A Supercharged Fluorescent Protein as a Versatile Probe for Homogeneous DNA Detection and Methylation Analysis

Supercharged proteins are a new class of functional proteins with exceptional stability and potent ability to deliver bio‐macromolecules into cells. As a proof‐of‐principle, a novel application of supercharged proteins as a versatile biosensing platform for nucleic acid detection and epigenetics analysis is presented. Taking supercharged green fluorescent protein (ScGFP) as the signal reporter, a simple turn‐on homogenous method for DNA detection has been developed based on the polyionic nanoscale complex of ScGFP/DNA and toehold strand displacement. This assay shows high sensitivity and potent ability to detect single‐base mismatch. Furthermore, combined with bisulfite conversion, this ScGFP‐based assay was further applied to analyze site‐specific DNA methylation status of genomic DNA extracted from real human colon carcinoma tissue sample with ultrahigh sensitivity (4 amol methylated DNA).     

Modified Nucleotides for Discrimination between Cytosine and the Epigenetic Marker 5‐Methylcytosine

5‐Methyl‐2′‐deoxycytosine, the most common epigenetic marker of DNA in eukaryotic cells, plays a key role in gene regulation and affects various cellular processes such as development and carcinogenesis. Therefore, the detection of 5mC can serve as an important biomarker for diagnostics. Here we describe that modified dGTP analogues as well as modified primers are able to sense the presence or absence of a single methylation of C, even though this modification does not interfere directly with Watson–Crick nucleobase pairing. By screening several modified nucleotide scaffolds, O⁶‐modified 2′‐deoxyguanosine analogues were identified as discriminating between C and 5mC. These modified nucleotides might find application in site‐specific 5mC detection, for example, through real‐time PCR approaches.     

Catalytic Hairpin Assembly‐Programmed DNA Three‐Way Junction for Enzyme‐Free and Amplified Electrochemical Detection of Target DNA

DNA three‐way junctions (DNA 3WJ) have been widely used as important building blocks for the construction of DNA architectures and dynamic assemblies. Herein, we describe for the first time a catalytic hairpin assembly‐programmed DNA three‐way junction (CHA‐3WJ) strategy for the enzyme‐free and amplified electrochemical detection of target DNA. It takes full advantage of the target‐catalyzed hairpin assembly‐induced proximity effect of toehold and branch‐migration domains for the ingenious execution of the strand displacement reaction to form the DNA 3WJ on the electrode surface. A low detection limit of 0.5 pM with an excellent selectivity was achieved for target DNA detection. The developed CHA‐3WJ strategy also offers distinct advantages of simplicity in probe design and biosensor fabrication, as well as enzyme‐free operation. Thus, it opens a promising avenue for applications in bioanalysis, design of DNA‐responsive devices, and dynamic DNA assemblies.     

Associating and Dissociating Nanodimer Analysis for Quantifying Ultrasmall Amounts of DNA

The amplification‐ and enzyme‐free quantification of DNA at ultralow concentrations, on the order of 10–1000 targets, is highly beneficial but extremely challenging. To address this challenge, true detection signals must be reliably discriminated from false or noise signals. Herein, we describe the development of associating and dissociating nanodimer analysis (ADNA) as a method that enables a maximum number of detection signals to be collected from true target‐binding events while keeping nonspecific signals at a minimum level. In the ADNA assay for ultralow target concentrations, Au nanoprobes on a lipid micropattern were monitored and analyzed in situ, and newly defined dissociating dimers, which are eventually decoupled into monomers again, were incorporated into the detection results. Tens to thousands of DNA copies can be reliably quantified with excellent single‐base‐mismatch differentiation capability by this non‐enzymatic, amplification‐free ADNA method.     

Harnessing Effective Molarity to Design an Electrochemical DNA‐based Platform for Clinically Relevant Antibody Detection

Easy‐to‐use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi‐step, time‐consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity‐driven electrochemical DNA‐based detection of target antibodies. We show a highly selective, signal‐on DNA‐based sensor that takes advantage of antibody‐binding‐induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.     

