Electrogenerated chemiluminescence DNA biosensor based on hairpin DNA probe labeled with ruthenium complex

A highly selective electrogenerated chemiluminescence (ECL) biosensor for the detection of target single-strand DNA (ss-DNA) was developed using hairpin DNA as the recognition element and ruthenium complex as the signal-producing compound. The ECL-based DNA biosensor was fabricated by self-assembling the ECL probe of thiolated hairpin DNA tagged with ruthenium complex on the surface of a gold electrode. In the absence of target ss-DNA, the ECL probe immobilized on the surface of the electrode was in the folded configuration in which its termini were held in close proximity to the electrode, and thus a strong ECL signal could be generated. In the presence of target ss-DNA, a stem-loop of the ECL probe on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the tag moving away from the electrode surface, which in turn decreased the ECL signal. The ECL intensity of the DNA biosensor generated a "switch off" mode, which decreased with an increase of the concentration of target DNA, and a detection limit of 9 x 10(-11) M complementary target ss-DNA was achieved. Single mismatched target ss-DNA was effectively discriminated from complementary target ss-DNA. The effect of different loop lengths of the hairpin DNA on the selectivity of the ECL DNA biosensor has been investigated. This work demonstrated that the sensitivity and specificity of an ECL DNA biosensor could be greatly improved using a hairpin DNA species which has an appropriate stem and loop length as the recognition element.     

CdS nanocrystal-based electrochemiluminescence biosensor for the detection of low-density lipoprotein by increasing sensitivity with gold nanoparticle amplification

Mercaptoacetic acid (RSH)-capped CdS nanocrystals (NCs) was demonstrated to be electrochemically reduced during potential scan and react with the coreactant S2O8(2-) to generate strong electrochemiluminescence (ECL) in aqueous solution. Based on the ECL of CdS NCs, a novel label-free ECL biosensor for the detection of low-density lipoprotein (LDL) has been developed by using self-assembly and gold nanoparticle amplification techniques. The biosensor was prepared as follows: The gold nanoparticles were first assembled onto a cysteamine monolayer on the gold electrode surface. This gold nanoparticle-covered electrode was next treated with cysteine and then reacted with CdS NCs to afford a CdS NC-electrode. Finally, apoB-100 (ligand of LDL receptor) was covalently conjugated to the CdS NC-electrode. The modification procedure was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy, respectively. The resulting modified electrode was tested as ECL biosensor for LDL detection. The LDL concentration was measured through the decrease in ECL intensity resulting from the specific binding of LDL to apoB-100. The ECL peak intensity of the biosensor decreased linearly with LDL concentration in the range of 0.025-16 ng mL-1 with a detection limit of 0.006 ng mL-1. The CdS NCs not only showed high ECL intensity and good biocompatibility but also could provide more binding sites for apoB-100 loading. In addition, the gold nanoparticle amplification for protein ECL analysis was applied to the improvement of the detection sensitivity. Thus, the biosensor exhibited high sensitivity, good reproducibility, rapid response, and long-term stability.     

Preparation and characterization of an electrogenerated chemiluminescence sensor by electrochemical grafting of 4-nitrophenyl diazonium salts onto a glass carbon electrode

An ECL sensor was fabricated by immobilization of a tris(2,2'-bipyridyl)ruthenium (II) complex (Ru(bpy)3(2+)) to an amine group-modified GC electrode (NH2-GC electrode). Here, the NH2-GC electrode was prepared by electrochemical reduction of a nitro group-modified GC electrode in 0.1 M KCl ethanol solution under H2 gas, which was followed by electrochemical grafting of 4-nitrophenyl diazonium salts in 0.1 M NBu4BF4 acetonitrile solution onto the GC electrode. The prepared ECL sensor was successfully confirmed via cyclic voltammetry, contact angle, scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and ECL spectrometry. The contact angle for the surface of the GC electrode, NO2-GC electrode, and NH2-GC electrod was 88.4 degrees, 67.4 degrees, and 52.4 degrees, respectively. The stability of the ECL sensor was investigated under continuous cyclic potential scanning for 55 cycles and the ECL intensity remained at 55%. The prepared ECL electrode can be expected to immobilize enzymes for preparation of the ECL biosensor to detect target molecules.     

