detect and discriminate one or more analytes in a single reaction while providing results in a short timeframe can significantly impact the fields of diagnostics, theragnostic, biomarker discovery, small molecules detection, drug detection, environmental pollution monitoring and protein-protein interaction studies.

Aspects of the present invention that allow the method according to the present disclosure to achieve the advantageous results, such as rapid one-step detection of multiple antigens, small molecules, proteins, and antibodies with high sensitivity, can be as follows:

- utilizing a nucleic acid-based nanosensor 10 (Fig. 2 and 3) as the detection platform. The nanosensor 10 comprises single-stranded nucleic acid arms 11 with specific binding sites 16 for unique binding moieties 17 (e.g., antibodies, aptamers, etc.) and also may provide primers (oligos) 15;

- binding moieties 17: these elements are responsible for binding to the target analytes 18 and are bound to nucleic acid structures of the arms 11. The binding moieties 17 are elements capable of simultaneously binding to the nucleic acid sensor structures and the target analytes 18 of interest. They can be various types of molecules, including antibodies, single-chain antibodies, antibody variable regions, affibodies, proteins, DNA/RNA mono and multivalent aptamers, peptide nucleic acids (PNAs), or antigens, small molecules, alone or combined together. The binding moieties 17 may be chemically modified at 3 ’-OH end with for example one or more of nucleoside analogue, 2 ',3 '-dideoxy cytidine (2-3’ ddC), 3’ inverted 2-deoxyribothymidine (dT), 3’ C3 spacer, 3’ amino, and 3’ phosphorylation to avoid polymerase binding;

- proximity-induced nucleic acid (e.g. DNA) amplification: the presence of a specific analyte induces an increase in the proximity’ of nucleic acid arms 1 1 bound to the binding moieties 17. This proximity-based interaction favors the annealing of anchor sequences 20 present in the arms 11 at a defined temperature;

- isothermal second strand synthesis 23: the interaction of anchor sequences 20 exposes 3 ’ priming portions 21 and arm template sequences 19 to DNA polymerase enzyme(s) 12, 13 This allows polymerase to perform isothermal second strand synthesis 23 of DNA amplicons at a specific temperature;

- PCR amplification (Fig. 5, phase E and Fig. 7): after isothermal polymerization, DNA primers 15 (oligos) recognize portions of the newly synthesized DNA during the PCR amplification, leading polymerase 12 or 14 to DNA replication through thermal cycling;

- post-PCR analysis: analyzing post-PCR melting curves, and/or probe fluorescence signals, to discriminate multiple target analytes (18) in case of multiple target analytes (18) detection (this enables the discrimination of multiple analytes in a single reaction without the need for amplicon sequencing); and/or analyzing post-PCR melting curves, and/or probe fluorescence signals intensity', to quantify of target analyte(s) ( 18), in case of quantitative detection;

- buffer solution: the buffer solution is a component that allows for the recognition between the nanosensor’s binding moieties 17 and the target analyte 18 of interest. It may contain specific chemicals that promote target binding and DNA polymerase activity;

- specific thermal protocol: the method according to the present disclosure may employ a specific thermal cycle that enables simultaneous binding of the binding moieties 17 to the target analyte 18, isothermal polymerase polymerization, and PCR amplification, all within a single reaction;

- chemical modifications 22: the arms 11 may be chemically modified to reduce self-annealing and non-specific annealing interactions, which helps to increase assay specificity and reduce background signals;

-multiple binding moieties: the use of multiple binding moieties 17 per single target significantly increases the test’s sensitivity and specificity;

- rapid results: the entire detection process, from sample dispensing to post-PCR analysis, can be completed in less than 90 minutes, or even less than 60 minutes.

The reaction solution used in the embodiments described herein, for one-step detection of one or more analytes through probes proximity-induced PCR, may comprise the following components:

- buffer solution: this may be a buffer composed of water, HEPES (hydroxyethylpiperazine ethane sulfonic acid, a buffering agent), MgCh (magnesium chloride), dNTPs (deoxyribonucleotide triphosphates), KC1, NaCl, Tris-HCl and may have a specific pH range. The buffer solution may facilitate the recognition and interaction between the nanosensor’s binding moieties 17 and the target analytes 18;

- binding moieties 17 as described above;

- arms 11 : at least two single-stranded nucleic acid structures capable of binding to at least one binding moiety 17. Arms 1 1 have an intrinsic affinity to each other due to the presence of complementary anchor sequence 20. Arms 11 may have specific chemical modifications 22 that modulate their affinity to each other and their capability to form secondary structures. Each arm 11 has a specific binding portion 16 for a unique binding moiety 17 and a peculiar template sequence 19; the nanosensor 10 for a certain target analyte exploits the coupling of a target specific binding moiety bound to an arm with a distinctive template sequence (characterized by a specific melting temperature or probe complementarity) to achieve target discrimination;

- anchor sequences 20: arms have at least one anchor sequence, responsible for the interaction between arms. Different anchor sequences have low affinity to each other, but the affinity increases when arms are in close proximity;

- arms 3’ priming portions 21 : proximal to arm anchor sequences 20, these portions expose 3 ’-OH chemical groups. These arm portions can trigger polymerase isothermal polymerization of a new DNA strand. Arms 3’ priming portions 21 recruit polymerases 12 or 13 only when anchor sequences 20 of at least two different arms 11 are interacting in close proximity;

- arm template sequence 19: a single-stranded nucleic acid sequence contained within the arm 11 that acts as a template for isothermal second strand synthesis 23;

- DNA primers 15 (oligos): DNA oligonucleotides complementary to the newly synthesized DNA sequences, starting from the 3 ’ priming portions 21 after anchor sequences’ 20 interaction;

- fluorescent dyes/probes: nucleic acid intercalating dyes or nucleic acid-based probes containing a quencher and a fluorophore for real-time PCR amplification fluorescence readout. The fluorescence intensity increases after each PCR cycle and is correlated, e.g. directly proportional, to the amount of amplified nucleic acid.

