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Vascular endothelial growth factor (VEGF) is a crucial signaling protein for the tumor growth and metastasis, which is also acted as the biomarkers for various diseases. In this research, we fabricate an aptamer-antibody sensor for point-of-care test of VEGF. Firstly, target VEGF is captured by antibody immobilized on the microplate, and then binds with aptamer to form the sandwich structure. Next, with the assist of glucose oxidase (GOx)-functionalized ssDNAs, hybridization chain reaction occurs using the aptamer as the primer. Thus, GOx are greatly gathered on the microplate, which catalyzes the oxidization of glucose, leading to the pH change. As a result, the detect limit at a signal-to-noise was estimated to be 0.5 pg/mL of target by pH meter, and 1.6 pg/mL of VEGF was able to be distinguished by naked eyes. Meanwhile, this method has been used assay VEGF in the serum with the satisfactory results.

Poly(glutamic acid) (P-GLU)/carboxyl functionalized multiwalled carbon nanotubes (MWCNTs-COOH)/polyvinyl alcohol (PVA) modified glassy carbon electrode (GCE) has been successfully prepared and the electrochemical behavior of procaterol hydrochloride (ProH) was studied. The results show that the as-prepared modified electrode exhibits a good electrocatalytic property towards the oxidation of ProH in 0.2M phosphate buffer solution (PBS) (pH 6.0) due to the enhanced oxidation peak current at ~+0.59V. Under optimal reaction conditions, the oxidation peak current of ProH is proportional to its concentration in the linear dynamic ranges of 0.060 - 8.0μM (R = 0.9974), with a detection limit of 8.0 × 10-9M. Finally, this method was efficiently used for the determination of ProH in tablets and human urine with recoveries of 88.5~98.7% and 89.2 ~ 108.0%, respectively.

Gold nanoparticles (AuNPs) have been frequently utilized for the construction of diverse colorimetric biosensors. Normally, AuNPs with sharp edges could have better sensitivity. However, the poor monodipersity of AuNPs with sharp edges seriously confines their utility for colorimetric biosensing. Herein, we demonstrate the utility of highly uniform gold nanobipyramids (Au NBPs) for ultrasensitive colorimetric detection of H5N1 virus. The proposed method is based on the fact that alkaline phosphatase (ALP) could catalyze the decomposition of 4-aminophenyl phosphate (4-APP) to generate 4-aminophenol (4-AP), which would then reduce silver nitrate to metal silver and then deposited on Au NBPs. The metal silver shell coated on the Au NBPs changed the refractive index of gold and thus resulted in a blue shift of longitudinal localized surface plasmon resonance (LSPR) and accompanied a vivid color change. This method exhibited a higher sensitivity than that of other Au NPs such as gold nanorods due to the high-i...

Herein, an electrochemiluminescence (ECL) biosensor for ultrasensitive and specific detection of Kras mutant genes has been developed on the basis of the high discrimination capability of locked nucleic acid (LNA) and dual signal amplification techniques including DNA walkers and hyperbranched rolling circle amplification (HRCA).

A novel electrochemiluminescence (ECL) biosensor was developed for high sensitive and selective detection of miRNA-21 based on the efficient and specific toehold-mediated strand displacement (TMSD) amplification with Ru(phen)

Thermometers, one of the most commonly used instruments at home, are normally adapted to measure temperature directly with high accuracy but rarely adopted to act as readout in the biosensors. It is necessary to find some ways to establish a relationship between the concentration of the target and the temperature change. In this study, a common thermometer was used as readout to develop a convenient immunosensor. The designed immunosensor comprises three components, including target recognition area, water flow system, and exothermic reaction bottle. The capture antibody for the target [carcinoembryonic antigen (CEA) was selected as a model target] was preloaded on the bottom of the recognition area. In the presence of CEA, a sandwich-type structure was formed between the capture antibody, CEA, and biotinylated detection antibody. Then, the streptavidin-functionalized platinum nanoparticles were labeled on the detection antibody due to biotin-avidin interaction. The captured platinum nanoparticles can effectively catalyze the decomposition of H

A copper(II)-based two-dimensional metal-organic framework with nanosheet structure (CuBDC NS) that possesses peroxidase (POx) mimicking activity was prepared. In the presence of hydrogen peroxide, the system catalyses the oxidation of terephthalic acid to a blue-fluorescent product (excitation = 315 nm; emission = 425 nm). Pyrophosphate has a very strong affinity for Cu2+ ion and blocks the POx-mimicking activity of the CuBDC NS. If, however, inorganic pyrophosphatase is present, the POx mimicking activity is gradually restored because pyrophosphate is hydrolyzed. The findings were used to design a method for the determination of the activity of inorganic pyrophosphatase by fluorometry. Fluorescence increases linearly in the 1–50 mU·mL−1 inorganic pyrophosphatase activity range. The limit of detection is 0.6 mU·mL−1 (S/N = 3).

Background Liquid biopsy based on 5-hydroxymethylcytosine (5hmC) signatures of plasma cell-free DNA (cfDNA) originating from tumor cells provides a novel approach for early diagnosis in hepatocellular carcinoma (HCC). Here, we sought to develop a reliable model using cfDNA 5hmC signatures and protein biomarkers for diagnosis and prognosis of HCC. Patients and methods We carried out genome-wide 5hmC sequencing of cfDNA samples collected from 165 healthy volunteers, 62 liver cirrhosis (LC) patients and 135 HCC patients. A sensitive 5hmC diagnostic model was developed based on 5hmC signatures selected by sparse Partial Least Squares Discriminant Analysis and cross-validation to define the weighted diagnostic score (wd-score). Then, we combined protein biomarkers with the wd-score to build a more robust score (HCC score) by logistic regression. Results The distribution pattern of differential 5hmC regions could clearly distinguish HCC patients, LC patients and healthy volunteers. The wd-score based on 64 5hmC signatures in cfDNA achieves 93.24% of area under the curve (AUC) to distinguish HCC patients from non-HCC patients, and the HCC score by combing protein biomarkers achieves 92.75% of AUC to distinguish HCC patients from LC patients. Meanwhile, the HCC score showed high capacity for screening high recurrence risk patients after receiving surgical resection, and appeared to be an independent indicator for both relapse-free survival (P = 0.00865) and overall survival (P = 0.000739). Furthermore, the values of the HCC score in patients' longitudinal plasma samples were positively associated with tumor burden dynamics during follow-up. Conclusion We have developed and validated a novel non-invasive liquid biopsy strategy for HCC diagnosis, prognosis and surveillance during HCC progression., Highlights • A novel liquid biopsy approach was developed for HCC diagnosis, prognosis, and surveillance. • Novel HCC score defined here showed excellent capacity in distinguishing HCC patients from liver cirrhosis patients. • HCC score could predict the early recurrence risk for HCC patients after receiving surgical operation. • HCC score could sensitively monitor tumor burden and progression of HCC in longitudinal.

