Rapid, sensitive, and quantitative detection of pathogenic DNA at the point of care through microfluidic electrochemical quantitative loop-mediated isothermal amplification

Single-step DNA detection: a microfluidic electrochemical loop mediated isothermal amplification platform is reported for rapid, sensitive, and quantitative detection of pathogen genomic DNA at the point of care. DNA amplification was electrochemically monitored in real time within a monolithic microfluidic device, thus enabling the detection of as few as 16 copies of Salmonella genomic DNA through a single-step process in less than an hour.     

Fluorescence Detection of Single‐Nucleotide Polymorphisms with a Single,Self‐Complementary,Triple‐Stem DNA Probe

Singled out for its singularity : In a single‐step, single‐component, fluorescence‐based method for the detection of single‐nucleotide polymorphisms at room temperature, the sensor is comprised of a single, self‐complementary DNA strand that forms a triple‐stem structure. The large conformational change that occurs upon binding to perfectly matched (PM) targets results in a significant increase in fluorescence (see picture; F=fluorophore, Q=quencher).

     

Electrochemical Label‐Free Nucleotide Sensors

Numerous researchers have devoted a great deal of effort over the last few decades to the development of electrochemical oligonucleotide detection techniques, owing to their advantages of simple design, inherently small dimensions, and low power requirements. Their simplicity and rapidity of detection makes label‐free oligonucleotide sensors of great potential use as first‐aid screening tools in the analytical field of environmental measurements and healthcare management. This review article covers label‐free oligonucleotide sensors, focusing specifically on topical electrochemical techniques, including intrinsic redox reaction of bases, conductive polymers, the use of electrochemical indicators, and highly ordered probe structures.     

Electrochemical Detection of DNA Melting Curves by Means of Heated Biosensors

This article reports about the detection of DNA melting curves at heated electrochemical biosensors. Osmium tetroxide‐bipyridine‐labeled target oligonucleotides are hybridized with probe oligonucleotides immobilized on gold electrodes. Then, the gold electrode is successively heated in order to measure a complete melting curve consisting of alternating current voltammetric signals. Melting temperatures ?_m, determined at various ionic strengths and in dependence on different numbers of base pair mismatches, have been compared with those obtained by means of UV spectrophotometry. The proposed method holds great promise for the fast and easy parallel detection of nucleic acids sequences on selectively heated electrode arrays. A stringent hybridization temperature can be easily adjusted in order to discriminate base pair mismatches.     

High Sensitive Electrochemical Detection of Sequence‐Specific DNA Using Low Current Voltammetry

In this article, we report on efforts to construct a high sensitive electrochemical sensor with immobilized sandwich‐type DNA borne ferrocene (Fc) head for sequence‐specific DNA detection using ultramicroelectrode and low current voltammetry. Based on the difference in deformability between the bending rigid complementary DNA double helix and its anomalous flexile mismatches, the fully complementary target can be distinguished from mismatched targets including the single‐base mismatched target. Detection limit estimated as the amount of DNA is observed to be 100 fM via low current voltammetry. The method offers great promise of high sensitivity and selectivity simultaneously for effective gene identification.     

Single‐Molecule Mechanochemical Sensing Using DNA Origami Nanostructures

While single‐molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single‐molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7‐tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet‐derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single‐molecule sensing with improved throughput.     

Rapid Non‐Crosslinking Aggregation of DNA‐Functionalized Gold Nanorods and Nanotriangles for Colorimetric Single‐Nucleotide Discrimination

Gold nanoparticles modified with DNA duplexes are rapidly and spontaneously aggregated at high ionic strength. In contrast, this aggregation is greatly suppressed when the DNA duplex has a single‐base mismatch or a single‐nucleotide overhang located at the outermost surface of the particle. These colloidal features emerge irrespective of the size and composition of the particle core; however, the effects of the shape remain unexplored. Using gold nanorods and nanotriangles (nanoplatelets), we show herein that both remarkable rapidity in colloidal aggregation and extreme susceptibility to DNA structural perturbations are preserved, regardless of the shape and aspect ratio of the core. It is also demonstrated that the DNA‐modified gold nanorods and nanotriangles are applicable to naked‐eye detection of a single‐base difference in a gene model. The current study corroborates the generality of the unique colloidal properties of DNA‐functionalized nanoparticles, and thus enhances the feasibility of their practical use.     

