Label-free Electrochemical Sensor Research Articles

Label-free simultaneous detection of quinolone antibiotic residues using an origami paper–based electrochemical immunosensor

Label-free electrochemical sensors have gained popularity as point-of-care devices because they do not require complicated labeling procedures. However, they have difficulties detecting multiple analytes simultaneously. In this study, we developed a novel label-free electrochemical immunosensor on an origami paper–based analytical device (oPAD) for the simultaneous detection of quinolone antibiotics, in which norfloxacin (Nor) and enrofloxacin (Enr) were used as a model. The oPAD was designed with two antibody zones, each zone contains different antibodies. The oPAD has two thionine- and ferrocenecarboxylic acid-coated reagent zones, as redox species, which will generate the response current signals for the detection of quinolone antibiotics. The simultaneous current responses of thionine and ferrocenecarboxylic acid decreased proportionately when exposed to Nor and Enr concentrations, respectively. Under optimal conditions, this device provided linear range at concentrations of 0.01–10 µg/mL for the detection of Nor and Enr and limit of detection was calculated as 2.02 and 1.70 ng/mL, respectively. Moreover, this device demonstrates performance comparable to standard methods for the simultaneous detection of Nor and Enr in milk, honey, and fish. Therefore, label-free electrochemical immunosensors provide a highly potent, inexpensive, and portable method for detecting multiple quinolone antibiotics simultaneously on a single device.

A label-free electrochemical sensor for the detection of two kinds of targets based on CRISPR/Cas12a system

In recent years, electrochemical sensors for nucleic acid testing based on CRISPR/Cas12a technology have attracted widespread interest due to their ease of operation and high detection sensitivity. Here, a label-free electrochemical sensor amplified by primer exchange reaction (PER), hybridization chain reaction (HCR) and CRISPR/Cas12a system was developed for analyzing miRNA- 155 and PCB 77. In the presence of targets, PER was initiated, whose products could be used to trigger the trans-cleavage activity of Cas12a for the single-stranded DNA (ssDNA) on the electrode. Afterwards, with the amplification of HCR on the electrode and electrostatic adsorption of Ru(NH3)63+, the concentration of the targets could be indicated by the DPV intensity with a low background signal. The detection limits for miRNA- 155 and PCB 77 were estimated to be 67 aM and 6.9 pg/L, respectively. This work provides a versatile method for sensitive detection of different targets using the same crRNA and can be used in various biochemical investigations.

Cascaded recycling amplification mediated in situ synthesis of silver nanoclusters for the construction of sensitive and label-free electrochemical sensor

A growing number of studies have shown the crucial role of microRNA (miRNA) sensing in cancer clinical diagnosis and prognosis research. In this study, a label-free and enzyme-free electrochemical sensor was presented to achieve sensitive determination of miRNA 122, which was constructed based on in situ synthesis of silver nanoclusters (AgNCs) mediated by cascaded recycling amplification. The efficient and cascaded recycling amplification was initiated by the target miRNA 122, resulting in the release of signal probe with rich cytosine bases, which would be loaded on the sensing interface for in situ synthesis of silver nanoclusters, leading to dramatically increased current for quantitative analysis. This method achieved selective and robust miRNA 122 sensing in human serum samples with a detection limit down to 27 aM. The proposed work would provide an innovative method for determining different biomarkers, holding great potential for boosting the development of clinical diagnosis and drug investigation.