Mesoporous silica nanoparticles (MSN) based controlled release system had been coupled with diverse detection technologies to establish biosensors for different targets. Chemiluminescence (CL) system of luminol/H2O2 owns the characters of simplicity, low cost and high sensitivity, but the targets of which are mostly focused on some oxidants or which can participate in a chemical reaction that yields a product with a role in the CL reaction. In this study, chemiluminescent detection technique had been coupled with mesoporous silica-based controlled released system for the first time to develop a sensitive biosensor for the target which does not cause effect to the CL system itself. Cocaine had been chosen a model target, the MSN support was firstly loaded with glucose, then the positively charged MSN interacted with negatively charged oligonucleotides (the aptamer cocaine) to close the mesopores of MSN. At the present of target, cocaine binds with its aptamer with high affinity; the flexible linear aptamer structured will become stems structured through currently well-defined non-Waston-Crick interactions and causes the releasing of entrapped glucose into the solution. With the assistant of glucose oxidase (GOx), the released glucose can react with the dissolved oxgen to produce gluconic acid and H2O2, the latter can enhance the CL of luminol in the NaOH solution. The enhanced CL intensity has a relationship with the cocaine concentration in the range of 5.0-60μM with the detection limit of 1.43μM. The proposed method had been successfully applied to detect cocaine in serum samples with high selectivity. The same strategy can be applied to develop biosensors for different targets.

The electronic balance, one of the most common pieces of equipment in the laboratory, is normally used to directly measure the weight of a target with high accuracy. However, little attention has been paid to the extension of its applications. In this study, an electronic balance was used as a readout to develop a novel aptasensor for protein quantification for the first time. Thrombin was selected as a model target, and its two aptamers recognizing different sites of the protein were used (one aptamer was immobilized on the surface of magnetic microparticles and the other aptamer was functionalized with platinum nanoparticles). The two aptamers were specifically bound with the thrombin to form a sandwich structure; thus, the platinum nanoparticles were linked to the magnetic microparticles, and they were separated by a magnet easily. The captured platinum nanoparticles effectively catalyzed the decomposition of H2O2, generating a large volume of O2 to discharge a certain amount of water in a drainage dev...

A novel signal-amplified impedimetric immunosensing strategy was successfully developed for ultrasensitive detection of low-abundance proteins (carcinoembryonic antigen, CEA, used as a model) based on hybridization chain reaction (HCR)-stimulated formation of DNAzyme concatamers on nanogold particle accompanying enzyme-triggered biocatalytic precipitation. The assay was carried out on capture antibody-modified electrode by using gold nanoparticle heavily functionalized with initiator strand and detection antibody as the signal-transduction tag with a sandwich-type immunosensing format. In the presence of target CEA, the formed immunocomplex underwent an unbiased strand-displacement reaction by the initiator strand on the gold nanoparticle between two auxiliary single-stranded DNA with the hemin aptamer. Upon hemin introduction, numerous DNAzyme molecules were formed on the concatamers and nanogold particle, which could catalyze 4-chloro-1-naphthol to produce an insoluble precipitation on the electrode, thereby resulting in the amplification of impedimetric signal. Under the optimal conditions, the immuno-HCR assay exhibited good impedimetric responses for the detection of target CEA in the working range from 1.0 pg mL(-1) to 20 ng mL(-1) with a detection limit of 0.42 pg mL(-1). In addition, the immuno-HCR assay was validated by measuring six human serum specimens for target CEA, receiving a highly matched correction between the obtained results by the immuno-HCR assay and the commercialized ELC-based immunoassay method.

Prostate-specific antigen (PSA), as a glycoprotein enzyme encoded in humans by the KLK3 gene, is one of the most important biomarkers for the diagnosis and prognosis of prostate cancer. Herein, a new electrochemical immunosensor for sensitive determination of PSA was designed by using redox and catalysis 'all-in-one' infinite coordination polymer (PtNP@ICP) as signal tag on the polyamidoamine dendrimers modified electrode interface. To construct such 'all-in-one' PtNP@ICP nanostructures, the coordination polymerization was fully carried between metal ions and polydentate bridging ligands, and the PtNP was encapsulated into the ICP in the process of polymerization. The prepared PtNP@ICP nanocatalyst was characterized by transmission electron microscope (TEM), energy dispersive X-ray spectrometry (EDX), ultraviolet and visible (UV-vis) spectrophotometry and Fourier transform infrared spectroscope (FTIR). And the synthesized PtNP@ICP was utilized as signal tag for the label of PSA. With a sandwich-type immunoassay format, the conjugated signal tag on the transducer increased with the increasing PSA concentration in the sample thus enhancing the signal of the electrochemical immunosensor due to the catalytic reduction toward H2O2 of the enveloped PtNP. Under optimal conditions, the current was proportional to the logarithm of PSA concentration ranging from 0.001 to 60 ng/mL. The detection limit (LOD) was 0.3 pg/mL at 3 sB. The immunosensor displayed an acceptable reproducibility, stability and selectivity. In addition, the methodology was evaluated with human serum specimens receiving good correlation with results from commercialized enzyme-linked immunosorbent assay (ELISA) method.

