Ultrasensitive electrochemical detection of nucleic acid based on the isothermal strand-displacement polymerase reaction and enzyme dual amplification

We describe an ultrasensitive electrochemical detection of DNA protocol based on the isothermal strand-displacement polymerase reaction (ISDPR) and enzyme dual amplifications. Target DNA triggered an ISDPR to produce numerous bi-functionalized duplex DNA complexes. Following an immuno-magnetic collection via an immunoreaction between the attached digoxin on the duplex DNA and the anti-digoxin antibody on the magnetic bead, horseradish (HRP) tracers were bound to the duplex DNA through a biotin–streptavidin interaction. The quantification of DNA was realized by square wave voltammetric detection of the enzymatic products with a screen-printed gold electrode. The voltammetric response was proportional to the concentration of DNA in the range of 0.1 fM–0.5 pM, and the limit of detection was estimated to be 0.06 fM. The new protocol showed great promise for simple, cost-effective, and quantitative gene analysis.     

Quantum-dots based electrochemical immunoassay of interleukin-1α

We describe a quantum-dot (QD, CdSe@ZnS) based electrochemical immunoassay to detect a protein biomarker, interleukin-1α (IL-1α). QD conjugated with anti-IL-1α antibody was used as a label in an immunorecognition event. After a complete sandwich immunoreaction among the primary IL-1α antibody (immobilized on the avidin-modified magnetic beads), IL-1α, and the QD-labeled secondary antibody, QD labels were attached to the magnetic-bead surface through the antibody-antigen immunocomplex. Electrochemical stripping analysis of the captured QDs was used to quantify the concentration of IL-1α after an acid-dissolution step. The streptavidin-modified magnetic beads and the magnetic separation platform were used to integrate a facile antibody immobilization (through a biotin/streptavidin interaction) with immunoreactions and the isolation of immunocomplexes from reaction solutions in the assay. The voltammetric response is highly linear over the range of 0.5–50 ng ml⁻¹ IL-1α, and the limit of detection is estimated to be 0.3 ng ml⁻¹ (18 pM). This QD-based electrochemical immunoassay shows great promise for rapid, simple, and cost-effective analysis of protein biomarkers.     

CtrA gene based electrochemical DNA sensor for detection of meningitis

Electrochemical DNA sensor has been fabricated by immobilizing thiolated single stranded oligonucleotide (ssDNA) probe onto gold (Au) coated glass electrode for meningitis detection using hybridization with complementary DNA (CtrA) in presence of methylene blue (MB). These electrodes (ssDNA/Au and dsDNA/Au) have been characterized using atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), electrochemical impedance spectroscopy (EIS) and cyclic voltammetric (CV) technique. The DNA/Au electrode can detect the complementary DNA in the range of 7–42 ng/μl in 5 min (hybridization) with response time 60 s and electrode is stable for about 4 months when stored at 4 °C. The sensitivity of dsDNA/Au electrode is 115.8 μA/ng with 0.917 regression coefficient (R).     

Quantum dot-based electrochemical DNA biosensor using a screen-printed graphite surface with embedded bismuth precursor

This work reports the development of screen-printed quantum dots (QDs)-based DNA biosensors utilizing graphite electrodes with embedded bismuth citrate as a bismuth precursor. The sensor surface serves both as a support for the immobilization of the oligonucleotide and as an ultrasensitive voltammetric QDs transducer relying on bismuth nanoparticles. The utility of this biosensor is demonstrated for the detection of the C634R mutation through hybridization of the biotin-tagged target oligonucleotide with a surface-confined capture complementary probe and subsequent reaction with streptavidin-conjugated PbS QDs. The electrochemical transduction step involved anodic stripping voltammetric determination of the Pb(II) released after acidic dissolution of the QDs. Simultaneously with the electrolytic accumulation of Pb on the sensor surface, the embedded bismuth citrate was converted in situ to bismuth nanoparticles enabling ultra-trace Pb determination. The biosensor showed a linear relationship of the Pb(II) peak current with respect to the logarithm of the target DNA concentrations from 0.1 pmol L^− 1 to 10 nmol L^− 1, and the limit of detection was 0.03 pmol L^− 1. The biosensor exhibited effective discrimination between a single-base mismatched sequence and the fully complementary target DNA. These “green” biosensors are inexpensive, lend themselves to easy mass production, and hold promise for ultrasensitive bioassay formats.     

