Sensitive Electrochemical Assay Research Articles

Highly catalytic sulfur-doped and bimetal-coordinated CoFe(CN)5NO nanoparticles coupled with PER/HCR amplification cascades for sensitive electrochemical aptamer luteinizing hormone assay

Sensitive monitoring of luteinizing hormone (LH), a glycoprotein that regulates the synthesis of regulatory steroid hormones, can facilitate the diagnosis of various reproductive diseases. In this work, a new and highly catalytic Sulfur-doped and bimetal-coordinated CoFe(CN)5NO (denoted as S–CoFe(CN)5NO) nanoparticles are synthesized. Such material is further used to construct high performance sensing interface and coupled with primer exchange reaction (PER) and hybridization chain reaction (HCR) amplification cascades for sensitive electrochemical aptamer-based LH assay. Target LH molecules bind aptamer sequences in DNA duplex probes to liberate ssDNA strands, which initiate subsequent PER/HCR amplification cascades for the capture of many ferrocene (Fc)-tagged DNAs on sensing interface. S–CoFe(CN)5NO subsequently leads to catalytic oxidation of these Fc tags for yielding substantially magnified currents for realizing ultrasensitive assay of LH with the detection limit of 0.69 pM in range from 5 pM to 10 nM. Owing to the high specificity of aptamer, such sensor has high selectivity and can achieve low levels of LH assay in diluted serum samples. With the successful demonstration for detecting trace LH, such sensor can be easily extended as a universal aptamer-based electrochemical sensing method for monitoring various target analytes in the biomedical and biological fields.

Biomolecules-mediated electrochemical signals of Cu2+: Y-DNA nanomachines enable homogeneous rapid one-step assay of lung cancer circulating tumor cells

This study presents a straightforward efficient technique for extracting circulating tumor cells (CTCs) and a rapid one-step electrochemical method (45 min) for detecting lung cancer A549 cells based on the specific recognition of mucin 1 using aptamers and the modulation of Cu2+ electrochemical signals by biomolecules. The CTCs separation and enrichment process can be completed within 45 min using lymphocyte separation solution (LSS), erythrocyte lysis solution (ELS), and three centrifugations. Besides, the influence of various biomolecules on Cu2+ electrochemical signals is comprehensively discussed, with DNA nanospheres selected as the medium. Three single-stranded DNA sequences were hybridized to form Y-shaped DNA (Y-DNA), creating DNA nanospheres. Upon specific capture of mucin 1 by the aptamer, most DNA nanospheres could form complexes with Cu2+ (DNA nanosphere-Cu2+), significantly reducing the concentration of free Cu2+. Our approach yielded the limit of detection (LOD) of 2 ag/mL for mucin 1 and 1 cell/mL for A549 cells. 39 clinical blood samples were used for further validation, yielding results closely correlated with pathological, computed tomography (CT) scan findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve displayed an area under the curve (AUC) value of 0.960, demonstrating 100% specificity and 93.1% sensitivity for the assay. Taken together, our findings indicate that this straightforward and efficient pretreatment and rapid, highly sensitive electrochemical assay holds great promise for liquid biopsy-based tumor detection using CTCs.

A target-triggered ultra-sensitive aptasensor for simultaneous detection of Cd2+ and Hg2+ using MWCNTs-Au NPs modified electrode

A sensitive electrochemical assay for simultaneously detecting cadmium ion (Cd2+) and mercury ion (Hg2+) with the aptamer as recognition unit was established, in which methylene blue (MB) and target-triggered in-situ generated Ag nanoclusters (Ag NCs) were identified as signal reporters. Multi-walled carbon nanotubes and gold nanoparticles composites were prepared with polyethyleneimine to amplify electrical signals of screen-printed electrodes. Due to the particular base sequences, MB labeled Cd2+ aptamer paired with ssDNA through T-Hg-T structure with Hg2+. Notably, the C-rich structure in ssDNA acted as a template for the generation of Ag NCs, which could induce differential pulse voltammetry signals corresponding to Hg2+ concentrations. This electrochemical aptasensor exhibited detection limits of 94.01 pg/mL and 15.74 pg/mL for Cd2+ and Hg2+, respectively. The developed aptasensor allowed for practical application to tea and vegetable samples with satisfactory accuracy. This work possesses potential in developing biosensing technologies for simultaneous determination of multiple heavy metals.