An ultrasensitive electrochemiluminescence (ECL) aptamer sensor for protein (thrombin as an example) detection based on hyperbranched rolling circle amplification (HRCA) had been developed. A complementary single-strand DNA (CDNA) of the thrombin aptamer had been modified on the gold electrode firstly, and then hybridized with thrombin aptamer to make the aptamer immobilized on the electrode surface, in the presence of thrombin, aptamer–thrombin bioaffinity complexes formed and made thrombin aptamer leave the electrode surface. Thus, the linear padlock probe hybridized with the free CDNA on the electrode surface and circularized by Escherichia coli DNA ligase. Subsequently, the linear padlock probe was served as a template for the initiation of HRCA reaction, and a lot of dsDNA modified on the electrode surface. Then Ru(phen) 3 2+ (acted as the ECL indicator) intercalates specifically into double-stranded DNA (dsDNA) grooves to generate ECL signal. The ECL intensity of the system has a linear relationship with thrombin concentration in the range of 3.0–300 aM with a detection limit of 1.2 aM (S/N=3). The proposed method combines the high sensitivity of ECL, exponential amplification of HRCA for signal enhancement and high selectivity of aptamer.

Tungsten disulfide (WS 2 ) nanosheets were discovered to possess intrinsic peroxidase-like activity and catalyze the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a color reaction in the presence of H 2 O 2 . Based on this finding, a colorimetric method and a portable test kit for the visual detection of blood glucose have been developed by using glucose oxidase (GOx) and WS 2 nanosheets-catalyzed reactions. The linear range for glucose was ranged from 5 to 300 μM ( R 2 =0.999) with the detection limit of 2.9 μM. The portable test kit was successfully evaluated glucose levels in serum samples from normal persons and diabetes persons by the observable color change from pale yellow to yellow–green, blue–green.

A label-free and enzyme-free sensitive fluorescent detection of human immunodeficiency virus (HIV) deoxyribonucleic acid (DNA) based on isothermal hybridization chain reaction (HCR) was developed. A G-quadruplex sequence which was incorporated into one of the two hairpin probes was inactive in the absence of target DNA. However, at the presence of target DNA numerous G-quadruplexes along DNA nanowires were self-assembled through HCR. Using N-methyl mesoporphyrin IX (NMM) as the fluorophore, a "turn-on" fluorescent response would be achieved and detected as low as 0.5 nmol L(-1) of HIV DNA. This proposed method was applied to detect HIV DNA in biologic samples with satisfactory results.

Hypochlorite (ClO

A specific and label-free "on-off-on" luminescence biosensor based on a novel heterometallic cluster [Ag

A portable, affordable and simple detector is requested in a “Point-of-Care-Testing” (POCT) system. In this study, we exploited the potentialities of Differential Pressure Gauge (DPG) to the orientation of POCT technology. Alpha fetoprotein (AFP) was chosen as a model analyte that could specifically recognized by its antigen, and a tiny outfits equipped with a DPG was employed as the signal readout. Pt/SiO2 nanospheres were synthesized and modified with the detection antibody. In the presence of target, a sandwich of immunocomplex specifically formed and the Pt/SiO2 had been modified on the capture antibody. Which then can be dissolved to release plenty of Pt and the suspensions were transferred into a closed vial filled with appropriated amount of hydrogen peroxide. Subsequently, hydrogen peroxide was decomposed to produce oxygen, resulting in the enhancement of pressure in the closed vial and which can be detected by DPG easily. Under the optimized conditions, the read out signal from DPG had a direct relationship with AFP concentrations in the range of 10~200 ng/mL, and the detection limit was as low as 3.4 ng/mL. The proposed portable sensor had been successfully applied to detect AFP in serum samples with satisfactory results. This strategy holds a great promising in biological analysis as its convenient operations, reliable results and flexible apparatus.

g-C3N4 was found to possess intrinsic peroxidase-like activity, and could catalytically oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to produce a color reaction. Using g-C3N4 peroxidase-like catalytic activity and glucose oxidase (GOx), a colorimetric method for glucose detection in serum samples has been developed. The linear range for glucose was from 5 to 100 μM (R2=0.9987) and the limit of detection was as low as 1.0 μM. The proposed method was applied to detect glucose in serum samples by the naked eyes.

Owning to the characteristics such as high sensitivity and simplicity of apparatus, electrochemiluminescence (ECL) has become a powerful analytical technique and has been widely used. Ru(phen) 3 2+ can be intercalated into the grooves of dsDNA and act as an ECL probe efficiently, which has been applied to develop a sensitive ECL biosensor for folate receptor in this study. One ssDNA with a thiol group at its 3′ termini had been modified on the Au electrode first, and the other ssDNA with folic acid at its 3′ termini hybridized with the former one being modified on the electrode surface to form a dsDNA. In the absence of folate receptor, the 3′-terminus in the dsDNA region can be specificity hydrolyzed into mononucleotides by ExoIII and on dsDNA presents on the electrode surface, leading to the lower of ECL intensity detected. However, in the presence of the target (folate receptor), ExoIII failed to hydrolyze the dsDNA since the one 3′-terminus had been protected by the target and the other protected by the Au electrode, resulting in the enhancement of ECL intensity. The enhanced ECL intensity has a linear relationship with the logarithm of folate receptor concentration in the range of 0.66 nmol/L and 26.31 nmol/L with a detection limit of 0.1204 nmol/L. The proposed biosensor had been applied to detect HeLa cells concentration with satisfied results.