Magnetic nanoparticle-molecular imprinted polymer: A new impedimetric sensor for tributyltin detection

Recently, molecular imprinted polymers (MIPs) were extensively used for separation and identification of specific molecules, replacing expensive and unstable biological receptors. Nonetheless, their application in electrochemical sensors has not been sufficiently explored. Here we report the use of a MIP as a specific receptor in a new highly sensitive tributyltin (TBT) electrochemical sensor. The sensor combines the specificity, pre-concentration capability and robustness of molecular imprinted polymer attached onto magnetic nanoparticles with the quantitative outputs of impedimetric measurements. The proposed device detects TBT in a concentration range of 5 pM to 5 μM with a low limit of detection (5.37 pM), which is lower than the one recommended for TBT in sea water by the US Environmental Protection Agency (EPA). We believe that this new electrochemical sensor can play an important role in the monitoring of the quality of sea and fresh waters worldwide.     

Multiplex electrochemical immunoassay using gold nanoparticle probes and immunochromatographic strips

We describe a multiplex electrochemical immunoassay based on the use of gold nanoparticle (Au-NP) probes and immunochromatographic strips (ISs). The approach takes advantage of the speed and low cost of the conventional IS tests and the high sensitivities of the nanoparticle-based electrochemical immunoassays. Rabbit IgG (R-IgG) and human IgM (H-IgM) were used as model targets for the demonstration of the proof of concept. The Au-NPs based sandwich immunoreactions were performed on the IS, and the captured gold nanoparticle labels on the test zones were determined by highly sensitive stripping voltammetric measurement of the dissolved gold ions (III) with a carbon paste electrode. The detection limits are 1.0 and 1.5 ng ml⁻¹ with the linear range of 2.5–250 ng ml⁻¹ for quantitative detection of R-IgG and H-IgM, respectively. The total assay time is around 25 min. Such multiplex electrochemical immunoassay could be readily highly multiplexed to allow simultaneous parallel detection of numerous proteins and is expected to open new opportunities for protein diagnostics and biosecurity.     

An aptamer-based biosensor for sensitive thrombin detection

A sensitive aptamer-based sandwich-type sensor is presented to detect human thrombin using quantum dots as electrochemical label. CdSe quantum dots were labeled to the secondary aptamer, which were determined by the square wave stripping voltammetric analysis after dissolution with nitric acid. The aptasensor has a lower detection limit at 1 pM, while the sample consumption is reduced to 5 μl. The proposed approach shows high selectivity and minimizes the nonspecific adsorption, so that it was used for the detection of target protein in the human serum sample. Such an aptamer-based biosensor provides a promising strategy for screening biomarkers at ultratrace levels in the complex matrices.     

Magnetic beads-based bioelectrochemical immunoassay of polycyclic aromatic hydrocarbons

A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL⁻¹ was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.     

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.     

Efficient stripping voltammetric detection of organophosphate pesticides using NanoPt intercalated Ni/Al layered double hydroxides as solid-phase extraction

We developed a simple strategy for designing a sensitive electrochemical stripping voltammetric sensor for organophosphate pesticides (OPs) based on solid-phase extraction (SPE) using nanosized Pt intercalated Ni/Al layered double hydroxides (labeled as NanoPt-LDHs). By assembling NanoPt with LDHs together, the resulting NanoPt-LDHs are highly efficient to capture OPs. It dramatically facilitates the enrichment of OPs onto their surface and realizes the sensitive stripping voltammetric detection of methyl parathion (MP) as a model of OPs. The stripping analysis shows highly linear over MP concentration ranges of 0.001–0.15 and 0.3–1.0 μg mL^? 1 with a detection limit of 0.6 ng mL^–1 (S/N = 3). The combination of NanoPt, LDHs, SPE, and square-wave voltammetry (SWV) provides a fast, simple, and sensitive electrochemical method for OPs.     

End-labeling of peptide nucleic acid with osmium complex. Voltammetry at carbon and mercury electrodes

Peptide nucleic acid (PNA), the DNA mimic with electrically neutral pseudopeptide backbone, is intensively used in biotechnologies and particularly in single-base mismatch detection in DNA hybridization sensors. We propose a simple method of covalent end-labeling of PNA with osmium tetroxide, 2,2′-bipyridine (Os,bipy). Os,bipy-modified PNA (PNA–Os,bipy) produces voltammetric stripping peaks at carbon and mercury electrodes. Peak potential (E_p) of one of the anodic peaks of PNA–Os,bipy at the pyrolytic graphite electrode (PGE) differs from E_p of the reagent, allowing PNA–Os,bipy analysis directly in the reaction mixture. At the hanging mercury drop electrode (HMDE) the PNA–Os,bipy yields a catalytic peak Cat_p, in addition to the redox couples. Using Cat_p it is possible to detect purified PNA–Os,bipy down to 1 pM concentration at accumulation time 60 s. To our knowledge this is the highest sensitivity of the electrochemical detection of PNA.     