Label-free electrochemical biosensor based on Ng-PCL polymer signal amplification for the detection of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is the related antigen first extracted from embryonic tissue and is an acidic glycoprotein with human embryonic antigenic properties. It is an important biomarker for the detection of many malignancies. In this paper, we have developed a signal amplified sandwich-type electrochemical impedance biosensor based on naringin initiated poly(ε-caprolactone) (Ng-PCL) polymer for highly sensitive and rapid determination of CEA. The polymer Ng-PCL is a macromolecular polymer that can increase the sensitivity of biosensor. First, SH-functionalized CEA-aptamer 1 (Apt1) was attached to the electrode surface as a recognition probe by Au-S bonds in order to form a self-assembly monolayer (SAM). Then, the one end of CEA antigen could recognize the Apt1 modified on the electrode surface, the other end could recognize the COOH-functionalized CEA-aptamer 2 (Apt2) forming a sandwich structure. Finally, polymer Ng-PCL as a signal amplification unit, which was connected to Apt2 by ester bonds. As a result, the electron transfer in the electrode interface was hindered effectively and a label-free electrochemical impedance sensor was constructed successfully. The high sensitivity and selectivity of biosensor relied on the SAM, polymer Ng-PCL, and specific recognition of CEA. Under the optimum experimental conditions, we proposed electrochemical impedance biosensor had wide detection range between 5 pg/mL and 200 ng/mL, and the sensitivity can reach 0.675 pg/mL. Moreover, the electrochemical impedance biosensor performed high specificity, good stability and satisfactory reproducibility as well. Thus, this strategy will provide new path for the detection of other biomarkers in early clinical diagnosis.

Iron oxide/poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) glassy carbon electrode as a novel label-free electrochemical microRNA-21 sensor

MicroRNAs (miRNAs), a type of non-protein-coding and small RNAs, are encouraging molecular biomarkers for prior diagnosis of the certain diseases. Here, an electrochemical sensor based on iron oxide/poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate (Fe3O4/PEDOT:PSS) hybrid coated glassy carbon electrode (GCE) is reported to determine microRNA-21 (miRNA-21) as a biosensing platform based on hybridization event. The biosensor was characterized by cyclic voltammetry (CV) measurements. The Fe3O4/PEDOT:PSS demonstrated good sensing performance, including a wide detection range (0.1 pM-1 µM) and a low detection limit (0.023 pM) for potential medical applications.

Highly sensitive and label-free electrochemical sensor for menthol detection

Menthol is a monocyclic monoterpenoid alcoholic compound and generally used in foods as additive and in medicine as analgesic and antipruritic. Poor water solubility and high volatility of menthol limit its application and increase the difficulty of detection. Herein, we proposed a simple and label-free method to sensitively detect menthol using differential pulse voltammetry (DPV). To improve the detection sensitivity, silica microspheres (SiO2) were introduced for better modification and enrichment of ferrocene (Fc) on the electrode surface. Based on the inclusion action of Fc and β-cyclodextrins (β-CD), the electrochemical signal of Fc was inhibited due to the limitation of Fc oxidation by β-CD cavity. However, when menthol presented, the electrochemical signal of Fc has a remarkable recovery. The recovered electrochemical signal increased with the increasing concentration of menthol, and showed a good linear relationship. Thus, the proposed electrochemical sensor is available for quantitative detection of menthol with high sensitivity.

Rapid and Sensitive Detection of Sulfamethizole Using a Reusable Molecularly Imprinted Electrochemical Sensor.

Efficient methods for monitoring sulfonamides (SAs) in water and animal-source foods are of great importance to achieve environmental safety and protect human health. Here, we demonstrate a reusable and label-free electrochemical sensor for the rapid and sensitive detection of sulfamethizole based on an electropolymerized molecularly imprinted polymer (MIP) film as the recognition layer. To achieve effective recognition, monomer screening among four kinds of 3-substituted thiophenes was performed by computational simulation and subsequent experimental evaluation, and 3-thiopheneethanol was finally selected. MIP synthesis is very fast and green, and can be in situ fabricated on the transducer surface within 30 min in an aqueous solution. The preparation process of the MIP was characterized by electrochemical techniques. Various parameters affecting MIP fabrication and its recognition response were investigated in detail. Under optimized experimental conditions, good linearity in the range of 0.001-10 μM and a low determination limit of 0.18 nM were achieved for sulfamethizole. The sensor showed excellent selectivity, which can distinguish between structurally similar SAs. In addition, the sensor displayed good reusability and stability. Even after 7 days of storage, or being reused 7 times, higher than 90% of the initial determination signals were retained. The practical applicability of the sensor was also demonstrated in spiked water and milk samples at the nM determination level with satisfactory recoveries. Compared to relevant methods for SAs, this sensor is more convenient, rapid, economical, and eco-friendly, and had comparable or even higher sensitivity, which offered a simple and efficient method for SA detection.

