Label‐Free Bioelectronic Detection of Point Mutation by Using Peptide Nucleic Acid Probes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes with a label‐free protocol is described. The detection of PNA‐DNA and DNA‐DNA hybridizations were accomplished based on the oxidation signal of guanine by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE). It was observed that the oxidation signals of guanine obtained from the PNA and DNA probe modified CPEs were higher than those obtained from the PNA‐DNA and DNA‐DNA hybrid modified CPEs due to the accessible unbound guanine bases. The detection of hybridization between PNA probe and point mutation containing DNA target sequences was clearly observed due to the difference of the oxidation signals of guanine bases, because the point mutation was guanine nearly at the middle of the sequence. The effect of the DNA target concentration on the hybridization signal was also observed. The PNA probe was also challenged with excessive and equal amount of noncomplementary DNA and also mixtures of point mutation and target DNA.     

Voltammetric Reduction of a 4‐Nitroimidazole Derivative on a Multiwalled Carbon Nanotubes Modified Glassy Carbon Electrode

We report the electrochemical behavior of a 4‐nitroimidazole derivative, 1‐methyl‐4‐nitro‐2‐hydroxymethylimidazole (4‐NImMeOH), on glassy carbon electrode (GCE) modified with multiwalled carbon nanotubes (MWCNT). As dispersing agents, dimethylformamide (DMF) and water were used. The electrochemical response of the resulting electrodes was evaluated using linear sweep, cyclic and square‐wave voltammetry (LSV, CV and SWV). Several parameters such as medium pH, nature and concentration of the CNTs dispersion and accumulation time were tested. The optimal conditions determined for obtain better response were: pH 2, dispersion concentration=4 mg/mL of CNT in water, accumulation time=7 min. The MWCNT‐modified GCE exhibited attractive electrochemical properties producing enhanced currents with a significant reduction in the overpotential and good signal‐to‐noise characteristics, in comparison with the bare GCE. The modified electrode is highly repeatable for consecutive measurements, reaching a variation coefficient of 2.9% for ten consecutive runs.     

Selective Detection of Dopamine in Presence of Ascorbic Acid by Use of Glassy‐Carbon Electrode Modified with Amino‐β‐Cyclodextrin and Carbon Nanotubes

A glassy carbon electrode modified with per‐6‐amino‐β‐cyclodextrin (β‐CDNH₂) and functionalized single‐walled carbon nanotubes (SWCNT‐COOH) was elaborated. This structure was investigated for the detection of dopamine acid (DA) in presence of ascorbic acid (AA). The sensor behavior was studied by cyclic voltammetry, square wave voltammetry and electrochemical impedance spectroscopy. The analysis results show that the electrode modification with CD derivative improves the sensitivity and selectivity of the DA recognition; the electrochemical response was further improved by introduction of SWCNT‐COOH. The sensor shows good and reversible linear response toward DA within the concentration range of 7×10^?7–10^?4 M with a detection limit of 5×10^?7 M.     

Polymer‐Decorated Carbon Nanotubes as Transducers for Label‐Free Photonic Biosensors

A biosensor taking advantage of the optical properties of sorted carbon nanotubes has been developed. A polyfluorene polymer bearing azido groups was synthesized and used for the selective extraction of semi‐conducting nanotubes from the bulk population. The resulting polymer‐decorated nanotubes were then conjugated by click‐chemistry to a ligand unit (biotin), and the sensing properties of the biotinylated nanotubes were investigated by photoluminescence measurements, upon interaction with the streptavidin target.     

Electrochemical Estrogen Receptor α based Biosensor for Label‐Free Detection of Estradiol

We describe a new electrochemical biosensor based on estrogen receptor α (ER‐α) for label‐free detection of 17β‐estradiol, a model of endocrine‐disrupting compounds. ER‐α is coupled onto the gold electrode through its 6‐His tag and NTA‐copper complex. After interaction of estradiol with ER‐α, the biosensor presents a well‐defined peak at +500 mV due to estradiol oxidation (E17 peak). The linear range of detection is from 1 fM to 1 nM and the detection limit is 1 fM. Good selectivity was obtained for interfering substances at nanomolar level, for concentration of E17 up to 0.1 pM. The E17 was detected in hospital effluents.     

