Analytical Techniques for DNA Extraction

Analytical Techniques for desoxyribonucleic acid ExtractionDevelopment of deoxyribonucleic acid sensing elements for lavishlyly sensitive detection of sequence specific deoxyribonucleic acid has become crucial out-of-pocket to their extensive maskings in clinical diagnosis, pathogen detection, gene expression studies, and environmental monitoring.ref Along with complementary base-pair hybridization betwixt long oligonucleotide for DNA detection, several DNA sensors employ short oligonucleotide (10 base pair) to this goal. Ref Easley and co-workers constructed the electrochemical proximity assay (ECPA) for highly sensitive and highly discriminating quantitative detection of protein, where stain-induced DNA hybridization between 5, 7, or 10 complementary base system brings redox tag close to the sensor bug out resulting direct electrochemical readout.To date, many analytical techniques have been established for DNA detection, such as electrochemistry, fluorescence, surfac e plasmon resonance, chemiluminiscence, quartz crystal microbalance and so on. Ref Among these methods, electrochemical DNA (E-DNA) sensors have attracted everywheremuch attention owing to their reliability, simplicity, rapid response, broken cost and portability, down in the mouth sample consumption, ability to work in interlocking-multicomp one and only(a)nt samples and remarkably high sensitivity and selectivity.ref The basic formula of E-DNA sensor is based on immobilization of single isolated DNA analyze, a selective biological recognition element, on a sensor surface followed by incubation with sample containing the target biomolecules. When a target-induced molecular recognition event (hybridization) takes place the sensor translates that to a measur up to(p) electrochemical signal which is at one time correlated to the target concentration. In recent years, numerous enquiry groups have studied the performance of these sensors by investigating the offspring of immob ilized national structure and investigating surface density, nature of the redox reporter used, target length, ionic strength of buffer and modifying the frequency of the squ ar-wave voltammetry employed. ref Nevertheless, distance dependence of the redox tag coition to the electrode surface to achieve maximum signal has never been explored. As solid-phase hybridization is very distinct from that in solution-phase in terms of kinetics and thermodynamics, ref sensor performance may be sensitive to the location of the redox reporter because surface charge would likely alter the hybridization rate of negatively supercharged DNA which, in turn, alters the signaling properties of E-DNA sensors. Especially for short oligonucleotide (10 base pair) hybridization near surface the effect may lead to very receivable to their low binding energy which is not sufficient to overcome. Here, we describe a specificed study of the extent to which the location of the redox reporter base be var ied to achieve maximum signal within shorter response time in effort to contrive efficient E-DNA sensors with improved sensitivity.Prior to this work, these electrochemical DNA (E-DNA) and electrochemical, aptamer based (E-AB) sensors have been reported against specific DNA and RNA sequences,2 proteins,3,4 small molecules,5-7 and inorganic ions.8,9 Because all of the sensing components in the E-DNA/EAB platform are covalently connected to the interrogating electrode, the approach requires neither exogenous reagents nor labeling of the target. Likewise, because their signaling is connect to specific, binding-induced changes in the dynamics of the probe DNA (rather than changes in adsorbed mass, charge, etc.), these sensors function well when challenged with complex, contaminant-ridden samples such as business blood serum, soil extracts, and foodstuffs.5,7,9,10 These attributes render the E-DNA/E-AB platform an appealing approach for the specific detection of oligonucleotides and other targets that bind DNA or RNA.11-13In the above methods, electrochemical biosensors are much popular because of their simple instrumentation setup, low sample and reagent consumption as well as high sensitivity and selectivity (Wenetal.,2012 Lu etal.,2012 Wenetal.,2011 Farjamietal.,2011 Xia etal.,2010 Xiang andLu, 2012 Pei etal.,2011 Farjamietal.,2013 Liu etal.,2013b).Electrochemical methods,1,11 macrocosm simple, portable and low-cost, are particularly charismatic for DNA detection.1216Electrochemical methods have been used extensively in DNA detection assays, as summarized in recent review articles.15,16Among these protocols, the electrochemical biosensors have attracted particular attention in different fields owing to its small dimensions, easy operation, rapid response, low cost, high sensitivity and selectivity 10,11.Among these techniques, the electrochemical techniques have received great interests owing to its superior characteristics of rapid response, low-cost, sm all-size, simple operation, and good selectivity 13-16.Among