Fabrication of amperometric xanthine biosensors based on direct chemistry of xanthine oxidase

The construction of amperometric xanthine biosensor by immobilization of xanthine oxidase (XOD) on the multi-wall carbon nanotubes (CNTs) modified glassy carbon (GC) electrode surface was investigated. The direct chemistry of XOD was accomplished and the formal potential was about − 0.465 V (vs SCE). The heterogeneous electron transfer rate constant was evaluated to be 2.0 ± 0.3 s^− 1. The xanthine biosensor based on XOD entrapped in silica sol–gel (SG) thin film on CNTs-modified GC electrode surface was also investigated. The XOD still maintains its activity to xanthine. The amperometric response to xanthine showed a linear relation in the range from 0.2 µM to 10 µM and a detection limit of 0.1 µM (S/N = 3). The enzyme electrode retained 95% of its initial activity after 90 days of storage. The sensor exhibited high sensitivity, rapid response and good long-term stability.     

Electrochemiluminescence immunosensor based on CdSe nanocomposites

A novel strategy for the enhancement of electrochemiluminescence (ECL) was developed by combining CdSe nanocrystals (NCs), carbon nanotube-chitosan (CNT-CHIT), and 3-aminopropyl-triethoxysilane (APS). A label-free ECL immunosensor for the sensitive detection of human IgG (HIgG) was fabricated. The colloidal solution containing CdSe NCs/CNT-CHIT composite was first covered on the Au electrode surface to form a robust film, which showed high ECL intensity and good biocompatibility. After APS as a cross-linker was covalently conjugated to the CdSe NCs/CNT-CHIT film, the ECL intensity was greatly enhanced. And, an intensity about 20-fold higher than that of the CdSe NCs/CNT-CHIT film was observed. After antibody was bound to the functionalized film via glutaric dialdehyde (GLD), the modified electrode could be used as an ECL immunosensor for the detection of HIgG. The specific immunoreaction between HIgG and antibody resulted in the decrease in ECL intensity. The ECL intensity decreased linearly with HIgG concentration in the range of 0.02-200 ng mL(-1), and the detection limit was 0.001 ng mL(-1). The immunosensor has the advantages of high sensitivity, speed, specificity, and stability and could become a promising technique for protein detection.     

Highly sensitive electrogenerated chemiluminescence biosensor in profiling protein kinase activity and inhibition using gold nanoparticle as signal transduction probes

A novel electrogenerated chemiluminescence (ECL) biosensor using gold nanoparticles as signal transduction probes was described for the detection of kinase activity. The gold nanoparticles were specifically conjugated to the thiophosphate group after the phosphorylation process in the presence of adenosine 59-[c-thio] triphosphate (ATP-s) cosubstrate. Due to its good conductivity, large surface area, and excellent electroactivity to luminol oxidization, the gold nanoparticles extremely amplified the ECL signal of luminol, offering a highly sensitive ECL biosensor for kinase activity detection. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar, and lipid metabolism in the human body, was used as a model to confirm the proof-of-concept strategy. The as-proposed biosensor presented high sensitivity, low detection limit of 0.07 U mL(-1), wide linear range (from 0.07 to 32 U mL(-1)), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC(50) of ellagic acid, a PKA inhibitor, was estimated, which was in agreement with those characterized with the conventional kinase assay. While nearly no ECL signal change can be observed in the presence of Tyrphostin AG1478, a tyrosine kinase inhibitor, but not PKA inhibitor, shows its excellent performance in kinase inhibitor screening. The simple and sensitive biosensor is promising in developing a high-through assay of in vitro kinase activity and inhibitor screening for clinic diagnostic and drug development.     

