Developing a Nano‐Biosensor for DNA Hybridization Using a New Electroactive Label

Development of electrochemical DNA hybridization biosensors based on carbon paste electrode (CPE) and gold nanoparticle modified carbon paste electrode (NGMCPE) as transducers and ethyl green (EG) as a new electroactive label is described. Electrochemical impedance spectroscopy and cyclic voltammetry techniques were applied for the investigation and comparison of bare CPE and NGMCPE surfaces. Our voltammetric and spectroscopic studies showed gold nanoparticles are enable to facilitate electron transfer between the accumulated label on DNA probe modified electrode and electrode surface and enhance the electrical signals and lead to an improved detection limit. The immobilization of a 15‐mer single strand oligonucleotide probe on the working electrodes and hybridization event between the probe and its complementary sequence as a target were investigated by differential pulse voltammetry (DPV) responses of the EG accumulated on the electrodes. The effects of some experimental variables on the performance of the biosensors were investigated and optimum conditions were suggested. The selectivity of the biosensors was studied using some non‐complementary oligonucleotides. Finally the detection limits were calculated as 1.35×10^?10 mol/L and 5.16×10^?11 mol/L on the CPE and NEGCPE, respectively. In addition, the biosensors exhibited a good selectivity, reproducibility and stability for the determination of DNA sequences.     

An introduction to electrochemical DNA biosensors

Electrochemical DNA biosensors exploit the affinity of single-stranded DNA for complementary strands of DNA and are used in the detection of specific sequences of DNA with a view towards developing portable analytical devices. Great progress has been made in this field but there are still numerous challenges to overcome. This review for researchers new to the field describes the components of electrochemical DNA biosensors and the important issues in their design. Methods of transducing DNA binding events are discussed along with future directions for DNA biosensors.     

Bioelectrochemistry for sensing amino acids,peptides, proteins and DNA interactions

Oxidative damage to peptides, proteins and DNA is considered to be one of the major causes of cancer and age-related diseases. The interaction of biomolecules, peptides, proteins, nucleic acids and pharmaceuticals with solid electrode surfaces is not only a fundamental phenomenon but also a key to important and novel analytical sensing applications in biosensors, biotechnology, medical devices and drug-delivery schemes. Electrochemical methods can provide insight into the redox mechanisms and the electron-transfer reactions of a variety of fundamental biological processes.     

Bioconjugation techniques for microfluidic biosensors

We have evaluated five bioconjugation chemistries for immobilizing DNA onto silicon substrates for microfluidic biosensing applications. Conjugation by organosilanes is compared with linkage by carbonyldiimidazole (CDI) activation of silanol groups and utilization of dendrimers. Chemistries were compared in terms of immobilization and hybridization density, stability under microfluidic flow-induced shear stress, and stability after extended storage in aqueous solutions. Conjugation by dendrimer tether provided the greatest hybridization efficiency; however, conjugation by aminosilane treated with glutaraldehyde yielded the greatest immobilization and hybridization densities, as well as enhanced stability to both shear stress and extended storage in an aqueous environment. Direct linkage by CDI activation provided sufficient immobilization and hybridization density and represents a novel DNA bioconjugation strategy. Although these chemistries were evaluated for use in microfluidic biosensors, the results provide meaningful insight to a number of nanobiotechnology applications for which microfluidic devices require surface biofunctionalization, for example vascular prostheses and implanted devices. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA hybridization detection at heated electrodes

The detection of DNA hybridization is of central importance to the diagnosis and treatment of genetic diseases. Due to cost limitations, small and easy-to-handle testing devices are required. Electrochemical detection is a promising alternative to evaluation of chip data with optical readout. Independent of the actual readout principle, the hybridization process still takes a lot of time, hampering daily use of these techniques, especially in hospitals or doctor's surgery. Here we describe how direct local electrical heating of a DNA-probe-modified gold electrode affects the surface hybridization process dramatically. We obtained a 140-fold increase of alternating current voltammetric signals for 20-base ferrocene-labeled target strands when elevating the electrode temperature during hybridization from 3 to 48 degrees C while leaving the bulk electrolyte at 3 degrees C. At optimum conditions, a target concentration of 500 pmol/L could be detected. Electrothermal regeneration of the immobilized DNA-probe strands allowed repetitive use of the same probe-modified electrode. The surface coverage of DNA probes, monitored by chronocoulometry of hexaammineruthenium(III), was almost constant upon heating to 70 degrees C. However, the hybridization ability of the probe self-assembled monolayer declined irreversibly when using a 70 degrees C hybridization temperature. Coupling of heated electrodes and highly sensitive electrochemical DNA hybridization detection methods should enhance detection limits of the latter significantly.     