Highly sensitive electrochemical assay based on strand displacement polymerization-assisted CRISPR/Cas12a collateral cleavage

In this work, a novel strand displacement polymerization strategy is designed with a single hairpin-structured DNA probe acting as both of primer and template. After target miRNA-induced structural transitions, extension and displacement reactions occur at the dimer with recycled target miRNA. The generated double-stranded products further activate CRISPR/Cas12a collateral cleavage of substrate DNA at the electrode surface, which can be reflected by the silver stripping peak variations. By analyzing the reduced peak intensity, the concentration of miRNA can be determined. This method combines the strand displacement amplification and CRISPR/Cas12a’s shearing capability. Thus a highly sensitive electrochemical biosensor is established with the limit of detection as low as 31 aM. It may also inspire new ideas of electrochemical platforms integrating CRISPR/Cas system for biological studies and clinical applications.

Metallo-base pair-mediated DNA probe for label-free, amplified and catalytic electrochemical detection of metallothionein

Metallothionein (MT) is an important biomarker for the early diagnosis of heavy metal toxicity and malignancy and the detection and quantification of MT therefore has important biomedical significances. In this work, we present a new metallo-base pair-mediated DNA probes for label-free and sensitive electrochemical assay of MT via hybridization chain reaction (HCR) and catalytic redox recycling signal amplifications. The DNA probe is immobilized on the gold electrode to prepare the sensor. Due to the strong binding capability between MT and the metal ions in the DNA probes, the presence of the MT target molecules leads to both disruption of the duplex structure of the DNA probes and the release of ssDNA strands, which trigger subsequent HCR formation of long dsDNA polymers on the electrode from two hairpins. Large amounts of [Ru(NH3)6]Cl3 can then be captured onto the dsDNA polymers to show substantially enhanced catalytic currents with co-existence of K3[Fe(CN)6] for highly sensitive assay of MT down to 0.15 nM in the range of 0.5–500 nM. The sensor also shows the capability for monitoring MT in diluted human serum samples, indicating its great potential for detecting various metal ion binding proteins and small molecules for different application purposes.

A highly sensitive electrochemical magneto-genosensing assay for the specific detection of a single nucleotide variation in the KRAS oncogene in human plasma

Liquid biopsy is a non-invasive and efficient technique for the detection of tumor biomarkers in biological fluids, which currently represents a new frontier in theranostics and precision medicine. Among mutations of the KRAS oncogene, p.G12D single nucleotide variation in the KRAS gene plays a central role in the early diagnosis and therapeutic treatment of colorectal cancer. In this context, we developed a highly sensitive magneto-genosensing assay based on PNA capture probes immobilized on magnetic microbeads. To detect the mutation of the KRAS oncogene, two capture probe sequences recognizing wild-type and p.G12D tumor DNA were used in association with a DNA signaling probe allowing for electrochemical detection using an enzyme conjugate. The assay conditions were optimized using a 32 full-factorial design, obtaining an outstanding specificity, evidenced by a remarkably lower (>95%) signal of the singly-mismatched- compared to that of fully-complementary-DNA. Ultra-high sensitivity was achieved in 10-fold diluted human plasma reaching detection limits of 818 fM for the p.G12D and 1.8 pM for the wild-type targets. The genosensing assay was tested on genomic tumor DNA samples provided by IRCCS-Regina Elena National Cancer Institute and finally integrated into a smart portable multichannel potentiostat capable of performing up to four simultaneous acquisitions. The developed magneto-genosensing assay demonstrated portability, simplicity, and high sensitivity, showing good potential as theranostic tool for personalized medicine in oncology.