Electrogenerated chemiluminescence. 67. Dependence of light emission of the tris(2,2')bipyridylruthenium(II)/tripropylamine system on electrode surface hydrophobicity

We describe the effect of electrode surface hydrophobicity on the electrochemical behavior and electrogenerated chemiluminescence (ECL) of Ru(bpy)3(2+) (bpy = 2,2'-bipyridyl)/tripropylamine (TPrA) system. Gold and platinum electrodes were modified with different thiol monolayers. The hydrophobicity of the electrode surfaces changed with different terminal groups of the thiol molecules. The oxidation rate of TPrA was found to be much larger at the modified electrode with a more hydrophobic surface. The adsorption of neutral TPrA species on this kind of surface was assumed to contribute to the faster anodic kinetics. Due to the rapid generation of the highly reducing radical, TPrA., ECL intensity increased significantly at more hydrophobic electrodes. This electrode surface effect in the ECL analytical system allows one to improve the detection sensitivity at low concentrations of Ru(bpy)3(2+). The surfactant effect on the ECL process was also examined and discussed based on the change of electrode hydrophobicity by the adsorption of surfactant species.     

ZnS nanoparticles electrodeposited onto ITO electrode as a platform for fabrication of enzyme-based biosensors of glucose

The electrochemical and photoelectrochemical biosensors based on glucose oxidase (GOD) and ZnS nanoparticles modified indium tin oxide (ITO) electrode were investigated. The ZnS nanoparticles were electrodeposited directly on the surface of ITO electrode. The enzyme was immobilized on ZnS/ITO electrode surface by sol–gel method to fabricate glucose biosensor. GOD could electrocatalyze the reduction of dissolved oxygen, which resulted in a great increase of the reduction peak current. The reduction peak current decreased linearly with the addition of glucose, which could be used for glucose detection. Moreover, ZnS nanoparticles deposited on ITO electrode surface showed good photocurrent response under illumination. A photoelectrochemical biosensor for the detection of glucose was also developed by monitoring the decreases in the cathodic peak photocurrent. The results indicated that ZnS nanoparticles deposited on ITO substrate were a good candidate material for the immobilization of enzyme in glucose biosensor construction.     

An enzyme flow immunoassay that uses beta-galactosidase as the label and a cellobiose dehydrogenase biosensor as the label detector

The aim was to develop a fast generic enzyme flow immunoassay (EFIA) using a beta-galactosidase (beta-GAL) label in combination with colorimetric detection as well as with a new amperometric biosensor as the label detector. The amperometric biosensor was previously developed within the group for the determination of diphenols in surface water samples. Antigen (Ag, analyte), tracer (Ag*, antigen labeled with beta-GAL), and antibody (Ab) were incubated off-line. After the equilibrium was reached, the sample was introduced into the flow system. The antibody complexes, AgAb and Ag*Ab, were trapped in a protein G column while the free unbound tracer was eluted and detected by an amperometric biosensor downstream after substrate reaction. The enzyme label beta-GAL converted the substrate 4-aminophenyl-beta-D-galactopyranoside (4-APG) into 4-aminophenol (4-AP), which subsequently was detected by a cellobiose dehydrogenase (CDH) modified solid graphite electrode. 4-AP was first oxidized at the electrode surface at +300 mV vs Ag/AgCl, and the formed 4-imino quinone (4-IQ) was reduced back to 4-AP by the CDH in the presence of cellobiose. By combining the EFIA with the CDH biosensor, the overall signal of one tracer molecule is amplified at two occasions, i.e., one enzyme label converts the substrate into many 4-AP molecules, and second these are further amplified by the CDH biosensor. The optimum conditions for the EFIA in terms of the molar ratio between tracer and beta-GAL, temperature, flow rate, etc., was investigated with colorimetric detection, using 2-nitrophenyl-beta-D-galactopyranoside (2-NPG) as the beta-GAL substrate. The performance of both the colorimetric and CDH biosensor detection was investigated and both methods were applied for determination of the model compound atrazine in spiked surface water samples. Detection limits of 0.056 +/- 0.008 and 0.038 +/- 0.007 microg L(-1) and IC50 values of 2.04 +/- 0.294 and 0.42 +/- 0.08 microg L(-1) were obtained for colorimetric and CDH detection, respectively. Matrix effects were less pronounced with the CDH biosensor than with colorimetric detection.     