Nanopore‐Based Sequencing and Detection of Nucleic Acids

Nanopore‐based techniques, which mimic the functions of natural ion channels, have attracted increasing attention as unique methods for single‐molecule detection. The technology allows the real‐time, selective, high‐throughput analysis of nucleic acids through both biological and solid‐state nanopores. In this Minireview, the background and latest progress in nanopore‐based sequencing and detection of nucleic acids are summarized, and light is shed on a novel platform for nanopore‐based detection.     

Microarray‐Based Detection of Dye‐Labeled DNA by SERRS Using Particles Formed by Enzymatic Silver Deposition

The growing interest in DNA diagnostics is addressed today by microarrays with fluoresence detection. In our approach, we utilize spatially defined arrays of short oligonucleotides on a modified glass surface. Surface enhanced resonance Raman scattering (SERRS) is used to obtain molecularly specific spectra of the Raman‐active dye‐labeled DNA. Nanoparticles produced by enzymatic silver deposition are used as SERS‐active substrate. They grow directly on the modified oligonucleotides and only in the spatially defined areas on the chip. Furthermore, they potentially offer several advantages for SERS detection. The nanoparticles are characterized and their ability for use as SERS‐ and SERRS‐active substrate is estimated. Three different Raman‐active dyes are investigated for their potential for involvement in sequence specific DNA analysis.     

Prostate Cancer Biomarker Detection with Carbon Nanotubes Modified Screen Printed Electrodes

DNA hypermethylation is an epigenetic alteration and a promising biomarker for early prostate cancer detection. Simple, sensitive, easy to handle and rapid detection methodologies are imperative for point of care diagnostics especially for cancer. Herein, we describe for the first time a regenerable and compatible electrochemical biosensor for detection of Glutathione S‐Transferase P‐1 (GSTP‐1) gene hypermethylation related to prostate cancer via DNA hybridization onto the disposable Carbon and Multi Walled Carbon Nanotubes (MWCNT) Screen Printed Electrodes (SPEs). In the study, capture probes were adsorbed onto the SPEs by simple passive adsorption and then hybridization was achieved by sending the complementary target onto the probe‐modified electrodes. The selectivity of the biosensor was proved by control studies. Differential Pulse Voltammetry (DPV) technique was used to detect hybridization via guanine oxidation signals changes. The total time of the optimized method was nearly 1h, measurements took for less than 1 min, and the biosensor response was stable up to 40 days of storage period at 4 °C. The main advantages of the biosensor are very low detection limit (picomolar range) and capability of reusing the biosensor for at least 3 times after very simple regeneration process that is a unique property to reduce the cost of the assay. In addition, this is the first study that demonstrates the detection of GSTP‐1 hypermethylation electrochemically by using SPEs in order to create point of care diagnostics. The optimum parameters for the biosensor, as well as its future prospects to enhance the performance of DNA biosensors were also presented.     

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.     

Batch Injection Analysis with Amperometric Detection for DNA Biosensing Applications

In this work, batch injection analysis with the amperometric detection (BIA‐AD), employing a detection cell designed to adapt a screen‐printed carbon electrode (SPCE) was used for the first time as a robust electroanalytical system for DNA biosensing applications. The sensitive amperometric detection was used to evaluate the structural changes in double‐stranded DNA (dsDNA) after UV‐C irradiation of its solution for a given time. Batching of DNA samples was performed by precise electronic pipette microinjection of an irradiated sample aliquot onto the unmodified activated SPCE surface incorporated in the BIA‐AD system. Using the optimized experimental conditions (40 μL of 1 mg mL^?1 dsDNA in a 0.1 M phosphate buffer of pH 7.4 sampled at the injection speed degree of 6 and detected at the potential of +1.5 V vs silver pseudo‐reference electrode), a time‐dependent response (gradual decrease of amperometric signal up to 58 % after 10 min of the irradiation) was found for the detection of damage to low molecular weight salmon sperm dsDNA. The advantages of this low‐dimensional and cost‐effective measuring system can be utilized not only for the quantification of DNA damage/degradation by UV irradiation, but they are also promising for studying other types of DNA interactions.     