Electrochemical biosensors for rapid pathogen detection

Rapid pathogen detection is an emerging issue in clinical, environmental, and food industry sectors. Biosensors can represent a solution to culture-based and molecular methods as they respond to sensitivity, specificity, and rapidity needs. Screen-printed electrodes have been used in association with nanoparticles to increase the signal and improve sensitivity reaching low numbers of the targets. Antibodies, DNA probes, and aptamers are mainly used to functionalize the working electrodes to ensure high specific pathogen detection by the use of voltammetry, impedance spectroscopy, amperometry, and conductivity. Electrochemical biosensors can be miniaturized to construct portable devices useful for in situ assays.     

Electrochemical DNA Biosensors Applicable to the Study of Interactions Between DNA and DNA Intercalators

Electrochemical DNA biosensors, based either on carbon paste electrode (CPE) or hanging mercury drop electrode (HMDE) were prepared. These biosensors were used in the study of interaction between double stranded DNA (dsDNA) and single stranded DNA (ssDNA) and acridine orange, a well known DNA intercalator. The different electrochemical behaviors were compared in the article.     

Signal enhancement of electrochemical DNA biosensors for the detection of trace heavy metals

Heavy-metal pollution has attracted intensive attention from the public because of the severe threats of heavy metals to the ecosystem and human health. Ultralow concentration of heavy metals in aquatic environment leads to the urgent needs of sensitive approaches for heavy-metal detection. Electrochemical DNA biosensors present outstanding superiority in convenience, selectivity, and sensitivity compared with conventional methods. To achieve the ultralow detection limit, efforts have been made to implement signal enhancement strategies to develop electrochemical DNA biosensors with enhanced sensing performance. This review focuses on the recent progress in signal enhancement strategies applied to electrochemical DNA biosensors for heavy-metal-ion detection including nicking enzyme–assisted amplification, the utilization of core–shell nanoparticles, and nanocomposites modification.     

Confinement effects on DNA hybridization in electrokinetic micro‐ and nanofluidic systems

Spatial confinement, within cells or micro‐ and nanofabricated devices, impacts the conformation and binding kinetics of biomolecules. Understanding the role of spatial confinement on molecular behavior is important for comprehending diverse biological phenomena, as well as for designing biosensors. Specifically, the behavior of molecular binding under an applied electric field is of importance in the development of electrokinetic biosensors. Here, we investigate whether confinement of DNA oligomers in capillary electrophoresis impacts the binding kinetics of the DNA. To infer the role of confinement on hybridization dynamics, we perform capillary electrophoresis measurements on DNA oligomers within micro‐ and nanochannels, then apply first‐order reaction dynamics theory to extract kinetic parameters from electropherogram data. We find that the apparent dissociation constants at the nanoscale (i.e., within a 100 nm channel) are lower than at the microscale (i.e., within a 1 μm channel), indicating stronger binding with increased confinement. This confirms, for the first time, that confinement‐based enhancement of DNA hybridization persists under application of an electric field.     

脱氧核糖核酸电化学传感器的原理及其应用

对电化学DNA传感器的组成及其在DNA损伤研究、环境污染监控、病原基因检测、基因疾病诊断和药物机理分析等方面的进行了总结，并对其发展趋势进行了评述。     

Lateral flow devices for nucleic acid analysis exploiting quantum dots as reporters

There is a growing interest in the development of biosensors in the form of simple lateral flow devices that enable visual detection of nucleic acid sequences while eliminating several steps required for pipetting, incubation and washing out the excess of reactants. In this work, we present the first dipstick-type nucleic acid biosensors based on quantum dots (QDs) as reporters. The biosensors enable sequence confirmation of the target DNA by hybridization and simple visual detection of the emitted fluorescence under a UV lamp. The ‘diagnostic’ membrane of the biosensor contains a test zone (TZ) and a control zone (CZ). The CZ always fluoresces in order to confirm the proper function of the biosensor. Fluorescence is emitted from the TZ, only when the specific nucleic acid sequence is present. We have developed two general types of QD-based nucleic acid biosensors, namely, Type I and Type II, in which the TZ consists of either immobilized streptavidin (Type I) or immobilized oligodeoxynucleotides (Type II). The control zone consists of immobilized biotinylated albumin. No purification steps are required prior to the application of the DNA sample on the strip. The QD-based nucleic acid biosensors performed accurately and reproducibly when applied to (a) the visual detection of PCR amplification products and (b) visual genotyping of single nucleotide polymorphisms (SNPs) in human genomic DNA from clinical samples. As low as 1.5 fmol of double-stranded DNA were clearly detected by naked eye and the dynamic range extended to 200 fmol. The %CV were estimated to be 4.3–8.2.     