Multichannel electrochemiluminescence of luminol in neutral and alkaline aqueous solutions on a gold nanoparticle self-assembled electrode

The electrochemiluminescence (ECL) behavior of luminol on a gold nanoparticle self-assembled electrode in neutral and alkaline pH conditions was studied under conventional cyclic voltammetry (CV). The gold nanoparticle self-assembled electrode exhibited excellent electrocatalytic property and redox reactivity to the luminol ECL system. In neutral solution, four ECL peaks were observed at 0.69, 1.03, -0.45, and -1.22 V (vs SCE) on the curve of ECL intensity versus potential. Compared with a bulk gold electrode, two anodic and one cathodic ECL peaks were greatly enhanced, and one new cathodic ECL peak appeared. In alkaline solution, two anodic ECL peaks were obtained at 0.69 and 1.03 V, which were much stronger than those on a bulk gold electrode. These ECL peaks were found to depend on gold nanoparticles on the surface of the electrode, potential scan direction and range, the presence of O(2) or N(2), the pH and concentration of luminol solution, NaBr concentration, and scan rate. The emitter of all ECL peaks was identified as 3-aminophthalate by analyzing the ECL spectra. The spatial distribution of the luminol ECL peaks on the gold nanoparticle self-assembled electrode was studied by CCD. The surface state of the gold nanoparticle self-assembled electrode was characterized by scanning electron microscopy (SEM) and UV-visible reflection spectra. The mechanism for the formation of these ECL peaks has been proposed. The results indicate that the gold nanoparticle self-assembled electrode could lead to novel ECL properties, and strong luminol ECL in neutral and alkaline solutions could be obtained on such an electrode, which is of great analytical potential.     

Prototype amperometric biosensor for sialic acid determination

This paper describes the first report on the development, characterization, and applications of a prototype amperometric biosensor for free sialic acid (SA). The sensor was constructed by the coimmobilization of two enzymes, i.e., N-acetylneuraminic acid aldolase and pyruvate oxidase, on a polyester microporous membrane, which was then mounted on top of a platinum disk electrode. The SA biosensor operation was based on the sequential action of the two enzymes to ultimately produce hydrogen peroxide, which was then detected by anodic amperometry at the platinum electrode. The surface of the platinum electrode was coated with an electropolymeric layer to enhance the biosensor selectivity in the presence of interfering oxidizable species. Optimization of the enzyme layer composition resulted in a fast and steady current response in phosphate buffer pH 7.2 at 37 degrees C. The limit of detection was 10 microM, and the response was linear to 3.5 mM (r = 0.9987). The prepared SA biosensors retained approximately 85% of their initial sensitivity after 8 days and showed excellent response reproducibility (CV = 2.3%). Utilization of a third enzyme, sialidase, expanded the scope of the present SA biosensor to determine bound sialic acid as well. The merits of the described biosensor allowed its successful application in determining SA in biological and pharmaceutical samples. The obtained results indicated that the presented SA biosensor should be a useful bioanalytical tool in several biological and clinical applications such as screening of SA as a nonspecific tumor marker as well as monitoring of tumor therapy.     