An electrochemical biosensor based on network-like DNA nanoprobes for detection of mesenchymal circulating tumor cells

The identification and detection of mesenchymal circulating tumor cells （mCTCs） is important for early warning of tumor metastasis. The majority of conventional detection methods for CTCs rely on the recognition of epithelial biomarkers, which is technically challenging for detecting CTCs with epithelial-mesenchymal transition (EMT)-induced phenotypic alteration. In this work, we have constructed a label-free biosensor for sensitive electrochemical assay of mCTCs. In our design, the capture probe can recognize the vimentin overexpressed on the surface of mCTCs with high specificity. Meantime, the network-like DNA nanoprobes with multiple G-quadruplex/hemin complexes and multiple cholesterol molecules can be grafted to the cell surface based on the high affinity between cholesterol molecules and cell membrane. Owing to the mimic horseradish peroxidase of G-quadruplex/hemin complexes, strong electrochemical responses will be obtained for sensitive quantification of mCTCs with a detection limit of 8 cell mL−1. Moreover, the biosensor can effectively overcome the interference of vimentin negative cells or secretory vimentin, and realize the recovery tests in whole blood with high accuracy, thereby may further promoting the diagnosis and personalized treatment of cancer in clinic.

Synthesis and characterization of Au-decorated graphene oxide nanocomposite for magneto-electrochemical detection of SARS-CoV-2 nucleocapsid gene

Fast and effective diagnosis is the first step in monitoring the current coronavirus 2 (CoV-2) pandemic. Herein, we establish a simple and sensitive electrochemical assay using magnetic nanocomposite and DNA sandwich probes to rapidly quantify the CoV-2 nucleocapsid (N) gene down to the 0.37 fM level. This assay uses a pair of specific DNA probes. The capture probe is covalently conjugated to Au-decorated magnetic reduced graphene oxide (AMrGO) nanocomposite for efficiently capturing target RNA. In contrast, the detection probe is linked to peroxidase for signal amplification. The probes target the COV-2 gene, allowing for specific magnetic separation, enzymatic signal amplification, and subsequent generation of voltammetric current with a total assay time of 45 min. The developed biosensor has high selectivity and can discriminate non-specific gene sequences. Synthetic COV-2 N-gene can be detected efficiently in serum and saliva, while 1-bp mismatch gene yielded a low response. The performance of the genosensor was good in an extensive linear range of 5 aM–50 pM. For synthetic N-gene, we achieved the detection limit of 0.37, 0.33, and 0.19 fM in human saliva, urine, and serum. This simple, selective, and sensitive genosensor could have various genetics-based biosensing and diagnostic applications.

Target-induced site-specific cleavage of phosphorothioate-modified hairpin DNA for amplified and label-free sensitive electrochemical myeloperoxidase assay

Abnormal myeloperoxidase (MPO) expression is associated with inflammation and neurodegenerative diseases. By using a new phosphorothioate (PT)-modified DNA hairpin probe, we fabricate a label-free and highly sensitive electrochemical sensor for monitoring MPO via integration of DNAzyme-assisted cleavage recycling with DNA polymerization reaction catalyzed by terminal deoxynucleotidyl transferase (TdT). The target MPO mediates the specific cleavage of PT-modified hairpin probes with assistance of hydrogen peroxide and chloride ion to initiate DNAzyme-based cyclic cleavage of substrate hairpin probes for the release of numerous trigger strands. Subsequent hybridizations of the trigger strands with the thiolated-hairpin probes on the electrode surface expose the 3′-OH termini of hairpin probes for triggering TdT–catalyzed in situ DNA polymerization formation of long ssDNAs containing multiple G-quadruplex repeats. Hemin is confined on the electrode surface via the formation of G-quadruplex/hemin complexes to produce highly enhanced current signals for the determination of MPO in a sensitive and label-free way. The proposed sensor exhibits a linear range of 0.1–100 ng mL−1 and achieves a low detection limit of 0.046 ng mL−1 for MPO. Such sensor also shows high selectivity against other interferents and shows the capability to detect MPO in diluted human serum samples.

