Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for sensitive detection of squamous cell carcinoma antigen on a portable pH meter by coupling enzyme-labeled hybridization chain reaction with two alternating hairpin DNA probes for the signal amplification.     

An electrochemiluminescent sensor for glucose employing a modified carbon nanotube paste electrode

A carbon nanotube paste (CNTP) electrode and a carbon nanotube paste/glucose oxidase (CNTP/GOx) electrode were prepared, and the electrochemiluminescent (ECL) behavior of luminol in the presence of glucose was investigated in detail at each of these electrodes. Compared to the classical carbon paste (CP) electrode, the CNTP electrode incorporating glucose oxidase greatly enhanced the response of the ECL sensor to glucose due to the electrocatalytic activity of the carbon nanotubes, the specificity of the enzymatic reaction, and the sensitivity of the luminol ECL reaction. Under optimal conditions, the electrode was found to respond linearly to glucose in the concentration range 1.0x10(-6) approximately 2.0x10(-3) mol/L, and the detection limit (defined as the concentration that can be detected at a signal-to-noise ratio of 3) was found to be a glucose concentration of 5.0x10(-7) mol/L. The method used to prepare the CNTP/GOx electrode was very convenient, and the electrode surface could be renewed in the case of fouling by simply polishing or cutting it to expose a new and fully active surface. The relative standard deviations (RSD) were found to be 6.8% and 8.9% for the CNTP electrode and the CNTP/GOx electrode (n=6). The electrode retained 95% of its initial response after two weeks.     

Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for the sensitive detection of squamous cell carcinoma antigen (SCCA; cervical carcinoma marker) on a portable pH meter coupling enzyme-labeled hybridization chain reaction (HCR) with two alternating hairpin DNA probes for the signal amplification. Initially, a sandwich-type immunoreaction was carried out between anti-SCCA capture antibody-conjugated magnetic bead and detection antibody/initiator strand-coated gold nanoparticle (AuNP). Then, the HCR reaction was readily executed between two glucose oxidase (GOx)-labeled hairpins through the initiator strand to form numerous GOx concatamers on the AuNP via the long nicked double-helix. The concatenated GOx oxidized glucose into gluconic acid and hydrogen peroxide, thus resulting in the pH change of the detection solution on a handheld pH meter. Several labeling protocols including GOx-antibody, GOx-AuNP-antibody and GOx-HCR-AuNP-antibody were investigated for detection of target SCCA, and improved analytical features were obtained with the immune-HCR assay. Under optimum conditions, the immune-HCR assay exhibited good pH responses for the determination of SCCA at a concentration as low as 5.7 pg/mL. Additionally, the immune-HCR assay had good precision and reproducibility, high specificity, and acceptable accuracy for analyzing human serum specimens.     

酶法合成的葡萄糖氧化酶-纳米金复合物的直接电化学与生物传感

将NaAuCl₄、葡萄糖氧化酶（GOx）和葡萄糖混合,借一步酶促反应制得吸附GOx的金纳米颗粒（AuNPs）,再通过滴干修饰法研制了Nafion/GOx-AuNPs修饰的玻碳（GC）电极,并考察了该酶电极上GOx的直接电化学和生物传感性能. 这种酶法合成的GOx-AuNPs复合物有良好的酶直接电化学活性,也保持了GOx的生物活性,似可归因于酶法合成的纳米金更接近酶氧化还原活性中心的缘故. 该酶电极在-0.4 V（vs. SCE）电位下,其稳态电流下降与葡萄糖浓度（0.5 4 mmol·L^-1）成正比,检测下限0.2 mol·L^-1.     

Direct electrochemistry of glucose oxidase immobilized on NdPO4 nanoparticles/chitosan composite film on glassy carbon electrodes and its biosensing application

The direct electrochemistry of glucose oxidase (GOx) immobilized on a composite matrix based on chitosan (CHIT) and NdPO(4) nanoparticles (NPs) underlying on glassy carbon electrode (GCE) was achieved. The cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the modified electrode. In deaerated buffer solutions, the cyclic voltammetry of the composite films of GOx/NdPO(4) NPs/CHIT showed a pair of well-behaved redox peaks that are assigned to the redox reaction of GOx, confirming the effective immobilization of GOx on the composite film. The electron transfer rate constant was estimated to be 5.0 s(-1). The linear dynamic range for the detection of glucose was 0.15-10 mM with a correlation coefficient of 0.999 and the detection limit was estimated at about 0.08 mM (S/N=3). The calculated apparent Michaelis-Menten constant was 2.5 mM, which suggested a high affinity of the enzyme-substrate. The immobilized GOx in the NdPO(4) NPs/CHIT composite film retained its bioactivity. Furthermore, the method presented here can be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.     