Ultrasensitive immunoassay of protein biomarker based on electrochemiluminescent quenching of quantum dots by hemin bio-bar-coded nanoparticle tags

A hemin bio-bar-coded nanoparticle probe labeled antibody was designed by the assembly of antibody and alkylthiol-capped bar-code G-quadruplex DNA on gold nanoparticles and the interaction of hemin with the DNA to form a G-quadruplex/hemin bio-bar-code. An ultrasensitive immunoassay method was developed by combining the labeled antibody with an electrochemiluminescent (ECL) immunosensor for protein. The ECL immunosensor was constructed by a layer-by-layer modification of carbon nanotubes, CdS quantum dots (QDs), and capture antibody on a glassy carbon electrode. In air-saturated pH 8.0 PBS the immunosensor showed a carbon-nanotube-enhanced cathodic ECL emission of QDs. Upon the formation of immunocomplex, the ECL intensity decreased owing to the consumption of ECL coreactant in bio-bar-code electrocatalyzed reduction of dissolved oxygen. Using α-fetoprotein as model analyte, the quenched ECL could be used for immunoassay with a linear range of 0.01 pg mL(-1) to 1 ng mL(-1) and a detection limit of 1.0 fg mL(-1). The wide detection range and high sensitivity resulted from the enhanced ECL emission and highly efficient catalysis of the bio-bar-code. The immunosensor exhibited good stability and acceptable fabrication reproducibility and accuracy, showing great promise for clinical application.     

Electrogenerated chemiluminescence. 66. The role of direct coreactant oxidation in the ruthenium tris(2,2')bipyridyl/tripropylamine system and the effect of halide ions on the emission intensity

We describe the electrogenerated chemiluminescence (ECL) processes of the Ru(bpy)3(2+) (bpy = 2,2'-bipyridyl)/ tripropylamine (TPrA) system at glassy carbon, platinum, and gold electrodes. The electrochemical behavior of TPrA on different electrode materials and its influence on the ECL process are demonstrated. At glassy carbon electrodes, the direct oxidation of TPrA began at approximately 0.6 V vs SCE and exhibited a broad irreversible anodic peak. Two ECL waves were observed, one in the potential region more negative than 1.0 V vs SCE and one at more positive potentials. The first ECL process apparently occurs without the electrogeneration of Ru(bpy)3(3+), in contrast to that of the second ECL wave. At Pt and Au electrodes, however, the formation of surface oxides significantly blocked the direct oxidation of TPrA. An ECL wave below 1.0 V did not appear at Pt and was very weak at gold. The ECL peaks at potentials of 1.1-1.2 V were also much weaker than those observed at the glassy carbon electrode. These results showed that the direct oxidation of TPrA played an important role in the ECL processes. Therefore, the enhancement of the TPrA oxidation current might lead to an increase in the ECL intensity. Small amounts of halide species were found to inhibit the growth of surface oxides on Pt and gold electrodes and led to an obvious increase of TPrA oxidation current. The anodic dissolution of gold in halide-containing solution was also important in activating the gold electrode surface. The electrochemical catalytic effect of bromide further promoted the oxidation of TPrA. A halide effect on ECL at Pt and Au electrodes was also evident. The most effective enhancement of ECL was observed at Au electrode in a bromide-containing solution. This effect was also found in an commercial flow-through instrument (IGEN) and provided a simple way to improve the detection sensitivity at low concentrations of Ru(bpy)3(2+).     

Biosensor for asparagine using a thermostable recombinant asparaginase from Archaeoglobus fulgidus

Asparaginase from the hyperthermophilic microorganism Archaeoglobus fulgidus was cloned and expressed in Escherichia coli as a fusion protein with a polyhistidine tail. After heat treatment to denature most of the native E. coli proteins, the enzyme was purified by an immobilized metal ion affinity chromatography method. The activity of the enzyme was determined by monitoring the change in ammonium concentration in solution. It was found that the enzyme is thermostable at temperatures as high as 85 degrees C. The KM for L-asparagine was 8 x 10(-5) and 5 x 10(-6) M at 37 and 70 degrees C, respectively. The catalytic activity for L-asparagine was 5-fold higher than for D-asparagine. The enzyme was immobilized in front of an ammonium-selective electrode and used to develop a biosensor for asparagine. The biosensor had a detection limit of 6 x 10(-5) M for L-asparagine. Unlike a sensor based on asparaginase from E. coli, the biosensor based on recombinant asparaginase from A. fulgidus demonstrated higher stability.     

