Surface-confined DNA Research Articles

Programming and monitoring surface-confined DNA computing

DNA-based molecular computing has evolved to encompass a diverse range of functions, demonstrating substantial promise for both highly parallel computing and various biomedical applications. Recent advances in DNA computing systems based on surface reactions have demonstrated improved levels of specificity and computational speed compared to their solution-based counterparts that depend on three-dimensional molecular collisions. Herein, computational biomolecular interactions confined by various surfaces such as DNA origamis, nanoparticles, lipid membranes and chips are systematically reviewed, along with their manipulation methodologies. Monitoring techniques and applications for these surface-based computing systems are also described. The advantages and challenges of surface-confined DNA computing are discussed.

DNA Nanomachine with Multitentacles for Integral Processing of Nanoparticles and Its Application in Biosensing.

DNA nanomachines developed by DNA nanotechnology, an attractive branch of nanoengineering, have been widely applied in drug delivery, biomarker detection, etc. However, the existing DNA nanomachines are mainly conducted in solution systems and the application of surface-confined DNA nanomachines is limited due to the less variety and lower efficiency than those in a solution. The efficiency is greatly limited due to the fact that the surface-confined substances cannot freely perform the Brownian movement, which is not conducive to performance improvement. In this work, we proposed a DNA nanomachine with multitentacles based on the multivalent effect and confirmed that the processing efficiency of multitentacles is much higher than that of a single tentacle by kinetic experiments. Interestingly, a multitentacle DNA nanomachine enables stable capture and integral processing of nanoparticles through only one collision between the surface and the nanoparticle, avoiding the loss of efficiency caused by repeated collisions. In addition, a multitentacle DNA nanomachine-based immunoassay exhibits comparable sensitivity to traditional enzyme-linked immunosorbent assay (ELISA) methods in practical applications. Therefore, it is believed that the construction and application of a multitentacle DNA nanomachine expand the application of DNA nanomachines in interfacial biosensing systems, e.g., ELISA and electrochemical biosensors, and provide ideas for the design of other nanomachines working on the interface.

High-performance biosensing based on autonomous enzyme-free DNA circuits.

Nucleic acids are considered not only extraordinary carriers of genetic information but also are perceived as the perfect elemental materials of molecular recognition and signal transduction/amplification for assembling programmable artificial reaction networks or circuits, which are similar to conventional electronic logic devices. Among these sophisticated DNA-based reaction networks, catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and DNAzyme represent the typical nonenzymatic amplification methods with high robustness and efficiency. Furthermore, their extensive hierarchically cascade integration into multi-layered autonomous DNA circuits establishes novel paradigms for constructing more different catalytic DNA nanostructures and for regenerating or replicating diverse molecular components with specific functions. Various DNA and inorganic nanoscaffolds have been used to realize the surface-confined DNA reaction networks with significant biomolecular sensing and signal-regulating functions in living cells. Especially, the specific aptamers and metal-ion-bridged duplex DNA nanostructures could extend their paradigms for detecting small molecules and proteins in even living entities. Herein, the varied enzyme-free DNA circuits are introduced in general with an extensive explanation of their underlying molecular reaction mechanisms. Challenges and outlook of the autonomous enzyme-free DNA circuits will also be discussed at the end of this chapter.

A surface-confined DNA assembly enabled target recycling amplification for multiplexed electrochemical DNA detection

A novel and enzyme-free electrochemical sensor was designed for multiplexed DNA detection based on a surface-confined DNA assembly enabled target recycling amplification. In this designed system, two hairpin DNA probes (hairpin 1 and hairpin 2) were immobilized on the surface of gold nanoparticles/graphene/nafion modified glassy carbon electrode (AuNPs/Gr/Nf/GCE). In the presence of two DNA targets, the hairpin structures of hairpin 1 and hairpin 2 opened through the complementary hybridization, resulting in the formation of hairpin 1: target DNA 1 and hairpin 2: target DNA 2. When they met methylene blue (MB)-labeled hairpin 3 (hairpin 3-MB) and ferrocene (Fc)-labeled hairpin 4 (hairpin 4-Fc), DNA cyclic amplification was activated, and the DNA targets were released and could be reused for the next recycling. Meanwhile, coupled with the utilization of positively charged gold nanoparticles ((+)AuNPs), multiplexed DNA detection was realized in the concentration range from 10 fM to 1.5 pM with the detection limits of 3.6 fM and 2.5 fM for target DNA 1 and target DNA 2, respectively. Moreover, satisfactory results were obtained when detecting DNA targets in human serum samples, suggesting that this proposed sensing platform is promising for the applications in complex biological samples.