This work reports on a simple and feasible colorimetric immunoassay with signal amplification for sensitive determination of prostate-specific antigen (PSA, used as a model) at an ultralow concentration by using a new enzyme-chromogenic substrate system. We discovered that glucose oxidase (GOx), the enzyme broadly used in enzyme-linked immunosorbent assay (ELISA), has the ability to stimulate in situ formation of squaric acid (SQA)-iron(III) chelate. GOx-catalyzed oxidization of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III), which can rapidly (1 min) coordinate with the SQA. Formation of the iron-squarate complex causes the color of the solution to change from bluish purple to bluish red accompanying the increasing absorbance with the increment of iron(III) concentration. On the basis of the SQA-iron(III) system, a new immunoassay protocol with GOx-labeled anti-PSA detection antibody can be designed for the detection of target PSA on capture antibody-functionalized magnetic immunosensing probe, monitored by recording the color or absorbance (λ = 468 nm) of the generated SQA-iron(III) chelate. The absorbance intensity shows to be dependent on the concentration of target PSA. A linear dependence between the absorbance and target PSA concentration is obtained under optimal conditions in the range from 1.0 pg mL(-1) to 30 ng mL(-1) with a detection limit (LOD) of 0.5 pg mL(-1) (0.5 ppt) estimated at the 3Sblank level. The sensitivity displays to be 3-5 orders of magnitude better than those of most commercialized human PSA ELISA kits. In addition, the developed colorimetric immunoassay was validated by assaying 12 human serum samples, receiving in good accordance with those obtained by the commercialized PSA ELISA kit. Importantly, the SQA-based immunosensing system can be further extended for the detection of other low-abundance proteins or biomarkers by controlling the target antibody.

A novel portable biosensor for sensitive and selective detection of platelet-derived growth factor-BB (PDGF-BB) had been developed based on the specific recognition between aptamer and protein using a personal glucose meter (PGM) as readout. In the presence of PDGF-BB, the primary aptamer of PDGF-BB which is bound to the surface of streptavidin magnespheres paramagnetic particles (SA-PMPs) reacts quantitatively with invertase-functionalized secondary aptamer of PDGF-BB to form a stable complex, resulting in the attachment of invertase on the SA-PMPs. Subsequently, the invertase catalyzes the hydrolysis of sucrose to produce a large amount of glucose and quantitative readout by the PGM. The enhanced signal of the PGM has a relationship with the concentration of PDGF-BB in the range of 1.0 × 10(-14) M~3.16 × 10(-12) M, and the detection limit is 2.9 fM. The proposed portable biosensor had been successfully applied to assay the PDGF-BB in saliva samples.

A new digital multimeter (DMM)-based immunosensing system was designed for quantitative monitoring of biomarker (prostate-specific antigen, PSA used in this case) by coupling with an external capacitor and an enzymatic catalytic reaction. The system consisted of a salt bridge-linked reaction cell and a capacitor/DMM-joined electronic circuit. A sandwich-type immunoreaction with target PSA between the immobilized primary antibody and glucose oxidase (GO x )-labeled detection antibody was initially carried out in one of the two half-cells. Accompanying the sandwiched immunocomplex, the conjugated GO x could catalyze the oxidation of glucose, simultaneously resulting in the conversion of [Fe(CN) 6 ] 3− to [Fe(CN) 6 ] 4− . The difference in the concentrations of [Fe(CN) 6 ] 3− /[Fe(CN) 6 ] 4− in two half-cells automatically produced a voltage that was utilized to charge an external capacitor. With the closing circuit switch, the capacitor discharged through the DMM, which could provide a high instantaneous current. Under the optimal conditions, the resulting currents was indirectly proportional to the concentration of target PSA in the dynamic range of 0.05–7 ng mL −1 with a detection limit (LOD) of 6 pg mL −1 . The reproducibility, precision, and selectivity were acceptable. In addition, the methodology was validated by analyzing 12 clinical serum specimens, receiving a good accordance with the referenced values for the detection of PSA.

A new sandwich-type electrochemical immunosensor based on nanosilver-doped bovine serum albumin microspheres (Ag@BSA) with a high ratio of horseradish peroxidase (HRP) and detection antibody was developed for quantitative monitoring of biomarkers (carcinoembryonic antigen, CEA, used in this case) by coupling enzymatic biocatalytic precipitation with tyramine signal amplification strategy on capture antibody-modified glassy carbon electrode. Two immunosensing protocols (with and without tyramine signal amplification) were also investigated for the detection of CEA and improved analytical features were acquired with tyramine signal amplification strategy. With the labeling method, the performance and factors influencing the electrochemical immunoassay were studied and evaluated in detail. Under the optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005-80 ng mL(-1) toward CEA standards with a low detection limit of 5.0 pg mL(-1). Intra- and inter-assay coefficients of variation were below 11%. No significant differences at the 0.05 significance level were encountered in the analysis of 6 clinical serum specimens and 6 spiked new-born cattle serum samples between the electrochemical immunoassay and the commercialized electrochemiluminescent immunoassay method for the detection of CEA.

A versatile, ultrasensitive immunosensor for detection of influenza virus was designed by combining silver nanoparticles (Ag NPs) labeled antibodies with indirect fluorescence. A new technology using Ag-S covalent binding was applied for antibody labeling. Influenza A (H1N1) virus, as a subtype of influenza A virus that was the most common cause of human influenza (flu), was acted as the target antigen using sandwich type-immunoreactions on the high binding ELISA plates. The antibody-labeled Ag NPs were then released by acid solution to produce Ag(+) which can catalyze o-phenylenediamine (OPDA) oxidation to produce fluorescence for highly sensitive detection. Under the optimal conditions, it shows good linear relationship between fluorescence intensity and the logarithm of the concentration of H1N1 over the range of 1.0×10(-12)-1.0×10(-8) g mL(-1) with a detection limit (LOD, 3σ) of 1.0×10(-13) g mL(-1). Results indicated that the proposed method give a good sensitivity and simple operation for detecting the influenza virus. This work also provided a promising potential for antigen detection by Ag NPs labeled, and the steps were easy to handle.