GOx@ZIF‐8(NiPd) Nanoflower: An Artificial Enzyme System for Tandem Catalysis

We report a facile approach to prepare an artificial enzyme system for tandem catalysis. NiPd hollow nanoparticles and glucose oxidase (GOx) were simultaneously immobilized on the zeolitic imidazolate framework 8 (ZIF‐8) via a co‐precipitation method. The as‐prepared GOx@ZIF‐8(NiPd) nanoflower not only exhibited the peroxidase‐like activity of NiPd hollow nanoparticles but also maintained the enzymatic activity of GOx. A colorimetric sensor for rapid detection of glucose was realized through the GOx@ZIF‐8(NiPd) based multi‐enzyme system. Moreover, the GOx@ZIF‐8(NiPd) modified electrode showed good bioactivity of GOx and high electrocatalytic activity for the oxygen reduction reaction (ORR), which could also be used for electrochemical detection of glucose.     

固载于壳聚糖-纳米金复合膜修饰玻碳电极上的葡萄糖氧化酶的直接电化学研究及其生物传感应用

通过将葡萄糖氧化酶固载于壳聚糖-纳米金复合膜内所构置的传感器,实现了葡萄糖氧化酶的直接电化学,并采用循环伏安法与电化学阻抗法对修饰电极进行了表征。研究表明：在除氧缓冲溶液中,葡萄糖氧化酶-壳聚糖-纳米金复合膜修饰电极表现出一对良好的氧化还原峰,这对峰归因于葡萄糖氧化酶的氧化还原,证明葡萄糖氧化酶被成功固载于复合膜内。电子传递速率常数为15.6 s-1,说明葡萄糖氧化酶的电活性中心与电极之间的电子传递很快。将壳聚糖与纳米金相结合还提高了葡萄糖氧化酶在复合膜内的稳定性并保持其生物活性,并可以用于葡萄糖检测。计算得到其表观米氏常数为10.1 mmol·L-1。而且,该生物传感器可以用于血样中葡萄糖含量的测定。     

An electrochemically preanodized screen-printed carbon electrode for achieving direct electron transfer to glucose oxidase

Here we report the unique property of a preanodized screen-printed carbon electrode (SPCE1) that can allow direct electron transfer (DET) reaction of glucose oxidase (GOx). The GOx can be immobilized in the composite of oxygen functionalities and edge plane sites generated during preanodization without additional cross-linking agents. The electron transfer rate of GOx is greatly enhanced to 4.38 s⁻¹ as a result of the conformational change of GOx in the microenvironment enabling the accessibility of active site for GOx to the electrode. The analytical versatility is further improved with the aid of Nafion film. As a consequence, the as-prepared electrode can be used as a glucose biosensor and the number of potential foreign species is then restricted by molecular size, permeation and/or (bio)chemical reaction. Most importantly, the disposable nature of the proposed electrode is expected to promote the DET-related researches.     

Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions

A Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4'-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15% of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12% of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.     

Glucose and lactate biosensors prepared by a layer-by-layer deposition of concanavalin A and mannose-labeled enzymes: electrochemical response in the presence of electron mediators.

An electrochemical response of glucose and lactate biosensors which were prepared by coating a platinum electrode with a thin film composed of concanavalin A and mannose-labeled glucose oxidase (GOx) or lactate oxidase (LOx) was evaluated in the presence of ferrocene derivatives as electron mediator. Both glucose and lactate biosensors showed catalytic current to glucose and lactate, respectively, in cyclic voltammetry, suggesting that the ferrocene derivatives can mediate electron transport smoothly from the reduced forms of GOx and LOx in the thin films to the electrode. Among the three kinds of ferrocene derivatives used, ferrocenylmethanol was found to be the most suitable electron mediator because of its low oxidation potential. The glucose and lactate sensors gave useful calibration graphs, in which higher detection limits were reached as compared with those observed when the sensors were operated in the absence of electron mediator.     

