Bispecific Antibody Detection Using Antigen-Conjugated Synthetic Nucleic Acid Strands.

We report here the development of two different sensing strategies based on the use of antigen-conjugated nucleic acid strands for the detection of a bispecific antibody against the tumor-related proteins Mucin1 and epidermal growth factor receptor. Both approaches work well in serum samples (nanomolar sensitivity), show high specificity against the two monospecific antibodies, and are rapid. The results presented here demonstrate the versatility of DNA-based platforms for the detection of bispecific antibodies and could represent a versatile alternative to other more reagent-intensive and time-consuming analytical approaches.

Synthetic Antigen-Conjugated DNA Systems for Antibody Detection and Characterization.

Antibodies are among the most relevant biomolecular targets for diagnostic and clinical applications. In this Perspective, we provide a critical overview of recent research efforts focused on the development and characterization of devices, switches, and reactions based on the use of synthetic antigen-conjugated DNA strands designed to be responsive to specific antibodies. These systems can find applications in sensing, drug-delivery, and antibody-antigen binding characterization. The examples described here demonstrate how the programmability and chemical versatility of synthetic nucleic acids can be used to create innovative analytical tools and target-responsive systems with promising potentials.

Electrochemical Cell-Free Biosensors for Antibody Detection.

We report here the development of an electrochemical cell-free biosensor for antibody detection directly in complex sample matrices with high sensitivity and specificity that is particularly suitable for point-of-care applications. The approach is based on the use of programmable antigen-conjugated gene circuits that, upon recognition of a specific target antibody, trigger the cell-free transcription of an RNA sequence that can be consequently detected using a redox-modified probe strand immobilized to a disposable electrode. The platform couples the features of high sensitivity and specificity of cell-free systems and the strength of cost-effectiveness and possible miniaturization provided by the electrochemical detection. We demonstrate the sensitive, specific, selective, and multiplexed detection of three different antibodies, including the clinically-relevant Anti-HA antibody.

Programmable Cell-Free Transcriptional Switches for Antibody Detection.

We report here the development of a cell-free in vitro transcription system for the detection of specific target antibodies. The approach is based on the use of programmable antigen-conjugated DNA-based conformational switches that, upon binding to a target antibody, can trigger the cell-free transcription of a light-up fluorescence-activating RNA aptamer. The system couples the unique programmability and responsiveness of DNA-based systems with the specificity and sensitivity offered by in vitro genetic circuitries and commercially available transcription kits. We demonstrate that cell-free transcriptional switches can efficiently measure antibody levels directly in blood serum. Thanks to the programmable nature of the sensing platform, the method can be adapted to different antibodies: we demonstrate here the sensitive, rapid, and cost-effective detection of three different antibodies and the possible use of this approach for the simultaneous detection of two antibodies in the same solution.

Programmable, Multiplexed DNA Circuits Supporting Clinically Relevant, Electrochemical Antibody Detection.

Current health emergencies have highlighted the need to have rapid, sensitive, and convenient platforms for the detection of specific antibodies. In response, we report here the design of an electrochemical DNA circuit that responds quantitatively to multiple specific antibodies. The approach employs synthetic antigen-conjugated nucleic acid strands that are rationally designed to induce a strand displacement reaction and release a redox reporter-modified strand upon the recognition of a specific target antibody. The approach is sensitive (low nanomolar detection limit), specific (no signal is observed in the presence of non-targeted antibodies), and selective (the platform can be employed in complex media, including 90% serum). The programmable nature of the strand displacement circuit makes it also versatile, and we demonstrate here the detection of five different antibodies, including three of which are clinically relevant. Using different redox reporters, we also show that the antibody-responsive circuit can be multiplexed and responds to different antibodies in the same solution without crosstalk.