Clamped Hybridization Chain Reactions for the Self-Assembly of Patterned DNA Hydrogels.

DNA hydrogels hold great potential for biological and biomedical applications owing to their programmable nature and macroscopic sizes. However, most previous studies involve spontaneous and homogenous gelation procedures in solution, which often lack precise control. A clamped hybridization chain reaction (C-HCR)-based strategy has been developed to guide DNA self-assembly to form macroscopic hydrogels. Analogous to catalysts in chemical synthesis or seeds in crystal growth, we introduced DNA initiators to induce the gelation process, including crosslinked self-assembly and clamped hybridization in three dimensions with spatial and temporal control. The formed hydrogels show superior mechanical properties. The use of printed, surface-confined DNA initiators was also demonstrated for fabricating 2D hydrogel patterns without relying on external confinements. This simple method can be used to construct DNA hydrogels with defined geometry, composition, and order for various bioapplications.

A Surface‐Confined Proton‐Driven DNA Pump Using a Dynamic 3D DNA Scaffold

A proton-driven molecular pump is devised using a surface-confined dynamic 3D DNA scaffold. A dynamic DNA tetrahedral nanostructure is designed by incorporating a pH-sensitive i-motif sequence in one edge, which serves as the scaffold to ensure highly ordered orientation and spatial isolation of this nanomachine on the macroscopic gold surface. It is found that the switching ability of this dynamic tetrahedron is fully maintained on the surface. Importantly, this proton-driven nanomachine can reversibly pump water and ferricynide in response to pH variation in solution.

A Quercetin Biosensor Based on Chitosan-Entrapped Carbon Nanotube Paste Electrode Coated with DNA.

This paper presents an easy, fast, low cost, and sensitive approach for the electrochemical determination of quercetin based on its intercalation into DNA double helix. Electrochemical studies of the interaction between quercetin and DNA showed a decrease in peak currents with a reduction in redox reversibility of quercetin in the presence of the DNA. The electrochemical behavior of quercetin at a chitosan-entrapped carbon nanotube paste electrode coated with DNA was studied. A considerable increase was observed in the oxidation signal of quercetin at the DNA-coated electrode compared with a DNA-free electrode, indicating the preconcentration of quercetin due to its interaction with the surface-confined DNA layer. After optimizing the main experimental parameters influencing the biosensor response, its performance was evaluated from an analytical point of view. Two linear dependences of the anodic peak current of quercetin on its concentration were observed in the ranges of 0.40-7.50 and 7.50-30.0 μmol/L, with LOD and LOQ of 0.039 and 0.13 μmol/L, respectively. The proposed biosensor was successfully applied to the analysis of black and green tea extracts for their quercetin content.

Electrical potential-assisted DNA hybridization. How to mitigate electrostatics for surface DNA hybridization.

Surface-confined DNA hybridization reactions are sensitive to the number and identity of DNA capture probes and experimental conditions such as the nature and the ionic strength of the electrolyte solution. When the surface probe density is high or the concentration of bulk ions is much lower than the concentration of ions within the DNA layer, hybridization is significantly slowed down or does not proceed at all. However, high-density DNA monolayers are attractive for designing high-sensitivity DNA sensors. Thus, circumventing sluggish DNA hybridization on such interfaces allows a high surface concentration of target DNA and improved signal/noise ratio. We present potential-assisted hybridization as a strategy in which an external voltage is applied to the ssDNA-modified interface during the hybridization process. Results show that a significant enhancement of hybridization can be achieved using this approach.

Electrochemical determination of biophenol oleuropein using a simple label-free DNA biosensor

Oleuropein (Ole), naturally occurring phenolic compound found in olive products, is well known for its benefits for human health. In the present work, a simple, sensitive and rapid determination of Ole was achieved using a label-free electrochemical DNA biosensor. The application was related to the molecular interaction between Ole and double-stranded DNA (dsDNA). So, the voltammetric behavior of Ole at the surface of a DNA-immobilized chitosan-modified carbon paste electrode was studied using differential pulse voltammetry (DPV) where the oxidation peak current of Ole was measured as an analytical signal. A considerable increase was observed in the oxidation signal of Ole at the DNA-coated electrode compared with the DNA-free electrode, indicating the pre-concentration of Ole due to the interaction with the surface-confined DNA layer. In order to use the proposed sensor for real samples, different parameters affecting Ole signal such as, immobilization time and potential, accumulation time and pH, and stripping pH were optimized. Under optimized experimental conditions, a linear concentration range of 0.30–12μmolL−1 with a detection limit of 0.090μmolL−1 was obtained for Ole determination. The proposed biosensor was successfully applied to the determination of Ole in olive leaf extract and human serum samples.

