EP-4739793-A1 - DNA-ENZYME CONJUGATES FOR SPECIFIC AND ULTRA-SENSITIVE DETECTION OF A TARGET ANALYTE, AND METHOD THEREOF

Abstract

The present invention relates to a method for the detection and quantification of target analytes such as, for example, antibodies, proteins, enzymes, small molecules, or ions directly in complex matrices of samples, with very high specificity and sensitivity. In particular, said method is preferably based on the use of a programmable DNA-based circuit which, in the presence of a specific target analyte, triggers the release of an enzyme-conjugated DNA filament through a DNA filament displacement reaction. Said enzymeconjugated DNA filament is then detected using suitable optical, electrochemical, or enzymatic methods.

Inventors

• RICCI, FRANCESCO
• RANALLO, Simona
• DIAZ FERNANDEZ, Ana

Assignees

• Consorzio Interuniversitario Istituto Nazionale di Biostrutture e Biosistemi
• Ricci, Francesco
• Ranallo, Simona
• Diaz Fernandez, Ana

Dates

Publication Date

20260513

Application Date

20240703

Claims (9)

1. 1 . A method for the detection and quanti fication of target molecular analytes , with high sensitivity and speci ficity, directly in complex matrices of samples , said method comprising the following phases : - activating a DNA-based circuit through induction by said target analytes , giving a functional bi- molecular complex capable of forming a link with a DNA-enzyme conj ugated duplex ; - forming said link between said functional bi- molecular complex and said DNA-enzyme conj ugated duplex ; - triggering a filament displacement reaction giving the release of a DNA-enzyme conj ugated filament , wherein the amount of DNA-enzyme conj ugated filament released is proportional to the concentration of the target analyte which induces the displacement reaction; - detecting and quanti fying the released filament , through known electrochemical , or optical , or colorimetric methods , in the presence of the related enzymatic substrate .
2. 2 . The method according to claim 1 , wherein said target molecular analytes are selected from the group comprising, or consisting of, antibodies and/or proteins and/or enzymes and/or small molecules and/or ions or others .
3. 3. The method according to claim 2, wherein said molecular analytes are selected from antibodies.
4. 4. The method according to any one of claims from 1 to 3, wherein said complex matrices of samples are selected from the group comprising, or consisting of, plasma, or serum, or blood, or saliva, or sweat, or analogues .
5. 5. The method according to any one of the preceding claims, wherein said DNA-based circuit is formed of two DNA filaments conjugated with recognition elements of the analyte.
6. 6. The method according to any one of the preceding claims, for simultaneous and independent detection of multiple different target analytes.
7. 7. The method according to any one of the preceding claims, wherein the sensitivity of the method is within the low pico-molar or lower (1-10 fM) range.
8. 8. The method according to any one of the preceding claims, the duration of which is less than 70 minutes.
9. 9. Use of the method according to any one of the preceding claims for detecting and quantifying target molecular analytes, with high sensitivity and specificity, directly in complex matrices of samples at the point of care.

Description

DESCRIPTION Annexed to Patent Application for INDUSTRIAL INVENTION entitled : "DNA-ENZYME CONJUGATES FOR SPECIFIC AND ULTRA-SENSITIVE DETECTION OF A TARGET ANALYTE, AND METHOD THEREOF" By: Ana Diaz Fernandez, Calle Pardies, 8, 33430, Candas (ES) {25% property share) ; Simona Ranallo, Via Cerenzia, 47, 00178 Roma (IT) , {25% property share) ; Francesco Ricci, Via Cimone, 132, 00141 Roma (IT) , {48% property share) ; Istituto Nazionale Biostrutture e Biosistemi I.N.B.B., Viale delle Medaglie d'Oro, 305, 00136 Roma (IT) , {2% property share) . Appointed Inventors: Ana DIAZ FERNANDEZ, Simona RANALLO, Francesco RICCI . DESCRIPTION Technical Field of the Invention The present invention relates to a method for the detection and quantification of target analytes such as, for example, antibodies and/or proteins and/or enzymes and/or small molecules and/or ions, or other ones, with very high specificity and sensitivity, directly in complex matrices of samples. In particular, said method is preferably based on the use of a programmable DNA-based circuit which, in the presence of a specific target analyte, triggers the release of an enzyme-conjugated DNA filament through a DNA filament displacement reaction. Said enzyme-con ugated DNA filament is then detected using suitable optical, electrochemical, or enzymatic methods. Description of the Prior Art The detection of target analytes such as, for example, antibodies and/or proteins and/or enzymes and/or small molecules and/or ions, or so on, plays a fundamental role in the diagnosis of a large number of diseases, such as, for example, infections, autoimmune diseases, and oncologic diseases. In addition to being important as disease markers, the antibodies (like, for example, monoclonal and/or bispecific antibodies) and proteins are being increasingly used also as drugs within therapeutic contexts [1] . As is well known in the art, standard analytical methods employed for detecting such types of markers are, for example, based on complex processes requiring multiple wash steps, or on the use of multiple reagents (like, e.g., the enzyme-linked immunosorbent assay, commonly referred to as ELISA) , or on qualitative or semi- quantitative methods like, for example, lateral flow tests (usually, LET) . ELISA assays are both sensitive and quantitative, but require time-consuming measurements to be carried out in a laboratory through the use of special instruments, so that the applicability of this technique for "point-of-care" diagnostics is rather limited. In turn, lateral flow tests are fast and easy to use, but their essentially qualitative (or, in the best of cases, semi- quantitative) nature limits the possibility to benefit from quantitative information, which may sometimes be very important. In recent times, sensors based on synthetic DNA have acquired increasing importance as promising analytical tools for fast, economical and quantitative measurement of a wide range of analytes, such as, for example, nucleic acids, proteins, antibodies, and small molecules. The unique characteristics of synthetic nucleic acids, which include low cost, easy synthesis, and high capability of predicting base-pair interaction, have been exploited for programming switches, circuits and devices that can be employed in applications for detecting molecular targets and/or for releasing drugs. In particular, the chemical versatility of synthetic nucleic acids has been exploited to conjugate different molecules, such as, for example, antigens, small molecules, peptides, proteins, enzymes, antibodies, etc., as well as "tag" (or "label") molecules capable of providing a signal that can be measured (e.g., using optical or electrochemical methods) , in order to develop devices which can detect and monitor a wide range of molecular targets, including, for example, antibodies [2] . As a demonstration of the importance of such systems for clinical applications, several classes of DNA-based sensors have been developed so far in the industry, wherein the detection of specific antibodies is based on optical and electrochemical methods [3'4] . In a recent embodiment, the present inventors have proposed a novel programmable approach for the detection of antibodies and/or other analytes, which employs a DNA-based circuit. Said circuit is based on the use of a so called reaction of displacement of a DNA filament, and has been engineered in such a way as to induce the release of a DNA filament modified with an optical or electrochemical reporter only in the presence of a specific target antibody. More particularly, said circuit uses three synthetic elements: a DNA duplex and two single-stranded DNAs conjugated with an antigen and designed in such a way that, upon formation of a divalent bond with a target antibody, they come in close vicinity to each other and, as a consequence, can hybridize to form a functional bi-molecular complex. Said resulting functional bi-molecular complex, as it binds with the