these approaches, electrochemical methods have been shown to be superior over the other existing measurement systems,11 because electrochemical transduction possesses a potential allowing the development of rapid, simple, low-cost, and portable devices.12-14As an alternative to conventional techniques, electrochemical DNA biosensors have attracted healthy interest owing to their intrinsic advantages, including good portability, fast response, and remarkably high sensitivity (Sun etal.,2010). More crucially, a number of DNA biosensors have been developed and extensively applied for the determination of biomarkers (Huang etal.,2014).Microfabrication technology has enabled the development of electrochemical DNA biosensors with the capacity for sensitive and sequence-specific detection of nucleic acids.1-5 The ability of electrochemical sensors to directly identify nucleic acids in complex mixtures is a significant advantage over approaches such as polymerase chain reaction (PCR) that require target purification and amplification.Electrochemical DNA sensors are reliable, fast, simple, and cost- in effect(p) devices that convert the hybridization occurring on an electrode surface into an electrical signal by means of direct or indirect methods.the electrochemical DNA (E-DNA) sensor is one of them. This sensor platform, the electrochemical equivalent of optical molecular beacons, exhibits notable sensitivity, specificity and operational convenience whilst also being fully electronic, reusable and able to work in complex, contaminant-rich samples 4-6.Compared with other transducers, electrochemical ones received particular interest due to a rapid detection and great sensitivity. Combining the characteristics of DNA probes with the capacity of direct and label-free electrochemical detection represents an attractive solution in many different fields of application, such as rapid monitoring of pollutant age nts or metals in the environment, investigation and military rating of DNA-drug interaction instruments, detection of DNA base damage in clinical diagnosis, or detection of specific DNA sequences in human, viral, and bacterial nucleic acids 2-8.The determination victimization electrochemical biosensor methods has attracted much interest because of their simple instrumentation, high specificity, sensitivity, rapid, and is two-a-penny with potential for applications in molecular sensing devices.Amongst the electrochemical transducers, carbon electrodes such as smooth carbon, carbon fibre, graphite, or carbon black exhibit several unique properties.Recent engineering advances have enabled the development of electrochemical DNA biosensors with molecular symptomatic capabilities (2, 8, 18, 33, 47). Electrochemical DNA biosensors offer several advantages compared to alternative molecular detection approaches, including the ability to analyze complex body fluids, high sensitivity, compa tibility with microfabrication technology, a low provide requirement, and compact instrumentation compatible with portable devices (18, 48). Electrochemical DNA sensors consist of a recognition layer containing oligonucleotide probes and an electrochemical signal transducer. A well-established electrochemical DNA sensor strategy involves sandwich hybridization of target nucleic acids by capture and detector probes (5, 7, 46, 50).First reported in 2003, electrochemical DNA (E-DNA) biosensors are reagentless, single-step sensors comprised of a redox-reporter-modified nucleic acid probe attached to an interrogating electrode.1 Originally used for the detection of DNA29 and RNA10 targets, the platform has since been expanded to the detection of a wide range of small molecules,11,12 inorganic ions,13,14 and proteins,12,1517 including antibodies,18,19 via the introduction of aptamers and nucleic-acid-small molecule and nucleic-acid-peptide conjugates as recognition elements (reviewed in refs 20 and 21).Irrespective of their specific target, all of these sensors are predicated on a common mechanism binding alters the efficiency with which the attached redox reporter approaches the electrode due to either the steric bulk of the target or the changes in the conformation of the probe.1,12,18 Given this mechanism, these sensors are quantitative, single-step (washfree), and selective enough to perform well even in complex clinical samples.12,15 They are likewise supported on micrometer- scale electrodes22 and require only inexpensive, hand-held driving electronics (analogous to the home glucose meter23), suggesting they are well suited to applications at the point-of-care.Among these, the electrochemical detection of DNA hybridization appears promising due to its rapid response time, low cost, and suitability for mass production.11,12 The E-DNA sensor,13-16 which is the electrochemical equivalent of an optical molecular beacon,17-20 appears to be a particularly promisin g approach to oligonucleotide detection because it is rapid, reagentless, and operationally convenient.21,22 The E-DNA sensor is