A Novel Electrochemical DNA Biosensor Fabricated with Layer‐by‐Layer Covalent Attachment of Multiwalled Carbon Nanotubes and Gold Nanoparticles

A novel DNA biosensor has been fabricated for the detection of DNA hybridization based on layer‐by‐layer (LBL) covalent assembly of gold nanoparticles (GNPs) and multiwalled carbon nanotubes (MWCNTs). The stepwise LBL assembly process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored by differential pulse voltammetry (DPV) measurement of the intercalated doxorubicin, and the factors influencing the performance of the DNA hybridization was investigated in detail. The signal was linearly changed with target DNA concentration increased from 0.5 to 0.01 nM, and had a detection limit of 7.5 pM (signal/noise ratio of 3). In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Label‐free DNA Hybridization Based on Coupling of a Heated Carbon Paste Electrode with Magnetic Separations

The technique of electrically heated carbon paste electrodes is applied for label‐free detection of DNA hybridization after magnetic isolation. Coupling of both techniques leads to highly selective and sensitive detection of DNA hybridization. Minimal contributions of nonhybridized DNA are thus coupled to an enhanced signal. Application of elevated temperatures during the accumulation step yields a max. 6‐fold enhancement of guanine oxidation signals compared to room temperature deposition. Use of noncomplementary nucleic acids gives no significant peaks at room or elevated temperatures. The noise level remained unaffected. Variation of the target concentration results in linear calibration plots under hot accumulation conditions.     

DNA‐Mediated Copper Nanoparticle Formation on Dispersed Single‐Walled Carbon Nanotubes

A new and facile method for the preparation of single‐walled carbon nanotubes (SWCNTs) decorated with Cu nanoparticles (CuNPs) formed on a double‐stranded DNA template in aqueous solution has been developed. A specially designed synthetic DNA sequence, containing a single‐stranded domain for the dispersion of carbon nanotubes and double‐stranded domains for the selective growth of CuNPs, was utilized. The final SWCNT/CuNP hybrids were characterized using fluorescence spectroscopy and transmission electron microscopy. The analyses clearly demonstrated the selective formation of uniform CuNPs on the carbon nanotube scaffold.     

Direct DNA Hybridization on the Single‐Walled Carbon Nanotubes Modified Sensors Detected by Voltammetry and Electrochemical Impedance Spectroscopy

Carboxylic acid functionalized single‐walled carbon nanotubes modified graphite sensors (SWCNT‐PGEs) were developed for electrochemical monitoring of direct DNA hybridization related to specific sequence of Hepatitis B virus, which substantially enhance the electrochemical transduction resulting from guanine oxidation signal comparison to bare PGEs. The performance characteristics of DNA hybridization on disposable CNT‐PGE were explored measuring the guanine signal in terms of optimum analytical conditions; probe and target concentration, hybridization time, and selectivity. The voltammetric results were also complemented with electrochemical impedance spectroscopy (EIS), that was used to characterize the successful construction of carbon nanotubes modification onto the surface of PGEs.     

Detection of Human Interleukine‐2 Gene Using a Label‐Free Electrochemical DNA Hybridization Biosensor on the Basis of a Non‐Inosine Substituted Probe

The human interleukine‐2 gene (hIL‐2) is detected with a label‐free DNA hybridization biosensor using a non‐inosine substituted probe. The sensor relies on the immobilization of a 20‐mer antisense single strand oligonucleotide (chIL‐2) related to the human interleukine‐2 gene on the pencil graphite electrode (PGE) as a probe. The guanine oxidation signal was monitored using anodic differential pulse voltammetry (ADPV). The electrochemical pretreatment of the polished PGE at 1.80 V for 5 min is suggested. Then, 5 min immobilization at 0.50 V was found as the optimum condition for immobilization of the probe. The electrochemical detection of hybridization between chIL‐2 and hIL‐2 as a target was accomplished. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Diagnostic performance of the biosensor is described and the detection limit is found 36 pg/μL.     