A Glucose Biosensor Based on Immobilization of Glucose Oxidase into 3D Macroporous TiO2

3D macroporous TiO₂ inverse opals have been derived from a sol‐gel procedure using polystyrene colloidal crystals as templates. EDS and SEM showed a face‐centered cubic (FCC) structure TiO₂ inverse opal was obtained. Glucose oxidase (GOx) was successfully immobilized on the surface of indium‐tin oxide (ITO) electrode modified by TiO₂ inverse opal (TiO_2(IO)). Electrochemical properties of GOx/TiO_2(IO)/ITO electrode were characterized by using the three electrodes system. The result of cyclic voltammetry showed that a couple of stable and well‐defined redox peaks for the direct electron transfer of GOx in absence of glucose, and the redox peak height enhanced in presence of 0.1 μM glucose. Compare with the ordinary structured GOx/TiO₂/ITO electrode, inverse opal structured GOx/TiO_2(IO)/ITO electrode has a better respond to the glucose concentration change. Under optimized experimental conditions of solution pH 6.8 and detection potential at 0.30 V versus saturated calomel electrode (SCE), amperometric measurements were performed. The sensitivity and the detection limit of glucose detection was 151 μA cm^?2 mM^?1 and 0.02 μM at a signal‐to‐noise ratio of 3, respectively. The good response was due to the good biocompatibility of TiO₂ and the large effective surface of the three‐dimensionally ordered macroporous structure.     

Amperometric Biosensor Coupled to Capillary Electrophoresis for Glucose Determination

A novel glucose biosensor has been fabricated and employed as the amperometric detector of a capillary electrophoresis (CE) system. (±)-1-Ferrocenylethylamine and chitosan were successively modified on a 500-µm diameter disc platinum electrode by dip-coating. The modified electrode was subsequently immersed in glucose oxidase (GOx) solution to entrap the enzyme in the chitosan membrane. The primary amino groups of 1-ferrocenylethylamine, GOx, and chitosan were cross-linked by glutaraldehyde to obtain a biosensing membrane so as to reduce leaching of 1-ferrocenylethylamine and GOx. The electrochemical behavior of the target biosensor was investigated. It was demonstrated that the investigated biosensor features fast response, high stability, long lifetime, and ideal compatibility with the CE system. When CE was employed to introduce a glucose plug into the surface of the biosensor, the current response was linear to the glucose concentration in the range of 0.0025 to 2.5 mM with a detection limit of 1.2 µM (S/N = 3) at a working potential of +0.6 V (vs. SCE). The CE-biosensor system was applied to the determination of the glucose level in human serum. The results were satisfactory and in good agreement with the hospital assay results.     

Highly sensitive electrogenerated chemiluminescence biosensor in profiling protein kinase activity and inhibition using a multifunctional nanoprobe

We presented a novel electrogenerated chemiluminescence (ECL) biosensor for monitoring the activity and inhibition of protein kinases based on signal amplification using enzyme-functionalized Au NPs nanoprobe. In this design, the biotin-DNA labeled glucose oxidase/Au NPs (GOx/Au NPs/DNA-biotin) nanoprobes, prepared by conjugating Au NPs with biotin-DNA and GOx, were bound to the biotinylated anti-phosphoserine labeled phosphorylated peptide modified electrode surface through a biotin−avidin interaction. The GOx assembled on the nanoprobe can catalyze glucose to generate H₂O₂ in the presence of O₂ while the ECL reaction occurred in the luminol ECL biosensor. At a higher concentration of kinase, there are more nanoprobes on the electrode, which gives a higher amount of GOx at the electrode interface and thus higher electrocatalytic efficiency to the luminol ECL reaction. Therefore, the activity of protein kinases can be monitored by ECL with high sensitivity. 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 present proof-of-concept strategy. The as-proposed biosensor presents high sensitivity, low detection limit of 0.013 U mL⁻¹, wide linear range (from 0.02 to 40 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 IC₅₀ of ellagic acid, a typical PKA inhibitor, was estimated, which is in agreement with those obtained using the conventional kinase assay. 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.     