The combination of these components in the reaction solution may enable the rapid and sensitive detection of multiple antigens, small molecules, proteins, and antibodies in a one-step reaction format.

A further aspect may be the specific formulation of the buffer solution. The buffer solution may be designed to facilitate the recognition between the nanosensor’s binding moieties 17 and the target analytes 18. It may enable the binding moieties 17 to interact wdth the specific target analytes 18 in the sample, triggering the subsequent proximity-induced PCR amplification.

The specific formulation of the buffer solution may allow the following advantages:

- enabling rapid reaction: the composition of the buffer solution allows for efficient and rapid binding interactions between the nanosensor’s binding moieties 17 and the target analytes 18. This facilitates the initiation of PCR amplification in a timeefficient manner;

- enhancing sensitivity: the buffer solution may contribute to enhancing the sensitivity of the assay by ensuring strong and specific binding interactions between the binding moieties 17 and target analytes 18. This can lead to higher signal amplification during the PCR process;

- compatibility with in purified and also crude samples: the buffer solution may be formulated to be compatible with various crude and also purified samples, such as whole blood, blood serum, urine, saliva, etc. (in this regards the use of serum albumin blocking agents such as caffeine , theophylline, theobromine, chlorpromazine, hemin may be contemplated), ensuring that the assay can be applied to a wide range of diagnostic scenarios, enabling direct detection of target analytes in these samples without extensive purification or preparation;

- stability and reproducibility: the buffer solution specific composition may contribute to the stability and reproducibility of the assay, ensuring consistent results across different samples and experiments.

According to embodiments, the buffer solution may be a specialized solution designed to enable the recognition and interaction between the nanosensor binding moieties 17 and the target analytes 18 in a one-step one or more analytes detection assay.

The buffer solution may be composed of specific chemical components and reagents, selected to create an optimal environment for the binding moieties 17 to bind to the target analytes 18 and trigger subsequent PCR amplification.

In yet another embodiment, the buffer solution may include a source of monovalent or bivalent cations. For example, a chloride containing monovalent ion or bivalent ions can be used. As a source of monovalent cations, potassium ions can be used. K⁺ can be obtained from potassium salts, e.g. potassium chloride, in particular potassium chloride at a concentration of 0.1 M. As source of bivalent cations magnesium or manganese ions can be used. Mg²' can be obtained from magnesium salts, e.g. magnesium chloride. Examples are: TrisHCl and/or NaCl and/or KC1 and/or NH4CI and possibly in some cases MgCh as well.

The composition of the buffer solution may typically include:

- water: provides the solvent for the buffer and ensures compatibility with samples;

- HEPES (4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid): acts as a buffering agent, maintaining a stable pH range for efficient enzymatic reactions;

- magnesium chloride (MgCh): essential for the PCR reaction, as magnesium ions are cofactors for DNA polymerases, enabling efficient polymerization of DNA strands;

- deoxyribonucleotide triphosphates (dNTPs): serve as the building blocks for DNA synthesis during PCR amplification;

- other potential additives and/or stabilizing agents, depending on the specific requirements of the assay.

Among additives or stabilizing agents, the buffer solution may include for instance, one or more of: Betaine, Bovine Serum Albumin (BSA), TMAC (Tetramethylammonium Chloride), DMSO, Gelatin, Acetamide, Formamide, DTT (dithiothreitol), Glycerol, beta-mercaptoethanol (BME), a nonionic surfactant, such as polysorbate-type nonionic surfactant (e.g. Polysorbate 20, such as Tween 20).The buffer solution may also include detergents, in particular non-ionic detergents, such as for instance IGEPAL CA630 or Nonidet-P40 (NP40) or Triton.

In possible examples, the buffer solution may therefore include: Tris HC1, KC1, Betaine, BSA, TMAC, DMSO, Gelatin, Syto 9, Acetamide, Formamide, NaCl, MgCh, DTT, Tween 20, HEPES, IGEPAL CA630, NH4CI, Glycerol, BME, dNTPs, primers. In such examples, the final concentration of such components in the buffer solution may be as follows (concentration ranges are indicated):

- Tris HC1: 0.01-0.10 M.

- KC1: 0.01-0.10 M.

- Betaine: 0.1-1 M.

- BSA: 0.01-0.10 mg/mL.

- TMAC: 0.01-0.10 M.

- DMSO: 0.3%-10% w/w.

- Gelatin: 0.05-0.10 mg/mL.

- Syto 9: 1-6 pM.

- Acetamide: 0.5%-8% w/w.

- Formamide: 0.3%-4% w/w.

- NaCl: 10-700 mM.

- MgCh: 0.75-10 mM.

- DTT: 0.5-5 mM.

- Tween 20: 0.05%-0.5% w/w.

- HEPES: 5-70 mM.

- IGEPAL CA630: 0.01%-10% w/w. - NH₄C1: 2-70 mM.

- Glycerol: 0.5%-9% w/w.

- BME: 0.1-5 mM.

- dNTPs: 10-80 pM.

- Primers: 100-1000 nM.

The pH of the buffer solution may be carefully controlled within a specific range, optimizing the binding affinity between the binding moieties and the target analytes, as well as the efficiency of the PCR amplification process. The pH may range from 6 to 12.

The buffer solution may be instrumental for the successful performance of the assay, providing the necessary⁷ conditions for proximity-induced PCR amplification and the subsequent post-PCR melting curve analysis for discrimination of multiple analytes in a single reaction.

The specific concentrations and ratios of the components in the buffer solution may vary based on the particular requirements and characteristics of the nanosensor and the PCR-based assay.

Thus, one aspect of the present disclosure may be the specific formulation and combination of the identified solution’s components, particularly the buffer solution, along with the design and integration of various elements.

The buffer solution, which may include specific chemical components such as HEPES, MgCE, dNTPs, and may have a controlled pH range, e.g. in a range of 6 to 12, may enable the recognition and interaction between the nanosensor binding moieties and the target analytes. This buffer, in combination with the other elements in the reaction solution, may allow for a rapid one-step one or more analytes detection assay.