Design of a Boron-Doped Diamond Microcell Grafted with HRP for the Sensitive and Selective Detection of Ochratoxin A

Ochratoxin A (OTA) is considered the most toxic member of the ochratoxin group. Herein, a novel label-free electrochemical sensor based on the horseradish peroxidase (HRP) enzyme is developed for OTA detection. The HRP enzyme was covalently immobilized on the working electrode of a planar boron-doped diamond (BDD) electrochemical microcell previously covered with diazonium film and grafted with single-walled carbon nanotubes (SWCNTs). Each surface modification step was evaluated by cyclic voltammetry and scanning electron microscopy. Square wave voltammetry was used for the detection of OTA. The linear working range of the biosensors ranged between 10−14 and 0.1 M, with a limit of detection (LOD) of 10 fM, an RSD equal to 5%, and a sensitivity of 0.8 µA per decade. In addition, the sensor showed good selectivity in the presence of OTA analogs; it was validated in samples such as corn, feed, and wheat. The metrological performance of the present sensor makes it a good alternative for OTA detection.

A novel signal amplification strategy for label-free electrochemical DNA sensor based on the interaction between α-cyclodextrin and ferrocenyl indicator

The synthesized ferrocene appended naphthalimide derivative (FND) exhibited great binding ability toward dsDNA, while its usage as the electrochemical hybridization indicator was restricted by the poor water solubility. Herein, a simple and effective signal amplification strategy for FND based label-free DNA biosensors was developed based on the interaction between FND and cyclodextrin. α-Cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) were helpful to amplify the signal of the DNA biosensor, while the signal amplification effect of α-CD was better than that of β-CD and γ-CD. Under the optimum conditions, there was a 3-fold increase in the sensitivity of the DNA biosensor after the addition of α-CD. The interaction between FND and α-/β-/γ-CD was investigated by differential pulse voltammetry and fluorescence experiment. Experimental results showed that α-CD not only minimized the impact on the electrochemical activity of FND but also improved the dispersity of FND in aqueous solution. That was why the proposed biosensor showed higher sensitivity in the presence of α-CD. This strategy was universal for other ferrocenyl indicators with similar structures as used in this work.

An ultrasensitive bacterial imprinted electrochemical sensor for the determination of Lactobacillus rhamnosus GG

An ultrasensitive label-free electrochemical sensor based on a homemade imprinted polypyrrole (PPy) polymer film was prepared to achieve quantitative determination of Lactobacillus rhamnosus GG (LGG). The LGG-imprinted polymer (LIP) film was deposited on a portable screen-printed electrode (SPE) via electropolymerization, which constituted an independent integrated system. The main preparation parameters of the LIP sensor were investigated to obtain optimal performance. Under optimized conditions, the peak current response of the LIP sensor showed a linear relationship with the logarithmic value of LGG concentration in the range from 101 to 109 CFU mL−1 and a detection limit of 5 CFU mL−1. The proposed LIP sensor has achieved efficient, ultrasensitive, highly selective, and cost-effective detection of LGG and can be further developed for practical applications in the quality inspection and development of probiotic products.

A homogeneous label-free electrochemical aptasensor based on an omega-like DNA nanostructure for progesterone detection.