Label-free bioelectronic detection of aptamer–protein interactions

We demonstrate for the first time the utility of nucleic acid aptamers for electrochemical detection of proteins. Highly specific and sensitive label-free detection of the target protein is achieved by combining aptamer-coated magnetic beads and chronopotentiometric stripping measurements of the captured protein (in connection to the intrinsic electroactivity of the protein). Lysozyme has thus been detected selectively in a mixture containing a large excess of six proteins and amino acids (both electroactive and non-electroactive), with a detection limit of 350 fmol (7 nM). While aptamer-based electronic sensors are in their infancy, such devices offer attractive opportunities for electrochemical detection of proteins and for developing proteomic chips.     

Zeptomolar detection of Hg2 + based on label-free electrochemical aptasensor: One step closer to the dream of single atom detection

An ultra-sensitive and highly selective electrochemical label-free aptasensor is proposed for the quantitation of Hg^2 + based on the hybridization/dehybridization of double-stranded DNA (dsDNA) on a gold electrode. Thiol-substituted single-stranded DNA (ssDNA) is self-assembled on the gold electrode surface through the SAu interaction. The hybridization of ssDNA with complementary DNA (cDNA) and the consequences of dehybridization in the presence of mercury ions are followed through differential pulse voltammetry (DPV) responses using a [Fe(CN)₆]^{3 −/4 −} redox probe. The formation of a thymine–Hg^2 +–thymine (T–Hg^2 +–T) complex is the key to producing a highly selective and sensitive aptasensor for Hg^2 + determination. Specifically, the present electrochemical aptasensor is able to quantify Hg^2 + ions in concentrations from 5 zeptomolar (zM) to 55 picomolar (pM) with a limit of detection of 0.6 zM, close to the dream of single atom detection, without requiring a complicated procedure or expensive materials.     

Microfluidic chip-based aptasensor for amplified electrochemical detection of human thrombin

In this paper, a microchip-based sandwich-type aptasensor is developed for the detection of human thrombin. The SH-aptamer/thrombin/alkaline phosphatase-functionalized aptamer (ALP-aptamer) system was constructed in the microfluidic channels. And the substrate solution containing 4-aminophenyl phosphate (p-APP) was introduced to the microchannels for the end-column electrochemical detection. The on-chip aptasensor has a broad linear response range of 1–100 pM with a detection limit of 1 pM, which shows high sensitivity and specificity. The system was then applied to detect thrombin in human serum sample. Therefore, the on-chip aptasensor has a great promise for detecting and screening ultratrace levels of biomarkers in the complex matrices.     

Sensitive detection of thyroid stimulating hormone by inkjet printed microchip with a double signal amplification strategy

In this paper, we reported a sensitive electrochemical immunosensor coupling protein A/G@magnetic beads and an ALP-based enzymatic-electrochemical reaction on the inkjet printing microchips for the determination of thyroid stimulating hormone.     

DNA surface nanopatterning by selective reductive desorption from polycrystalline gold electrode

Selective electrochemical desorption was employed to pattern polycrystalline gold electrodes with thiolated DNA. The sacrificial thiol 3-mercaptopropionic acid (3-MPA) was selectively desorbed from the crystallographic plane Au(1 1 1) to revealed bare gold domains, surrounded by SAMs of 3-MPA present on the adjacent low index planes Au(1 1 0) and Au(1 0 0). Thiolated DNA sequences were further immobilised on the revealed Au(1 1 1) and the hybridisation efficiency towards complementary and non-complementary sequences evaluated. All derivatisation steps were followed by cyclic voltammetry and faradaic electrochemical impedance spectroscopy. Successful hybridisation resulted in large drops in resistance to charge transfer, attributed to the extension of the DNA surface duplex into solution resulting in an increased diffusion of electrochemical probes to the electrode surface. The results demonstrated the feasibility of the method to generate a DNA sensor able to efficiently discriminate between complementary and non-complementary sequences with good reproducibility.     

Selective determination of trichloroacetic acid using silver nanoparticle coated multi-walled carbon nanotubes

Silver nanoparticle coated multi-walled carbon nanotubes (Ag/MWCNT) were prepared and used to fabricate a modified electrode. The Ag/MWCNT composites were observed by a transmission electron microscope (TEM), and the electrochemical properties of the Ag/MWCNT composite modified glassy carbon electrode were characterized by electrochemical measurements. The results showed that these composites had a favorable catalytic ability for the reduction of trichloroacetic acid (TCAA). Square wave voltammetric (SWV) technique was applied to detect TCAA. Under optimum conditions, the voltammetric determination of TCAA was performed with a linear range of 5.0 × 10^? 6–1.2 × 10^? 4 mol L^? 1 and a detection limit of 1.9 × 10^? 6 mol L^? 1 (S/N = 3).     