Perspectives on electrochemical biosensing of COVID-19

Rapid detection of human coronavirus disease 2019, termed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or COVID-19 infection, is urgently needed for containment strategy owing to its unprecedented spreading. Novel biosensors can be deployed in remote clinical settings without central facilities for infection screening. Electrochemical biosensors serve as analytical tools for rapid detection of viral structure proteins, mainly spike (S) and nucleocapsid (N) proteins, human immune responses, reactive oxygen species, viral ribonucleic acid, polymerase chain reaction by-products, and other potential biomarkers. The development of point-of-care testing devices is challenging due to the requirement of extensive validation, a time-consuming and expensive step. Together with specific biorecognition molecules, nanomaterial-based biosensors have emerged for the fast detection of early viral infections.     

Microfluidic integration for electrochemical biosensor applications

Electrochemical biosensors are particularly suitable for miniaturization and integration in microfluidic devices. Applications include the detection of whole cells, cell components, proteins, and small molecules to address tasks in the fields of diagnostics and food and environmental control. Microfluidic setups range from simple channels for sample transport to channels with integrated sensing electrodes to highly sophisticated platforms with additional elements for sample preparation. The design of the microfluidics depends on both the type of detection and on the application and sample material. This review summarizes recent work on electrochemical biosensors with integrated microfluidics with the focus on developments for real sample applications, particularly those including measurements with real sample media.     

An Electrochemical Study of the Surface Hybridization Process of Morpholino‐DNA: Thermodynamics and Kinetics

Morpholino (MO) is a neutral analogue of DNA, which shows promise in the development of DNA biosensors and diagnostic devices. The present study explores the hybridization process of a surface‐attached MO 22‐mer with 10‐mer and 20‐mer DNA targets on a gold electrode. The melting process of the MO‐DNA duplex at the electrode/buffer interface is recorded using cyclic voltammetry. These results show that the length of target DNA, the binding location of the target DNA on the surface‐immobilized MO chain, and electrostatic forces from neighbouring duplexes all modulate the stability and hybridization kinetics of the DNA targets with the MO probes. Melting temperatures for immobilized MO‐DNA duplexes are found to be insensitive to ionic strength, provided the duplexes do not have a linker. Although the melting temperature does not shift appreciably with ionic strength, the maximum hybridization yield does. This somewhat surprising observation is considered to originate from an electrostatic limit on the extent of attainable hybridization. It is also reported that hybridization tends to initiate at the upper half of MO probes.     

Electrical switching of DNA monolayers investigated by surface plasmon resonance

The switching of DNA monolayers between a "lying" and a "standing" state initiated by applying electric field, and the subsequent DNA hybridization at different states were investigated in a contactless, label-free mode by surface plasmon resonance (SPR) technique. The results showed that the strength of the electric field and surface coverage could influence the switching of DNA monolayers. In addition, it was found that DNA hybridization efficiency could be enhanced or decreased when DNA probes stood straight up or lay flat on the gold surface, depending on the potential of the gold substrate. The enhancement of DNA hybridization efficiency reached the maximum when surface coverage reached 5.87 x 10(12) molecules/cm(2) and the potential of gold substrate was more negative than -0.7 V (versus ITO-coated glass). The research may be helpful for the construction of sensitive biosensors, biochips, and nanoscale electronic devices.     

Carbon film electrodes for oxidase-based enzyme sensors in food analysis

Carbon film resistor electrodes have been evaluated as transducers for the development of multiple oxidase-based enzyme electrode biosensors. The resistor electrodes were first modified with Prussian Blue (PB) and then covered by a layer of covalently immobilized enzyme. Electrochemical impedance spectroscopy was used to characterize the interfacial behaviour of the Prussian Blue modified and enzyme electrodes; the spectra demonstrated that the access of the substrates is essentially unaltered by application of the enzyme layer. These enzyme electrodes were used to detect the substrate of the oxidase (glucose, ethanol, lactate, glutamate) via reduction of hydrogen peroxide at +50 mV versus Ag/AgCl in the low micromolar range. Response times were 1-2 min. Finally, the glucose, ethanol and lactate electrochemical biosensors were used to analyse complex food matrices—must, wine and yoghurt. Data obtained by the single standard addition method were compared with a spectrophotometric reference method and showed good correlation, indicating that the electrodes are suitable for food analysis.     