Tyrosine-Conjugation Method for Evaluating BACE1 Activity Based on Silver Nanoparticles/Metal-Organic Framework Composites as Electrochemical Tags.

Beta-site secretase (BACE1) catalyzes the cleavage of amyloid precursor protein (APP), which process ultimately lead to plaque deposition in the brain of Alzheimer's disease (AD). Thus, accurate monitor of BACE1 activity is essential to screen inhibitors for AD treatment. This study develops a sensitive electrochemical assay for probing BACE1 activity based on silver nanoparticles (AgNPs) and tyrosine conjugation as tags and a marking method, respectively. An APP segment is firstly immobilized on aminated microplate reactor. Cytosine (C) rich sequence-templated AgNPs/Zr-based metal-organic framework (MOF) composite is modified by phenol groups, and then the prepared tag (ph-AgNPs@MOF) is captured in microplate surface by the conjugation reaction of phenolic groups between tyrosine and tag. After cleavage by BACE1, the solution containing ph-AgNPs@MOF tags is transferred to the screen-printed graphene electrode (SPGE) surface for voltammetric detection of AgNP signal. This sensitive detection for BACE1 provided an excellent linear relationship between 1 to 200 pM with a detection limit of 0.8 pM. Furthermore, this electrochemical assay is successfully applied for screening of BACE1 inhibitors. This strategy is also verified to be used for evaluation of BACE1 in serum samples.

Incorporating quaternary mesoporous nanospheres and DNA stochastic nanowalkers into a signal amplified switch: A highly sensitive electrochemical assay for APE1

In this work, we engineered a quaternary mesoporous nanosphere by boron- and phosphorus-codoped Pd-Pt alloy ([email protected]@Pt) to prepare a DNA stochastic nanowalker for catalysis efficiency and local concentration enhancement, which can be applied as an excellent signal amplified switch to electrochemically detect apurinic endonuclease 1 (APE1) with the assist of a highly efficient proximity ligation assay (PLA)-based protein conversion strategy. Specifically, the synthetic [email protected]@Pt nanomaterial showed excellent catalytic performance for electroactive substances (ferrocene) due to the desirable pore confinement effect and abundant active sites. Moreover, it can be employed to program a DNA stochastic nanowalker to significantly improve the detection sensitivity and response time. In addition, by designing a highly efficient PLA-based protein converting strategy, the target APE1 can be efficiently recognized to concurrently convert APE1 to enormous DNA outputs, which promoted the detection selectivity and sensitivity for APE1 in the range of 1 fg/mL to 1 pg/mL with a detection limit of 0.45 fg/mL. Although the [email protected]@Pt-based DNA stochastic nanowalker driven by the biological enzyme whose activity may be affected by the environment, the as-prepared electrochemical biosensor based on the [email protected]@Pt-based DNA stochastic nanowalker has still achieved good detection performance, and possesses enormous potential for the applications of new-type DNA nanomachines in biosensing, bioimaging and clinical diagnosis.

Rapid and Sensitive Electrochemical Assay of Cefditoren with MWCNT/Chitosan NCs/Fe2O3 as a Nanosensor.

In this research, a glassy carbon electrode (GCE) modified by MWCNT/chitosan NCs/Fe2O3 was prepared for the determination of the cephalosporin antibiotic cefditoren (CFT) using adsorptive stripping differential pulse and cyclic voltammetry techniques. The effects of pH, the scan rate, the deposition potential, the accumulation time, and modification agents on the determination of CFT were analyzed. The results showed that the modified electrode significantly increased the oxidation peak current of CFT. Under optimized conditions, the MWCNT/chitosan NCs/Fe2O3/GCE nanosensor exhibited a linear response between 0.2 µM and 10 µM toward CFT. The limit of detection and quantification were determined to be 1.65 nM and 5.50 nM, respectively. Model drugs (cefdinir, cefpodoxime, cephalexin, and ceftazidime compounds) were used to enlighten the CFT oxidation mechanism. Moreover, the nanosensor was used to analyze CFT in a pharmaceutical dosage form and commercial deproteinated human serum samples. The accuracy of these methods was proven in the recovery studies, with values of 96.98 and 98.62% for the pharmaceutical dosage form and commercial deproteinated human serum sample, respectively.