Electrocatalytic detection of NADH and glycerol by NAD(+)-modified carbon electrodes

The electrochemical oxidation of the adenine moiety in NAD+ and other adenine nucleotides at carbon paste electrodes gives rise to redox-active products which strongly adsorb on the electrode surface. Carbon paste electrodes modified with the oxidation products of NAD+ show excellent electrocatalytic activity toward NADH oxidation, reducing its overpotential by about 400 mV. The rate constant for the catalytic oxidation of NADH, determined by rotating disk electrode measurements and extrapolation to zero concentration of NADH, was found to be 2.5 x 10(5) M-1 s-1. The catalytic oxidation current allows the amperometric detection of NADH at an applied potential of +50 mV (Ag/AgCl) with a detection limit of 4.0 x 10(-7) M and linear response up to 1.0 x 10(-5) M NADH. These modified electrodes can be used as amperometric transducers in the design of biosensors based on coupled dehydrogenase enzymes and, in fact, we have designed an amperometric biosensor for glycerol based on the glycerol dehydrogenase (GlDH) system. The enzyme GlDH and its cofactor NAD+ were co-immobilized in a carbon paste electrode using an electropolymerized layer of nonconducting poly(o-phenylenediamine) (PPD). After partial oxidation of the immobilized NAD+, the modified electrode allows the amperometric detection of the NADH enzymatically obtained at applied potential above 0 V (Ag/AgCl). The resulting biosensor shows a fast and linear response to glycerol within the concentration range of 1.0 x 10(-6)-1.0 x 10(-4) M with a detection limit of 4.3 x 10(-7) M. The amperometric response remains stable for at least 3 days. The biosensor was applied to the determination of glycerol in a plant-extract syrup, with results in good agreement with those for the standard spectrophotometric method.     

Positive potential operation of a cathodic electrogenerated chemiluminescence immunosensor based on luminol and graphene for cancer biomarker detection

In this work, we report a cathodic electrogenerated chemiluminescence (ECL) of luminol at a positive potential (ca. 0.05 V vs Ag/AgCl) with a strong light emission on the graphene-modified glass carbon electrode. The resulted graphene-modified electrode offers an excellent platform for high-performance biosensing applications. On the basis of the cathodic ECL signal of luminol on the graphene-modified electrode, an ECL sandwich immunosensor for sensitive detection of cancer biomarkers at low potential was developed with a multiple signal amplification strategy from functionalized graphene and gold nanorods multilabeled with glucose oxidase (GOx) and secondary antibody (Ab(2)). The functionalized graphene improved the electron transfer on the electrode interface and was employed to attach the primary antibody (Ab(1)) due to it large surface area. The gold nanorods were not only used as carriers of secondary antibody (Ab(2)) and GOx but also catalyzed the ECL reaction of luminol, which further amplified the ECL signal of luminol in the presence of glucose and oxygen. The as-proposed low-potential ECL immunosensor exhibited high sensitivity and specificity on the detection of prostate protein antigen (PSA), a biomarker of prostate cancer that was used as a model. A linear relationship between ECL signals and the concentrations of PSA was obtained in the range from 10 pg mL(-1) to 8 ng mL(-1). The detection limit of PSA was 8 pg mL(-1) (signal-to-noise ratio of 3). Moreover, the as-proposed low-potential ECL immunosensor exhibited excellent stability and reproducibility. The graphene-based ECL immunosensor accurately detected PSA concentration in 10 human serum samples from patients demonstrated by excellent correlations with standard chemiluminescence immunoassay. The results suggest that the as-proposed graphene ECL immunosensor will be promising in the point-of-care diagnostics application of clinical screening of cancer biomarkers.     