Electrochemical detection of quantum dots by stabilization of electrogenerated copper species

The determination of quantum dots is of particular interest as they are frequently used as labels in electrochemical biosensors. In this paper, we describe a method for the detection of very low concentrations of quantum dots using the voltammetric response of copper in ammonia solution. Copper species electrogenerated on the electrode surface are stabilized by the nanoparticles, preventing their oxidation by dissolved oxygen, and a relationship between the concentration of the nanoparticles and the copper voltammetric response can thus be obtained. The reported method shows a linear range between 0.05 and 2 nM of quantum dots, with a limit of detection in the order of 9 × 10⁷ nanoparticles. This method could be employed to improve the detection limit of electrochemical biosensors using quantum dots as labels.     

Cyclic voltammetric detection of chemical DNA damage induced by styrene oxide in natural dsDNA layer-by-layer films using methylene blue as electroactive probe

In this work, an electrochemical sensor for detecting damage of natural double-stranded DNA (dsDNA) was constructed based on PSS/{PDDA/dsDNA}₃ layer-by-layer films, where PSS was poly(styrene sulfonate) and PDDA was poly(diallyldimethyl ammonium). When the PSS/{PDDA/dsDNA}₃ films assembled on pyrolytic graphite (PG) electrodes were immersed into methylene blue (MB) solution and loaded MB into the films, the formed PSS/{PDDA/dsDNA}₃-MB films in blank buffers at pH 7.0 showed a reversible cyclic voltammetric (CV) peak pair at −0.23 V vs. SCE for MB redox couple. In blank solutions, the MB loaded into the films was released gradually from the films, but the complete reloading of MB into the films could be realized by immersing the films into MB solution again, indicating the good reversibility of MB incorporation. However, after incubation in the solution of known genotoxic agent styrene oxide (SO), the damaged PSS/{PDDA/dsDNA}₃-MB films could not return to their original and fully-loaded state with reloading of MB, and showed smaller CV peak currents than those of intact PSS/{PDDA/dsDNA}₃-MB films. The relative peak current ratio, I_p,I/I_p,III, where I_p,I was the anodic peak current of intact DNA films in blank buffers after fully loading with MB and I_p,III was that of SO-damaged DNA films after reloading with MB, increased linearly with the incubation time with SO in the range of 5–40 min with a damage rate of 0.0099 min⁻¹. While the formation of SO adducts with dsDNA had no substantial effect on the dsDNA conformation, the steric hindrance of SO adducts with guanine or adenine blocked the intercalation of MB into the base pairs of dsDNA, resulting in the decrease of CV peak currents of loaded MB. The specific intercalation of MB into dsDNA base pairs and the sensitive electrochemical response of MB, combined with the unique feature of loading reversibility of MB in the DNA layer-by-layer films, make the difference in CV response between the intact and damaged dsDNA films become pronounced in the “loading/release/reloading” procedure. This may provide a new and general approach for constructing a biosensor for screening genetoxic chemicals in vitro.     

Evaluation of a platinum electrode modified with hydroxyapatite in the lead(II) determination in a square wave voltammetric procedure

The behavior of a modified carbon platinum electrode (Pt) for lead(II) determination by square wave voltammetry (SWV) was studied. The modified electrode is obtained by electrodeposition of hydroxyapatite (HAP) on the surface of a bare platinum electrode. The new electrode (HAP/Pt) revealed interesting electroanalytical detection of lead(II) based on the adsorption of this metal onto hydroxyapatite under open circuit conditions. After optimization of the experimental and voltammetric conditions, the best voltammetric responses-current intensity and voltammetric profile were obtained in 0.2 mol L^?1 KNO₃ with: 30 min accumulation time, 5 mV pulse amplitude and 1 mV s^?1 scan rate. The observed detection (DL, 3σ) and quantification (DL, 10σ) limits in pure water were 2.01 × 10^?8 and 6.7 × 10^?7 mol L^?1, respectively. The reproducibility of the proposed method was determined from five different measurements in a solution containing 2.2 × 10^?6 mol L^?1 lead(II) with a coefficients of variation of 2.08%.The electrochemical of hydroxyapatite at platinum surfaces was characterized, after calcinations 900 °C, by X-ray diffraction, infrared spectroscopy, chemical and electrochemical analysis.