A novel homogeneous label-free electrochemical aptamer sensor for the detection of progesterone was prepared by combining a well-designed omega (Ω)-like DNA (Ω-DNA) nanostructure, with an isothermal cycling amplification strategy based on the highly efficient exonuclease III (Exo III). The omega-like (Ω) DNA is composed of two oligonucleotide strands: DNA1 and DNA2. The Pro aptamer triggers a chain displacement reaction of Ω-DNA nanostructures, forms a new double-stranded DNA structure (aptamer precursor-DNA2), and releases DNA1. Then, Exo III selectively cleaves the DNA duplex and releases the Pro aptamer to participate in a new displacement reaction. Meanwhile, the released DNA1 strands gain access to the strongly bound hemin, forming a hemin/G-quadruplex (DNAzyme). In the presence of hydrogen peroxide (H2O2), differential pulse voltammetry (DPV) was used to detect the current signal from the oxidation of o-phenylenediamine (OPD) to aminoazobenzene (DAP) catalyzed by DNAzyme. However, the amount of released DNA1 from the Ω-DNA nanostructures is reduced in the presence of the target Pro, and the DPV signal declines because of the small amount of DNAzyme formed. The developed electrochemical aptasensor has a wide dynamic linear relationship in the range of 1 pg mL-1 to 10 ng mL-1 under optimal conditions. Its detection limit is down to 0.3 pg mL-1, providing a potential platform for a sensitive Pro assay among electrochemical assays.

DNAzyme-driven bipedal DNA walker for label-free and signal-on electrochemical detection of amyloid-β oligomer

As a common technique for detecting AβO, the enzyme-linked immunosorbent assay (ELISA) method is time-consuming, high in cost, and poor in stability. Therefore, it is necessary to develop a highly sensitive, method-simple and low-cost method for the selective detection of AβO. Here, we created a novel signal-on and label-free electrochemical aptamer sensor for the detection of AβO based on a DNAzyme-driven DNA bipedal walking strategy. Compared with common DNA walkers, bipedal DNA walkers exhibit larger walking areas and faster walking kinetics, and provide higher amplification efficiency. The DNAwalker is powered by an Mg2+-dependent DNAzyme, and the binding-induced DNAwalker continuously clamps the MB, unlocking several active G-quadruplex-forming sequences. These G-quadruplexes can be further combined by hemin to generate a G-quadruplex/heme complex, resulting in an amperometric signal, resulting in a broad proportional band from 0.1 pM to 1 nM and an excellent detection range of 46 fM. A bipedal DNA walker aptamer sensor can detect human serum AβO with remarkable specificity, high reproducibility and practical application value.

Enhanced Bisphenol A Sensing in Water Using 1-D H Exchanged -MnO-Based Impedimetric Sensor

Bisphenol A (BPA) is a known endocrine disruptor with estrogen-like effects, which causes damage to human organs. BPA can affect the immune, reproductive, and neuroendocrine systems and is also carcinogenic. Herein, highly selective electrochemical sensing of BPA using H+-exchanged <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\alpha $ </tex-math></inline-formula> -MnO2 in water under room conditions was developed. H+-exchanged <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\alpha $ </tex-math></inline-formula> -MnO2 was synthesized via ion exchange. The synthesized material was characterized using an X-ray diffractometer (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS), which confirms a successful synthesis of 1-D H+-exchanged <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\alpha $ </tex-math></inline-formula> -MnO2. A nano-enabled, label-free electrochemical sensor was fabricated by employing synthesized materials. The sensor prominently showed high sensitivity of 0.64 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\text{k}\Omega $ </tex-math></inline-formula> /nM/mm2 and a lower limit of detection (LoD) of 16 nM. The equivalent Randel circuit was modeled for electron transfer kinetics evaluation. Additionally, the shelf life analysis confirmed the stability and reproducibility of the designed sensor over 30 days. Moreover, the sensor is highly selective toward BPA against other organic compounds. So, H+-exchanged <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\alpha $ </tex-math></inline-formula> -MnO2-based BPA sensor provides a feasible and novel avenue to detect BPA in water under ambient conditions for health safety purposes.