The specific formulation of the buffer solution and the optimized combination of components may enable quick binding interactions between the binding moieties and the target analytes, initiating isothermal nucleic acid polymerization, amplicons generation andamplification process in a time-efficient manner, thus providing a rapid analyte detection.

The composition of the buffer solution may contribute to enhancing the sensitivity of the assay, leading to strong and specific binding interactions between the binding moieties and target analytes, resulting in higher fluorescence signal amplification during PCR.

The specific design and formulation of the buffer solution may facilitate the one-step reaction format, eliminating the need for complex and time-consuming experimental steps, as required in existing products based on proximity principles and PCR amplification.

The combination of the buffer solution and other optimized components may therefore allow the assay to provide results in less than 90 minutes, even less than 60 minutes, thus providing a short analysis time, making it significantly faster than existing techniques that require hours to produce results.

The combination of the aforementioned aspects may enable the present invention to provide a simple, rapid, and highly sensitive method for the quantitative detection and discrimination of multiple antigens, small molecules, proteins, and antibodies in a one-step reaction format. Unlike existing technologies that require multiple experimental steps and extended analysis times, the present invention offers a faster and more efficient solution with in low abundant analytes detection and diagnostic settings.

When the method according to embodiments described herein is carried out, the reaction starts when a sample is dosed inside the buffer solution which contains some or all of the above-mentioned elements.

If the target analyte 18 of interest is present, it binds the binding moieties 17 causing an increase of the proximity of nucleic acid arms 11 bound to the binding moieties 17 (Fig. 4, phase A). Arms local concentration increase favors the interaction among arm anchor sequences 20 at a defined temperature, which may range from 15 °C to 50°C.

Anchor sequences 20 annealing exposes 3’ arm priming portion 21 and arm template sequence 19 to polymerase enzyme 12 or 13 which has a high affinity for these structures (Fig. 4, phase B). It may be DNA polymerase enzyme 12, able to perform the isothermal nucleic acid synthesis and PCR amplification, or alternatively isothermal nucleic acid polymerase 13 that works e.g. at room temperature. Room temperature according to embodiments described herein might mean a temperature of between 15° to 50 °C.

Polymerase enzyme 12 or 13 starts arm second strand DNA isothermal synthesis 23 at a defined temperature which may be between 15°C and 50°C creating DNA amplicons (Fig. 4, phase C).

Second strand DNA 23 is then the newly synthesized nucleic acid portion complementary to arm templates 19 and polymerized starting from arms 3’ priming portions 21.

Amplicons are composed by arm template sequence 19 and anchor portion 20 annealed.

After DNA second strand synthesis 23, PCR thermal cycling starts. Thermal cycling is a process involving heating and cooling that creates the conditions necessary for amplicons replication. PCR thermal cycling may be divided into two or three phases with specific temperature ranges: denaturation phase which ranges from 90°C to 100, annealing phase from 50°C to 68°C, extension phase from 55°C to 80°C, in some cases annealing and extension phases can be performed in one single step protocol at a defined temperature ranging from 50 to 80°C (Fig. 5, phase E and Fig. 7). It may be used the same polymerase enzyme 12, able to perform the isothermal nucleic acid (e.g. DNA) synthesis and PCR amplification, or alternatively hot-start nucleic acid (e.g. DNA) polymerase 14 that is activated after heat treatment.

PCR amplification can be repeated for 10-60 cycles.

After isothermal polymerization (second strand DNA synthesis 23, Fig. 4, phase D and Fig. 6) the DNA primers 15 (oligos) present in the reaction solution recognize portions of the newly synthetized DNA during the annealing/extension PCR phase thus allowing PCR amplification (Fig. 5, phase E and Fig. 7). Since each target analyte 18 is bound by a specific binding moiety' 17 which is coupled with a specific arm template sequence 19 w'hich has a distinctive melting temperature and/or is complementary' to specific fluorescent probes, post-PCR melting curves analysis, and/or fluorescent probe analysis, allow correlating a specific melting temperature and/or a specific fluorescent intensity⁷ at a defined wavelength to a specific analyte thus creating the possibility to design multiplexing assays where multiple analytes can be discriminate analyzing melting curve and/or probe hydrolysis derived fluorescence. Fluorescence intensity⁷, during PCR cycling, increases directly proportional to the amount of target analyte 18 present in the reaction making the assay suitable for quantitative analysis; moreover, post- PCR analysis such as melting curve fluorescent intensity⁷ measurement can also be used for analyte 18 quantification alone or combined with fluorescence intensity derive from amplification curves during PCR cycling.

Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA during heating ramps. As the temperature is raised, double strand DNA begins to dissociate leading to intercalating dye or fluorescent probes release thus creating a drop in fluorescence which is peculiar for each amplicon. The temperature at which 50% of DNA is denatured is known as the melting temperature.

Fig. 1 is used to illustrate the structure of nucleic acid arms 11 that may be used in the present disclosure. The arms 11 may comprise different portions with distinct features, including the binding portion 16, which can be directly and chemically conjugated with at least one binding moiety 17 or annealed, directly or indirectly, through multiple hydrogen bonds to at least one binding moiety 17. Each arm 11 can bind different binding moieties 17; however, multiple binding moieties 17 against the same target 18 are all coupled to the same nanosensor 10. The template sequence 19 in the arm 11 serves as a polymerase template for the second strand synthesis 23, allowing the creation of double-stranded DNA amplicons with a distinctive melting temperature, or different fluorescent probes complementarity. The arm 11 also contains anchor sequences 20 that allow interaction with other arms 11.

