Sensitive estimation of total cholesterol in blood using Au nanowires based micro-fluidic platform

Determination of cholesterol level in blood is important in clinical applications. In this work, modified Au nanowires-electrochemical biosensor based on MEMS micro-fluidic platform is proposed for estimating total cholesterol in blood. This sensor consists of "aligned" Au nanowires as working electrode, platinum counter electrode deposited on the silicon platform and Ag/AgCl (3M KCl) reference electrode. The "aligned" Au nanowires are immobilized with cholesterol oxidase and cholesterol esterase using specific covalent chemistry. Further, Au nanowires promotes better electron transfer between the enzymes and electrodes, because of their large surface to volume ratio, small diffusion time, large electrical conductivity and their aligned nature. The modified Au nanowires showed a stable calibration line and a quasi-linear relationship between cholesterol level and current response in the range of 1-6 mM (in steps of 1 mM over the baseline blood serum). The sensitivity of the modified electrode was found to be about 69 nA/mM with good storage and interference stability.     

Polyaniline protected gold nanoparticles based mediator and label free electrochemical cortisol biosensor

Polyaniline protected gold nanoparticles (PPAuNPs) were electrophoretically deposited onto a gold electrode, and utilized to fabricate an electrochemical cortisol biosensor. Cortisol specific monoclonal antibody (C-Mab) was covalently immobilized onto the surface of a PPAuNP/Au electrode using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. BSA was employed for blocking nonspecific adsorption on the electrode surface. PPAuNP formation and BSA/C-Mab/PPAuNP/Au electrode fabrication were characterized using transmission electron microscopy, atomic force microscopy and electrochemical impedance techniques, respectively. Cyclic voltammetry and differential pulse voltammetric techniques were used to determine the cortisol concentration in a phosphate buffer saline (PBS) solution. Results confirmed that the PPAuNP based electrode was stable during repeated scans and exhibited repeatable redox peaks. Further, the BSA/C-Mab/PPAuNP/Au electrode in the PBS buffer accurately detected cortisol in the range of 1 pM-100 nM with a sensitivity of 1.63 μAM(-1). The biosensor was found to be selective against BSA and 17-α-hydroxy progesterone. This research establishes the feasibility of using a PPAuNP based matrix for a label and mediator free electrochemical biosensor for cortisol, a stress biomarker.     

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35 °C when operated at a scan rate of 50 mV s⁻¹. Linear range, response time, and detection limit were 0.1–500 μM, 6 s, and 0.1 μM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.     

Amperometric biosensor for hydrogen peroxide based on horseradish peroxidase onto gold nanowires and TiO2 nanoparticles

An electrochemical biosensor for determination of hydrogen peroxide (H₂O₂) was fabricated, based on the electrostatic immobilization of horseradish peroxidase (HRP) with one-dimensional gold nanowires (Au NWs) and TiO₂ nanoparticles (nano-TiO₂) on a gold electrode. The nano-TiO₂ can give a biocompatible microenvironment and compact film, and the Au NWs can provide fast electron transferring rate and greatly add the amount of HRP molecules immobilized on the electrode surface. Au NWs were characterized by ultraviolet–visible spectra and transmission electron microscope. The electrode modification process was probed by cyclic voltammetry and electrochemical impedance spectroscopy. Chronoamperometry was used to study the electrochemical performance of the resulting biosensor. Under optimal conditions, the linear range for the determination of H₂O₂ was from 2.3 × 10⁻⁶ to 2.4 × 10⁻³ M with a detection limit of 7.0 × 10⁻⁷ M (S/N = 3). Moreover, the proposed biosensor showed superior stability and high sensitivity.     