Sequence Specific Electrochemical DNA Detection Based on Solution‐Phase Competitive Hybridization

We report a novel electrochemical method for detecting sequence‐specific DNA based on competitive hybridization that occurs in a homogeneous solution phase instead of on a solution‐electrode interface as in previously reported competition‐based electrochemical DNA detection schemes. The method utilizes the competition between the target DNA (t‐DNA) and a ferrocene‐labeled peptide nucleic acid probe (Fc‐PNA) to hybridize with a probe DNA (p‐DNA) in solution. The neutral PNA backbone and the electrostatic repulsion between the negatively‐charged DNA backbone and the negatively‐charged electrode surface are then exploited to determine the result of the competition through measurement of the electrochemical signal of Fc. Upon the introduction of the t‐DNA, the stronger hybridization affinity between the t‐DNA and p‐DNA releases the Fc‐PNA from the Fc‐PNA/p‐DNA hybrid, allowing it to freely diffuse to the negatively charged electrode to produce a significantly enhanced electrochemical signal of Fc. Therefore, the presence of the t‐DNA is indicated by the appearance or enhancement of the electrochemical signal, rendering a signal‐on DNA detection, which is less susceptible to false positive and can produce more reliable results than signal‐off detection methods. All the competitive hybridizations occur in a homogeneous solution phase, resulting in very high hybridization efficiency and therefore extremely short assay time. This simple and fast signal‐on solution‐competition‐based electrochemical DNA detection strategy has promising potential to find application in fields such as nucleic acid‐based point‐of‐care testing.     

Development of a Novel Electrochemical Biosensor for Detection and Discrimination of DNA Sequence and Single Base Mutation in dsDNA Samples Based on PNA‐dsDNA Hybridization – a new Platform Technology

In spite of the extensive attention paid on the development of various DNA detection strategies, very few studies have been reported regarding direct detection of DNA sequence and mutation in dsDNA. Here, we describe the feasibility of detection and discrimination of target DNA sequences and single base mutations (SBM) directly in double‐stranded oligonucleotides and PCR products without the need for denaturation of the target dsDNA samples. This goal was achieved by employing a peptide nucleic acid (PNA) chain, self‐assembled on the gold electrode as a probe, which binds to dsDNA and forms PNA‐dsDNA hybrid.     

A fully portable microchip real‐time polymerase chain reaction for rapid detection of pathogen

Point‐of‐care detection for pathogen is of critical need for wide epidemic warning and medical diagnosis. In this work, we have designed and developed a fully portable and integrated microchip based real‐time polymerase chain reaction machine for rapid pathogen detection. The instrument consists of three functional components including heating, optical, and electrical modules, which are integrated into a portable compact box. The microchip is consumable material replaceable to meet various detection needs. Consequently, we demonstrated the outstanding performance of this portable machine for rapid detection of Salmonella and Escherichia coli O157:H7 with the advantage of time‐saving (～25 min), less samples consumption, portability, and user‐friendly operation.     

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.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.     

SNPs Feasibility of Nonlabeled Oligonucletides on Using Electrochemical Sensing

We have demonstrated an electrochemical gene chip protocol for the SNPs detection of nonlabeled DNA. Using an array consisting of streptavidin‐modified gold electrodes, probe DNA were attached through the application of a direct electric field. Electrochemical response changes originating from the hybridization of nucleic acids to protein‐bound nucleic acids using soluble mediators in K₃Fe(CN)₆ solution could then be observed. The electrochemical protocol developed showed high sensitivity and good reproducibility in the detection of DNA hybridization. Significant changes in electrochemical signals were also observed when using target DNA with a single base mismatch, indicating the applicability of this method to single nucleotide polymorphisms (SNPs) detection.     

A microchip electrophoretic assay for DNA methyltransferase activity based on methylation‐sensitive endonuclease DpnⅡ