Prostate-specific antigen (PSA), one of the indications of possible prostate malignancy, is used as a biomarker for the diagnosis and prognosis of prostate cancer. Herein, we develop a new homogeneous potentiometric immunoassay for sensitive detection of low-concentration PSA without the need of sample separation and washing step. Two nanostructures including positively charged polyethyleneimine-poly(styrene-co-acrylic acid) (PEI-PSAA) nanospheres and negatively charged gold nanoparticles conjugated with anti-PSA antibody (Ab-AuNP) were first synthesized by using mulsifier-free emulsion copolymerization and wet chemistry method, respectively. Thereafter, the as-prepared PEI-PSAA was used as a pseudo hapten for the construction of immunosensing probe based on an electrostatic interaction between PEI-PSAA and Ab-AuNP. Upon target introduction, the added PSA competed with PEI-PASS for Ab-AuNP based on a specific antigen–antibody interaction, and displaced Ab-AuNP from PEI-PASS. The dissociated PEI-PASS was captured through the negatively charged Nafion- modified electrode, thereby resulting in the change of membrane potential. The fabrication process was characterized by using high-resolution transmission electron microscope (HRTEM), scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX), surface plasmon resonance (SPR) and dynamic laser scattering (DLS) technique. Under optimal conditions, the output signal was indirectly proportional to the concentration of target PSA in the sample and exhibited a dynamic range from 0.1 to 50 ng/mL with a detection limit (LOD) of 0.04 ng/mL. Intra- and inter-assay coefficients of variation (CVs) were 6.8 and 7.5%, respectively. In addition, the methodology was evaluated for analysis of 12 clinical serum samples and showed good accordance between the results obtained by the developed immunosensing protocol and a commercialized enzyme-linked immunosorbent assay (ELISA) method.

Three new mononuclear copper(II) complexes, [Cu(L2)](2+) (1), [Cu(acac)(L)](+) (2), and [Cu(acac-Cl)(L)](+) (3) (L = 2-(4-pyridine)oxazo[4,5-f]1,10-phenanthroline (4-PDOP); acac = acetylacetone; acac-Cl = 3-chloroacetylacetone), have been synthesized and characterized by elemental analysis, high resolution mass spectrometry (Q-TOF), and IR spectroscopy. Two of the complexes were structurally characterized by single-crystal X-ray diffraction techniques. Their interactions with DNA were studied by UV-vis absorption and emission spectra, viscosity, thermal melting, DNA unwinding assay and CD spectroscopy. The nucleolytic cleavage activity of the compounds was carried out on double stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment in the presence and absence of an oxidant (H2O2). Active oxygen intermediates such as hydroxyl radicals and hydrogen peroxide generated in the presence of L and complexes 1-3 may act as active species for the DNA scission. The cytotoxicity of the complexes against HepG2 cancer cells was also studied.

Molybdenum disulfide (MoS2) has attracted increasing research interest recently due to its unique physical, optical and electrical properties, correlated with its 2D ultrathin atomic-layered structure. Until now, however, great efforts have focused on its applications such as lithium ion batteries, transistors, and hydrogen evolution reactions. Herein, for the first time, MoS2 nanosheets are discovered to possess an intrinsic peroxidase-like activity and can catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to produce a color reaction. The catalytic activity follows the typical Michaelis-Menten kinetics and is dependent on temperature, pH, H2O2 concentration, and reaction time. Based on this finding, a highly sensitive and selective colorimetric method for H2O2 and glucose detection is developed and applied to detect glucose in serum samples. Moreover, a simple, inexpensive, instrument-free and portable test kit for the visual detection of glucose in normal and diabetic serum samples is constructed by utilizing agarose hydrogel as a visual detection platform.

In this study, a hyperbranched rolling circle amplification (HRCA)-based colorimetric biosensor for carcinoembryonic antigen (CEA) is developed with high sensitivity and specificity. A CEA aptamer can bind with its target (CEA) to form a complex due to their high affinity, and the introduced CDNA cannot hybridize with the aptamer. Thus, free CDNA can propagate the HRCA reaction to form a large number of single-stranded DNA (ss-DNA). ss-DNA can be easily adsorbed onto AuNPs and prevent salt-induced AuNPs aggregation, which causes the change in the color of the system. It is found that the absorbance intensity ratio (A520/A660) has a linear relationship with the concentration of the target in the range of 5 pM-0.5 nM, and the detection limit is as low as 2 pM (S/N = 3).

Chiral recognition based on enantioselective sensors is superior to conventional chromatographic enantioseparation techniques in terms of simplicity and rapidity. Normally, highly specific enantioselective receptors are used for the fabrication of enantioselective sensors. However, to date there only limited number of highly specific chiral selectors are reported, which greatly confines the development of enantioselective sensors. Herein, we demonstrate the feasibility of using relatively weak chiral selectors to construct an enantioselective biosensor for accurate chiral discrimination of enantiomers. The detection of racemic mixture of ( R )- and ( S )-1,2,3,4-Tetrahydro-1-naphthylamine (TNA) was demonstrated as an example. The sensor was made up of a dual-channel microfluidic chip. One channel of the chip was modified with human serum albumin (HSA), which was reported to be a weak chiral selector for TNA; while the other channel was modified with a monoclonal anti-TNA antibody, which was a non-enantioselective TNA receptor. A portable localized surface plasmon resonance (LSPR) detection system was integrated with the microfluidic chip to accomplish the signal collection. Our investigation revealed that the combination of LSPR responses obtained from the two channels can be used for quantitative discrimination of the ( R )- and ( S )-TNA. The limit of detection was found to be 150 nM for ( R )-TNA and 100 nM for ( S )-TNA. The feasibility of use relatively weak chiral selectors could potentially promote the development of various enantioselective sensors.

Development of simple but sensitive biosensor for influenza detection is highly important in immediate and effective clinical treatment. In this study, a sensitive colorimetric immunosensor which combines the advantages of high selectivity of immunoassay and simplicity of colorimetric detection has been developed to detect influenza virus H5N1 based on enzyme-encapsulated liposome. Biotin-tagged liposome encapsulated with large amount of horseradish peroxidase (HRP) was firstly synthesized. In the presence of H5N1, H5N1 co-bound with the capture antibody and the biotinylated detection antibody to form sandwich immunocomplex. Subsequently, the HRP-encapsulated liposome was introduced to conjugate with the detection antibody through biotin-avidin-biotin linkage. Upon the addition of substrate (mixture of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2), the liposome was directly lysed to release large amount of HRP by TMB. The released HRP catalyzed the H2O2-mediated oxidation of TMB, resulting in color change of the system, which was observed by naked eyes or UV-vis spectra. The result showed that the absorption intensity enhanced with the increase of H5N1 concentration ranging from 0.1 to 4.0 ng/mL, and the detection limit was calculated to be 0.04 ng/mL. The sensitivity of the proposed biosensor is much higher than that of conventional enzyme-linked immunosorbent assay method. The proposed immunosensor is relatively simple, low-cost, sensitive, and selective without using any sophisticated instruments, therefore it may have a promising prospect for detecting targets in clinical medicine, food safety analysis, and environmental monitoring.

