CE combined with rolling circle amplification for sensitive DNA detection

Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.     

Biosensor for multiplex detection of two DNA target sequences using enzyme-functionalized Au nanoparticles as signal amplification

Multiplex electrochemical detection of two DNA target sequences in one sample using enzyme-functionalized Au nanoparticles (AuNPs) as catalytic labels for was proposed. This DNA sensor was fabricated using a “sandwich” detection strategy, involving two kinds of capture probes DNA immobilized on glassy carbon electrode (GCE), and hybridization with target DNA sequences, which further hybridized with the reporter DNA loaded on the AuNPs. The AuNP contained two kinds of DNA sequences, one was complementary to the target DNA, while the other was noncomplementary to the target. The noncomplementary sequences were linked with horseradish peroxidase (HRP) and alkaline phosphatase (ALP), respectively. Enhanced detection sensitivity was obtained where the AuNPs carriers increased the amount of enzyme molecules per hybridization. Electrochemical signals were generated from the enzymatic products produced from the substrates catalyzed by HRP and ALP. Under optimal conditions, a 33-mer sequence could be quantified over the ranges from 1.5 × 10⁻¹³ to 5.0 × 10⁻¹² M with a detection limit of 1.0 × 10⁻¹³ M using HRP-AuNP as labels, and a 33-mer sequence could be quantified over the ranges from 4.5 × 10⁻¹¹ M to 1.0 × 10⁻⁹ M with a detection limit of 1.2 × 10⁻¹¹ M using ALP-AuNP as labels.     

Enzyme-based electrochemical biosensor for sensitive detection of DNA demethylation and the activity of DNA demethylase

A novel electrochemical method is developed for detection of DNA demethylation and assay of DNA demethylase activity. This method is constructed by hybridizing the probe with biotin tagged hemi-methylated complementary DNA and further capturing streptavidin tagged alkaline phosphatase (SA-ALP) to catalyze the hydrolysis reaction of p-nitrophenyl phosphate. The hydrolysate of p-nitrophenol (PNP) is then used as electrochemical probe for detecting DNA demethylation and assaying the activity of DNA demethylase. Demethylation of target DNA initiates a degradation reaction of the double-stranded DNA (dsDNA) by restriction endonuclease of BstUI. It makes the failed immobilization of ALP, resulting in a decreased electrochemical oxidation signal of PNP. Through the change of this electrochemical signal, the DNA demethylation is identified and the activity of DNA demethylase is analyzed with low detection limit of 1.3 ng mL⁻¹. This method shows the advantages of simple operation, cheap and miniaturized instrument, high selectivity. Thus, it provides a useful platform for detecting DNA demethylation, analyzing demethylase activity and screening inhibited drug.     

MCE‐electrochemical detection for following interactions of ssDNA and dsDNA with methylene blue

The interaction between the organic dye, methylene blue and DNA has been studied by MCE with electrochemical detection. Interaction produces two different signals, one corresponding to free methylene blue and other, for the complex methylene blue–DNA. The hybridization between a ssDNA and a complementary sequence, specific to the severe acute respiratory syndrome virus, has been performed and studied in a thermoplastic olefin polymer of amorphous structure CE‐microchip with an end‐channel gold wire detector. Moreover, studies with a longer dsDNA, an expression vector involved in the transitory or stable expression in mammals cells, pFLAG‐CMV4, has also been performed.     

Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification

Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo⁻), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.     

Apolipoprotein B100 analysis in microchip with electrochemical detection

Apolipoprotein B100(apoB-100) is a major protein of the cholesterol-rich low-density lipoprotein(LDL) and reflects a better assessment of total atherogenic burden to the vascular system than LDL.In this work,a simple and sensitive method has been developed to determine picoliter apoB-100s using the PMMA microfluidic chip coupled with electrochemical detection system. This method performs very well with a detectable linear range of 1-800 pg/mL and a detection limit of 1 pg/mL.A real serum sample has further been detected by this microchip-based biosensor.The results show that this kind of method is practicable and has the potential application in clinical analysis and diagnosis.     

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

New DNA Technology and the DNA Biosensor

Abstract

Recently there has been an increasing demand to produce systems for the detection of specific DNA sequences that are amenable to non-specialised laboratories. This demand has led to many innovative and novel approaches to DNA analysis which may collectively be termed ′new DNA technology′. Here, we review these advances in relation to their applicability for the production of a DNA biosensor. The present state of the art is described and future possibilities are considered.     

Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Aunano-CNT/PANnano films

A polyaniline nanofibers (PAN_nano)/carbon paste electrode (CPE) was prepared via dopping PAN_nano in the carbon paste. The nanogold (Au_nano) and carbon nanotubes (CNT) composite nanoparticles were bound on the surface of the PAN_nano/CPE. The immobilization and hybridization of the DNA probe on the Au_nano-CNT/PAN_nano films were investigated with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator, and electrochemical impedance spectroscopy (EIS) using [Fe(CN)₆]^3−/4− as redox probe. The voltammetric peak currents of MB increased dramatically owing to the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films, and then decreased obviously owing to the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA). The electron transfer resistance (R_et) of the electrode surface increased after the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films and rose further after the hybridization of the probe DNA. The remarkable difference between the R_et value at the DNA-immobilized electrode and that at the hybridized electrode could be used for the label-free EIS detection of the target DNA. The loading of the DNA probe on Au_nano-CNT/PAN_nano films was greatly enhanced and the sensitivity for the target DNA detection was markedly improved. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene and the polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from transgenically modified beans were determined with this label-free EIS DNA detection method. The dynamic range for detecting the PAT gene sequence was from 1.0 × 10⁻¹² mol/L to 1.0 × 10⁻⁶ mol/L with a detection limit of 5.6 × 10⁻¹³ mol/L.     

A highly efficient and versatile microchip capillary electrophoresis method for DNA separation using gold nanoparticle as a tag

A highly efficient and versatile method for DNA separation using Au nanoparticles (Au NPs) as a tag based on microchip capillary electrophoresis (MCE) was developed. The thiol-modified DNA-binding Au NPs were utilized as a tag. Target DNA was sandwiched between Au NPs and probe DNA labeled with horseradish peroxidase (HRP). In electrophoresis separation, the difference in electrophoretic mobility between free probe and probe-target complex was magnified by Au NPs, which enabled the resulting mixture to be separated with high efficiency by microchip capillary electrophoresis. Horseradish peroxidase was used as a catalytic label to achieve sensitive electrochemical DNA detection via fast catalytic reactions. With this protocol, 27-mer DNA fragments with different sequences were separated with high speed and high resolution. The proposed method was critical to achieve improved DNA separations in hybridization analyses.     

Fabrication of tunable microreactor with enzyme modified magnetic nanoparticles for microfluidic electrochemical detection of glucose

A microfluidic device was designed for amperometric determination of glucose by packing enzyme modified magnetic nanoparticles (MNPs) in its microchannel as an enzyme microreactor. Glucose oxidase was covalently attached to the surface of MNPs and localized in the microchannel by the help of an external magnetic field, leading to a tunable packing length. By changing the length of microreactor from 3 to 10 mm, the performance for glucose detection was optimized. The optimal linear range to glucose was from 25 μM to 15 mM with a detection limit of 11 μM at a length of 6 mm. The inter- and intra-day precisions for determination of 1.0 mM glucose were 0.8% and 1.7%, respectively, and the device-to-device reproducibility was 95.6%. The enzyme reactor remained its 81% activity after three-week storage. Due to the advantages of the device and fracture sampling technique, serum samples could be directly sampled through the fracture to achieve baseline separation from ascorbic acid, and proteins in the samples did not interfere with the detection. This work provided a promising way for pretreatment-free determination of glucose with low cost and excellent performance.     

Graphene oxide-based biosensor for sensitive fluorescence detection of DNA based on exonuclease III-aided signal amplification

Based on the super fluorescence quenching efficiency of graphene oxide and exonuclease III aided signal amplification, we develop a facile, sensitive, rapid and cost-effective method for DNA detection. In the presence of target DNA, the target-probe hybridization forms a double-stranded structure and exonuclease III catalyzes the stepwise removal of mononucleotides from the blunt 3′ termini of probe, resulting in the recycling of the target DNA and signal amplification. Therefore, our proposed sensor exhibits a high sensitivity towards target DNA with a detection limit of 20 pM, which was even lower than previously reported GO-based DNA sensors without enzymatic amplification, and provides a universal sensing platform for sensitive detection of DNA.     

Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy

A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwich-type assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH₃)₆]³⁺, RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was non-methylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL⁻¹ to 10 U mL⁻¹, with a detection limit down to 0.03 U mL⁻¹. Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence.     