Electrochemical DNA biosensor for the detection of TT and Hepatitis B virus from PCR amplified real samples by using methylene blue

DNA biosensors based on nucleic acid hybridization processes are rapidly being developed towards the goal of rapid and inexpensive diagnosis of genetic and infectious diseases. Electrochemical transducers are often being used for detecting the DNA hybridization event, due to their high sensitivity, small dimensions, low cost, and compatibility with microfabrication technology. In this study, an electrochemical biosensor for the voltammetric detection of DNA sequences related to the Hepatitis B virus (HBV) and TT virus (TTV) from polymerase chain reaction (PCR) amplified real samples is described for the first time. The biosensor relies on the immobilization of the 21- or 24-mer single stranded oligonucleotides (probe) related to the HBV and TTV sequences and hybridization of these oligonucleotides with their complementary sequences (target) at carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (MB) as the hybridization indicator. As a result of the interaction between MB and the bound guanine bases of hybrid at CPE surface, the MB signal decreased, when it was compared with the MB signal, which was observed with probe modified CPE. The difference between the MB signals, obtained from the hybrid modified and the probe modified CPE is used to detect the DNA sequences of the infectious diseases from PCR amplified real samples. Numerous factors affecting the target hybridization and indicator binding reactions are optimized to maximize the sensitivity.     

Point‐of‐Care Smartphone‐based Electrochemical Biosensing

Point‐of‐care (PoC) biosensors offer promising solutions to today's adverse and costly healthcare issues by moving diagnostic tools closer to the patient. The ubiquity of smartphones has brought about an emergence of PoC devices, which leverage the smartphone's capabilities, enabling the creation of low‐cost and portable biosensors. Electrochemical biosensors are well suited for PoC testing since the transducers can be miniaturized and inexpensively fabricated. This review paper discusses recent developments in smartphone‐based electrochemical biosensors for PoC diagnostics. These peripherals utilize the various connectivity options (for example proprietary ports, audio headphone‐jack, or wireless radio) to offload functionality to the smartphone. The smartphone‐based implementations of various electrochemical techniques, such as amperometry, potentiometry, and impedance spectroscopy are explored. Major challenges include reducing power, area, and cost of measurement circuitry, while maintaining adequate performance for PoC diagnostic applications.     

Allele-specific genotyping by using guanine and gold electrochemical oxidation signals

Electrochemical DNA biosensors have become strong candidates for DNA based analysis. Allele-specific genotyping is also one of the important research areas, where electrochemical approaches provide promising advances. Recently reported two methods based on electrochemical guanine and colloidal gold (Au) nanoparticle oxidation signals are reviewed and compared with the existing genotyping methods in this report.     

Electrical biochip technology—a tool for microarrays and continuous monitoring

Based on electrical biochips made in Si-technology cost effective portable devices have been constructed for field applications and point of care diagnosis. These miniaturized amperometric biosensor devices enable the evaluation of biomolecular interactions by measuring the redox recycling of ELISA products, as well as the electrical monitoring of metabolites. The highly sensitive redox recycling is facilitated by interdigitated ultramicroelectrodes of high spatial resolution. The application of these electrical biochips as DNA microarrays for the molecular diagnosis of viral infections demonstrates the measurement procedure. Self-assembling of capture oligonucleotides via thiol-gold coupling has been used to construct the DNA interface on-chip. Another application for this electrical detection principle is continuous measuring with bead-based biosensors. Here, paramagnetic nanoparticles are used as carriers of the bioanalytical interface in ELISA format. A Si-micromachined glucose sensor for continuous monitoring in interstitial fluid ex vivo shows the flexibility of the electrical platform. Here the novel approach is a pore membrane in micrometer-dimensions acting as a diffusion barrier. The electrochemical detection takes place in a cavity containing glucose oxidase and a Pt-electrode surface. The common hydrogen peroxide detection, together with Si technology, enable precise differential measurements using a second cavity.