A low-fouling and reusable biosensor for the sensitive electrochemical assay of tuberculosis gene

A low-cost, portable, and reusable biosensor for the rapid and selective quantitation of tuberculosis (TB) gene with low expression levels in early-stage serum specimens is highly desirable but remains a challenge. Herein, a novel biosensor was fabricated using the layer-by-layer assembly of gold nanoparticles (Au NPs), polyethylenimine (PEI) and chondroitin sulfate (CS). The synergistic effect of PEI and CS endows this biosensor antifouling properties towards single proteins, even towards complex biological systems. Before and after the incubation in 10% serum, only 6.4% peak current change of DPV response was obtained. To the best of our knowledge, a few examples have been reported on the successful application of CS in mitigating biofouling. Next, a special gene only representing TB was modified on CS-modified electrode, and this biosensor showed a fast response with good linear concentration dependence on the target DNA in the range 10−14 M to 10−10 M. More importantly, this biosensor based on the reversible bonding of DNA double strand could be reused for at least four times after the regeneration of urea without any passivation, which further reduced the cost of the entire strategy. To some extent, this indicates that the CS-modified biosensor has certain practical application prospect and can be extended to fabricate similar biosensors for the detection of other diseases.

Simple Design Concept for Dual-Channel Detection of Ochratoxin A Based on Bifunctional Metal-Organic Framework.

A simple fluorescence and electrochemical dual-channel biosensor based on bifunctional Zr(IV)-based metal-organic framework (Zr-MOF) was proposed to detect Ochratoxin A (OTA). The bifunctional Zr-MOF, with photoluminescence properties and enormous electroactive ligands, was exploited to load OTA-specific aptamers for designing signal probes, greatly simplifying the probe-fabrication process and improving sensing reliability. Upon specific recognition of aptamer toward OTA, the anchored probe was released from the sensing interface into the reaction solution. In this circumstance, the increased amount of the signal probe in reaction solution led to an enhanced fluorescence response, while the decreased amount of the signal probe on the sensing interface resulted in a diminished electrochemical response. According to the dual-channel signal change with increasing OTA concentration, the visual fluorescence strategy was established for intuitive OTA detection, and meanwhile, sensitive electrochemical assay with a detection limit of 0.024 pg/mL was also achieved with the help of one-step electrodeposition as a sensing platform. Moreover, the proposed dual-channel assay has been successfully applied to determine OTA levels in corn samples with rapid response, superior accuracy, and high anti-interference capability, providing a promising method for food safety monitoring.

Amplified electrochemical biosensing based on bienzymatic cascade catalysis confined in a functional DNA structure

Herein, an amplified and renewable electrochemical biosensor was developed via bienzymatic cascade catalysis of glucose oxidase (GOx) and horseradish peroxidase (HRP), which were confined in a functional Y-shaped DNA nanostructure oriented by a dual-thiol-ended hairpin probe (dSH-HP) with a paired stem as a rigid scaffold and unpaired loop as enclosed binding platform. For proof-of-concept assay of sequence-specific biomarker DNA related to Alzheimer's disease (aDNA), GOx and redox ferrocene-modified HRP (Fc@HRP) were chemically conjugated in two enzyme strands (GOx-ES1 and Fc@HRP-ES2), respectively. The repeated recycling of aDNA was powered by the displacement of GOx-ES1 by aDNA and exonuclease III (ExoIII)-assisted cleavage reaction for amplified output of numerous GOx-ES1 as dependent transducers, together with Fc@HRP-ES2 which was simultaneously hybridized with dSH-HP to assemble this DNA structure. Rationally, the bienzymatic cascade catalysis was motivated through GOx-catalyzed glucose oxidization to in situ generate hydrogen peroxide (H2O2) and overlapped HRP-catalyzed H2O2 decomposition to promote the electron transfer, producing significantly enhanced electrochemical signal of Fc with an ultrahigh sensitivity down to 0.22 fM of aDNA. Benefited from the unique design of dSH-HP-oriented bienzymatic cascades, this one-step strategy without non-specific blockers passivation was simple and renewable, and would pave a promising avenue for sensitive electrochemical assay of biomolecules.