According to embodiments, the arms 11 may be divided into different portions, each with distinct features:

- binding moiety binding portion 16: this portion of the arm 11 contains the sequence that can be directly and chemically conjugated with at least one binding moiety 17 (e.g., an antibody or aptamer) or annealed, directly or indirectly, through multiple hydrogen bonds to at least one binding moiety 17. In case of indirect annealing, a nucleic acid based or PNA based adapter contiguous with the binding moiety may be used. Each arm 11 can bind different binding moieties 17coupled to the same target 18. This means that multiple binding moieties 17 against the same target 18 are all coupled to the arms 11 of the same nanosensor 10;

- template sequence 19: this portion serves as the polymerase template during the isothermal phase, which typically ranges from 15°C to 50°C. The template sequence 19 is the segment that will be copied by polymerase enzymes 12 or 13 during the isothermal phase. It acts as the template for the synthesis of the second strand of DNA 23, which is the reverse complement of the template sequence 19. Importantly, the template sequence 19 portion contains a unique nitrogenous base sequence that will generate double-stranded DNA amplicons with a distinctive melting temperature, or different probes complementarity;

- arm anchor 20 sequence: this sequence may range from 2 to 12 bases and may contain a segment that is totally or partially complementary to the anchor sequence 20 of another arm 11. The arm anchor sequences 20 may play a crucial role in the mechanism of action by enabling the interaction between different arms 11 ;

- arm 3’ priming portion 21: this portion of the arm 11 exposes a 3’ OH chemical group, which is important for polymerase binding. Polymerase binding to the 3’ priming portion is possible only when the arm anchor sequences 20 interact together due to their complementarity. This interaction is a key aspect in initiating the first isothermal polymerization process (second strand synthesis 23);

- chemical modifications 22: Fig. 1 shows a specific chemical modification 22, denoted as SAMRS (self-avoiding molecular recognition system) such as for example 2-aminopurine-2’ -deoxyriboside (A*), 2’-deoxy-2-thiothymidine (T*), 2 ’-deoxy inosine (G*) and N4-ethyl-2’ -deoxy cytidine which prevents arm selfrecognition and the formation of unwanted nucleic acid secondary structures. These modifications 22 ensure the proper functioning of the nanosensor 10 and prevent potential interference such as secondary structures formation, self- annelaing, and dimers formation.

Fig. 2 and 3 is used to describe a mechanism of action of the nucleic acid-based nanosensor 10 used in embodiments of the present disclosure. When the target analyte 18 of interest is present, it binds to the binding moieties 17, causing an increase in the proximity of nucleic acid arms 11 bound to the binding moieties 17. This increase in local concentration favors the interaction of anchor sequences 20 present in the arms 11 at a defined temperature. Anchor sequence annealing exposes the 3’ priming portion 21 and arm template sequence 19 to the polymerase (e.g. polymerase 12 or isothermal polymerase 13), initiating arm second strand DNA synthesis 23 at a defined temperature. After isothermal polymerization, the DNA oligos, or primers 15, recognize portions of the newly synthesized DNA during the annealing/extension PCR phase, allowing PCR amplification via polymerase enzyme (e.g. polymerase 12 or hot-start polymerase 14). PCR cycling creates conditions for exponential amplification of the amplicons, and intercalating dyes and/or probes generate a fluorescent signal which is directly proportional to the initial analyte amount.

Continuing with describing the mechanism of action in greater detail using Fig.

2 and 3:

- binding moieties 17: the binding moieties 17 (e.g., antibodies or aptamers) may be designed to specifically bind the target analyte 18 of interest (e.g. a protein or biomolecule). When the target analyte 18 binds to the binding moieties 17, it causes an increase in the proximity of the nucleic acid arms 11 that are bound to these binding moieties 17;

- arm anchor sequences 20 annealing: the increased local concentration of arms 11 leads to a higher chance of interaction among the arm anchor sequences 20 of different arms 11. As a result, the arm anchor sequences 20 anneal, bringing together the 3’ priming portion 21 and the template sequence 19 of the arms 11;

- polymerase binding and second strand synthesis 23: the polymerase enzyme (e.g. polymerase 12 or isothermal polymerase 13), which has a high affinity for the annealed arm anchor sequences 20, can now bind to the 3’ priming portion 21 and the template sequence 19 of the arms 11. This initiates the synthesis of the second strand of DNA 23 using the template sequence 19 as a guide;

- annealing/extension PCR phase: after isothermal polymerization (second strand DNA synthesis 23), DNA primers 15 or oligos (short DNA sequences) present in the reaction solution, after DNA denaturation phase, recognize specific portions of the newly synthesized DNA during the annealing/extension phase of the PCR. This recognition allows for the selective amplification of the target DNA amplicons via polymerase enzyme (e.g. polymerase 12 or hot-start polymerase 14);

- PCR cycling and exponential amplification: the PCR cycling involves repeated cycles of denaturation, annealing, and extension. During each cycle, the DNA amplicons generated by the nanosensor 10, following the presence of the analyte 18, undergo exponential amplification, leading to a detectable fluorescent signal.

Overall, the embodiments described using Figs. 1 and 2 enable a fast, sensitive and specific detection of the target analyte 18 of interest through the amplification of nucleic acid amplicons, resulting in a measurable signal that indicates the presence of the target analyte 18.

According to aspects of the present disclosure, embodiments utilize nucleic acid arms 11 coupled to one or multiple binding moieties 17 as the core sensing elements. Each nucleic acid arm 11 comprises distinct portions, as illustrated using Figs.1, 2 and 3. The binding moiety’ binding portion 16 can directly and chemically conjugate with at least one binding moiety 17 or anneal, directly or indirectly, through hydrogen bonds.

The template sequence 19 within each nucleic acid arm 11 serves as the polymerase template during the isothermal phase, typically ranging from 15 °C to 50°C. This template sequence 19 contains a unique nitrogenous base sequence that generates double-stranded DNA amplicons with a distinctive melting temperature or complementary to specific fluorescent probes with a peculiar emission wavelength. As explained above, for different analyte 18 discrimination it may also be contemplated to use, at the same time, both melting curve analysis and fluorescent probe intensity analysis.

The arm anchor sequence 20, that may be ranging e.g. from 2 to 12 bases, contains a segment that is totally or partially complementary to the anchor sequence 20 of another arm 11. When the target analyte 18 binds to the binding moieties 17, the proximity of nucleic acid arms 11 increases, promoting the interaction among arm anchor sequences 20 and facilitating the annealing of complementaiy sequences.