Highly sensitive voltammetric detection of DNA hybridization in sandwich format using thionine-capped gold nanoparticle/reporter DNA conjugates as signal tags

A highly sensitive sandwich DNA detection method based on voltammetric detection of thionine-capped gold nanoparticle (AuNP)/reporter DNA conjugate tags on gold particle-modified screen-printed carbon electrode (SPCE) was developed. The SPCE was modified with gold particle by electrodeposition of gold on SPCE surface. The DNA sensor was prepared by self-assembly of a thiolated DNA probe on gold particle-modified SPCEs. The sandwich-type system was formed by specific recognition of biosensor surface-confined probe DNA to target DNA, followed by attachment of thionine-capped AuNPs/reporter DNA conjugates. The biosensor is very sensitive because of the large number of electroactive thionine molecules in the thionine-capped AuNPs/reporter DNA conjugates. Under optimal conditions, the dynamic detection range of target DNA was from 1.0 × 10−16 to 1.0 × 10−14 mol L−1, and the detection limit was 0.5 × 10−16 mol L−1. The DNA sensor exhibited selectivity against single-base mismatched DNA.

Voltammetric behavior of uric acid on carbon paste electrode modified with salmon sperm dsDNA and its application as label-free electrochemical sensor

A simple and sensitive label-free electrochemical DNA biosensor was proposed for the rapid determination of uric acid (UA) using a carbon nano tube paste electrode (CNTPE) modified with salmon sperm dsDNA. At first, the interaction between UA and the DNA was studied using differential pulse voltammetry (DPV). The addition of the DNA to UA solution resulted in a decrease in the peak current of UA and at the same time, a positive shift in the peak potential indicating an intercalative interaction. Then, the voltammetric response of a DNA-immobilized CNTPE was investigated for the determination of UA. The immobilization of the DNA was carried out using acid-functionalized carbon nanotubes and studied using Fe(CN)63−/Fe(CN)64− redox indicator. Compared with unmodified CNTPE, the oxidation signal of UA showed a significant increase at the DNA-coated electrode, and shifted to more positive potentials attributed to the pre-concentration of UA at the electrode surface due to interaction with the surface-confined DNA layer. This interaction was used for the fabrication of a simple and sensitive biosensor for determining UA. After the optimization of operational parameters, a linear dependence of the peak current on the UA concentration was observed in the range of 7.0×10−7 to 1.1×10−4molL−1, with the detection and quantification limits of 1.8×10−7 and 5.8×10−7molL−1, respectively. The proposed biosensor was successfully applied to validate its capability for the analysis of UA in human serum and urine samples.

Scaffolded biosensors with designed DNA nanostructures

In addition to its fundamental function as a genetic code carrier, the utilization of DNA in various material applications has been actively explored over the past several decades. DNA is intrinsically an excellent type of self-assembly nanomaterial owing to its predictable base-pairing, high chemical stability and the convenience it possesses for synthesis and modification. Because of these unparalleled properties, DNA is widely used as excellent recognition elements in biosensors and as unique building blocks in nanodevices. A critical challenge in surface-based DNA biosensors lies in the reduced accessibility of target molecules to the DNA probes arranged on heterogeneous surfaces, especially when compared to probe–target recognition in homogeneous solutions. To improve the recognition abilities of these heterogeneous surface-confined DNA probes, much effort has been devoted to controlling the surface chemistry, conformation and packing density of the probe molecules, as well as the size and geometry of the surface. In this review, we aim to summarize the recent progress on the improvement of the probe–target recognition properties by introducing DNA nanostructure scaffolds. A range of new strategies have proven to provide a significantly enhanced range in the spatial positioning and the accessibility of the probes to the surface over previously reported linear structures. We will also describe the applications of DNA nanostructure scaffold-based biosensors.