黄曲霉毒素氧化酶/壳聚糖-单壁碳纳米管/聚邻苯二胺修饰电极对杂色曲霉素的检测

将黄曲霉毒素氧化酶(AFO)固定在壳聚糖(CS)-单壁碳纳米管(SWCNTs)杂交膜中,组装在聚邻苯二胺(POPD)修饰的金电极(Au)表面,制备了对杂色曲霉素(ST)敏感的生物传感器(AFO/CS-SWCNTs/POPD/Au)。运用原子力显微镜(AFM)、傅立叶变换红外光谱(FT-IR)和交流阻抗技术(EIS)对电极组装过程进行了表征。循环伏安法研究表明,AFO在修饰电极上发生了准可逆的氧化还原反应,是表面控制过程,其式量电位为-0.436V(vs.Ag/AgCl),说明包埋在CS-SWCNTs中的AFO和电极之间发生了直接电子传递。AFO修饰电极对ST具有明显的电催化作用,其表观米氏常数appKM为7.13μmol/L,催化电流与ST浓度在10～310ng/mL范围内呈线性关系,相关系数为0.997,检出限为3ng/mL(S/N=3),响应时间小于10s。组装的生物传感器具有较好的稳定性与重现性,连续检测20ng/mL的ST标准溶液11次,电流值RSD为3.9%;放置一个月后,其电流响应值仍为初始值的85.6%。该方法具有较高的选择性和灵敏度,应用于实际样品检测时,其回收率在87.6%～105.5%之间...     

Electrochemically deposited chitosan hydrogel for horseradish peroxidase immobilization through gold nanoparticles self-assembly

A new strategy for immobilization of horseradish peroxidase (HRP) has been presented by self-assembling gold nanoparticles on chitosan hydrogel modified Au electrode. From a mildly acidic chitosan solution, a chitosan film is electrochemically deposited on Au electrode surface via a negative voltage bias. This process is accompanied by the hydrogen evolution reaction, and the released hydrogen gas made the deposited chitosan film with porous structure, which facilitates the assembly of gold nanoparticles and HRP. The resulting substrates were characterized by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The immobilized HRP displayed an excellent catalytic property to the reduction of H2O2 in the presence of methylene blue mediator. The resulting biosensor (HRP-modified electrode) showed a wide dynamic range of 8.0 microM-15 mM H2O2, and the linear ranges were 8.0 microM-0.12 mM and 0.50-12 mM, with a detection limit of 2.4 microM estimated at a signal-to-noise ratio of 3. Moreover, the biosensor remained about 85% of its original sensitivity after four weeks' storage.     

Development of DNA electrochemical biosensor based on covalent immobilization of probe DNA by direct coupling of sol-gel and self-assembly technologies

A new procedure for fabricating deoxyribonucleic acid (DNA) electrochemical biosensor was developed based on covalent immobilization of target single-stranded DNA (ssDNA) on Au electrode that had been functionalized by direct coupling of sol-gel and self-assembled technologies. Two siloxanes, 3-mercaptopropyltrimethoxysiloxane (MPTMS) and 3-glycidoxypropyltrimethoxysiloxane (GPTMS) were used as precursors to prepare functionally self-assembly sol-gel film on Au electrode. The thiol group of MPTMS allowed assembly of MPTMS sol-gel on gold electrode surface. Through co-condensation between silanols, GPTMS sol-gel with epoxide groups interconnected into MPTMS sol-gel and enabled covalent immobilization of target NH(2)-ssDNA through epoxide/amine coupling reaction. The concentration of MPTMS and GPTMS influenced the performance of the resulting biosensor due to competitive sol-gel process. The linear range of the developed biosensor for determination of complementary ssDNA was from 2.51 x 10(-9) to 5.02 x 10(-7)M with a detection limit of 8.57 x 10(-10)M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of target ssDNA on self-assembled sol-gel matrix could serve as a versatile platform for DNA immobilization and fabrication of biosensors.     

Direct electrochemistry of horseradish peroxidase immobilized on a colloid/cysteamine-modified gold electrode

Direct electron transfer of immobilized horseradish peroxidase on gold colloid and its application as a biosensor were investigated by using electrochemical methods. The Au colloids were associated with a cysteamine monolayer on the gold electrode surface. A pair of redox peaks attributed to the direct redox reaction of horseradish peroxidase (HRP) were observed at the HRP/Au colloid/cysteamine-modified electrode in 0.1 M phosphate buffer (pH 7.0). The surface coverage of HRP immobilized on Au colloid was about 7.6 x 10(-10) mol/cm(2). The sensor displayed an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The calibration range of H(2)O(2) was 1. 4 microM to 9.2 mM with good linear relation from 1.4 microM to 2.8 mM. A detection limit of 0.58 microM was estimated at a signal-to-noise ratio of 3. The sensor showed good reproducibility for the determination of H(2)O(2). The variation coefficients were 3. 1 and 3.9% (n = 10) at 46 microM and 2.8 mM H(2)O(2), respectively. The response showed a Michaelis-Menten behavior at higher H(2)O(2) concentrations. The K(app)(M) value for the H(2)O(2) sensor was found to be 2.3 mM.     