The 3’ priming portion 21 of each arm 11 exposes a 3’ OH chemical group, essential for polymerase binding. Polymerase binding to the 3 ' priming portion 21 is only possible when arm anchor sequences 20 interact together due to their complementarity. This interaction triggers the polymerase 12, 13 to initiate the synthesis of the second strand of DNA 23 using the template sequence as a guide.

Embodiments of the method according to the present disclosure may operate as follows, as described using Fig. 2 and 3:

- binding moiety’ 17 interaction: the binding moieties 17, which are specifically selected to target the analyte of interest, bind to the target analyte 18;

- increased proximity of nucleic acid arms 11 : the binding of the target analyte 18 causes an increase in the proximity of the nucleic acid arms 1 1 , bringing them closer together; - annealing of arm anchor sequences 20: the increased local concentration of arms 11 leads to a higher chance of interaction among the arm anchor sequences 20 of different arms 11. This results in the annealing of complementary sequences;

- polymerase binding and second strand synthesis 23: the polymerase enzyme 12, 13 binds to the 3’ priming portion 21 of the nucleic acid arms 11, initiated by the annealing of arm anchor sequences 20. The polymerase 12, 13 then starts the synthesis of the second strand of DNA 23 using the template sequence 19 as a guide;

- PCR amplification: after the isothermal polymerization (second strand DNA synthesis 23), DNA oligos, or primers 15 present in the reaction recognize specific portions of the newly synthesized DNA after a PCR denaturation phase and during the annealing/ extension phase of the PCR. This recognition allows for the selective amplification of the target DNA amplicons via polymerase enzyme (e.g. polymerase 12 or hot-start polymerase 14).

According to the present disclosure, embodiments of the method described herein operate through a multi-phase mechanism in a one-step reaction.

Possible embodiments may contemplate using the same DNA polymerase enzyme 12, with differential activities at defined temperatures, wherein such same polymerase enzyme may a low activity' during isothermal phase between 15°C and 42°C and high activity over 42°C.

In other embodiments, two different polymerases may be used, i.e. isothermal nucleic acid (e.g. DNA) polymerase 13 and hot-start nucleic acid (e.g. DNA) polymerase 14. For instance, Fig. 6 and 7 are used to describe embodiments where such two different polymerases are used. For example, Fig. 6 shows a phase of the method corresponding to phase D of Fig. 4 where however such two different polymerases are used, in particular isothermal DNA polymerase 13 for isothermal amplification. Of course, also phases A, B and C of Fig. 4 may be implemented accordingly using such two different polymerases 13, 14 instead of polymerase 12. Fig. 7 shows a phase of PCR amplification analogous to phase E of Fig. 5, where however the hot-start DNA polymerase 14 is used instead of polymerase 12.

In embodiments of the method, it may be employed an isothermal DNA polymerase 13 that works at room temperature to synthesize new DNA from a 3’- OH DNA free end and template. A hot-start DNA polymerase 14 may also be used, which can synthesize DNA from DNA primers 15 and a 3 ’-OH of the 3’ priming portion 21 , following heat activation at 98°C for a few seconds. DNA arms 11 with partially complementary anchor sequences 20 are utilized, along with binding moieties 17 (e.g. PNA, peptides, proteins, antibodies, etc.) for target analyte 18 detection.

The method leverages both local concentration increases and PCR-based amplification for highly sensitive and quantitative protein, antibody, small molecules and antigen detection.

When the target analyte 18 is present, the DNA arms 11 colocalize in a confined space, leading to the annealing of anchor sequences 20 present in both arms 11 (Fig. 4, phase A).

Since isothermal polymerase 13 is active at e.g. room temperature, it binds to the 3 ’-OH template created after anchor sequence 20 annealing, enabled by the presence of the target analyte 18.

Isothermal polymerase 13 initiates second DNA strand synthesis from the overlapping structure formed by the DNA arms 11 , generating the first amplicons. Primers 15 cannot perform the annealing with any sequence before this phase because they are complementary only to the newly synthesized DNA portions.

After the isothermal phase, temperature is increased to 98°C, activating the hot- start polymerase 14 and thereby also inactivating the isothermal polymerase 13. DNA primers 15 complementary to the newly synthesized DNA portions can now match with the first amplicons during the annealing PCR phase, starting the amplification process.

Through e.g. 15-45 PCR thermal cycles, potent amplification is achieved, detectable using e.g. fluorescent dyes/probes.

Of course, the above description may apply also to embodiments where the same polymerase 12 is used to perform isothermal DNA synthesis and PCR amplification phases.

Preliminary results obtained by the Applicant demonstrate the ability of the disclosed methods to detect 100 nm of Trastuzumab monoclonal antibody, showing a shift of e.g. seven PCR cycles over the background signal. The method disclosed herein works in complex matrices such as up to 10% blood serum and 1%, 5%, and 10% culture media for trastuzumab production. Further, the Applicant also found that different binding moieties may be used for the same target according to embodiments described herein.

The disclosed method is also effective in LB (Lysogeny Broth) bacterial culture containing overnight bacterial growth.

The disclosed method holds the potential to achieve unprecedented sensitivity⁷ in less than 60 minutes, capable of detecting 10-1,000 single antigens or antibodies per reaction.

Preliminary results obtained by the Applicant indicate the disclosed method as the only PCR-based one-step platform for protein, antigen, small molecules and antibody detection.

The technology of the disclosed method has the capability to reach unprecedented sensitivity levels.

The streamlined process of the disc losed method ensures results in minutes from sample to result.

The disclosed method can be employed with the majority of the real-time PCR machines available on the market.

Preliminary results obtained by the Applicant confirm the system’s efficacy generally in samples and also in complex biological matrices, such as blood serum and culture media for protein and antibody production.

The embodiments disclosed herein presents a breakthrough in highly sensitive antibodies, antigens, small molecules and protein detection. Through the utilization of DNA polymerases, DNA arms, and binding moieties, the platform achieves highly sensitive and quantitative results within a one-step reaction. The preliminary results demonstrate the capability of the disclosed method to detect low concentrations of analytes also in complex biological matrices. This innovative technology has the potential to be disruptive in the field of antigen and protein detection, offering a streamlined and rapid approach for researchers and clinicians alike.