Sensitive chemiluminescence detection of wild-type p53 protein captured by surface-confined consensus DNA duplexes

A novel chemiluminescence (CL) biosensor for sensitive detection of wild-type p53 protein has been proposed. The wild-type p53 protein in solution was captured by highly specific consensus double-stranded (ds) oligonucleotides (ODNs) preimmobilized onto a gold plate. The cysteine residues on the exterior of the wild-type p53 molecules were then derivatized with N-biotinoyl-N′-(6-maleimidohexanoyl) hydrazide (biotin-Mi) for the attachment of streptavidin-horseradish peroxidase (SA-HRP) complex. The attached HRP molecules could catalyze the CL reaction between luminol and H2O2, producing an enhanced CL signal. The CL intensity was dependent on the surface coverage of the HRP molecules, which was related to the concentration of wild-type p53 protein. Under the optimal experimental conditions, the CL intensity increased linearly with the concentration of wild-type p53 protein from 0.01 to 0.5nM. The detection limit was estimated to be 3.8pM. The proposed method has been successfully utilized for the assay of wild-type p53 protein in normal and cancer cell lysates. The sensing protocol is sensitive, cost-effective, and holds great promise for clinical diagnosis.

Probe accessibility effects on the performance of electrochemical biosensors employing DNA monolayers

Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here we demonstrate that the density of the DNA probe monolayer on the gold electrode is a crucial determinant of the final signalling of such devices. We do so using redox modified single-stranded and double-stranded DNA probes attached to the surface of a gold electrode and measuring the rate of digestion in the presence of a non-specific nuclease enzyme. We demonstrate that accessibility of DNA probes for binding to their macromolecular target is, as expected, improved at lower probe densities. However, with double-stranded DNA probes, even at the lowest densities investigated, a significant fraction of the immobilized probe is inaccessible to nuclease digestion. These results stress the importance of the accessibility issue and of probe density effects when DNA-based sensors are used for detection of macromolecular targets.

Microfluidic Electrochemical Aptameric Assay Integrated On-Chip: A Potentially Convenient Sensing Platform for the Amplified and Multiplex Analysis of Small Molecules

Aptamers are artificial oligonucleotides that have been widely employed to design biosensors (i.e., aptasensors). In this work, we report a microfluidic electrochemical aptamer-based sensor (MECAS) by constructing Au-Ag dual-metal array three-electrode on-chip for multiplex detection of small molecules. In combination with the microfluidic channels covering on the glass chip, different targets are transported to the Au electrodes integrated on different positions of the chip. These electrodes are premodified by different kinds of aptamers, respectively, to fabricate different sensing interfaces which can selectively capture the corresponding target. It is an address-dependent sensing platform; thus, with the use of only one electrochemical probe, multitargets can be recognized and detected according to the readout on a corresponding aptamer-modified electrode. In the sensing strategy, the electrochemical probe, [Ru(NH(3))(6)](3+) (RuHex), which can quantitatively bind to surface-confined DNA via electrostatic interaction, was used to produce chronocoulometric signal; Au nanoparticles (AuNPs) were used to improve the sensitivity of the sensor by amplifying the detection signals. Moreover, the sensing interface fabrication, sample incubation, and electrochemical detection were all performed in microfluidic channels. By using this detection chip, we achieved the multianalysis of two model small molecules, ATP, and cocaine, in mixed samples within 40 min. The detection limit of ATP was 3 × 10(-10) M, whereas the detection limit of cocaine was 7 × 10(-8) M. This Au-Ag dual metal electrochemical chip detector integrated MECAS was simple, sensitive, and selective. Also it is similar to a dosimeter which accumulates signal upon exposure. It held promising potential for designing electrochemical devices with high throughput, high automation, and high integration in multianalysis.

Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E. coli DNA ligase

An electrochemical method for nicotinamide adenine dinucleotide (NAD +) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5′-SH and 3′-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold–thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD +, E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD +-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD + range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD + from its analogues.