Highly sensitive electrochemical assay for selective detection of Aminotriazole based on TiO2/poly(CTAB) modified sensor

This research work describes the electrochemical activity of aminotriazole (ATZ) by constructing titanium-dioxide nanoparticles (TiO 2 ) and cetyltrimethylammonium bromide (CTAB) based carbon paste electrode (TiO 2 /CTAB-CPE). Square wave voltammetric technique was employed to investigate herbicide, ATZ in the soil as well as in water samples. The characterization of TiO 2 nanoparticles was achieved using XRD and TEM techniques. Owing to the supreme sensing properties, TiO 2 /CTAB-CPE was recognized as sensitive for the detection of ATZ as the sensor demonstrated improved catalytic characteristics and peak current in presence of pH 7.0 of 0.2 PBS i.e., phosphate buffer solution through cyclic voltammetric (CV) along with square wave voltammetric (SWV) techniques. The peak current for the modified CPE observed to be 18. 92 μ A with peak potential 0.912 V and for the bare carbon paste electrode (BCPE) the peak current is 4 μ A with the peak potential at 0.975 V. The impact of electro-kinetic parameters including heterogeneous rate constant, scan rate, transfer coefficient, pH, activation energy, accumulation time, temperature effect, and also the number of electrons and protons entailed in the ATZ electro-oxidation was resolved. The detection limit and the quantification limit of the ATZ was found to be 2.53 nM and 8.44 nM respectively. The proposed method proved to be a propitious procedure for precise determination of the ATZ herbicide in water and soil samples. • Electrochemical detection of ATZ herbicide was achieved by CTAB and TiO 2 composite. • The electrochemical process was diffusion controlled with two-electron transfer. • Successfully used the sensor for the analysis of ATZ in water and soil samples.

Electrochemical Sensing of Exosomal MicroRNA Based on Hybridization Chain Reaction Signal Amplification with Reduced False-Positive Signals.

MicroRNAs (miRNAs) in cancer cell-derived exosomes are important cancer biomarkers. Herein, a sensitive hybridization chain reaction (HCR) electrochemical assay was fabricated for the detection of exosomal microRNA-122 (miR-122). The hairpin DNA (hpDNA) probes were first immobilized on the surface of a gold electrode. In the presence of miR-122, the hairpin structure of the hpDNA could be opened and triggered the HCR through the cross-opening and hybridization of two helper DNA hairpins. Long nicked double helixes generated from HCR are used to capture more RuHex and increase the signal of differential pulse voltammetry (DPV). In this assay, the density of the hpDNA probes on the surface of the gold electrode was precisely controlled by the simultaneous immobilization of hpDNA and short 12 nucleotides single-stranded DNA (S-12), providing a very high amplification efficiency. More importantly, the false positive signal could be reduced or completely eliminated by applying exonuclease I (Exo I) before the introduction of target miR-122. Under optimal conditions, the assay offers very high sensitivity with an attomolar level detection limit, a linear range with 9 orders of magnitude, and specificity in single mismatch discrimination. This sensitive electrochemical assay could successfully evaluate the miR-122 concentration in different cancer-derived exosomes, indicating its potential use in cancer diagnostics.

