No abstract

The recent surge of effort in nucleic acid-based electrochemical (EC) sensors has been fruitful, and some have even shown real-time quantification of drugs in the blood of living animals. Yet there remains a need for more generalizable EC platforms for the detection of multiple classes of clinically relevant targets. Our group has recently reported a nucleic acid nanostructure that permits simple, economical, and generalizable EC readout of a wide range of analytes (small molecules, peptides, large proteins, or antibodies). The DNA nanostructure is built through on-electrode enzymatic ligation of three oligonucleotides for attachment, binding, and signaling. However, the signaling mechanism predominantly relies on tethered diffusion of methylene blue at the electrode surface, limiting the detection of larger proteins that have no readily available small molecule binding partners. In this study, we adapted the nanostructure sensor to quantify larger proteins in a more generic manner, through conjugating the proteins minimized antibody-binding epitope to the central DNA strand of the nanostructure (DNA-peptide conjugate). This concept was verified using creatine kinase (CK-MM), an important biomarker of muscle damage, myocardial infarction, overexertion/rhabdomyolysis, or neuromuscular disorders where clinical outcomes could be improved with rapid sensing. DNA-epitope conjugates permitted a competitive immunoassay protocol at the electrode surface for quantifying CK protein. Square-wave voltammetry (SWV) signal suppression was proportional to the amount of surface-bound antibody with a limit of detection (LOD) of 5 nM and a response time as low as 3 minutes, and displacement of antibody by native CK-MM protein analyte could also be assayed. CK was quantified from the LOD of 14 nM up to 100 nM, overlapping well with the normal (non-elevated) human clinical range of 3 37 nM, and the sensor response was validated in 98% human serum. While a need for improved DNA-epitope conjugate purification was identified, overall this approach not only allows the detection of a generic protein- or peptide-binding antibody, but it also should facilitate future quantitative EC readout of various clinically relevant protein analytes that were previously inaccessible to EC techniques.

Antibodies have long been recognized as clinically relevant biomarkers of disease. The onset of a disease often stimulates antibody production at low quantities, making it crucial to develop sensitive, specific, and easy-to-use antibody assay platforms. Antibodies are also extensively used as probes in bioassays, and there is a need for simpler methods to evaluate specialized probes such as antibody-oligonucleotide (AbO) conjugates. Previously, we have demonstrated that thermofluorimetric analysis (TFA) of analyte-driven DNA assembly can be leveraged to detect protein biomarkers using AbO probes. A key advantage of this technique is its ability to circumvent autofluorescence arising from biological samples, which otherwise hampers homogenous assays. The analysis of differential DNA melt curves (dF/dT) successfully distinguishes the signal from background and interferences. Expanding the applicability of TFA further, here-in we demonstrate a unique proximity based TFA assay for antibody quantification which is functional in 90% human plasma. We show that conformational flexibility of the DNA-based proximity probes is critically important for optimal performance in these assays. To promote stable, proximity-induced hybridization of the short DNA strands, substitution of polyethylene glycol (PEG) spacers in place of ssDNA segments led to improved conformational flexibility and sensor performance. Finally, by applying these flexible spacers to study AbO conjugates directly, we validate this modified TFA approach as a novel tool to elucidate the probes valency, clearly distinguishing between monovalent and multivalent AbOs and reducing the reagent amounts by 12-fold

Antibodies have long been recognized as clinically relevant biomarkers of disease. The onset of a disease often stimulates antibody production at low quantities, making it crucial to develop sensitive, specific, and easy-to-use antibody assay platforms. Antibodies are also extensively used as probes in bioassays, and there is a need for simpler methods to evaluate specialized probes such as antibody-oligonucleotide (AbO) conjugates. Previously, we have demonstrated that thermofluorimetric analysis (TFA) of analyte-driven DNA assembly can be leveraged to detect protein biomarkers using AbO probes. A key advantage of this technique is its ability to circumvent autofluorescence arising from biological samples, which otherwise hampers homogenous assays. The analysis of differential DNA melt curves (dF/dT) successfully distinguishes the signal from background and interferences. Expanding the applicability of TFA further, here-in we demonstrate a unique proximity based TFA assay for antibody quantification which is functional in 90% human plasma. We show that conformational flexibility of the DNA-based proximity probes is critically important for optimal performance in these assays. To promote stable, proximity-induced hybridization of the short DNA strands, substitution of polyethylene glycol (PEG) spacers in place of ssDNA segments led to improved conformational flexibility and sensor performance. Finally, by applying these flexible spacers to study AbO conjugates directly, we validate this modified TFA approach as a novel tool to elucidate the probes valency, clearly distinguishing between monovalent and multivalent AbOs and reducing the reagent amounts by 12-fold

Biomarker detection is vitally important for disease diagnosis. Critical biomarkers which are present in ultra-low concentrations in body fluids have been detected with immunoassays such as ELISA and improved versions such as Alpha-LISA and digital ELISA. Although these techniques have fM-pM range detection limits, the requirement of special instruments, reagents, and labor-intensive workflows have directed researchers to develop simpler assay techniques. The ideal alternative assay will be cost effective, with simple mix-and-read workflows, yet will not compromise its specificity and sensitivity. Analyte-driven hybridization of short complementary DNA strands have gained substantial attention for these reasons. Molecular pincer assays and proximity ligation assays (PLA) have successfully demonstrated sensitivities comparable to ELISA, using antibody-DNA conjugates. These techniques rely on fluorescence detection, which is sensitive and specific, but is challenging in terms of miniaturization and point-of-care (POC) use. Electrochemical (EC) detection, on the contrary, can be easily miniaturized and employs simple instrumentation with high sensitivity. We have previously introduced the electrochemical proximity assay (ECPA), where insulin and thrombin have been detected at fM or pM concentrations, respectively. ECPA leverages target driven hybridization of a methylene blue (redox reporter) tagged DNA to a thiolated DNA attached to a gold surface. In this work, we have developed a novel assay for antibody detection, leveraging the ECPA concept. Here, simultaneous binding of recognition elements to paratopes of the antibody results in hybridization of methylene blue (MB) tagged DNA to thiolated-DNA (see schematic), and generated current is proportional to the antibody concentration. Using free-solution, thermoflourimetric analysis (TFA) of similar strands, we showed that probe flexibility is very important for sensor response, where ssDNA with polyethylene glycol (PEG) linkers improved sensitivities significantly. Using information from TFA, we used the same modification and demonstrated that analyte-dependent EC current was increased after ssDNA attachments to the electrode were substituted with more flexible PEG linkers (see data). This modification was introduced to both the MB-DNA and thiolated-DNA, allowing direct antibody sensing of 45 nM. With optimization in the near future, this new ECPA-based antibody sensor should be useful for clinical monitoring of the immune response. Figure 1