DNA methylation is a significant epigenetic modification and the methods for the detection of DNA methyltransferase (MTase) activity are important due to aberrant methylation closely related to the occurrence of cancer. In this study, a simple and rapid microchip electrophoresis (ME) coupled with LED‐induced fluorescence (LEDIF) method was presented for the detection of Dam MTase activity. This strategy was based on methylation‐sensitive endonuclease DpnⅡ which could recognize the same specific site 5′‐GATC‐3′ with Dam MTase in double‐stranded DNA (dsDNA). The adenines in the specific site could be methylated by Dam MTase, then the special site could not be digested by DpnⅡ. Both methylated dsDNA and unmethylated dsDNA could be analyzed by ME‐LEDIF after stained by SYBR gold. The results showed the fluorescence intensities of methylated dsDNA were directly proportional to Dam MTase activities in the range of 0.5–20 U/mL with a detection limit of 0.12 U/mL. Furthermore, the method could successfully be applied to evaluation experiments of Dam MTase inhibitors. The results confirmed the ME‐LEDIF method is a promising approach for inhibitors screening of DNA MTase and development of anticancer drugs     

Efficient Preparation of Large‐Sized Rings of Single‐Stranded DNA through One‐Pot Ligation of Multiple Fragments

Circular single‐stranded DNA (c‐ssDNA) has significant applications in DNA detection, the development of nucleic acid medicine, and DNA nanotechnology because it shows highly unique features in mobility, dynamics, and topology. However, in most cases, the efficiency of c‐ssDNA preparation is very low because polymeric byproducts are easily formed due to intermolecular reaction. Herein, we report a one‐pot ligation method to efficiently prepare large c‐ssDNA. By ligating several short fragments of linear single‐stranded DNA (l‐ssDNA) in one‐pot by using T4 DNA ligase, longer l‐ssDNAs intermediates are formed and then rapidly consumed by the cyclization. Since the intramolecular cyclization reaction is much faster than intermolecular polymerization, the formation of polymeric products is suppressed and the dominance of intramolecular cyclization is promoted. With this simple approach, large‐sized single‐stranded c‐ssDNAs (e.g., 200‐nt) were successfully synthesized in high selectivity and yield.     

Nucleic acid and SNP detection via template‐directed native chemical ligation and inductively coupled plasma mass spectrometry

Detection of nucleic acids and single nucleotide polymorphisms (SNPs) is of pivotal importance in biology and medicine. Given that the biological effect of SNPs often is enhanced in combination with other SNPs, multiplexed SNP detection is desirable. We show proof of concept of the multiplexed detection of SNPs based on the template‐directed native chemical ligation (NCL) of PNA‐probes carrying a metal tag allowing detection using ICP‐MS. For the detection of ssDNA oligonucleotides (30 bases), two probes, one carrying the metal tag and a second one carrying biotin for purification, are covalently ligated. The methodological limit of detection is of 29 pM with RSD of 6.7% at 50 pM (n = 5). Detection of SNPs is performed with the combination of two sets of reporter probes. The first probe set targets the SNP, and its yield is compared with a second set of probes targeting a neighboring sequence. The assay was used to simultaneously differentiate between alleles of three SNPs at 5‐nM concentration.     

Homogeneous DNA Detection Based on Fluorescence Quenching by Nanoparticles in Single-step Format :Target-Induced Configuration Transform

A new strategy for homogeneous detection of DNA hybridization in single-step format was developed based on fluorescence quenching by gold nanoparticles. The gold nanoparticle is functionalized with 5’-thiolated 48-base oligonucleotide (probe sequence), whose 3’-terminus is labeled with fluorescein (FAM), a negatively charged fluorescence dye. The oligonucleotide adopts an extended configuration due to the electrostatic repulsion between negatively charged gold nanoparticle and the FAM-attached probe sequence. After addition of the complementary target sequence, specific DNA hybridization induces a conformation change of the probe from an extended structure to an arch-like configuration, which brings the fluorophore and the gold nanoparticle in close proximity. The fluorescence is efficiently quenched by gold nanoparticles. The fluorescence quenching efficiency is related to the target concentration, which allows the quantitative detection for target sequence in a sample. A linear detection range from 1.6 to 209.4 nmol/L was obtained under the optimized experimental conditions with a detection limit of 0.1 nmol/L. In the assay system, the gold nanoparticles act as both nanoscaffolds and nanoquenchers. Furthermore, the proposed strategy, in which only two DNA sequences are involved, is not only different from the traditional molecular beacons or reverse molecular beacons but also different from the commonly used sandwich hybridization methods. In addition, the DNA hybridization detection was achieved in homogenous solution in a single-step format, which allows real-time detection and quantification with other advantages such as easy operation and elimination of washing steps.