Target-Responsive Structural Switching for Nucleic Acid-Based Sensors

Interest in the development of sensitive, selective, rapid, and cost-effective biosensors for biomedical analysis, environmental monitoring, and the detection of bioterrorism agents is rapidly increasing. A classic biosensor directly transduces ligand-target binding events into a measurable physical readout. More recently, researchers have proposed novel biosensing strategies that couple ligand-induced structural switching of biomolecules with advanced optical and electronic transducers. This approach has proven to be a highly general platform for the development of new biosensors. In this Account, we describe a series of electrochemical and optical nucleic acid sensors that use target-responsive DNA structures. By employing surface-confined DNA structures with appropriate redox labels, we can monitor target-induced structural switching of DNA or aptamer-specific small molecule probes by measuring electrochemical currents that are directly associated with the distance between the redox label and the electrode surface. We have also demonstrated significant improvements in sensing performance through optimization of the DNA self-assembly process at electrode surfaces or the introduction of nanomaterial-based signal amplification. Alternatively, gold nanoparticles interact differently with folded and unfolded DNA structures, which provides a visual method for detecting target-induced structural switching based on the plasmonic change of gold nanoparticles. This novel method using gold nanoparticles has proven particularly suitable for the detection of a range of small-molecule targets (e.g., cocaine) and environmentally toxic metal ions (e.g., Hg(2+)). Rational sequence design of DNA aptamers improves the sensitivity and increases the reaction kinetics. Recently, we have also designed microfluidic devices that allow rapid and portable mercury detection with the naked eye. This Account focuses on the use of bulk and nanoscale gold and DNA/aptamer molecules. We expect that researchers will further expand the analyte spectrum and improve the sensitivity and selectivity of nucleic acid sensors using functional biomolecules, such as DNAzymes, peptide aptamers and engineered proteins, and nanomaterials of different sizes, dimensions and compositions, such as carbon nanotubes, graphene, silicon nanowires, and metal nanoparticles or nanorods.

A sensitive ligase-based ATP electrochemical assay using molecular beacon-like DNA

A sensitive and selective ligase-based signal-on electrochemical sensing method for adenosine-5′-triphosphate (ATP) detection had been developed using molecular beacon (MB)-like DNA. In this method, the biotin-tagged MB-like DNA was self-assembled onto a gold electrode to form a stem-loop structure by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of ATP, two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the ATP-dependent T4 DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a significant conformational change in this surface-confined DNA structure, which in turn released the biotin from the surface allowing free exchange of electrons with the electrode generating a measurable electrochemical signal (eT ON). The resulting change in electron transfer efficiency was readily measured by differential pulse voltammetry at target ATP concentrations as low as 0.05 nM and with linear response range from 0.1 to 1000 nM. Moreover, it was also able to discriminate ATP from its analogues. The proposed method had been successfully applied to the determination of ATP in the Escherichia coli O157:H7 extracts of water samples, and the linear response was found between the concentrations of 10 3 and 10 7 cfu/mL.

Nanoscale Architecture Dictates Detection Profile of Surface-Confined DNA by MALDI-TOF MS

The design and fabrication of various nanoscale architectures for the effective MALDI-TOF MS analysis of surface-confined DNA are reported. The nanoscale architectures are derived from layer-by-layer assemblies of negatively charged nanoparticles and positively charged polymers. An important finding is that porous structures comprising bottom SiO(2) nanoparticle layers and top Au nanoparticle layers enable the observation of both covalently bound (with Au-S bond) and hybridized oligonucleotides. The achievement of this unprecedented detection capability is attributed to the unique combination of a nanoporous dielectric film and discrete noble metal nanoparticles. The conclusion is supported by control experiments performed on a variety of single and multilayered Au nanoparticle structures, where only the hybridized oligonucleotides in a two- or three-strand system could be detected.

A sensitive, label free electrochemical aptasensor for ATP detection

A sensitive, label free electrochemical aptasensor for small molecular detection has been developed in this work based on gold nanoparticles (AuNPs) amplification. This aptasensor was fabricated as a tertiary hybrid DNA–AuNPs system, which involved the anchored DNA (ADNA) immobilized on gold electrode, reporter DNA (RDNA) tethered with AuNPs and target-responsive DNA (TRDNA) linking ADNA and RDNA. Electrochemical signal is derived from chronocoulometric interrogation of [Ru(NH 3) 6] 3+ (RuHex) that quantitatively binds to surface-confined DNA via electrostatic interaction. Using adenosine triphosphate (ATP) as a model analyte and ATP-binding aptamer as a model molecular reorganization element, the introduction of ATP triggers the structure switching of the TRDNA to form aptamer–ATP complex, which results in the dissociation of the RDNA capped AuNPs (RDNA–AuNPs) and release of abundant RuHex molecules trapped by RDNA–AuNPs. The incorporation of AuNPs in this strategy significantly enhances the sensitivity because of the amplification of electrochemical signal by the RDNA–AuNPs/RuHex system. Under optimized conditions, a wide linear dynamic range of 4 orders of magnitude (1 nM–10 μM) was reached with the minimum detectable concentration at sub-nanomolar level (0.2 nM). Those results demonstrate that our nanoparticles-based amplification strategy is feasible for ATP assay and presents a potential universal method for other small molecular aptasensors.