Visible light induced photoelectrochemical biosensing based on oxygen-sensitive quantum dots

A visible light induced photoelectrochemical biosensing platform based on oxygen-sensitive near-infrared quantum dots (NIR QDs) was developed for detection of glucose. The NIR QDs were synthesized in an aqueous solution, and characterized with scanning electron microscopy and X-ray photoelectron spectroscopy. The as-prepared NIR QDs were employed to construct oxygen-sensitive photoelectrochemical biosensor on a fluorine-doped tin oxide (FTO) electrode. The oxygen dependency of the photocurrent was investigated at as-prepared electrode, which demonstrated the signal of photocurrent is suppressed with the decreasing of oxygen. Coupling with the consumption of oxygen during enzymatic reaction, a photoelectrochemical strategy was proposed for the detection of substrate. Using glucose oxidase (GOx) as a model enzyme, that is, GOx was covalently attached to the surface of CdTe QDs, the resulting biosensor showed the sensitive response to glucose. Under the irradiation of visible light of a wavelength at 505 nm, the proposed photoelectrochemical method could detect glucose ranging from 0.1 mM to 11 mM with a detection limit of 0.04 mM. The photoelectrochemical biosensor showed a good performance with high upper detection limit, acceptable stability and accuracy, providing an alternative method for monitoring biomolecules and extending the application of near-infrared QDs.     

An Amperometric Glucose Biosensor Based on Poly (Pyrrole‐2‐Carboxylic Acid)/Glucose Oxidase Biocomposite

A newly developed amperometric glucose biosensor based on graphite rod (GR) working electrode modified with biocomposite consisting of poly (pyrrole‐2‐carboxylic acid) (PCPy) particles and enzyme glucose oxidase (GOx) was investigated. The PCPy particles were synthesized by chemical oxidative polymerization technique using H₂O₂ as initiator of polymerization reaction and modified covalently with the GOx (PCPy‐GOx) after activation of carboxyl groups located on the particles surface with a mixture of N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). Then the PCPy‐GOx biocomposite was dispersed in a buffer solution containing a certain amount of bovine serum albumin (BSA). The resulting biocomposite suspension was adsorbed the on GR electrode surface with subsequent solvent airing and chemical cross‐linking of the proteins with glutaraldehyde vapour (GR/PCPy‐GOx). It was determined that the current response of the GR/PCPy‐GOx electrodes to glucose measured at +300 mV vs Cl reference electrode was influenced by the duration of the PCPy particles synthesis, pH of the GOx solution used for the PCPy particles modification and the amount of immobilized PCPy‐GOx biocomposite. An optimal pH of buffer solution for operation of the biosensor was found to be 8.0. Detection limit was determined as 0.039 mmol L⁻¹ according signal to noise ratio (S/N: 3). The proposed glucose biosensor was tested in human serum samples.     

Electrochemical glucose biosensor based on silver nanoparticles/multiwalled carbon nanotubes modified electrode

A novel glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) on Ag nanoparticles-decorated multiwalled carbon nanotube (AgNP-MWNT) modified glass carbon electrode (GCE). The AgNP-MWNT composite membrane showed an improving biocompatibility for GOx immobilization and an enhancing electrocatalytic activity toward reduction of oxygen due to decoration of AgNPs on MWNT surfaces. The AgNPs also accelerated the direct electron transfer between redox-active site of GOx and GCE surface because of their excellent conductivity and large capacity for protein loading, leading to direct electrochemistry of GOx. The glucose biosensor of this work showed a lower limit of detection of 0.01 mM (S/N?=?3) and a wide linear range from 0.025 to 1.0 mM, indicating an excellent analytical performance of the obtained biosensor to glucose detection. The resulting biosensor exhibits good stability and excellent reproducibility. Such bionanocomposite provides us good candidate material for fabrication of biosensors based on direct electrochemistry of immobilized enzymes.     

