US-20260126433-A1 - Cell-Free Transcriptional Electrochemical Biosensors for Detecting Molecular Analytes, and Method Thereof

US20260126433A1US 20260126433 A1US20260126433 A1US 20260126433A1US-20260126433-A1

Abstract

The present invention relates to a cell-free transcriptional electrochemical biosensor and to the use of the same for detecting specific molecular analytes, such as specific antibodies, proteins, small molecules, nucleic acids, and derivatives thereof, in complex arrays of biological samples, such as plasma, serum, blood, saliva, sweat, and the like, wherein said biosensor is based on the activation of the transcription of a specific RNA strand, induced by recognition with the analyte. The invention further relates to a method for the detection of specific molecular analytes in complex arrays of biological samples, said method being based on the use of said cell-free transcriptional electrochemical biosensor.

Inventors

Francesco Ricci
Simona Ranallo
Sara Bracaglia

Assignees

Francesco Ricci
Simona Ranallo
Sara Bracaglia
CONSORZIO INTERUNIVERSITARIO ISTITUTO NAZIONALE DI BIOSTRUTTURE E BIOSISTEMI

Dates

Publication Date: 20260507
Application Date: 20231017
Priority Date: 20221018

Claims (8)

1 . A method for the electrochemical detection, with high sensitivity and specificity, of target molecular analytes directly in complex sample arrays, said method comprising: the activation of an initially incomplete, i.e., inactive, DNA gene circuit, induced by a target analyte, wherein said analyte is selected from the group comprising, or consisting of, antibodies, proteins, small molecules, nucleic acids, derivatives thereof; the transcription of a final RNA sequence by said activated DNA gene circuit; the detection of said transcribed RNA sequence by means of an electrochemical biosensor.
2 . The method according to claim 1 , wherein said molecular analytes are selected from antibodies.
3 . The method according to claim 1 , wherein said complex sample arrays are selected from the group comprising, or consisting of, plasma, serum, blood, saliva, sweat, and the like.
4 . The method according to claim 1 , wherein said activation of said gene circuit is carried out by forming a bimolecular complex between it and a pair of antigen-conjugated DNA strands following the formation of a bivalent bond between the target analyte (preferably, the antibody) and the two antigens.
5 . The method according to claim 1 , wherein said transcription of a final RNA sequence is carried out by means of a RNA polymerase and nucleotides.
6 . The method according to claim 1 , wherein said electrochemical biosensor is constituted by a screen-printed silver electrode on which a redox probe complementary to the transcribed RNA sequence has been previously immobilized.
7 . (canceled)
8 . (canceled)

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a cell-free transcriptional electrochemical biosensor and to the use of the same for detecting specific molecular analytes, such as, for example, specific antibodies, proteins, small molecules, nucleic acids, or derivatives thereof, in complex arrays of biological samples, such as, for example, plasma, serum, blood, saliva, sweat, and the like, wherein said biosensor is based on the activation of the transcription of a specific RNA strand, induced by recognition with the analyte. The invention further relates to a method for the detection of specific molecular analytes in complex arrays of biological samples, said method being based on the use of said cell-free transcriptional electrochemical biosensor. DESCRIPTION OF THE KNOWN ART Antibody detection is important in several clinical contexts, in that it provides useful information on present and past infections, and can also provide information about the clinical outcomes when treating and monitoring autoimmune diseases and cancer. Reference assays and methods, also called gold standards, which are commonly used for detecting antibodies and proteins, are selectable, for example, among the following: a) ELISA (Enzyme Linked Immunosorbent Assay) and b) LFT (Lateral Flow Test, or “lateral flow immunochromatographic assay”, or “rapid test”). ELISA assays are very sensitive, and are widely used for measuring antibodies, antigens and clinically relevant chemical substances. However, they are based on a multi-phase procedure, the execution of which may take a long time, sometimes longer than actually tolerable. Moreover, ELISA tests require many reagents, and this often makes them very costly. On the other hand, lateral flow immunological tests (LFT) are used in so-called point-of-care diagnostics because they are cheap and fast. However, they can only provide qualitative or semi-quantitative results. By way of example, WO 2017/147486 describes the electrochemical detection of DNA amplification sequences, thanks to the use of a simple disposable electrochemical sensor suitable for measuring a DNA/RNA sequence by using methylene blue. The subject matter of WO 2017/147486 is not an example of cell-free technology. In recent times, the so-called “synthetic biology” has been proposed as an alternative technique which can be used in order to overcome the above-mentioned limitations. Synthetic biology is based on a biomolecular engineering approach for in-vitro reproduction of processes that would otherwise only occur within cells. Actually, there is an ever increasing need for expanding the medical monitoring and diagnostics of human diseases and, from this point of view, the devices of synthetic biology might provide diagnostic methods with new potentialities, e.g. by creating sensors with new functions, thereby broadening the range of testable analytes (or targets) and improving the sensitivity and specificity of the same. The so-called cell-free biosensors utilize several biological components, such as, for example, nucleic acids (DNA) and proteins (enzymes). DNA acts as a template for the production of specific RNA sequences, while enzymes (e. g. a polymerase) catalyze the process of formation of RNA (transcription) and/or of other proteins (translation). In cell-free biosensors, the recognition of the target analyte by a DNA sequence induces the transcription or the expression of the final biomolecule (called output biomolecule; for example, a RNA or a protein). In recent years, cell-free biosensors have been developed which can detect specific sequences of messenger RNA (mRNA), small molecules and proteins, including antibodies (by the way, also due to the researches by the present inventors)(1-6). Recently, Patino Diaz Aitor et al. have proposed a different approach that exploits a cell-free transcription system for detecting specific antibodies, as described in their article entitled: “Programmable cell-free transcriptional switches for antibodies detection”, Journal of The American Chemical Society, vol. 144, n. 13, 22 Mar. 2022, pp. 5820-5826 (cited herein for reference and available in its entirety at the address https://pubs.acs.org/doi/10.1021/jacs.1c11706). This article describes a programmable transcriptional switch based on a gene circuit designed to adopt a stem-loop conformation that prevents the transcription of an aptamer to a RNA capable of promoting a fluorescence signal (so called light-up signal), because the promoter region (so called promoter) is hidden inside this stem-loop structure and cannot be recognized by the RNA polymerase. In order to obtain a change in the conformation of the gene circuit induced by the antibody, two DNA strands conjugated with the specific antigen are used which, following the formation of a divalent bond with a target antibody, are brought into close mutual proximity (are co-located) and can hybridize to form a functional bimolecular complex. This co