Amperometric quantification of polar organic solvents based on a tyrosinase biosensor

A novel amperometric biosensor for quantification of the electrochemically inert polar organic solvents based on tyrosinase electrode was preliminarily reported. The biosensor was fabricated by simply syringing an aqueous solution of tyrosinase/PVAVP (PVAVP: copolymer of poly(vinyl alcohol) grafting with 4-vinylpyridine) onto glassy carbon electrode surface followed by drying the modified electrode at +4 degrees C in a refrigerator. The current generated from electrochemical reduction of quinone is a probe signal. The biosensor can be used for quantification of polar organic solvents, and its mechanism was characterized with in situ steady-state amperometry-quartz crystal microbalance experiments. The detection limit, sensitivity, and dynamic range for certain organic solvents are dependent on the kind and concentration of the substrate probe and the hydrophobicity of the immobilization matrix. The response time for all the tested organic solvents is less than 2 min.     

Versatile electrochemiluminescence assays for cancer cells based on dendrimer/CdSe-ZnS-quantum dot nanoclusters

In this work, a novel dendrimer/CdSe-ZnS-quantum dot nanocluster (NC) was fabricated and used as an electrochemiluminescence (ECL) probe for versatile assays of cancer cells for the first time. A large number of CdSe-ZnS-quantum dots (QDs) were labeled on the NCs due to the many functional amine groups within the NCs, which could significantly amplify the QD's ECL signal. Capture DNA was specially designed as a high-affinity aptamer to the target cell; a novel ECL biosensor for cancer cells was directly accomplished by using the biobarcode technique to avoid cross-reaction. Moreover, magnetic beads (MBs) for aptamers immobilization were combined with the dendrimer/QD NCs probe for signal-on ECL assay of cancer cells, which greatly simplified the separation procedures and favored for the sensitivity improvement. In particular, a novel cycle-amplifying technique using a DNA device on MBs was further employed in the ECL assay of cancer cells, which greatly improved the sensitivity. To the best of our knowledge, this is the first study that the novel dendrimer/QD NCs probe combined with a DNA device cycle-amplifying technique was employed in the ECL assays of cells. Excellent discrimination against target and control cells is demonstrated, indicating that the ECL assays have great potential to provide a sensitive, selective, cost-effective, and convenient approach for early and accurate detection of cancer cells.     

Electrodeposition of chitosan-glucose oxidase biocomposite onto Pt-Pb nanoparticles modified stainless steel needle electrode for amperometric glucose biosensor

A glucose biosensor was fabricated by electrodepositing chitosan (CS)-glucose oxidase(GOD) biocomposite onto the stainless steel needle electrode (SSN electrode) modified by Pt–Pb nanoparticles (Pt–Pb/SSN electrode). Firstly, Pt–Pb nanoparticles were deposited onto the SSN electrode and then CS-GOD biocomposite was co-electrodeposited onto the Pt–Pb/SSN electrode in a mixed solution containing p-benzoquinone (p-BQ), CS and GOD. The electrochemical results showed that the Pt–Pb nanoparticles can accelerate the electron transfer and improve the effective surface area of the SSN electrode. As a result, the detection range of the proposed biosensor was from 0.03 to 9 mM with a current sensitivity of 0.4485 μA/mM and a response time of 15 s. The Michaelis constant value was calculated to be 4.9837 mM. The cell test results indicated that the electrodes have a low cytotoxicity. This work provided a suitable technology for the fabrication of the needle-type glucose biosensor.     

Highly selective detection of single-nucleotide polymorphisms using a quartz crystal microbalance biosensor based on the toehold-mediated strand displacement reaction

Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.