Rapid separation of strychnine and brucine on a dynamically modified poly(dimethylsiloxane) microchip followed by electrochemical detection

A method has been developed for rapidly separating and detecting strychnine and brucine using a poly(dimethysiloxane) (PDMS) microchip and electrochemical (EC) detection. PDMS microchannels dynamically modified by Brij35 are shown to be more efficient than native ones. The two analytes are well separated within 90 s in 70 mmol/L acetate buffer (pH 5.5) containing 0.01% (v/v) Brij35. Detection limits were found to be 1.0 μmol/L for strychnine and 0.2 μmol/L for brucine at S/N=3. The method was used to determine trace strychnine and brucine in rat serum, and the results obtained correlate well with those obtained via high-performance liquid chromatography (HPLC).      

Simplified current decoupler for microchip capillary electrophoresis with electrochemical and pulsed amperometric detection

There is a need to develop broadly applicable, highly sensitive detection methods for microchip CE that do not require analyte derivatization. LIF is highly sensitive but typically requires analyte derivatization. Electrochemistry provides an alternative method for direct analyte detection; however, in its most common form, direct current (DC) amperometry, it is limited to a small number of easily oxidizable or reducible analytes. Pulsed amperometric detection (PAD) is an alternative waveform that can increase the number of electrochemically detectable analytes. Increasing sensitivity for electrochemical detection (EC) and PAD requires the isolation of detection current (nA) from the separation current (muA) in a process generally referred to as current decoupling. Here, we present the development of a simple integrated decoupler to improve sensitivity and its coupling with PAD. A Pd microwire is used as the cathode for decoupling and a second Au or Pt wire is used as the working electrode for either EC or PAD. The electrode system is easy to make, requiring no clean-room facilities or specialized metallization systems. Sensitive detection of a wide range of analytes is shown to be possible using this system. Using this system we were able to achieve detection limits as low as 5 nM for dopamine, 74 nM for glutathione, and 100 nM for glucose.     

Fabrication of SU-8 based microchip electrophoresis with integrated electrochemical detection for neurotransmitters

A new SU-8 based microchip capillary electrophoresis (MCE) device has been developed for the first time with integrated electrochemical detection. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology has allowed the monolithic integration in the device of the electrochemical detection system based on platinum electrodes. The fabrication of the chips presented in this work is totally compatible with reel-to-reel techniques, which guarantee a low cost and high reliability production. The influence of relevant experimental variables, such as the separation voltage and detection potential, has been studied on the SU-8 microchip with an attractive analytical performance. Thus, the effective electrical isolation of the end-channel amperometric detector has been also demonstrated. The good performance of the SU-8 device has been proven for separation and detection of the neurotransmitters, dopamine (DA) and epinephrine (EP). High efficiency (30,000-80,000 N/m), excellent precision, good detection limit (450 nM) and resolution (0.90-1.30) has been achieved on the SU-8 microchip. These SU-8 devices have shown a better performance than commercial Topas (thermoplastic olefin polymer of amorphous structure) microchips. The low cost and versatile SU-8 microchip with integrated platinum film electrochemical detector holds great promise for high-volume production of disposable microfluidic analytical devices.     

Current strategies for electrochemical detection of DNA with solid electrodes

A review of current strategies aimed at detecting nucleic acids (NA) using NA-modified solid electrodes reveals the versatility and potential of electrochemical detection in this field. What emerged at the beginning of 90s as a very promising detection system in DNA technology is now resulting in the first commercial devices. Many aspects of the experimental design, for example surface immobilisation and detection schemes, are outlined and evaluated. Although most approaches use hybridisation as the recognition reaction, those not based on hybridisation are also included. As is finally shown, great advances have been achieved, although further developments are required if electrochemical devices are to be suitable for routine measurement.Abbreviations A Adenine - a.c.V Alternating current voltammetry - Amp. Amperometry - Apo E Apolipoprotein E - BCB Brilliant cresyl blue - BLM Bilayer lipid membrane - bp Base pairs - BPPG Basal planar pyrolytic graphite - bpy 2,2-Bipyridine - C Cytosine - CMV Citomegalovirus - CPE Carbon paste electrode - CPSA Constant-current chronopotentiometric stripping analysis - Cronoamp. Chronoamperometry - CSPE Carbon screen-printed electrode - CV Cyclic voltammetry - dmb 4,4-Dimethyl-2,2-bipyridine - dmphen Dimethylphenanthroline - DPPZ Dipyrido[3,2-a.2,3-c]phenazine - DPV Differential pulse voltammetry - dsDNA Double-stranded DNA - EtBr Ethidium bromide - Fc Ferrocene - Fc-CA Ferrocenecarboxaldehyde - Fc-CTPPZ Di[1-ferrocenecarbamoylpropyl)tetrahydropyrazine-4-(propylcarbamoylpyridine)]phenazine - Fc-NH₂ Aminoferrocene - FET Field-effect transistor - G Guanine - GCE Glassy carbon electrode - HBV Hepatitis B virus - HGH1 Human growth hormone 1 - HIV Human immunodeficiency virus - HRP Horseradish peroxidase - HOPGE Highly oriented pyrolytic graphite electrode - IPA Intermittent pulse amperometry - ISFET Ion-selective field-effect transistor - ITO Tin-doped indium oxide electrode - LSV Linear sweep voltammetry - Nano-Au Au nanoparticles - NMP Nucleotide monophosphate - ODN Oligodeoxynucleotide - ox. Oxidation - PCR Polymerase chain reaction - PET Poly(ethylene terephthalate) - PGE Pyrolytic graphite electrode - phen 1,10-Phenanthroline - PNA Peptide nucleic acid - Ppy Polypyrrole - (PQQ)GDH Pyrroline quinone glucose dehydrogenase - red. Reduction - SAM Self-assembled monolayer - SBP Soybean peroxidase - SNP Single nucleotide polymorphism - ssDNA Single-stranded DNA - SWV Square-wave voltammetry - T Thymine - Th Thionine - TTV TT virus - Ep Difference of peak potential     