Binding Moieties

Binding moieties 17 that may be used in the present disclosure can be antibodies, mono and multivalent aptamers single-chain antibodies, antibody variable regions, affibodies, proteins, DNA/RNA aptamers, peptide nucleic acids (PNAs), or antigens, small molecules. The binding moieties may be chemically bound to nucleic acid arms to facilitate target binding. Alternatively, the nucleic acid binding moieties may be annealed, directly or indirectly, to the arms through hydrogen bonds. In case of indirect annealing, a nucleic acid based or PNA based adapter contiguous with the binding moiety may be used. In some particular embodiments multiple binding moieties can be bound to a single arm such as for example monovalent, bi-valent or trivalent aptamers. Different binding moieties may also be used for the same target.

Chemical Modifications of Nucleic Acid Arms

The nucleic acid arms 11 may include chemical modifications 22. They may be chemically modified with for example one or more of 2-aminopurine-2’- deoxyriboside (A*), 2’-deoxy-2-thiothymidine (T*), -deoxy inosine (G*) and N4-ethyl-2’ -deoxy cytidine to reduce self-annealing and non-specific annealing interactions. These chemical modifications 22 may enhance the specificity of the assay and reduce background signals, thereby increasing the assay’s sensitivity and reproducibility.

Agglutination Mechanism

In some embodiments, more than one binding moiety 17 may be coupled with a single nucleic acid arm 11 to favor agglutination mechanisms. This arrangement leads to a higher-specific interaction between the arms 11 when the analyte of interest is present. The affinity between the arms 11 is low in the absence of the target analyte 18, but it is strongly promoted by proximity when the target analyte 18 binds to the binding moieties 17.

Isothermal nucleic acid (e.g. DNA) Polymerization and PCR Amplification

To achieve isothermal nucleic acid (e.g. DNA) synthesis and PCR amplification in the same reaction, a peculiar chemical composition, specific thermal protocol, and specific enzymes may be used.

In embodiments, isothermal nucleic acid synthesis and PCR amplification phases may be performed by the same nucleic acid polymerase enzyme 12, with differential activities at defined temperatures, wherein such same polymerase enzyme 12 may have a low activity' during isothermal phase in a defined temperature range and high activity at higher temperature, for instance low activity between 15°C and 42°C and higher activity over 42°C. Examples of such kind of polymerase enzyme that can be used may be for instance: Taq DNA Polvmerase, DreamTaq DNA Polymerase, EasyTaq® DNA Polymerase, PerfectStart® Taq DNA Polymerase, AccuTaq™ LA DNA Polymerase, Gotaq G2 Flexi.

In other embodiments, an isothermal nucleic acid polymerase 13 for the isothermal nucleic acid synthesis and a hot-start nucleic acid polymerase 14 for the PCR amplification may be used. The isothermal nucleic acid polymerase 13 works at temperature ranging from 15°C to 50°C to synthesize nucleic acid and the hot- start nucleic acid polymerase 14 is activated after heat treatment ty pically ranging from 90°C to 100°C.

For instance, in such embodiments one isothermal polymerase 13 performs the second strand nucleic acid synthesis 23 starting from arm 3 ’ priming portions 21 and template sequences 19 at a temperature between 15°C and 50°C. Subsequently, the hot-start polymerase 14, activated at a temperature higher than 90°C, e.g. at 95°C or 98°C, performs amplicon amplification during thermal cycling. This allows both polymerase activities to occur in the same reaction solution and eliminates the need for separate buffers for different polymerases. Examples of isothermal polymerase enzymes that can be used may be for instance: IsoPol, IsoPol™ SD+ -, IsoPol® BST+, IsoFast™ Bst Polymerase, Bst 2.0 WarmStart® DNA Polymerase, Bsu DNA Polymerase, Large Fragment, phi29 DNA Polymerase, Bst DNA Polymerase. Isothermal polymerase enzymes may be used at a final concentration in the well of e.g. 2 U.

Examples of hot-start polymerase enzymes that can be used may be for instance: PerfectStart® Taq DNA Polymerase, VeriFi™ Hot Start Polymerase, Phusion Hot Start II DNA Polymerase, Phire Hot Start II DNA Polymerase, GoTaq® MDx Hot Start Polymerase, EpiMark® Hot Start Taq DNA Polymerase, Q5® Hot Start High-Fidelity DNA Polymerase, Hot Start Taq DNA Polymerase, Hot-Start Taq DNA Polymerase. Hot-start polymerase enzymes may be used at a final concentration in the well of e.g. 0.4 U.

Nucleic Acid Probes for Real-time PCR Amplification Fluorescence Readout

The nucleic acid probes used for real-time PCR amplification fluorescence readout could be labeled with CY5.5 or Alexa 680 fluorophores, which are far-red fluorophores highly detectable in blood serum. Of course, other fluorophores may be used, depending on the type of sample. These fluorophores enable sensitive and accurate detection of the amplified nucleic acid during PCR cycling. Alternatively, the fluorescence read-out of the real time PCR of both amplification and melting curve can be provided by using intercalating dyes, in particular including e.g. Syto 9.

EXAMPLES AND EXPERIMENTAL DATA

Applicant carried out experimental tests to show that embodiments described herein may be used for the detection of different target molecules, such as protein antigens, small molecules and antibodies, in crude and also purified samples, The methods demonstrated to detect target analytes in up to 10% of blood serum and raw bacteria liquid culture.

These methods were applied for the detection of different analyte classes, by exploiting different binding moieties (e.g. antigens, small molecules, antibodies, aptamers), reported in Table 1.

Table 1 : Binding moieties classes and detected target analytes

Target Analyte Binding Moiety Experimental Application

Antibody PNA-peptide Trastuzumab detection

Antibody Anti-antibody Trastuzumab detection

Antigen Antibody TNF-alpha detection

Antibody Antigen Digoxigenin detection

Antibody Aptamer Trastuzumab detection

Antibody detection

In a first exemplary' application, Trastuzumab, a monoclonal antibody used for breast cancer treatment, was quantitatively detected directly in crude biological samples, using three binding moieties different versions as possible implementing examples.