A label-free impedance-based electrochemical sensor based on self-assembled dendritic DNA nanostructures for Pb2+ detection

Here, a label-free impedance-based electrochemical sensor was developed for the quantitative detection of Pb2+. Using conductive gold nanomaterials as electrode substrate materials can provide sensors with larger specific surface area, action sites and excellent conductivity. DNA nanostructures are used for the determination of biomolecules due to their good properties. The Y-DNA structure is formed by the annealing of three DNA sequences, which acts as a stable structure and forms a dendritic structure in combination with the hybrid chain reaction. In the presence of the target Pb2+, it induces the conversion of specific aptamers into G-quadruplexes, resulting in HCR and Y-DNA loading on the electrodes and a significant change in the impedance value signal. Therefore, the proposed biosensor realizes the quantitative detection of Pb2+. Under the optimal experimental conditions, the concentration of Pb2+ exhibited a linear correlation range from 0.5 to1000 nmol/L with a limit of detection (LOD) of 0.38 nmol/L. The designed sensors have good recoveries in real samples (tap water and tea). This flexible experimental protocol has broad application prospects.

An enzyme-free and label-free electrochemical biosensor for polynucleotide kinase

Polynucleotide kinase (PNK) plays a significant role in the cell signaling pathway and the metabolism damage of cellular nucleic acid. Therefore, it is greatly pivotal to detect the PNK activity and its inhibition. In this work, an enzyme-free and label-free electrochemical sensor for detecting the PNK activity is developed using a glassy carbon electrode (GCE) modified with gold nanoparticles (AuNPs) and zirconia (ZrO2) based on the PNK catalyzing the phosphorylation of the 5′-hydroxyl group of DNA. In the presence of PNK, the DNA was transformed to phosphorylated DNA (pDNA) which preferentially bound with ZrO2/AuNPs/GCE over the unphosphorylated DNA by virtue of the strong affinity between ZrO2 and the phosphate groups. Once the phosphorylated DNA was captured by the ZrO2/AuNPs/GCE, methylene blue (MB), an electroactive indicator, was loaded and produced a characteristic cathodic peak current. The cathodic peak current exhibited a linear correlation to the logarithm of PNK concentration ranging from 0.01 U/mL to 10 U/mL. Moreover, this method can be used to screen the PNK inhibitors and shows excellent performance in real sample analysis, thus holding great potential for further application biological researches and clinic diagnosis.

ZnO/α-MnO2 hybrid 1D nanostructure-based sensor for point-of-care monitoring of chlorinated phenol in drinking water

Herein, we have reported a highly selective electrochemical sensing of 2,4-dichlorophenol in water at room conditions. 1D hybrid nanostructure of ZnO/α-MnO2 nanowire by combining ZnO nanoparticles and α-MnO2 nanowire was developed using a facile ultrasound induced coprecipitation method. The developed hybrid nanostructured material has been characterized in detail using X-ray diffractometer, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy, which confirm a successful synthesis of the ZnO/α-MnO2 hybrid 1D nanostructure. A nano-enabled, label-free electrochemical sensor is fabricated by utilizing the synthesized ZnO/α-MnO2 hybrid 1D nanostructure on a printed contacts-based working electrodes. The sensor prominently showed high sensitivity (0.45 kΩ/nM/mm2) with an excellent limit of detection of 2 nM. The equivalent Randel circuit has been designed and modeled for understanding the evolution of electron transfer kinetics. The shelf life analysis of the fabricated sensor device revealed the stability and reproducibility of over 90 days. The sensor is found to be highly selective toward 2,4-dichlorophenol even in the presence of other organic compounds. The developed sensor provides a novel, fast, and economical approach to detect chlorinated phenolic pollutants in water at ambient conditions, beneficial for health and environmental safety.

A label-free electrochemical sensor constructed with layer-by-layer assembly of GCE-AuNPs-Q[7]·HAuCl4 for detection of diphenylamine.

Metal-organic frameworks (MOFs) including cucurbit[7]uril block (Q[7]·HAuCl4) were employed to develop a diphenylamine (DPA) sensor in electrochemical method, the presence of HAuCl4 improved the conductivity of the macrocyclic compound. To further enhance of the sensitivity, Au nanoparticles were inserted between the surface of glassy carbon electrode and Q[7]·HAuCl4 MOFs (GCE-AuNPs-Q[7]·HAuCl4). Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) were applied for evaluation on the electrochemical behavior. For the electrochemical inertness of DPA, a label-free electrochemical sensor in 5mM K3[Fe(CN)6] solution was achieved, to produce a limit of detection as low as 4.6µM in a linear range of 5-1000µM with good reproducibility, high stability and acceptable anti-interference ability. Application of the proposed electrode for the quantitative determination of DPA in tap water and apple juice confirms its real value.