Mercaptoethylpyrazine promoted electrochemistry of redox protein and amperometric biosensing of uric acid

Electrochemistry of microperoxidase-11 (MPx-11) anchored on the mixed self-assembled monolayer (SAM) of 2-(2-mercaptoethylpyrazine) (PET) and 4,4'-dithiodibutyric acid (DTB) on gold (Au) electrode and the biosensing of uric acid (UA) is described. MPx-11 has been covalently anchored on the mixed SAM of PET and DTB on Au electrode. MPx-11 on the mixed self-assembly exhibits reversible redox response characteristic of a surface confined species. The heterocyclic ring of PET promotes the electron transfer between the electrode and the redox protein. The apparent standard rate constant kapps obtained for the redox reaction of MPx-11 on the mixed monolayer is approximately 2.15 times higher than that on the single monolayer of DTB modified electrode. MPx-11 efficiently mediates the electrocatalytic reduction of H2O2. MPx-11 electrode is highly sensitive to H2O2 and it shows linear response for a wide concentration range. The electrocatalytic activity of the MPx-11 electrode is combined with the enzymatic activity of uricase (UOx) to fabricate uric acid biosensor. The bienzyme assembly is highly sensitive towards UA and it could detect UA as low as 2 microM at the potential of -0.1 V. The biosensor shows linear response with a sensitivity of 3.4+/-0.08 nA cm(-2) microM(-1). Ascorbate (AA) and paracetamol (PA) do not significantly interfere in the amperometric sensing of UA.     

Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry

A direct electrochemical DNA biosensor based on zero current potentiometry was fabricated by immobilization of ssDNA onto gold nanoparticles (AuNPs) coated pencil graphite electrode (PGE). One ssDNA/AuNPs/PGE was connected in series between clips of working and counter electrodes of a potentiostat, and then immersed into the solution together with a reference electrode, establishing a novel DNA biosensor for specific DNA detection. The variation of zero current potential difference (ΔE(zcp)) before and after hybridization of the self-assembled probe DNA with the target DNA was used as a signal to characterize and quantify the target DNA sequence. The whole DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. Under the optimized conditions, ΔE(zcp) was linear with the concentrations of the complementary target DNA in the range from 10nM to 1μM, with a detection limit of 6.9nM. The DNA biosensor showed a good reproducibility and selectivity. Prepared DNA biosensor is facile and sensitive, and it eliminates the need of using exogenous reagents to monitor the oligonucleotides hybridization.     

Sub-femtomolar electrochemical detection of DNA using surface circular strand-replacement polymerization and gold nanoparticle catalyzed silver deposition for signal amplification

A highly sensitive method was developed for detection of target DNA. This method combined circular strand-displacement polymerization (CSRP) with silver enhancement to achieve dual signal amplification. After molecular beacon (MB) hybridized with target DNA, the reporter gold nanoparticle (Au NPs) was attached to an electrode surface by hybridization between Au NP labeled primer and stem part of the MB to initiate a polymerization of DNA strand, which led to the release of target and another polymerization cycle. Thus the CSRP produced the multiplication of target-related reporter Au NPs on the surface. The Au NPs then catalyzed silver deposition for subsequent stripping analysis of silver. The dual signal amplification offered a dramatic enhancement of the stripping response. This signal could discriminate perfect matched target DNA from 1-base mismatch DNA. The dynamic range of the sequence-specific DNA detection was from 10(-16) to 10(-12)molL(-1) with a detection limit down to sub-femtomolar level. This proposed method exhibited an efficient amplification performance, and would open new opportunities for sensitive detection of other biorecognition events.     