Development and evaluation of electrochemical glucose enzyme biosensors based on carbon film electrodes

Electrochemical glucose enzyme biosensors have been prepared on carbon film electrodes made from carbon film electrical resistors. Evaluation and characterisation of these electrodes in phosphate buffer saline solution has been carried out with and without pretreatment by cycling in perchloric acid or at fixed applied potential. Both pretreatments led to a reduction in the carbon surface oxidation peak and enabled better detection of hydrogen peroxide in the pH range of 5-7. Glucose oxidase enzyme was immobilised on the carbon surface by mixing with glutaraldehyde, bovine serum albumin and with and without Nafion. The performance of these two types of electrode was similar, that containing Nafion being more physically robust. Linear ranges were up to around 1.5 mM, with detection limits 60 μM, and pretreatment of the carbon film electrode at a fixed potential of +0.9 V versus SCE for 5 min was found to be the most beneficial. Michaelis-Menten constants between 5 mM and 10 mM were found under the different experimental conditions. Coating the immobilised enzyme layer with a thin layer of Nafion was found to give similar results in the determination of glucose to mixing it but with benefits against interferences for the analysis of complex matrices, such as wine. Potentialities, for a short-term-use or disposable sensors, are indicated.     

Simultaneous electrochemical and electrochemiluminescence detection for microchip and conventional capillary electrophoresis

A simultaneous electrochemical (EC) and electrochemiluminescence (ECL) detection scheme was introduced to both microchip and conventional capillary electrophoresis (CE). In this dual detection scheme, tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) was used as an ECL reagent as well as a catalyst (in the formation of Ru(bpy)3(3+)) for the EC detection. In the Ru(bpy)3(2+)-ECL process, Ru(bpy)3(3+) was generated and then reacted with analytes resulting in an ECL emission and a great current enhancement in EC detection due to the catalysis of Ru(bpy)3(3+). The current response and ECL signals were monitored simultaneously. In the experiments, dopamine and three kinds of pharmaceuticals, anisodamine, ofloxacin, and lidocaine, were selected to validate this dual detection strategy. Typically, for the EC detection of dopamine with the presence of Ru(bpy)3(2+), a approximately 5 times higher signal-to-noise ratio (S/N) can be achieved than that without Ru(bpy)3(2+), during the simultaneous EC and ECL detection of a mixture of dopamine and lidocaine using CE separation. The results indicated that this dual EC and ECL detection strategy could provide a simple and convenient detection method for analysis of more kinds of analytes in CE separation than the single EC or ECL detection alone, and more information of analytes could be achieved in analytical applications simultaneously.     

Highly sensitive measurement of mepindolol in biological fluids using HPLC and electrochemical detection

Summary Mepindolol, a β-blocker agent, has an indolic structure which can undergo oxidation. A high-performance liquid chromatographic technique has been used to measure mepindolol in biological fluids using pindolol as the internal standard. The chromatography has been combined with electrochemical detection (coulometric detector). This method allows the determination of very low amounts of mepindolol with good precision and accuracy, the limit of quantification being 0.6 ng · ml⁻¹.