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A solid-state electrochemiluminescence (ECL) biosensor based on special ferrocene-labeled molecular beacon (Fc-MB) for highly sensitive detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene was developed successfully using Ru(bpy) ${{{2+\hfill \atop 3\hfill}}}$/2-(dibutylamino)ethanol (DBAE) as detecting pattern. Such a special sensor involves two main parts, an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Ruthenium (II) tris-(bipyridine) and Au nanoparticles (Ru(bpy) ${{{2+\hfill \atop 3\hfill}}}$-AuNPs) onto the Au electrode (AuE) surface. The molecular beacon probe in which the ferrocene tag could effectively quench the ECL of the Ru(bpy) ${{{2+\hfill \atop 3\hfill}}}$acted as ECL intensity switch. The molecular beacon probe was designed with special base sequence, which could hybridize with its complementary target DNA. In the absence of a target, the hairpin structure of the probe forced the ferrocene (Fc) into close proximity with the ECL substrate, thus reducing ECL intensity. Target binding allowed the Fc away from the ECL substrate and resulted in an obvious increment in ECL intensity due to the decreased Fc quenching effect. The effect of the amount of Ru(bpy) ${{{2+\hfill \atop 3\hfill}}}$ and the mixing procedure of Ru(bpy) ${{{2+\hfill \atop 3\hfill}}}$ and AuNPs solution on the fabrication of ECL film had been investigated. As a result, the change of ECL intensity had a direct relationship with the logarithm of PML/RARα fusion gene concentration in the range of 0.05-500 pM with a detection limit of 7 fM, and the developed biosensor possessed good molecular recognizability in human serum. Thus, the approach holds promise for the early diagnostics and prognosis monitoring of APL and other diseases. [ABSTRACT FROM AUTHOR]

A novel electrochemical method for the sequence-specific detection of double-stranded polymerase chain reaction (PCR) products of PML/RARα fusion gene in acute promyelocytic leukemia (APL) was described in detail. Based on a 'sandwich' sensing mode involving a pair of locked nucleic acids probes (capture probe and reporter probe), this DNA sensor exhibited excellent selectivity and specificity. The direct and quantitative analysis of double-stranded complementary was firstly performed by our sensor without the use of alkali, helicase enzymes, or denaturants. Finally, combining PCR technique with electrochemical detection scheme, PCR amplicons (191 bp) of the PML/RARα fusion gene were obtained and rapidly identified with a low detection limit of 79 fmol in the 100-μL hybridization system. The results clearly showed the power of sensor as a promising tool for the sensitive, specific, and portable detection of APL and other diseases. [ABSTRACT FROM AUTHOR]

Gold nanostructures and a Nafion modified screen-printed carbon electrode (Nafion/AuNS/SPCE) were developed to assess the cell viability of Parkinson's disease (PD) cell models. The electrochemical measurement of cell viability was reflected by catecholamine neurotransmitter (represented by dopamine) secretion capacity, followed by a traditional tetrazolium-based colorimetric assay for confirmation. Due to the capacity to synthesize, store, and release catecholamines as well as their unlimited homogeneous proliferation, and ease of manipulation, pheochromocytoma (PC12) cells were used for PD cell modeling. Commercial low-differentiated and highly-differentiated PC12 cells, and home-made nerve growth factor (NGF) induced low-differentiated PC12 cells (NGF-differentiated PC12 cells) were included in the modeling. This approach achieved sensitive and rapid determination of cellular modeling and intervention states. Notably, among the three cell lines, NGF-differentiated PC12 cells displayed the enhanced neurotransmitter secretion level accompanied with attenuated growth rate, incremental dendrites in number and length that were highly resemble with neurons. Therefore, it was selected as the PD-tailorable modeling cell line. In short, the electrochemical sensor can be used to sensitively determine the biological function of neuron-like PC12 cells with negligible destruction and to explore the protective and regenerative impact of various substances on nerve cell model. [ABSTRACT FROM AUTHOR]

Sepsis is a life-threatening multi-organ failure syndrome, with bacterial infections being the most common cause. Rapid Gram-typing is imperative to assist in antibiotic intervention. C-reactive protein (CRP) and lipopolysaccharide (LPS) are effective biomarkers for discerning the Gram type of bacteria but differ by several orders of magnitude in clinical detection, thereby impeding their simultaneous detection. And two independent methods are time-consuming and laborious. In this study, a dual-channel electrochemical biosensor was developed for simultaneous detection of LPS and CRP. Under optimal conditions, linear ranges of LPS (0.5–1000 pg/mL) and CRP (0.1–20 μg/mL) were obtained in line with the clinical evaluation scopes. In simulated sample tests, Gram-positive, Gram-negative, and healthy plasma samples were clearly distinguished by the developed biosensors, and these results were consistent with that of enzyme-linked immunosorbent assay (ELISA). In addition, the results of the plasma samples tested by the electrochemical biosensor matched those derived from blood cultures in the laboratory. Collectively, the electrochemical biosensor was expected to provide the scientific basis for the rapid Gram-typing and point-of-care detection of bacterial sepsis. [ABSTRACT FROM AUTHOR]