Sensitive detection of endonuclease activity and inhibition using gold nanorods

A sulfite oxidase (SO(X)) (EC 1.8.3.1) purified from Syzygium cumini leaves was immobilized onto carboxylated gold coated magnetic nanoparticles (Fe(3)O(4)@GNPs) electrodeposited onto the surface of a gold (Au) electrode through N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC)-N-hydroxy succinimide (NHS) chemistry. An amperometric sulfite biosensor was fabricated using SO(X)/Fe(3)O(4)@GNPs/Au electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode. The working electrode was characterized by Fourier Transform Infrared (FTIR) Spectroscopy, Cyclic Voltammetry (CV), Scanning Electron Microscopy (SEM) and Electrochemical Impedance Spectroscopy (EIS) before and after immobilization of SO(X). The biosensor showed optimum response within 2s when operated at 0.2V (vs. Ag/AgCl) in 0.1 M Tris-HCl buffer, pH 8.5 and at 35 °C. Linear range and detection limit were 0.50-1000 μM and 0.15 μM (S/N=3) respectively. Biosensor was evaluated with 96.46% recovery of added sulfite in red wine and 1.7% and 3.3% within and between batch coefficients of variation respectively. Biosensor measured sulfite level in red and white wines. There was good correlation (r=0.99) between red wines sulfite value by standard DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) method and the present method. Enzyme electrode was used 300 times over a period of 4 months, when stored at 4 °C. Biosensor has advantages over earlier biosensors that it has excellent electrocatalysis towards sulfite, lower detection limit, higher storage stability and no interference by ascorbate, cysteine, fructose and ethanol.     

A sensitive NADH and glucose biosensor tuned by visible light based on thionine bridged carbon nanotubes and gold nanoparticles multilayer

A NADH and glucose biosensor based on thionine cross-linked multiwalled carbon nanotubes (MWNTs) and Au nanoparticles (Au NPs) multilayer functionalized indium-doped tin oxide (ITO) electrode were presented in this paper. The effect of light irradiation on the enhancement of bioelectrocatalytic processes of the biocatalytic systems by the photovoltaic effect was investigated. This bioelectrode exhibited excellent catalytic activity of the oxidation towards dihydronicotinamide adenine dinucleotide (NADH). Most interesting, the performance of this NADH sensor could be tuned by the visible light. When the biosensor was performed in the dark, the anodic current increased linearly with NADH concentration over the range from 0.5 to 237 microM with detection limit 0.1 microM and sensitivity 17 nA microM(-1). The sensitivity became 115 nA microM(-1) with detection limit 0.05 microM with the light irradiation. Compared with the reaction in dark, the sensitivity increased around 7 folds while the detection limit decreased 2 folds. The glucose biosensor also exhibited the same behavior. The linear range was from 10 microM to 2.56 mM with the sensitivity of 7.8 microAmM(-1) and detection limit 5.0 microM in the dark. After the light irradiation, the linear range was from 1 microM to 3.25 mM with the sensitivity of 18.5 microA mM(-1) and detection limit 0.7 microM. It indicated a potential to provide an operational access to develop new kinds of photocontrolled dehydrogenase enzyme-based bioelectronics.     

A cytokine immunosensor for multiple sclerosis detection based upon label-free electrochemical impedance spectroscopy

A biosensor for the serum cytokine, interleukin-12 (IL-12), based upon a label-free electrochemical impedance spectroscopy monitoring is described. Overexpression of IL-12 has been correlated to the diagnosis of multiple sclerosis (MS). The prototype biosensor was fabricated on a disposable gold-coated silver ribbon electrode by immobilizing anti-IL-12 monoclonal antibodies (mAbs) onto the surface of the electrode. This technique was advantageous as the silver electrodes provided a more rigid and conductive substrate than thin gold foil electrodes and helped in obtaining more reproducible data when used with the electrode holder. Results indicate that IL-12 can be detected at physiological levels, <100 fM with p<0.05 in a label-free and real-time manner. The cost-effective approach described here can be used for diagnosis of diseases (like MS) with known biomarkers in body fluids and for monitoring physiological levels of biomolecules with healthcare, food, and environmental relevance.     

Self-assembled monolayer for low density lipoprotein detection

Heparin (HEP) has been covalently immobilized onto 4-aminothiophenol (ATP) self-assembled monolayer (SAM) deposited onto gold (Au)-coated glass plate for low density lipoprotein (LDL) detection. The HEP/ATP/Au and LDL/HEP/ATP/Au electrodes have been characterized using cyclic voltammetry (CV) and scanning electron microscopy (SEM). Surface plasmon resonance (SPR) measurements reveal that HEP/ATP/Au electrode is sensitive to detection of the LDL in the range 0.03 microM (10 mg/dl)-0.39 microM (130 mg/dl). The values of association and dissociation rate constants in the association phase calculated by kinetic analysis have been found to be k(a) = 9.67 x 10(1) M(-1) s(-1) and k(d) = 2.64 x 10(-4) s(-1).     