Direct Electrochemistry and Electrocatalysis Behaviors of Glucose Oxidase Based on Hyaluronic Acid-Carbon Nanotubes-Ionic Liquid Composite Film

Multi‐walled carbon nanotubes (MWNTs) were dispersed in the ionic liquid [BMIM][BF₄] to form a uniform black suspension. Based on it, a novel glucose oxidase (GOx)‐hyaluronic (HA)‐[BMIM][BF₄]‐MWNTs/GCE modified electrode was fabricated. UV‐vis spectroscopy confirmed that GOx immobilized in the composite film retained its native structure. The experimental results of EIS indicated MWNTs, [BMIM][BF₄] and HA were successfully immobilized on the surface of GCE and [BMIM][BF₄]‐MWNTs could obviously improve the diffusion of ferricyanide toward the electrode surface. The experimental results of CV showed that a pair of well‐defined and quasi‐reversible peaks of GOx at the modified electrode was exhibited, and the redox reaction of GOx at the modified electrode was surface‐confined and quasi‐reversible electrochemical process. The average surface coverage of GOx and the apparent Michaelis‐Menten constant were 8.5×10⁻⁹ mol/cm² and 9.8 mmol/L, respectively. The cathodic peak current of GOx and the glucose concentration showed linear relationship in the range from 0.1 to 2.0 mmol/L with a detection limit of 0.03 mmol/L (S/N=3). As a result, the method presented here could be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.     

Development of a Glucose Biosensor Using Advanced Electrode Modified by Nanohybrid Composing Chemically Modified Graphene and Ionic Liquid

Nanohybrids of chemically modified graphene (CMG) and ionic liquid (IL) were prepared by sonication to modify the electrode. The modified CMG‐IL electrodes showed a higher current and smaller peak‐to‐peak potential separation than a bare electrode due to the promoted electron transfer rate. Furthermore, the glucose oxidase (GOx) immobilized on the modified electrode displayed direct electron transfer rate and symmetrical redox potentials with a linear relationship at different scan rates. The fabricated GOx/CMG‐IL electrodes were developed selective glucose biosensor with respect to a sensitivity of 0.64 μA mM^?1, detection limit of 0.376 mM, and response time of <5 s.     

Direct electron transfer of glucose oxidase promoted by carbon nanotubes is without value in certain mediator-free applications

We have investigated the direct electron transfer (DET) promoted by carbon nanotubes (CNTs) on an electrode containing immobilized glucose oxidase (GOx) with the aim to develop a third-generation glucose biosensor and a mediator-free glucose biofuel cell anode. GOx was immobilized via chitosan (CS) on a glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWCNTs). Cyclic voltammetric revealed that the GOx on the surface of such an electrode is unable to simultaneously demonstrate DET with the electrode and to retain its catalytic activity towards glucose, although the MWCNTs alone can promote electron transfer between GOx and electrode. This is interpreted in terms of two types of GOx on the surface, the distribution and properties of which are quite different. The first type exhibits DET capability that results from the collaboration of MWCNTs and metal impurities, but is unable to catalyze the oxidation of glucose. The second type maintains its glucose-specific catalytic capability in the presence of a mediator, which can be enhanced by MWCNTs, but cannot undergo DET with the electrode. As a result, the MWCNTs are capable of promoting the electron transfer, but this is without value in some mediator-free applications such as in third-generation glucose biosensors and in mediator-free anodes for glucose biofuel cells.

基于WS2量子点材料固定葡萄糖氧化酶的直接电化学及其生物传感研究

采用水热法制备水溶性WS₂量子点（WS₂ QDs）材料,并将该材料进一步用于葡萄糖氧化酶（GOx）的有效固定,构建GOx/W₂ QDs/GCE传感界面. 采用透射电镜、紫外-可见光谱和电化学等方法对材料的形貌、GOx的固定化过程,以及传感器的直接电化学和电催化性能进行了表征. 结果表明,WS₂ QDs材料能够有效促进GOx与电极之间的直接电子转移. 并且,基于该传感器对葡萄糖良好的电催化作用,该方法有效实现了对葡萄糖的高灵敏检测,其线性范围为25 ~ 100 μmol·L^-1和100 ~ 600 μmol·L^-1,检测限为5.0 μmol·L^-1（S/N=3）. 该传感器具有良好的选择性、重现性和稳定性,可用于实际样品血糖的分析测定.