Direct visual detection of circulating microRNAs could be a big challenge due to the ultralow concentration of microRNAs in real samples. Herein, we combine the techniques of nuclease amplification and oriented assembly of gold nanoparticle dimers to realize visual detection of microRNAs in human serum with the naked eye.

Colorimetric enzyme-linked immunosorbent assay utilizing 3′-3-5′-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3′-3-5′-5-tetramethylbenzidine (TMB2+) can quantitatively etch gold nanoparticles. Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL.

3, 3', 5, 5'-tetramethylbenzidine (TMB) has been widely used as a chromogenic substrate for colorimetric immunoassays. Normally, the colorless TMB is oxidized into yellow TMB2+ (in acidic solution) to indicate the presence of the target molecules. However, this kind of monochromic intensity changes seriously confine the accuracy of visual inspection. Herein, we demonstrate for the first time that TMB2+ can quantitatively and efficiently etch gold nanorods (AuNRs). The addition of AuNRs into a solution containing different amount of TMB2+ generates vivid color responses as colorful as a rainbow, and the etching process can be finished within 90s. As a result, the exact concentration of TMB2+ can be easily distinguished with the naked eyes by the corresponding solution color. Based on this finding, we incorporate AuNRs into the well-developed, commercially available horseradish peroxidase (HRP)-TMB immunoassay system, so that it can be utilized for semiquantitative detection of a broad range of disease biomarkers with the naked eyes (termed 'NEQ-IA'). Carcinoembryonic antigen (CEA) and Prostate specific antigen (PSA) had been chosen as example targets to test the feasibility of the proposed biosensor. The results showed good accordance with the conventional methods. Because no sophisticated apparatus but human eyes are used as the readout, the proposed NEQ-IA could be a good supplementary to current state-of-the-art immunoassay methods for those applications that require the use of portable and affordable devices, for example, for the detection of disease biomarkers at home and in the field.

Telomerase is one of the most common markers of human malignant tumors, such as uterine, stomach, esophageal, breast, colorectal, laryngeal squamous cell, thyroid, bladder, and so on. It is necessary to develop some sensitive but convenient detection methods for telomerase activity determination. In this study, a label-free and ultrasensitive electrochemiluminescence (ECL) biosensor has been fabricated to detect the activity of telomerase extracted from HeLa cells. Thiolated telomerase substrate (TS) primer was immobilized on the gold electrode surface through gold-sulfur (Au-S) interaction and then elongated by telomerase specifically. Then, it was hybridized with complementary DNA to form double-stranded DNA (dsDNA) fragments on the electrode surface, and Ru(phen)3 2+ has been intercalated into the dsDNA grooves to act as the ECL probe. The enhanced ECL intensity has a linear relationship with the number of HeLa cells in the range of 5∼5000 and with a detection limit of 2 HeLa cells. The proposed ECL biosensor has high specificity to telomerase in the presence of common interferents. The relative standard deviations (RSDs) were

Here we designed a new electrochemical immunoassay protocol for determination of carcinoembryonic antigen (CEA) using nanoplatinum-enclosed gold nanocores (Pt@Au) as catalytically promoted nanolabels on the carbon nanospheres and graphene-modified immunosensor. The Pt@Au nanolabels were synthesized and functionalized with monoclonal anti-CEA antibodies and glucose oxidase (GOx). Using the functional Pt@Au nanolabels as molecular tags, the assay was implemented relative to glucose-hydroquinone system with a sandwich-type immunoassay. Initially, the added glucose was oxidized to gluconolactone and H(2)O(2) by the labeled GOx, and then the generated H(2)O(2) was reduced with the help of platinum nanoparticles, leading to the production of oxygen. The self-produced oxygen could promote the re-oxidation of the glucose, thus resulting in the dual amplification of the electrochemical signal. Several nanolabels, such as multiarmed star-like platinum nanowires, hollow platinum nanospheres and Pt@Au nanostructures, were investigated for CEA detection and improved analytical features were obtained with the Pt@Au nanostructures. Under optimal conditions, the Pt@Au-based immunoassay displayed a wide working range from 0.001 to 120 ng mL(-1) with a low detection limit of 0.5 pg mL(-1) CEA at 3s(B). Intra- and inter-assay coefficients of variation were10.9%. The system was evaluated with 10 clinical serum samples, receiving good accordance with results from enzyme-linked immunosorbent assay method.

Methods based on nanomaterial labels have been developed for electrochemical immunosensors and immunoassays, but most involved low sensitivity. Herein a novel class of molecular tags, nanogold–polyaniline–nanogold microspheres (GPGs), was first synthesized and functionalized with horseradish peroxidase-conjugated thyroid-stimulating hormone antibody (HRP-Ab 2 ) for sensitive electrochemical immunoassay of thyroid-stimulating hormone (TSH). X-ray diffraction, confocal Raman spectroscopy, scanning electron microscope and transmission electron microscope were employed to characterize the prepared GPGs. Based on a sandwich-type immunoassay format, the assay was performed in pH 5.0 acetate buffer containing 6.0 mmol L −1 H 2 O 2 by using GPG-labeled HRP-Ab 2 as molecular tags. Compared with pure polyaniline nanospheres and gold nanoparticles alone, the GPG hybrid nanostructures increased the surface area of the nanomaterials, and enhanced the immobilized amount of HRP-Ab 2 . Several labeling protocols comprising HRP-Ab 2 , nanogold particle-labeled HRP-Ab 2 , and polyaniline nanospheres-labeled HRP-Ab 2 , were also investigated for determination of TSH and improved analytical features were obtained by using the GPG-labeled HRP-Ab 2 . With the GPG labeling method, the effects of incubation time and pH of acetate buffer on the current responses of the immunosensors were also studied. The strong attachment of HRP-Ab 2 to the GPGs resulted in a good repeatability and intermediate precision down to 7%. The dynamic concentration range spanned from 0.01 to 20 μIU mL −1 with a detection limit (LOD) of 0.005 μIU mL −1 TSH at the 3 s B criterion. Significantly, no significant differences at the 0.05 significance level were encountered in the analysis of 15 spiking serum samples between the developed electrochemical immunoassay and the commercially available enzyme-linked immunosorbent assay (ELISA) method for determination of TSH.