Hemin/G-quadruplex based electrochemical sensor for highly sensitive detection of ATP in fish

A label-free, sensitive and reliable electrochemical sensor based on hemin/G-quadruplex (G4) DNAzyme was engineered to determine adenosine triphosphate (ATP) in fish. The nucleic acid sequence (DNA1) including the G4 sequence subunit and anti-ATP aptamer segment is anchored on Au electrodes. The nucleic acid sequence (DNA2) is composed of another G4 sequence fragment and anti-ATP aptamer subunit. The probes DNA1 and DNA2 self-assemble into the active hemin/G4 DNAzyme structure on Au electrodes upon exposure to the target ATP. The determination of the target ATP relied on an increase in the catalytic currents occurring on DNAzyme-anchored electrode via the electrochemical reduction of H2O2. The proposed strategy enables high-performance and specified determination of the target ATP with a detection limit of 0.04 μmol/L. Specifically, the proposed ATP strategy was further implemented to determine ATP in microorganisms in carp samples. Compared with the classical total viable count (TVC) assay, the proposed ATP assay was feasible to assess the freshness of carp, which expands the applications of hemin/G4 DNAzyme and holds promising potential for freshness assessment in food safety.

A simple label-free electrochemical aptamer sensor for detection of Hg2+ based on black phosphorus

In this work, a simple label-free aptamer-based electrochemical sensor was developed for determination of mercury ions (Hg2+) by polylysine (PLL) modified black phosphorus nanosheets (BP) and gold nanoparticles (AuNPs) as signal amplification strategy. The PLL-BP composite materials greatly improved electron transfer speed and electrochemical behavior of electrode in the detection of analytes. AuNPs can amplify the electrochemical signal and facilitate the binding of aptamers to the electrode. The selected aptamer (Apt) can bind specifically to Hg2+ to form a T-Hg2+-T structure, which we used to detect Hg2+. Experimental results showed that the prepared electrochemical aptamer sensor exhibited wide linear range from 1.0–500 nM, along with a low detection limit of 0.14 nM. Moreover, the electrochemical aptamer sensor has good specificity, stability, and repeatability. Therefore, this proposed electrochemical aptamer sensor provide a simple and sensitive method for the detection of Hg2+, which is expected to be used for the detection of trace amounts of Hg2+ in actual samples.

CRISPR-Cas12a-mediated label-free electrochemical aptamer-based sensor for SARS-CoV-2 antigen detection

Serological antigen testing has emerged as an important diagnostic paradigm in COVID-19, but often suffers from potential cross-reactivity. To address this limitation, we herein report a label-free electrochemical aptamer-based sensor for the detection of SARS-CoV-2 antigen by integrating aptamer-based specific recognition with CRISPR-Cas12a-mediated signal amplification. The sensing principle is based on the competitive binding of antigen and the preassembled Cas12a-crRNA complex to the antigen-specific aptamer, resulting in a change in the collateral cleavage activity of Cas12a. To further generate an electrochemical signal, a DNA architecture was fabricated by in situ rolling circle amplification on a gold electrode, which serves as a novel substrate for Cas12a. Upon Cas12a-based collateral DNA cleavage, the DNA architecture was degraded, leading to a significant decrease in impedance that can be measured spectroscopically. Using SARS-CoV-2 nucleocapsid antigen as the model, the proposed CRISPR-Cas12a-based electrochemical sensor (CRISPR-E) showed excellent analytical performance for the quantitative detection of nucleocapsid antigen. Since in vitro selection can obtain aptamers selective for many SARS-CoV-2 antigens, the proposed strategy can expand this powerful CRISPR-E system significantly for quantitative monitoring of a wide range of COVID-19 biomarkers.