Label-free assay of protein kinase A activity and inhibition in cancer cell using electrochemically-prepared AuNP/rGO nanohybrid electrode modified with C-Kemptide

A simple, label-free and sensitive electrochemical assay is described for the detection of protein kinase A (PKA) activity and inhibition in cancer cell by measuring the change in electrochemical impedance upon phosphorylation. The assay utilized gold nanoparticle (AuNP) and reduced graphene oxide (rGO) nanohybrid which was synthesized and deposited on the electrode from GO and Au3+ precursors by one-pot electrochemical synthesis. As-prepared AuNP/rGO electrode was employed to immobilize C-Kemptide peptide substrates which was phosphorylated in the presence of PKA and ATP. The resulting assay allowed effective, selective and sensitive monitoring of PKA activity according to the impedimetric change in the range of 0.1–500 U/mL, and the detection limit (LOD) is 53 mU/mL. It could also be used for the screening of protein kinase inhibitors. Furthermore, the assay could be applied for the evaluation of PKA activity and inhibition in HeLa cell samples. Therefore, the proposed assay provides one promising tool for PKA activity detection and inhibitor screening with excellent performance.

Immuno-DNA binding directed template-free DNA extension and enzyme catalysis for sensitive electrochemical DNA methyltransferase activity assay and inhibitor screening.

Sensitive and accurate determination of DNA methyltransferase (DNA Mtase) activity is highly pursued for understanding fundamental biological processes related to DNA methylation, clinical disease diagnosis and drug discovery. Herein, we propose a new electrochemical immuno-DNA sensing platform for DNA Mtase activity assay and inhibitor screening. After homogeneous DNA methylation by CpG methyltransferase (M.SssI Mtase), the methylated DNA can be specifically recruited onto an electrode via its immunological binding with the immobilized anti-5-methylcytosine antibody. The recruited methylated DNA was simultaneously used as a substrate to facilitate successive template-free DNA extension and enzyme catalysis for the dual-step signal amplification of DNA Mtase activity. The developed immuno-DNA sensing strategy effectively integrates solution-phase DNA methylation, surface affinity binding recognition, and successive template-free DNA extension and enzyme catalysis-based signal amplification, rendering a highly specific, sensitive and accurate assay of DNA Mtase activity. A low detection limit of 0.039 U mL-1 could be achieved with a high selectivity. It was also applied for efficient evaluation of various inhibitors. Current affinity recognition of the immobilized antibody with methylated DNA switches the sensing platform into a DNA operation interface, facilitating the opportunity for combining various DNA-based signal amplification strategies to improve the detection performance. It would be used as a general strategy for the analysis of DNA Mtase activity, inhibitors and more analytes, and is anticipated to show potential for applications in disease diagnosis and drug discovery.

Aptamer based determination of the cancer biomarker HER2 by using phosphate-functionalized MnO2 nanosheets as the electrochemical probe.

The authors report a sensitive electrochemical aptamer-based assay for the cancer biomarker human epidermal growth factor receptor-2 (HER2). It is based on the use of MnO2 nanosheets that were functionalized with phosphate and a HER2 binding aptamer and serve as the electrochemical probe. The assay follows a sandwich protocol. A peptide that can specifically recognize HER2 was immobilized on a gold electrode for the capture of HER2. Then, the functionalized MnO2 nanosheets were linked to the electrode via the binding between HER2 and HER2 aptamer on the MnO2 nanosheets. The reaction of phosphate and aptamer on the MnO2 nanosheets with molybdate leads to the formation of redox-active molybdophosphate. This results in dual signal amplification. The generated electrochemical current was measured at 0.22V (vs. Ag/AgCl). The assay allows HER2 to be determined in the 0.1 to 500pg·mL-1 concentration range, and the detection limit is as low as 0.05pg·mL-1. The assay was successfully applied for the detection of HER2 in spiked human serum samples. Graphical abstract Electrochemical detection of breast cancer biomarker human epidermal growth factor receptor-2 (HER2) is reported utilizing phosphate ions and aptamer functionalized MnO2 nanosheet as probe.