In this work, we developed a simple and general method for highly sensitive detection of proteins and small molecules based on cyclic enzymatic signal amplification (CESA) and hairpin aptamer probe. Our detection system consists of a hairpin aptamer probe, a linker DNA, two sets of DNA-modified AuNPs, and nicking endonuclease (NEase). In the absence of a target, the hairpin aptamer probe and linker DNA can stably coexist in solution. Then, the linker DNA can assemble two sets of DNA-modified AuNPs, inducing the aggregation of AuNPs. However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an aptamer probe-target complex. Then, the probe-target complex can hybridize to the linker DNA. Upon formation of the duplex, the NEase recognizes specific nucleotide sequence and cleaves the linker DNA into two fragments. After nicking, the released probe-target complex can hybridize with another intact linker DNA and the cycle starts anew. The cleaved fragments of linker DNA are not able to assemble two sets of DNA-modified AuNPs, thus a red color of separated AuNPs can be observed. Taking advantage of the AuNPs-based sensing technique, we are able to assay the target simply by UV-vis spectroscopy and even by the naked eye. Herein, we can detect the human thrombin with a detection limit of 50 pM and adenosine triphosphate (ATP) with a detection limit of 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. In addition, this method is general since there is no requirement of the NEase recognition site in the aptamer sequence. Furthermore, we proved that the proposed method is capable of detecting the target in complicated biological samples.

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL(-1). Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.

Single stranded DNA often forms stable secondary structures under physiological conditions. These DNA secondary structures play important physiological roles. However, the analysis of such secondary structure folded DNA is often complicated because of its high thermodynamic stability and slow hybridization kinetics. In this article, we demonstrate that Y-shaped junction probes could be used for rapid and highly efficient detection of secondary structure folded DNA. Our approach contained a molecular beacon (MB) probe and an assistant probe. In the absence of target, the MB probe failed to hybridize with the assistant probe. Whereas, the MB probe and the assistant probe could cooperatively unwind the secondary structure folded DNA target to form a ternary Y-shaped junction structure. In this condition, the MB probe was also opened, resulting in separating the fluorophores from the quenching moiety and emitting the fluorescence signal. This approach allowed for the highly sensitive detection of secondary structure folded DNA target, such as a tau specific DNA fragment related to Alzheimer's disease in this case. Additionally, this approach showed strong SNPs identifying capability. Furthermore, it was noteworthy that this newly proposed approach was capable of detecting secondary structure folded DNA target in cell lysate samples.

Two nanostructures including carbon nanospheres-graphene hybrid nanosheets (CNS-GNS) and hollow platinum nanospheres (HPtNS) were first synthesized by using direct electrolytic reduction and wet chemistry methods, respectively. Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of carcinoembryonic antigen (CEA) by using HPtNS-labeled horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA) as molecular tags and anti-CEA-assembled CNS-GPS as sensing probes. Compared with pure graphene nanosheets, the presence of carbon nanospheres on the graphene increased the surface coverage of the substrate, and enhanced the immobilized amount of primary antibodies. Several labeling protocols, such as HRP-anti-CEA, solid platinum nanoparticle-labeled HRP-anti-CEA, and hollow platinum nanospheres-labeled HRP-anti-CEA, were investigated for determination of CEA and improved analytical features were obtained with hollow platinum nanosphere labeling. With the HPtNS labeling method, the effects of incubation time and pH on the current responses of the immunosensors were also studied. The strong attachment of biomolecules to the CNS-GPS and HPtNS resulted in a good repeatability and intermediate precision down to 10.2%. The dynamic concentration range spanned from 0.001 ng mL(-1) to 100 ng mL(-1) CEA with a detection limit of 1.0 pg mL(-1) at the 3S(blank) level. No significant differences at the 0.05 significance level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of CEA.

A novel class of molecular tags, cadmium ion-doped magnetic poly(styrene-acrylic acid) nanospheres (Cd-MPSA), was first synthesized and functionalized with polyclonal rabbit anti-human luteinizing hormone antibodies (PAb(2)) for highly efficient electrochemical immunoassay of luteinizing hormone (LH). Transmission electron microscope (TEM) and Fourier transform infrared spectroscope (FTIR) were employed to characterize the prepared Cd-MPSA. By using Cd-MPSA-labeled PAb(2) as molecular tags, a novel sandwich-type immunoassay protocol was built for determination of LH on monoclonal mouse anti-human luteinizing hormone antibody (MAb(1))-functionalized gold electrode. The assay was carried out in pH 5.3 HAc-NaAc buffer solution by square wave voltammetry (SWV). The signal was obtained by the reduction of the doped cadmium ions in the Cd-MPSA. Under optimal conditions, the currents increased with the increasing LH level in the sample, and exhibited a linear range from 0.25 to 240 mIU mL(-1) with a detection limit of 0.08 mIU mL(-1) LH at 3s(B). The precision, reproducibility, and specificity were acceptable. No obvious difference was encountered in the analysis of spiking LH samples into newborn calf serum with the referenced values.