Sequence‐Specific Incorporation of Enzyme–Nucleotide Chimera by DNA Polymerases

DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High‐fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high‐fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template‐dependent DNA synthesis, despite this “cargo” being more than 100‐fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked‐eye detection of DNA and RNA at single nucleotide resolution.     

Functionalized Nanogap for DNA Read-Out: Nucleotide Rotation and Current-Voltage Curves

Functionalized nanogaps embedded in nanopores show a strong potential for enhancing the detection of biomolecules, their length, type, and sequence. This detection is strongly dependent on the features of the target biomolecules, as well as the characteristics of the sensing device. In this work, through quantum-mechanical calculations, we elaborate on representative such aspects for the specific case of DNA detection and read-out. These aspects include the influence of single DNA nucleotide rotation within the nanogap and the current-voltage (I-V) characteristics of the nanogap. The results unveil a distinct variation in the electronic current across the functionalized device for the four natural DNA nucleotides with the applied voltage. These also underline the asymmetric response of the rotating nucleotides on this applied voltage and the respective variation in the rectification ratio of the device. The electronic tunneling current across the nanogap can be further enhanced through the proper choice of an applied bias voltage. We were able to correlate the enhancement of this current to the nucleotide rotation dynamics and a shift of the electronic transmission peaks towards the Fermi level. This nucleotide specific shift further reveals the sensitivity of the device in reading-out the identity of the DNA nucleotides for all different configurations in the nanogap. We underline the important information that can be obtained from both the I-V curves and the rectification characteristics of the nanogap device in view of accurately reading-out the DNA information. We show that tuning the applied bias can enhance this detection and discuss the implications in view of novel functionalized nanopore sequencers.     

Electrochemical Analysis of Glycoprotein Samples Prepared on a Pneumatically‐controlled Microfluidic Device

In this work, we designed and manufactured a microfluidic device for isolation and purification of glycoprotein samples. The conceived sample preparation device was fabricated in polycarbonate by micro‐milling. The flow control and the fluid dosage into the micro‐channels was solved by equipping the device with integrated pneumatic valves. The biochemical functionality was provided by beaded support modified by molecules with affinity to glycoproteins which was stacked inside the micro‐channel reminiscent of packed affinity columns used in glycoprotein lectin assays. Unlabeled glycoproteins, namely fetuin, asialofetuin, and prostate‐specific antigen, were voltammetrically analyzed using catalytic peak H at silver amalgam electrode.     

Additional Base‐Pair Formation in DNA Duplexes by a Double‐Headed Nucleotide

We have designed and synthesised a double‐headed nucleotide that presents two nucleobases in the interior of a dsDNA duplex. This nucleotide recognises and forms Watson–Crick base pairs with two complementary adenosines in a Watson–Crick framework. Furthermore, with judicious positioning in complementary strands, the nucleotide recognises itself through the formation of a T:T base pair. Thus, two novel nucleic acid motifs can be defined by using our double‐headed nucleotide. Both motifs were characterised by UV melting experiments, CD and NMR spectroscopy and molecular dynamics simulations. Both motifs leave the thermostability of the native dsDNA duplex largely unaltered. Molecular dynamics calculations showed that the double‐headed nucleotides are accommodated in the dsDNA by entirely local perturbations and that the modified duplexes retain an overall B‐type geometry with the dsDNA unwound by around 25 or 60°, respectively, in each of the modified motifs. Both motifs can be accommodated twice in a dsDNA duplex without incurring any loss of stability and extrapolating from this observation and the results of modelling, it is conceivable that both can be multiplied several times within a dsDNA duplex. These new motifs extend the DNA recognition repertoire and may form the basis for a complete series of double‐headed nucleotides based on all 16 base combinations of the four natural nucleobases. In addition, both motifs can be used in the design of nanoscale DNA structures in which a specific duplex twist is required.     