In a first version, Trastuzumab detection was possible using small PNA- peptides as system binding moieties. The system was able to detect and distinguish different concentration of Trastuzumab, with a total of at least 10 PCR cycles of shift between 0 pg/ml and 20 pg/ml (100 nM) of Trastuzumab (Fig. 8). Different amount of Trastuzumab diluted in blood serum at known concentrations ranging from 0 to 100 nM were detected. As expected, a 3 PCR cycles-shift was observed for each 10-fold dilution (fluorescence threshold 100,000 delta Rn). The right panel in Fig. 8 reports mean and standard deviations of 10 experimental replicates.

In a second version, anti-antibodies were exploited as binding moieties for antibody detection; specifically, specific anti-Trastuzumab antibodies directly conjugated to the nucleic acid oligos have been used.

In a third version, Trastuzumab detection was successfully achieved using antitrastuzumab DNA aptamers as binding moieties; aptamers were matching through complementary sequences to nanosensor arms.

In a second exemplary application of antibody detection, the method of the present disclosure was successfully used to detect anti-Digoxigen antibody, through the conjugation of digoxigenin molecule with the nucleic acid oligos that anneal with the nucleic acid nanosensor arms (Fig. 9). Different amounts of anti- digoxigenin antibodies diluted in bacterial culture at known concentrations, ranging from 0 to 100 nM were detected (n-6 replicates). As expected, a 3 PCR cycles-shift was observed for each 10-fold dilution.

Antigen/protein detection

In a second exemplary application, Adalimumab and Infliximab, two monoclonal antibodies used for chronic disease treatment, were used as nanosensor binding moieties. They were conjugated to the nucleic acid oligos that anneal to DNA arm structures for TNF-alpha protein detection. It was possible to detect TNF-alpha protein using two Adalimumab conjugates, or two Infliximab conjugates, or one Adalimumab and one Infliximab conjugate for each DNA arm, obtaining a shift of about 9 PCR cycles between 0 and 100 nM of TNF-alpha (Fig. 10). Different amounts of TNF-alpha diluted in bacteria culture at known concentrations, ranging from 0 to 100 nM was detected (n=8 replicates). As expected, about a 3 PCR cycles-shift was observed for each 10-fold dilution.

In conclusion, such experimental data and examples showed that methods according to the present disclosure may be useful to detect different targets and also in various complex biological matrices, without requiring any purification step. The methods according to the present disclosure can be implemented in a user-friendly system with a ven high sensitivity' thus opening new scenarios in the field of biomarker discovery' and early diagnosis.

While the foregoing is directed to embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

1. A method for one-step highly sensitive detection of one or more analytes in samples, the method occurring in a one-pot PCR machine system, the method comprising:

- providing a reaction solution comprising: a buffer solution, single stranded nucleic acid arms (11), at least one polymerase enzyme (12; 13, 14), nucleic acid primers (15) complementary to reverse complement of arm template sequence (19) portions, possibly fluorescent intercalating dyes and/or fluorescent probes, possibly additives and stabilizers, each single stranded nucleic acid arm (11) containing a binding portion (16) for binding moieties (17) capable of simultaneously binding nucleic acid sensor structures and target analytes (18) of interest, a template sequence (19), a partially complementary anchor sequence (20), and a 3’ priming portion (21);

- adding an amount of a sample to said reaction solution;

2. The method of claim 1, wherein the binding portion (16) is configured to directly and chemically conjugate with at least one binding moiety (17) or anneal, directly or indirectly, through hydrogen bonds, such that the binding moieties (17) are chemically bound to the arms (11) or the binding moieties ( 17) are annealed, directly or indirectly, to the nucleic acid arms (11) through hydrogen bonds.

3. The method of claims 1 or 2, wherein the template sequence (19) serves as the polymerase template during said isothermal phase and generates double-stranded nucleic acid amplicons detectable and discriminable, because they are characterized by a distinctive melting temperature and/or associated to different fluorescent probe complementary sequences.

4. The method of any of claims 1 to 3, wherein the arm anchor sequence (20) is partially or totally complementary' to the anchor sequence (20) of another nucleic acid arm (11).

5. The method of any of claims 1 to 4, wherein the 3’ priming portion (21) exposes a 3’ OH chemical group for polymerase binding.

6. The method of any of claims 1 to 5, wherein each nucleic acid arm (11) is coupled to one or more binding moiety' (17), allowing target analyte (18) binding with high affinity and specificity.

7. The method of any of claims 1 to 6, wherein the binding moiety (17) is selected from the group consisting of antibodies, single-chain antibodies, antibody variable regions, affibodies, proteins, DNA/RNA aptamers, peptide nucleic acids (PNAs), antigens or small molecules.

8. The method of any of claims 1 to 7, wherein the arms (11) further comprise chemical modifications (22) to prevent arm self-recognition and nucleic acid secondary structure formation.

9. The method of any of claims 1 to 8, wherein the isothermal phase occurs at a temperature ranging from 15°C to 50°C.

10. The method of any of claims 1 to 9, wherein nucleic acid polymerase (12; 13) initiates the synthesis of the second strand of nucleic acid (23) using the template sequence (19) as a guide generating amplicon nucleic acid double stranded templates.

11. The method of any of claims 1 to 10, wherein nucleic acid amplicons undergo exponential amplification through PCR where nucleic acid primers (15) are able to recognize and bind the nucleic acid neo-synthetized portion of the amplicon.

12. The method of any of claims 1 to 11, wherein said reaction solution comprises an isothermal nucleic acid polymerase (13) for the isothermal nucleic acid synthesis and a hot-start nucleic acid polymerase (14) for the PCR amplification.

13. The method of claim 12, wherein said isothermal nucleic acid polymerase (13) works at temperature ranging from 15 °C to 50°C to synthesize nucleic acid and said hot-start nucleic acid polymerase (14) is activated as a consequence of an increase of temperature.