A novel enzyme-free sandwich electrochemical immunoassay with an ultrahigh sensitivity was developed for detection of alpha-fetoprotein (AFP, as a model analyte) using carbon nanotube-enriched gold nanoparticles (CNT-AuNPs) as nanolabels/nanocatalysts on anti-AFP/glutaraldehyde/thionine-modified glassy carbon electrodes (GCEs). The assays were carried out in a pH 8.0 acetic acid-buffered solution containing 6 mM p-nitrophenol (NP) and 6mM NaBH(4) after the formation of the sandwich-type immunocomplex. Initially, the NP molecules were reduced to p-aminophenol (AP) by the catalysis of the immobilized gold-nanoparticle labels on the CNT-AuNPs with the aid of NaBH(4), then the generated AP molecules were electrochemically oxidized to p-quinone imine (QI) by an electron mediator of thionine, and then the oxidized QI molecules were reduced back to APs by NaBH(4). The redox cycling of AP and QI continuously increased the signaling, leading to a high sensitivity. Compared with individual gold-nanoparticle labels, the immunosensor using CNT-AuNPs as labels displayed a wider linear range of 8.0×10(-7)-2.0×10(2) ng/mL with a lower detection limit (LOD) of 0.8 fg/mL AFP at a signal-to-noise ratio of 3, which was lower 6 orders than that of commercially available ELISA. Intra-and inter-assay coefficients of variation were below 10%. In addition, the assay was evaluated with clinical serum samples, and no significant differences at the 5% confidence level were encountered in the analysis of real samples between the proposed immunoassay and commercially available Roche 2010 Electrochemiluminescent Automatic Analyzer for determination of AFP.

Domoic acid (DA), a neurotoxic amino acid produced by some strains of phytoplankton, is responsible for the human toxic syndrome amnesic shellfish poisoning. This exocitotoxin results in neuronal degeneration and necrosis in specific regions of the hippocampus. Because DA accumulates mostly in shellfish, causing outbreaks in different countries, screening for DA has been carried out with various assays. Although bioassays and immunoassays have been developed, several liquid chromatographic methods for the determination of DA in different matrices such as shellfish, algae, or seawater have been reported. Additionally, other alternative methods such as capillary electrophoresis and capillary electrochromatography have been described. This paper summaries the toxicology, the chemistry, and the developed determination methods of DA.

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE-ESI-MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol-water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L -Ala- L -Ala (pI = 5.57), L -Leu- D -Leu (pI = 5.52), Gly- D -Phe (pI = 5.52) and Gly-Gly- L -Leu (pI = 5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2-2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45-3.57 and 4.93-6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.

A new electrochemical immunoassay protocol for the determination of carcinoembryonic antigen (CEA) in human serum was developed by means of immobilizing horseradish peroxidase-labeled anti-CEA antibodies (HRP-anti-CEA) on the nanogold/nano-Fe(3)O(4) particles-functionalized polyion complex (PIC) membrane. With a one-step immunoassay format, the formed immunocomplex inhibited partly the bioactive center of the immobilized HRP, and decreased the HRP toward the reduction of H(2)O(2). Nanogold-actuated nano-Fe(3)O(4) mimetic peroxidase was used for the sensitivity improvement of the electrochemical immunosensor with Prussian blue (PB) as mediators. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of 0.1-220 ng mL(-1) with a relative low detection limit (LOD) of 10 pg mL(-1) CEA (at 3σ). Intra- and inter-assay show good precisions with a coefficient of variation (CV) lower than 7.8% and 7.3%, respectively. No significant difference at the 95% confidence level was encountered in the analysis of 17 human serum specimens between the electrochemical immunosensor and automatic electrochemiluminescent (ECL) analyzer for the determination of CEA.

A new electrochemical immunoassay protocol for sensitive detection of alpha-fetoprotein (AFP, as a model) is designed using carbon nanoparticles (CNPs)-functionalized biomimetic interface as immunosensing probe and irregular-shaped gold nanoparticles (ISNGs)-labeled horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-ISNG) as trace label. The low-toxic and high-conductive CNPs provided a high capacity nanoparticulate immobilization surface and a facile pathway for electron transfer. In comparison with conventional label methods, i.e. spherical gold nanoparticles-labeled HRP-anti-AFP and HRP-labeled anti-AFP, the electrochemical immunosensor using HRP-anti-AFP-ISNGs as trace labels exhibited high bioelectrocatalytic response toward enzyme substrate and a wide dynamic range from 0.02 to 4.0 ng/mL with a low detection limit of 10 pg/mL toward AFP (at 3sigma). The developed immunoassay method showed good selectivity and acceptable reproducibility. Clinical serum samples with various AFP concentrations were evaluated by using the electrochemical immunosensor and the referenced enzyme-linked immunosorbent assay (ELISA), respectively, and received in good accordance with results obtained from these two methods.

A simple and effective method of capillary electrophoresis-amperometric detection (CE–AD) coupled with transient isotachophoresis (tITP) was developed for the trace determination of doping substances. Compared with the conventional capillary electrophoresis method, the maximum enhancement factor in terms of peak heights was up to 5500-fold when the tITP technique was adopted. Under the optimum conditions, the detection limit (S/N = 3) for methylephedrine (MDP), celiprolol (CEL), sotalol (SOT) and indapamide (IDP) were 4.2 × 10−14, 6.3 × 10−13, 5.8 × 10−14 and 9.5 × 10−13 mol L−1, respectively. The RSDs of four analytes were 1.0–2.3% for migration time and 2.6–3.8% for peak current, respectively. The proposed method was successfully applied to determine the contents of SOT and IDP in real urine sample, and the excretion curve of IDP within 48 h was also investigated. The recoveries of the four doping in urine ranged from 90.0 to 102%.

A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method.

A new electrochemical biosensor for the monitoring of ultratrace terbium based on the conformational change of DNA containing a single guanine (G)-rich stretch was described here. The biosensor was fabricated by immobilizing a thiolated DNA containing a single G-rich stretch on the gold surface as probe surface. The G-rich DNA probe was found to be capable of changing its configuration from flexible single-stranded structures to rigid tetramolecular G-quadruplex in the presence of terbium III, which provided a switchable charge transport path for the oxidation of [Fe(CN)6]4−. The switchable surface provided a sensing platform for the single-step and reagentless detection of Tb3+. Using this reusable electrochemical sensing platform, a simple, rapid, and selective biosensor for the determination of ultratrace terbium ions with a detection limit of 6.0 × 10−11 M has been developed. The success in the present biosensor served as a significant step toward the development of monitoring ultratrace Tb3+ in river water or seawater.