An FIA biosensor system for the determination of phosphate.

A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at 0.7 V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-1-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100 microM phosphate, with a minimum detectable concentration of 1.25 microM phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.     

A label-free electrochemical immunoassay for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and nonconductive polymer film

A simple and sensitive label-free electrochemical immunoassay electrode for detection of carcinoembryonic antigen (CEA) has been developed. CEA antibody (CEAAb) was covalently attached on glutathione (GSH) monolayer-modified gold nanoparticle (AuNP) and the resulting CEAAb-AuNP bioconjugates were immobilized on Au electrode by electro-copolymerization with o-aminophenol (OAP). Electrochemical impedance spectroscopy and cyclic voltammetry studies demonstrate that the formation of CEA antibody-antigen complexes increases the electron transfer resistance of [Fe(CN)(6)](3-/4-) redox pair at the poly-OAP/CEAAb-AuNP/Au electrode. The use of CEA antibody-AuNP bioconjugates and poly-OAP film could enhance the sensitivity and anti-nonspecific binding of the resulting immunoassay electrode. The preliminary application of poly-OAP/CEAAb-AuNP/Au electrode for detection of CEA was also evaluated.     

Electrochemical recognition for sugars on the chitosan-poly(diallyldimethylammonium chloride)-nano-Prussian blue/nano-Au/4-mercaptophenylboronic acid modified glassy carbon electrode

A new amperometric biosensor for the detection of sugars was prepared. A glassy carbon electrode was modified with Prussian blue (PB) nanoparticles protected by chitosan (CS) and poly(diallyldimethylammonium chloride) (PDDA), and then gold nanoparticles were assembled onto the electrode followed by the assembly of 4-mercaptophenylboronic acid (MPBA) onto the surface of gold nanoparticles through a sulfur–Au bond to fabricate a self-assembled biosensor. The PB nanoparticles protected by CS and PDDA were characterized using transmission electron microscopy and UV–vis absorption spectroscopy. The characterization of the self-assembled electrode was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The pK _a values of the MPBA monolayer before and after combining with sugars were determined. The fabricated electrode exhibited excellent performances for determining d(+)-glucose, d(+)-mannose, and d(−)-fructose on the basis of the change in i _p of the Fe(CN)₆^3−/4− ion in the presence of sugars.     

A novel glucose biosensor based on the immobilization of glucose oxidase onto gold nanoparticles-modified Pb nanowires

A novel glucose biosensor was developed, based on the immobilization of glucose oxidase (GOD) with cross-linking in the matrix of bovine serum albumin (BSA) on a Pt electrode, which was modified with gold nanoparticles decorated Pb nanowires (GNPs-Pb NWs). Pb nanowires (Pb NWs) were synthesized by an l-cysteine-assisted self-assembly route, and then gold nanoparticles (GNPs) were attached onto the nanowire surface through –SH–Au specific interaction. The morphological characterization of GNPs-Pb NWs was examined by transmission electron microscopy (TEM). Cyclic voltammetry and chronoamperometry were used to study and to optimize the electrochemical performance of the resulting biosensor. The synergistic effect of Pb NWs and GNPs made the biosensor exhibit excellent electrocatalytic activity and good response performance to glucose. The effects of pH and applied potential on the amperometric response of the biosensor have been systemically studied. In pH 7.0, the biosensor showed the sensitivity of 135.5 μA mM⁻¹ cm⁻², the detection limit of 2 μM (S/N = 3), and the response time <5 s with a linear range of 5–2200 μM. Furthermore, the biosensor exhibits good reproducibility, long-term stability and relative good anti-interference.     

Detection of ricin using a carbon nanofiber based biosensor

We report ricin detection using antibody and aptamer probes immobilized on a nanoelectrode array (NEA) consisting of vertically aligned carbon nanofibers (VACNFs). These biosensor chips are fabricated on a wafer scale using steps common in integrated circuit manufacturing. Electrochemical impedance spectroscopy is used to characterize the detection event and the results indicate that the electron transfer resistance changes significantly after the ricin protein binds to the probe. Further confirmation is obtained from evaluation of the electrode surface by atomic force microscopy which clearly shows a change in height from the bare electrode to the surface bound by the probe-protein.