Systems for detecting and quantifying nucleic acids
Inventors
Wang, Gangli • Wang, Tanyu • Merlin, Didier
Assignees
Georgia State University Research Foundation Inc • US Department of Veterans Affairs
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Publication Number
US-11156582-B2
Publication Date
2021-10-26
Expiration Date
2036-07-06
Abstract
This invention is based, in part, on our discovery of an essentially one-step, label-free system comprising a sensing unit having a redox current reporter and a nucleic acid sequence complementary to that of a target nucleic acid of interest or sufficiently complementary to that of the target nucleic acid or a sequence therein to specifically bind the target nucleic acid. The sensing unit is bound to an electroconductive substrate (e.g., a carbon- or metal-containing microelectrode (e.g., a gold microelectrode)), and the system includes a signal amplification mechanism that does not rely upon a redox enzyme and thereby overcomes a fundamental limitation of microelectrode DNA sensors that fail to generate detectable current in the presence of only small amounts of a target nucleic acid.
Core Innovation
This invention provides an essentially one-step, label-free system for detecting and quantifying target nucleic acids, particularly small RNAs such as microRNAs (miRNAs). The system comprises a sensing unit that includes a redox current reporter linked to a nucleic acid sequence complementary or sufficiently complementary to the target nucleic acid. The sensing units are bound to an electroconductive substrate, such as a carbon- or metal-containing microelectrode (e.g., gold microelectrode). The system includes a signal amplification mechanism that does not rely on a redox enzyme but instead utilizes a co-reactant (reductant or oxidant) in solution. Upon hybridization of the sensing unit's nucleic acid to the target nucleic acid, the redox current reporter is freed to cyclically react with the substrate and the co-reactant, resulting in in situ signal amplification through repeated electron transfer reactions.
The problem addressed arises from difficulties in detecting low-abundance nucleic acids such as circulating miRNAs, which are important disease biomarkers. Existing detection methods like qRT-PCR and microarray suffer from limitations including complexity, cost, time-consuming multi-step procedures, requirements for skilled personnel, low sensitivity for some microarray platforms, and inability to provide direct absolute quantitation. Micro- and nanoelectrode DNA sensors face challenges in generating detectable current signals in presence of only small amounts of target nucleic acids due to limited sensor surface area and signal amplification. Enzymatic amplification methods are not directly applicable to nucleic acid detection without additional complex steps, and existing nanomaterial-based strategies share similar limitations. There is a need for a sensitive, selective, fast, cost-effective, one-step system capable of low-level nucleic acid detection with absolute quantification, suitable for clinical and point-of-care applications.
The invention overcomes these challenges by coupling an electrochemical DNA sensor design with a novel chemical signal amplification approach. The co-reactant (e.g., tris-(2-carboxyethyl) phosphine hydrochloride (TCEP), ascorbic acid) present in the surrounding buffer chemically reduces oxidized redox current reporters (e.g., methylene blue linked to the nucleic acid probe) after their electrochemical oxidation at the electrode. This cycling amplifies the current signal during the detection period, enabling detection down to femtomolar and even attomolar levels within minutes. Additionally, a multi-step surface modification protocol passivates the electroconductive substrate, minimizing non-specific adsorption and background current, thereby improving selectivity and signal-to-noise ratio. The system supports multiple voltammetry detection modes and is capable of multiplexed, high-throughput nucleic acid screening. This provides a simpler, rapid, stable, regenerable, and broadly applicable platform for sensitive nucleic acid detection with direct quantitative readout.
Claims Coverage
The patent contains one independent claim directed to a system for nucleic acid detection, and independent claims directed to methods of using and making the system. The key inventive features focus on the specific system components and configuration enabling sensitive, label-free, one-step detection via electrochemical signal amplification without redox enzymes.
System comprising an electroconductive substrate with bound sensing units
The system includes an electroconductive substrate to which sensing units are directly bound. Each sensing unit comprises a single sequence-specific nucleic acid strand linked to a redox current reporter. The nucleic acid sequence is sufficiently complementary to the target nucleic acid or sequence therein to specifically bind it, and is bound directly (not via a separate strand) to the substrate.
Utilization of a coreactant to enable signal amplification
The system includes a coreactant in solution that interacts cyclically with the redox current reporter and the electrode to amplify the electrochemical signal. The coreactant can be a reductant that reduces oxidized redox reporters or an oxidant that oxidizes reduced redox reporters, enabling in situ signal amplification during detection without reliance on redox enzymes.
Specific characteristics of the redox current reporter and nucleic acid
The redox current reporter is linked to the sequence-specific nucleic acid and capable of undergoing oxidation or reduction at the electrode. The nucleic acid sequence is about 17-27 nucleotides in length and complementary to the target nucleic acid to allow specific hybridization and detection.
Electroconductive substrate characteristics and surface passivation
The substrate can be carbon- or metal-containing, fashioned as micro-, nano-, or macroelectrodes depending on size. The sensing units are covalently bound, for example through thiolate moieties. The system further comprises alkanethiolate moieties bound to the substrate to passivate unoccupied sites and reduce non-specific signals.
High throughput screening capability
The system can include multiple sensing units designed to detect different nucleic acids simultaneously. Levels of different nucleic acids detected by these plurality of sensors can together signify the presence of disease states, supporting multiplexed analysis.
Methods for using and making the system
Methods include detecting target nucleic acids by exposing biological samples to the system, and making the system by self-assembling sensing units on an electroconductive substrate followed by passivation with agents such as alkanethiols. Functional groups like sulfhydryl or alkyne on the sensing units enable binding to the substrate. Heating and cooling steps can be used during passivation to optimize surface modification.
Overall, the patent claims cover a label-free electrochemical detection system featuring a single-stranded nucleic acid sensing unit linked to a redox reporter bound to an electroconductive substrate, coupled with a co-reactant-based signal amplification mechanism without enzymes and optimized surface passivation. The scope includes various substrate sizes, redox chemistries, multiplexing capacity, and corresponding methods of making and use.
Stated Advantages
Enables one-step, label-free detection of nucleic acids with high sensitivity down to femtomolar and attomolar levels within minutes.
Signal amplification mechanism not reliant on redox enzymes, overcoming limitations of existing microelectrode DNA sensors.
Highly selective sensing units with excellent discrimination between target and mismatched sequences.
Minimized background and non-specific signals through multi-step surface modification and passivation.
Capability for multiplexed, high-throughput screening of multiple nucleic acid targets simultaneously.
The system exhibits regenerable sensing units with stable performance after multiple detection cycles and long-term storage.
Direct absolute quantification of target nucleic acids, suitable for clinical, point-of-care, and in-home applications.
Documented Applications
Detection and quantitation of circulating microRNAs (miRNAs) as disease biomarkers, including miR-122 associated with hepatocellular carcinoma and gastrointestinal disorders.
Diagnosis and prognosis of various cancers including diffuse large B cell lymphoma, breast cancer, colon cancer, gastric cancer, leukemia, lung cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, and squamous cell carcinoma by detecting relevant miRNA signatures.
Detection of target nucleic acids from pathogens in infections, including bacterial, fungal, or viral nucleic acids.
Use in biological samples such as blood, plasma, serum, saliva, cerebrospinal fluid, urine, and tissue biopsies from humans and other mammals.
Embedding in paper-based or polymer-based diagnostic tools such as lateral flow devices or printed electrode arrays for point-of-care testing.
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