Electrochemical DNA Biosensors Based on Palladium Nanoparticles Combined with Carbon Nanotubes

Palladium nanoparticles, in combination with multi‐walled carbon nanotubes (MWCNTs), were used to fabricate a sensitivity‐enhanced electrochemical DNA biosensor. MWCNTs and palladium nanoparticles were dispersed in Nafion, which were used to modify a glassy carbon electrode (GCE). Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. Due to the ability of carbon nanotubes to promote electron‐transfer and the high catalytic activities of palladium nanoparticles for electrochemical reaction of MB, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.2×10^?13 M.     

Applications of Carbon Nanotubes in Electrochemical DNA Biosensors

The discovery of carbon nanotubes (CNTs) about a decade ago has brought fascinating evolutions in electronics, material industry, as well as bio-techniques for DNA analysis, gene therapy, drug delivery etc. It has also dramatically promoted the development of DNA biosensing techniques, especially electrochemical DNA biosensor. The application of CNTs in electrochemical DNA biosensors includes two main aspects: on one hand, using CNTs as a novel substrate not only enables immobilization of DNA molecules but also serves as a powerful amplifier to amplify signal transduction event of DNA hybridization. On the other hand, CNTs can also be employed as a powerful carrier to pre-concentrate enzymes or electroactive molecules for electrochemical sensing of DNA hybridization as a novel indicator. In this review, we place emphasis on recent studies of CNTs-based electrochemical DNA biosensors based on these two aspects, with advantages and disadvantages of each aspect introduced herein.     

Electrochemical Detection of DNA Hybridization Based on the Probe Labeled with Carbon‐Nanotubes Loaded with Silver Nanoparticles

A sensitive electrochemical method for the detection of DNA hybridization based on the probe labeled with multiwall carbon‐nanotubes (MWNTs) loaded with silver nanoparticles (Ag‐MWNTs) has been developed. MWNTs were electroless‐plated with a large number of silver nanoparticles to form Ag‐MWNTs. Probe single strand DNA (ss‐DNA) with a thiol group at the 3′‐terminal labeled with Ag‐MWNTs by self‐assembled monolayer (SAM) technique was employed as an electrochemical probe. Target ss‐DNA with a thiol group was immobilized on a gold electrode by SAM technique and then hybridized with the electrochemical probe. Binding events were monitored by differential pulse voltammetric (DPV) signal of silver nanoparticles. The signal difference permitted to distinguish the match of two perfectly complementary DNA strands from the near perfect match where just three base pairs were mismatched. There was a linear relation between the peak current at +120 mV (vs. SCE) and complementary target ss‐DNA concentration over the range from 3.1×10^?14 to 1.0×10^?11 mol/L with a detection limit of 10 fmol/L of complementary target ss‐DNA. The proposed method has been successfully applied to detection of the DNA sequence related to cystic fibrosis. This work demonstrated that the MWNTs loaded with silver nanoparticles offers a great promising approach for sensitive detection of DNA hybridization.     

A New Indirect Electroanalytical Method to Monitor the Contamination of Natural Waters with 4‐Nitrophenol Using Multiwall Carbon Nanotubes

The electrochemical detection of the hazardous pollutant 4‐nitrophenol (4‐NP) at low potentials, in order to avoid matrix interferences, is an important research challenge. This study describes the development, electrochemical characterization and utilization of a multiwall carbon nanotube (MWCNT) film electrode for the quantitative determination of 4‐NP in natural water. Electrochemical impedance spectroscopy measurements showed that the modified surface exhibits a decrease of ca. 13 times in the charge transfer resistance when compared with a bare glassy carbon (GC) surface. Voltammetric experiments showed the possibility to oxidize a hydroxylamine layer (produced by the electrochemical reduction of 4‐NP on the GC/MWNCT surface) in a potential region which is approximately 700 mV less positive than that needed to oxidize 4‐NP, thus minimizing the interference of matrix components. The limit of detection for 4‐NP obtained using square‐wave voltammetry (0.12 μmol L^?1) was lower than the value advised by EPA. A natural water sample from a dam located in São Carlos (Brazil) was spiked with 4‐NP and analyzed by the standard addition method using the GC/MWCNT electrode, without any further purification step. The recovery procedure yielded a value of 96.5% for such sample, thus confirming the suitability of the developed method to determine 4‐NP in natural water samples. The electrochemical determination was compared with that obtained by HPLC with UV‐vis detection.     

Label‐Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label‐free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects.     