Efficient Discrimination of Single Nucleotide Polymorphisms (SNPs) Using Oligonucleotides Modified with C5‐Pyrene‐Functionalized DNA and Flanking Locked Nucleic Acid (LNA) Monomers

Oligodeoxyribonucleotides modified with 5‐[3‐(1‐pyrenecarboxamido)propynyl]‐2′‐deoxyuridine monomer X and proximal LNA monomers display higher affinity for complementary DNA, more pronounced increases in fluorescence emission upon DNA binding, and improved discrimination of SNPs at non‐stringent conditions, relative to the corresponding LNA‐free probes across a range of sequence contexts. The results reported herein suggest that the introduction of LNA monomers influences the position of nearby fluorophores via indirect conformational restriction, a characteristic that can be utilized to develop optimized fluorophore‐labeled probes for SNP‐discrimination studies.     

Specific Transport of Target Molecules by Motor Proteins in Microfluidic Channels

Direct transport powered by motor proteins can alleviate the challenges presented by miniaturization of microfluidic systems. There have been several recent attempts to build motor‐protein‐driven transport systems based on simple capturing or transport mechanisms. However, to achieve a multifunctional device for practical applications, a more complex sorting/transport system should be realized. Herein, the proof of concept of a sorting device employing selective capture of distinct target molecules and transport of the sorted molecules to different predefined directions is presented. By combining the bottom‐up functionality of biological systems with the top‐down handling capabilities of micro‐electromechanical systems technology, highly selective molecular recognition and motor‐protein‐based transport is integrated in a microfluidic channel network.     

DNA Dangling‐End‐Induced Colloidal Stabilization of Gold Nanoparticles for Colorimetric Single‐Nucleotide Polymorphism Genotyping

A single‐nucleotide polymorphism (SNP) detection method was developed by combining single‐base primer extension and salt‐induced aggregation of gold nanoparticles densely functionalized with double‐stranded DNA (dsDNA‐AuNP). The dsDNA‐AuNPs undergo rapid aggregation in a medium of high ionic strength, whereas particles having a single‐base protrusion at the outermost surface disperse stably, allowing detection of a single‐base difference in length by color changes. When SNP typing primers are used as analytes to hybridize to the single‐stranded DNA on the AuNP surface, the resulting dsDNA‐AuNP works as a visual indicator of single‐base extension. A set of four extension reaction mixtures is prepared using each of ddNTPs and subsequently subjected to the aggregation assay. Three mixtures involving ddNTP that is not complementary to the SNP site in the target produce the aggregates that exhibit a purple color. In contrast, one mixture with the complementary ddNTP generates the single‐base protrusion and appears red. This method could potentially be used in clinical diagnostics for personalized medicine.     

Selective Nascent Polymer Catch‐and‐Release Enables Scalable Isolation of Multi‐Kilobase Single‐Stranded DNA

Scalable methods currently are lacking for isolation of long ssDNA, an important material for numerous biotechnological applications. Conventional biomolecule purification strategies achieve target capture using solid supports, which are limited in scale and susceptible to contamination owing to nonspecific adsorption and desorption on the substrate surface. We herein disclose selective nascent polymer catch and release (SNAPCAR), a method that utilizes the reactivity of growing poly(acrylamide‐co‐acrylate) chains to capture acrylamide‐labeled molecules in free solution. The copolymer acts as a stimuli‐responsive anchor that can be precipitated on demand to pull down the target from solution. SNAPCAR enabled scalable isolation of multi‐kilobase ssDNA with high purity and 50–70 % yield. The ssDNA products were used to fold various DNA origami. SNAPCAR‐produced ssDNA will expand the scope of applications in nanotechnology, gene editing, and DNA library construction.