14. The method of any of claims 1 to 11, wherein the isothermal nucleic acid synthesis and PCR amplification phases are performed by the same polymerase enzyme (12), with differential activities at defined temperatures, wherein said same polymerase enzyme (12) has low activity' during the isothermal phase and high activity over a higher temperature, enabling both reactions to occur through a unique enzyme in the same buffer.

15. The method of any of claims 1 to 14, the method comprising:

- binding of binding moieties (17) to the target analyte (18) of interest;

- increased proximity of nucleic acid arms (11) due to binding moiety-analyte interaction;

- annealing of arm anchor sequences (20) and initiation of polymerase binding to 3’ priming portion (21);

- synthesis of the second strand of nucleic acid (23) using the template sequence (19) as a guide in the isothermal polymerase polymerization, generating amplicon nucleic acid double stranded templates;

- selective amplification of nucleic acid amplicons through Polymerase Chain Reaction (PCR) through nucleic acid primers (15) which are able to recognize and bind the nucleic acid neo-synthetized portion of the amplicon;

- measuring dyes or probes related fluorescence during real-time PCR cycling, which is correlated, e.g. directly proportional, to the amount of amplified nucleic acid amplicons, which are directly proportional to target analyte amount;

- analyzing post-PCR melting curves, and/or probe fluorescence signals, to discriminate multiple target analytes (18) in case of multiple target analytes (18) detection;

- analyzing post-PCR melting curves, and/or probe fluorescence signals intensity', to quantify of target analyte(s) (18), in case of quantitative detection.

16. The method of any of claims 1 to 15, the method comprising:

- providing a buffer solution containing water, possibly additives and stabilizers, such as HEPES, MgCh, dNTPs, TrisHCL, NaCl, KC1 and having a pH ranging from 6 to 12, fluorescent intercalating dyes and/or fluorescent probes;

- the binding moieties (17) being selected from the group consisting of antibodies, single-chain antibodies, antibody variable regions, affibodies, proteins, DNA/RNA aptamers, peptide nucleic acids (PNAs), small molecules and antigens;

- the arms (11) being possibly chemically modified with one or more 2- aminopurine-2’-deoxyriboside (A*), 2 ’-deoxy-2 -thiothymidine (T*), 2’- deoxyinosine (G*) and N4-ethy 1-2’ -deoxy cytidine to reduce self-annealing and non-specific annealing interactions;

17. The method of any of claims 1 to 16, wherein multiple binding moieties (17) are coupled to a single nucleic acid arm (11) to favor agglutination mechanisms.

18. The method of any of claims 1 to 17, wherein two nucleic acid arms (11) have anchor sequences (20) complementary to two distinct regions of a third arm, enabling a higher-specific interaction only when the target analyte (18) of interest is present.

19. The method of any of claims 1 to 18, wherein said reaction solution comprises fluorescent intercalating dyes and/or probes.

20. The method of any of claims 1 to 19, wherein nucleic acid probes used for realtime PCR amplification fluorescence readout are labeled with fluorophores suitable for detection in blood serum, in particular CY5.5 or Alexa 680 fluorophores, which are far-red fluorophores highly detectable in blood serum.

21. The method of any of claims 1 to 19, wherein the fluorescence read-out of the real time PCR of both amplification and melting curve can be provided by using intercalating dyes, in particular including Syto 9.

22. The method of any of claims 1 to 21, wherein adding an amount of a sample to said reaction solution includes mixing said amount of sample with said reaction solution, wherein if a target analyte (18) of interest is present, it binds to the binding moieties ( 17), inducing, by proximity, the interaction of nucleic acid arms (11); then polymerase enzyme (12; 13) initiates isothermal second-strand nucleic acid synthesis from the 3' priming portions (21) at a defined temperature; wherein during PCR thermal cycling, nucleic acid primers (15) recognize portions of the newly synthesized nucleic acid, allowing PCR amplification by means of polymerase (12; 14); wherein dyes or probes related fluorescence is measured during real-time PCR cycling, and it is correlated, e.g. directly proportional, to the amount of amplified nucleic acid amplicons, which are directly proportional to target analyte amount; wherein post-PCR melting curves, and/or probe fluorescence signals, are analyzed to discriminate multiple target analytes ( 18) in case of multiple target analytes (18) detection, and/or wherein post-PCR melting curves, and/or probe fluorescence signals intensity are analyzed to quantify target analyte(s) (18), in case of quantitative detection.

23. The method of any of claims 1 to 22, wherein the nucleic acid binding moieties (17) are chemically modified at 3’ end with one or more of 3' dideoxy-C, 3' Inverted dT, 3' C3 spacer, 3' amino, and 3' phosphory lation to avoid polymerase binding aiming to avoid RNA/DNA polymerase binding.

24. The method of any of claims 1 to 23, wherein one or more parts of said reaction solution can be mechanically separated from the other parts by means of physical layers or jellifying agents that can be disrupt by chemical and/or physical variation, allowing complementation of the reaction solution.

25. The method of any of claims 1 to 24, wherein said reaction solution can be provided in a freeze-dried or lyophilized format.

26. A kit for one-step highly sensitive detection of one or more analytes in samples, comprising:

- a buffer solution possibly containing also fluorescent dyes and/or probes, possibly additives and stabilizers;

- single stranded nucleic acid arms (11), each single stranded nucleic acid arm (11) containing a binding portion (16) for binding moieties (17) capable of simultaneously binding nucleic acid sensor structures and target analytes (18) of interest, a template sequence (T9), a partially complementary anchor sequence (20), and a 3’ priming portion (21);

- at least one polymerase enzyme (12; 13, 14);

- nucleic acid primers (15) complementary to reverse complement of arm template sequence (19) portions;

- fluorescent dyes and/or probes;

- possibly a PCR machine.

27. The kit according to claim 26, wherein said buffer solution, single stranded nucleic acid arms (11), at least one polymerase enzyme (12; 13, 14), nucleic acid primers (15), fluorescent dyes and/or probes, can be provided in a freeze-dried or lyophilized format.