Since microRNAs (miRNAs) are predictors of tumorigenesis, accurate identification and quantification of miRNAs with highly similar sequences are expected to reflect tumor diagnosis and treatment. In this study, a highly selective and sensitive electrochemiluminescence (ECL) biosensor was constructed for miRNAs determination based on Y-shaped junction structure equipped with locked nucleic acids (LNA), graphene oxide-based nanocomposite to enrich luminophores, and conductive matrix. Specifically, two LNA-modified probes were designed for specific miRNA recognition, that is, a dual-amine functionalized hairpin capture probe and a signal probe. A Y-shaped DNA junction structure was generated on the electrode surface upon miRNA hybridizing across the two branches, so as to enhance the selectivity. Carbon quantum dots-polyethylene imine-graphene oxide (CQDs-PEI-GO) nanocomposites were developed to enrich luminophores CQDs, and thus enhancing the ECL intensity. For indirect signal amplification, an electrochemically activated poly(2-aminoterephthalic acid) (ATA) film decorated with gold nanoparticles was prepared on electrode as an effective matrix to accelerate the electron transfer. The fabricated ECL biosensor achieved sensitive determination of miRNA-222 with a limit-of-detection (LOD) as low as 1.95 fM (S/N = 3). Notably, Y-shaped junction structures equipped with LNA probes endowed ECL biosensor with salient single-base discrimination ability and anti-interference capacity. Overall, the proposed Y-shaped ECL biosensor has considerable promise for clinical biomarker determination. [ABSTRACT FROM AUTHOR]

[Display omitted] • A microscale electrochemical DNA biosensor based on a miniaturized gold microelectrode (Au-Me) was developed. • Au-Me achieved sensitive determination of HFMD-related gene with a detection limit of 35 fM (S/N = 3). • The binding capability of HFMD-related gene probes towards target was measured by native PAGE and ITC. • The biosensor exhibited high specificity and anti-interference performance toward target DNA in complex serum milieu. Considering the spatially and temporally rapid spread of hand, foot and mouth disease (HFMD) incidence, point-of-care testing (POCT) is in great demand for HFMD early diagnosis to prevent outbreak. Herein, a miniaturized gold microelectrode (Au-Me), as a promising alternative to the traditional electrode due to its own essence of high mass transfer, high current density and rapid response, was developed to construct a sensitive, accurate and rapid biosensor suitable for POCT. Specifically, a sandwich-type electrochemical deoxyribonucleic acid (DNA) biosensor based on Au-Me was constructed to sensitively determine HFMD-related gene. The binding capabilities of probes towards the target sequences were investigated by native PAGE and isothermal titration calorimetry (ITC). The detection limit of the biosensor was as low as 35 fM (S/N = 3). Interaction binding between capture/signal probes and the target DNA had an association constant (K a) of ca. 1.1 × 109 M−1 via ITC. This biosensor exhibited excellent specificity and anti-interference performance toward target DNA in complex serum milieu, demonstrating the potential utility in clinical POCT applications for HFMD. [ABSTRACT FROM AUTHOR]

Abstract: An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer. [Copyright &y& Elsevier]

Abstract: A novel electrochemical DNA biosensor based on graphene-three dimensional nanostructure gold nanocomposite modified glassy carbon electrode (G-3D Au/GCE) was fabricated for detection of survivin gene which was correlated with osteosarcoma. The G-3D Au film was prepared with one-step electrochemical coreduction with graphite oxide and HAuCl4 at cathodic potentials. The active surface area of G-3D Au/GCE was 2.629cm2, which was about 3.8 times compared to that of a Au-coated GCE under the same experimental conditions, and 8.8 times compared to a planar gold electrode with a similar geometric area. The resultant nanocomposites with high conductivity, electrocatalysis and biocompatibility were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). A “sandwich-type” detection strategy was employed in this electrochemical DNA biosensor and the response of this DNA biosensor was measured by CV and amperometric current–time curve detection. Under optimum conditions, there was a good linear relationship between the current signal and the logarithmic function of complementary DNA concentration in a range of 50–5000fM with a detection limit of 3.4fM. This new biosensor exhibited a fast amperometric response, high sensitivity and selectivity and has been used in a polymerase chain reaction assay of real-life sample with a satisfactory result. [Copyright &y& Elsevier]

Abstract: This communication reports on a novel biosensor to study the hybridization specificity based on eriochrome cyanine R (ECR) monolayer film modified glassy carbon electrode (GCE). Differential pulse voltammetry (DPV) was used to monitor the hybridization reaction on the ECR-modified electrode. The decrease of the peak current of methylene blue (MB), an electroactive indicator, was observed upon hybridization of the probe with the target DNA. Electrochemical investigations indicated that ECR modified biosensor displayed a wide linear range of 5.0–200pM with a detection limit of 0.982pM. This new biosensor exhibited good selectivity for one-base mismatch and complementary sequence after hybridization in detecting promyelocytic leukemia/retinoic acid receptor α (PML/RARα) fusion gene in acute promyelocytic leukemia (APL). [Copyright &y& Elsevier]

Abstract: In this study, an enzyme-amplified electrochemical biosensor was developed for detection of the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia (APL). This new sensor employs a hairpin locked nucleic acids (LNAs) probe dually labeled with biotin and carboxyfluorescein molecule (FAM). The probe is immobilized at a streptavidin-modified electrode surface via the biotin–streptavidin bridge, and FAM serves as an affinity tag for the peroxidase conjugate binding. Initially, the immobilized hairpin probe was in the “closed” state in the absence of the target, which shielded FAM from being approached by the bulky anti-FAM-HRP conjugate due to the steric effect. Target binding opens the hairpin structure of the probe, the probe undergoes a significant conformational change, forcing FAM away from the electrode. As a result, the FAM label becomes accessible by the anti-FAM-HRP, and the target hybridization event can be sensitively transduced via the enzymatically amplified electrochemical current signal. This new biosensor demonstrates its excellent specificity for single-base mismatch and able to detect as little as 83fM target DNA even in the presence of human serum. We also employed this sensor to directly detect PCR real sample with satisfactory results. [Copyright &y& Elsevier]