An ultra-sensitive chemiluminescence immunosensor of carcinoembryonic antigen using HRP-functionalized mesoporous silica nanoparticles as labels

A novel chemiluminescence immunosensor using horseradish peroxidase (HRP)-functionalized mesoporous silica nanoparticles (MSN) as labels was developed, which increases the sensitivity of the chemiluminescence immunoassay. The enzyme-functionalized MSN were fabricated by simultaneous coimmobilization of HRP and the carcinoembryonic antigen antibody (anti-CEA) onto the surface of MSN using 3-aminopropyltriethoxysilane (APTES) as the linkage. Because the large surface area of MSN carriers increased the amount of HRP bound per sandwiched immunoreaction, the conjugates provided a much higher signal and increased sensitivity. This is an improvement over the traditional sandwich immunoassay which often has one or two enzyme molecules per antibody. This approach was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect CEA in practical samples. The analysis showed a linear response within the range of 0.1-40 ng/mL (r = 0.9912). The relative standard deviation (RSD) for 11 parallel measurements of 20 ng/mL CEA was 3.9%. The sensitivity of the immunosensor using MSN-HRP-Ab2 as labels was about 10-fold higher than that of traditional labels. These labels for immunosensors may provide many potential applications for detection of different biomolecules.     

A stable sandwich-type amperometric biosensor based on poly(3,4-ethylenedioxythiophene)-single walled carbon nanotubes/ascorbate oxidase/nafion films for detection of L-ascorbic acid

In this paper, a stable sandwich-type amperometric biosensor based on poly(3,4-ethylenedioxythiophene) (PEDOT)-single walled carbon nanotubes (SWCNT)/ascorbate oxidase (AO)/Nafion films for detection of l-ascorbic acid (AA) was successfully developed. PEDOT-SWCNT nanocomposite and Nafion films were used as inner and outer films, respectively. AO was immobilized between these two films. The PEDOT-SWCNT nanocomposite films were characterized by electrochemical impedance spectroscopy and scanning electron microscopy. The influence of detection potential and temperature on the biosensor performance was examined in detail. Despite the multilayer configuration, the biosensor exhibited a relatively fast response (less than 10 s) and a linear range from 1 μM to 18 mM (a correlation coefficient of 0.9974). The sensitivity of the biosensor was found to be 28.5 mA M⁻¹ cm⁻². Its experimental detection limit was 0.7 μM (S/N = 3) and the apparent Michaelis-Menten constant (K_m) was calculated to be 18.35 mM. Moreover, the biosensor exhibited good anti-interferent ability and excellent long-term stability. All the results showed that such sandwich-type PEDOT-SWCNT/AO/Nafion films could provide a promising platform for the biosensor designs for AA detection.     

Ochratoxin A immunoassay with surface plasmon resonance registration: Lowering limit of detection by the use of colloidal gold immunoconjugates

An immunoanalytical system was developed for the determination of ochratoxin A with the use of a surface plasmon resonance (SPR) sensor amplified by the anti-species antibody-colloidal gold particle (CGP) conjugate. The use of the binding of immune complexes to the CGP-anti-species antibody conjugate leads to the SPR signal amplification by a factor of more than 10 and results in the 60 pg/mL limit of detection of ochratoxin A with an assay time of 30 min. These characteristics are superior to those obtained both in the conventional enzyme immunoassay with the use of the same reagents and the SPR assay with unmodified antibodies and specific antibodies conjugated to colloidal gold.     

基于囊状银钯铂合金的无标记型癌胚抗原免疫传感器的研制

该文将所合成的中空囊状银钯铂合金和壳聚糖（CHIT）修饰于玻碳电极（GCE）表面,利用中空囊状银钯铂合金比表面积大、反应活性位点多、导电能力良好、催化性能优异和吸附能力强等优点将癌胚抗原的抗体（anti-CEA）固定到电极表面,从而制得高灵敏的无标记型癌胚抗原（CEA）免疫传感器。当抗体与抗原发生免疫反应形成复合物时,会降低银钯铂合金的电催化活性并增加传质阻力。采用示差脉冲伏安法（DPV）检测电化学探针铁氰化钾的响应电流信号的减小程度,实现对CEA的检测。实验考察了电极表面的电化学行为,并对免疫传感器的性能进行了研究。在最优的实验条件下检测癌胚抗原的线性范围为0.5-80.0 ng/mL,线性相关系数为0.989 0,检测下限为0.17 ng/mL。     

Rapid parallelized and quantitative analysis of five pathogenic bacteria by ITS hybridization using QCM biosensor

We developed a 2 × 5 model quartz crystal microbalance (QCM) DNA biosensor array for detection of five bacteria, which based on hybridization analysis of bacterial 16S-23S rDNA internal transcribed spacer (ITS) region. A pair of universal primers was designed for PCR amplification of the ITSs. The PCR products were analyzed by the biosensor. We used gold nanoparticles to amplify the frequency shift signals. Fifty clinical samples were detected by both the biosensor and conventional bacteria culture method. We found a linear quantitative relationship between frequency shift and logarithmic concentration of synthesized oligonucleotides or bacteria cells. The measurable concentration ranged from 10⁻¹² to 10⁻⁸ M for synthesized oligonucleotides and 1.5 × 10² to 1.5 × 10⁸ CFU/mL for bacteria. The 10⁻¹² M of synthesized oligonucleotides or 1.5 × 10² CFU/mL of Pseudomonas aeruginosa could be detected by the biosensor system. The detection could be completed within 5 h including the PCR amplification procedure. Compared with bacteria culture method, the detection sensitivity and specificity of the biosensor system were 94.12% and 90.91%, respectively. There was no significant difference between these two methods (P = 0.625 > 0.05). The biosensor system provides a rapid and sensitive method for parallelized and quantitative analysis of multiple pathogenic bacteria in clinical diagnosis.     