comprised of a redox-modified stemloop probe that is immobilized on the surface of a gold electrode via self-assembled monolayer chemistry. In the absence of a target, the stem-loop holds the redox moiety in proximity to the electrode, producing a large Faradic current. Upon target hybridization, the stem is broken and the redox moiety moves out-of-door from the electrode surface. This produces a readily measurable reduction in current that can be related to the heading and concentration of the target sequence. Both E-DNA sensors13-16 and related sensors based on the binding-induced folding of DNA aptamers23-28 have been extensively studied in recent years. Nevertheless, key issues in their fabrication and use have not yet been explored in detail.Electrochemical biosensors, combining the sensitivity of electroanalytical methods with the inherent bio-sele ctivity of the biological component, have found extensive application in diverse fields because of their high sensitivity with relatively simple and low-cost measurement systems.1 For example, by assembling artful target-responsive DNA architectures on the electrode surface, a series of electrochemical bioanalysis methods have been proposed for the sensing of specific biomarkers, such as DNA and proteins.2-5 The typical sensing schemes of these designs involve the immobilization of an efficient probe on the electrode surface, incubation with target biomolecules, and measurement of the output electrochemical signal.6,7A wide variety of nanomaterials including metal nanoparticles, oxide nanoparticles, quantum dots, carbon nanotubes, graphene and even hybrid nanomaterials have found attractive application in electrochemical biosensing, such as detection of DNA, proteins and pathogens and the design of biological nanodevices (bacteria/cells).14,15Electrochemical transducers offer broad opportunities in DNA sensor design due to simple experiment protocols, inexpensive and mostly commercially available equipment.Among various detection methods, the electrochemical approach attracted much attention due to its rapidness, low cost, high sensitivity and compatibility with portability 10,11. The E-DNA sensor 12,13, an electrochemical method derived from the optical molecular beacon14,15, is particularly promising because it is reagentlessness andoperation convenience. In brief, the E-DNA sensor is composed of a redox-modified hairpin-like stem-loop DNA probe that is immobilized on the electrode surface. Without a target, the stem-loop structure holds the redox probe close to the electrode surface, pro-ducing a large current. Upon hybridization with a target, the stem is opened and the redox label moves away from the electrode surface and the current is decreased. This current change is directly related to the target DNA concentration.Many different versions of the E-DNA sensor have been reported to date 7-9. A popular construct of this type of sensors is a folding-based E-DNA sensor comprised of a redox-labeled DNA stem-loop probe covalently attached to a gold disk electrode. In the absence of a target, the stem-loop conformation holds the redox label in close proximity to the electrode, facilitating electron transfer. In the presence of and binding to a complementary DNA target, hybridization forces the redox tag farther from the electrode, impeding electron transfer and producing an observable reduction in redox current 4-6.In this approach, a single-stranded DNA (ssDNA) probe is immobilized on a surface and exposed to a sample containing the specific complementary target sequence, which is captured by forming a double-stranded DNA(dsDNA) molecule. This recognition event (hybridization) is then transduced into a readable signal.In this strategy, the target is anchored to the sensor surface by the capture probe and detected by hybridization with a detector probe linked to a reporter function. Detector probes coupled to oxidoreductase reporter enzymes allow amperometric detection of redox signals by the sensor electrodes (28, 34). When a fixed potential is applied between the working and reference electrodes, enzyme-catalyzed redox activity is detected as a measurable electrical current (11, 16, 27). The current amplitude is a direct materialisation of the number of target-probe-reporter enzyme complexes anchored to the sensor surface. Because the initial step in the electrochemical detection strategy is nucleic acid hybridization rather than enzyme-based target amplification, electrochemical sensors are able to directly detect target nucleic acids in clinical specimens, an advantage over nucleic acid amplification techniques, such as PCR.Electrochemical methods are typically inexpensive and rapid methods that allow distinct analytes to be detected in a highly sensitive and selective manner 22-25. Although electrochemical DN A sensors utilize a range of distinct chemistries, they all take advantage of the nanoscale interactions among the target present in solution, the recognition layer, and the solid electrode