Electrochemical Treatment of Glassy Carbon for Label‐Free Detection of DNA Bases and Neurotransmitters

Electrochemically reduced glassy carbon (r‐GC) showed a superior electrochemical sensing performance, compared to oxidized GC (ox‐GC) and untreated GC for the oxidation of 4 DNA bases and neurotransmitters (epinephrine, norepinephrine and serotonin). r‐GC exhibited not only the largest current intensities of all redox biomolecules, but also displayed an excellent selectivity in detecting coexisting redox biomolecules. The enhanced performance of r‐GC was attributed to the improved surface cleanliness of electrode and its catalytic surface functional groups. The results presented herein imply that simple electrochemical treatments are a viable method to produce sensitive and selective electrodes for label‐free biosensing.     

Label‐Free and Ultra‐Low Level Detection of Salmonella enterica Serovar Typhimurium Using Electrochemical Impedance Spectroscopy

An immunosensor for rapid and low level detection of the bacterial pathogen Salmonella enterica Serovar Typhimurium was designed and developed based upon label‐free electrochemical impedance spectroscopy and correlated to viable cell counts. The immunosensor was fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode by immobilizing monoclonal antibody (MAb) specific against Salmonella typhimurium cell surface lipopolysaccharide (LPS) onto the surface of the electrode. Use of mass‐fabricated and electroplated PCB electrodes allowed for disposable, highly sensitive, and rapid detection of Salmonella in an aqueous environment. Results demonstrate that in purified solution, Salmonella can be detected as low as 10 CFU in a 100 μL volume and label‐free and rapid manner in fewer than 90 s. The cost effective approach described here can be used for detection of pathogens with relevance for healthcare, food, and environmental applications.     

Synthesis of Nucleobase‐Functionalized Carbon Nanotubes and Their Hybridization with Single‐Stranded DNA

For the first time ssDNA (25‐aptamer of mixed dA, dT, dG, and dC) was wrapped around functionalized single‐walled carbon nanotubes (SWCNTs), whose external surfaces were attached to multiple triazole‐(ethylene glycol)‐dA ligands. This method of hybridization involved the formation of hydrogen bonds between dT of ssDNA and dA of functionalized SWCNTs. It deviates from the reported π–π stacking between the nucleobases of DNA and the external sidewalls of nanotubes. The structural properties of the functionalized SWCNTs and its ssDNA complex were characterized by spectroscopic (including CD and Raman), thermogravimetric, and microscopic (TEM) methods. The results thus obtained establish a new platform of DNA delivery by use of nanotubes as a new vehicle with great potential in biomedical applications and drug development.     

Label‐Free Detection of Bisphenol A Using a Potentiometric Immunosensor

A potentiometric immunosensor for the label‐free detection of bisphenol A (BPA) was developed by covalently immobilizing a polyclonal antibody (PAb) onto the surface of a carboxylated poly(vinyl chloride) (PVC‐COOH) membrane. The immunosensor was characterized using scanning electron microscopy (SEM), X‐ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy. The immobilization of PAb and its interaction with antigen (Ag) and BPA were also confirmed by quartz crystal microbalance (QCM) studies. Experimental parameters affecting the immuno‐interaction between PAb and its Ag or BPA were examined in terms of pH, antibody concentration, and temperature. The immunosensor showed a specific recognition of BPA with less interference than 4.8% from other common phenolic compounds. A calibration plot was obtained between 1.0 and 30.0 ng/mL and the detection limit was determined to be 0.6 ng/mL. The proposed immunosensor was applied for a real water sample spiked with BPA and the recoveries were in the range between 102.5 and 105.7%.     

Label‐Free Electrochemical Imaging of Latent Fingerprints on Metal Surfaces

A label‐free electrochemical method based on scanning electrochemical microscopy (SECM) has been developed to image latent fingerprints with high resolution on five kinds of metal surfaces (platinum, gold, silver, copper and stainless steel), as it could measure the minor conductivity differences of the substrate surface and avoid the interference of the background‐color. The images of sebaceous fingerprints on clean metals were revealed by SECM with ferrocene methanol acting as a redox mediator to detect the topology of the fingerprint deposits in constant‐height feedback mode. Inhibition of electrochemical processes on areas of the surface masked by the insulating fingerprint residues generated a negative image of the fingerprint.