surface. This has led to the development of simple signal transducers for the electrochemical detection of DNA hybridization by using an inexpensive analyzer. DNA hybridization can be detected electrochemically by using various strategies that exploit the electrochemistry of the redox reaction of reporters 26 and enzymes immobilized onto an electrode surface 27, direct or catalytic oxidation of DNA bases 28-31, electrochemistry of nanoparticles 32-35, conducting polymers (CPs) 35-37, and quantum dots 38.E-DNA sensors, the electrochemical analog of optical molecular beacons e.g.,1-4, are based on the hybridization-induced folding of an electrode-bound, redox-tagged DNA probe. In their original implementation, the concentration of a target oligonucleotide is recorded when it hybridizes to a stem-l oop DNA probe, leading to the formation of a rigid, double stranded duplex that sequesters the redox tag from the interrogating electrode 1. Follow-on E-DNA architectures have dispensed with the stem-loop probe in favor of linear probes, leading to improved binding thermodynamics and, thus, improved gain 5, as well as strand-invasion, hairpin and pseudoknot probes producing signal-on sensors 6-8. Because E-DNA sensors are reagentless, electronic (electrochemical) and highly selective (they perform well even when challenged directly in complex, multicomponent samples such as blood serum or soil) e.g., 9, E-DNA sensors appear to be a promising and appealing approach for the sequence-specific detection of DNA and RNA see, e.g., 10,11.E-DNA signaling arises due to hybridization-linked changes in the rate, and thus efficiency, with which the redox moiety collides with the electrode and transfers electrons.To design efficient DNA-electrochemical biosensors, it is essential to know the str ucture and to understand the electrochemical characteristics of DNA molecules.Motivated by the potential advantages of the E-DNA sensing platform, numerous research groups have explored their fabrication and optimization over the past decade. Specifically, efforts have been made to improve the platforms signal gain (change in signal upon the addition of saturating target) by optimizing the frequency of the square-wave potential rampemployed,11 the density with which the target-recognizing probes packed onto the electrode,11,24 probe structure,25 the redox reporter employed,26 and the nature of the monolayer coating the electrode.25Contributing to these studies, we describe here a more comprehensive study of the extent to which the square-wave voltammetric approach itself can be optimized to achieve maximum signal gain. Specifically, we have investigated the effect of varying the square-wave frequency, amplitude, and potential step-size on the gain of E-DNA sensors, evaluating each line of reasoning as a function of the others as well as of the structure of the E-DNA probe, its packing density, the nature of its redox-reporter, and the monolayer chemistry used to coat the sensing electrode.E-DNA sensors are a reagentless, electrochemical oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely certified on the nature of the self-assembled monolayer coating the electrode. We have investigated this enquire by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and deaf(p) alkane thiols.The signaling mechanism of E-DNA sensors is linked to a bindingspecific change in the flexibility of the redox-tagged probe upon hybridization, the relatively rigid target/probe duplex hampers the conflict of the electroche mical tag thus decreasing the observable amperometric signal 5,12. This, in turn, suggests that E-DNA signaling may be sensitive to changes in surface chemistry which, due to surface charge and steric bulk effects, would likely alter the dynamics of a negatively charged DNA probe. However, despite rapid growth in the E-DNA belles-lettres reviewed in 13 the extent to which surface chemistry affects E-DNA signaling has not been established all previous E-DNA sensors were fabricated using hydroxyl-terminated alkane thiol self-assembled monolayers (SAMs) e.g.,1,3,5,7,9. Here we address this question and describe a study of E-DNA sensors fabricated using co-adsorbates of various lengths and charges in an effort to further optimize E-DNA performance.For example, while it is likely that the signaling properties of these sensors depend sensitively on the density of immobilized probe DNA molecules on the sensor surface (measured in molecules of probe per square centimeter) see, e.g., refs 5 and 29-36, no systematic study of this effect has been reported.Similarly, while it appears that the size of the target and the location of the recognition element within the target sequence affect signal suppression,24 this effect, too, has seen relatively little study. Here we detail the effects of probe surface density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of the E-DNA sensor.However, the sensitivity is one of the most important limiting factors for the development of electrochemical DNA biosensors.