Electrochemical performances of B doped and undoped diamond-like carbon (DLC) films deposited by femtosecond pulsed laser ablation for heavy metal detection using square wave anodic stripping voltammetric (SWASV) technique

Pure diamond-like carbon (DLC) thin films and boron-doped DLC thin films have been deposited on silicon substrates using femtosecond pulsed laser. The amorphous carbon materials (DLC), have been deposited at room temperature by ablating graphite targets with an amplified Ti:sapphire laser of 800 nm wavelength and a pulse duration of 150 fs in high vacuum conditions. Doping with boron has been performed by ablating alternatively graphite and boron targets.In this study, the DLC films were used as working electrodes for the electrochemical detection of trace heavy metals namely, Cd²⁺, Pb²⁺, Ni²⁺ and Hg²⁺, by using square wave anodic stripping voltammetry (SWASV) technique. Four metals were detected at −1.3 V deposition potential, and 90 s deposition time. The DLC films have been characterized by multiwavelength Raman spectrometry and high resolution scanning electron microscopy. The effect of the boron doping on the electrochemical behavior has been shown. The a-C:B 8%/Si₃N₄ electrode gives the more sensitive detection. The four metals are detected simultaneously with a detection limit of 1 μg/L or 2 μg/L and a dynamic range from 1 or 2 to 25 μg/L for every metal, as presented in third table of this article. The different sensitivities obtained are 6.2, 20.0, 1.2 and 6.6 μA/ppb or μA μg⁻¹ L for Cd²⁺, Pb²⁺, Ni²⁺ and Hg²⁺, respectively.     

Cu chemosensing behaviour of self-organized micro-array structures of a donor-acceptor bichromophoric compound anchored onto Ag nanoisland films

This work reports on the Cu²⁺ chemosensing behaviour of self-organized micro-array structures of a novel donor-acceptor bichromophoric compound anchored onto Ag nanoisland films. The system exhibits quenching of the fluorescence in the presence of Cu²⁺ ions, with detection range extending from 2 × 10⁻⁸ M up to 3 × 10⁻⁶ M and limit of detection (LOD) of 8 × 10⁻⁹ M. The quenching of fluorescence is accompanied by a quenching of SERS signal from the metal-organic structure, which is consistent with an electron transfer between the copper cation and the organic moiety. The self-organization property of the sensing complexes into micrometric arrays offers great potential for miniaturization and future development of Cu²⁺ detection systems based on real-time observation of fluorescence or SERS quenching by fluorescence microscopy or microRaman spectroscopy.     

All fiber-optic sensing of light using side-polished fiber overlaid with photoresponsive liquid crystals

This study presents a highly sensitive, all-fiber sensor for in situ detecting light. A fiber-optic light sensing platform was created by overlaying an in-line side-polished fiber (SPF) with a photoresponsive liquid crystal (P-LC) consisting of an azobenzene dye, a chiral dopant, and a nematic LC. The resulting P-LC overlaid SPF light sensor is sensitive to three different light sources, including 380 nm light emitting diode (LED), mercury lamp, and office ceiling lights. Under the light illumination, the energy of irradiation from short wavelengths of light (<450 nm) initiates the trans-to-cis photoisomerization of azobenzene. The photochemical LC-phase transition induced by the generated cis-moiety of azobenzene changes the refractive index of LC-overlaid side-polished area. Light illumination increased the attenuation of the input laser signal. After turning off the illumination, the attenuation returned to its original value, allowing the fiber-optic light sensor to be reused. The sensitivity of the resulting fiber-optic light sensor was 0.16 dB/(μW/cm²) with a detection limit of 5 μW/cm² and 0.06 dB/lx with a detection limit of 45 lx when a 380 nm LED and office ceiling lights were used as illumination sources, respectively. The detection limit increased from 45 to 12 lx when P-LC containing 20 wt% azobenzene was used as light sensing material. The proposed fiber-based light sensor has potential use in harsh environments, such as severely humid and corrosive environments, which could damage mechanical and electronic light sensors.     

A label-free amperometric immunosensor based on horseradish peroxidase functionalized carbon nanotubes and bilayer gold nanoparticles

In this work, a novel label-free amperometric immunosensor has been constructed for detecting α-1-fetoprotein (AFP) based on nanocomposite of horseradish peroxidase (HRP) labeled carbon nanotubes (CNTs). First, the gold nanoparticles (AuNPs) were electrodeposited on the surface of the glass carbon electrode by electrochemical reduction of gold chloride tetrahydrate (HAuCl₄) to immobilize horseradish peroxidase labeled carbon nanotubes (HRP-CNTs). Then HRP-CNTs bioconjugate was immobilized on the surface of the electrodeposited AuNPs layer by the combination of forces (coordination and electrostatic force). Subsequently, it was immersed into gold colloidal nanoparticles (GNPs) solution, which was used to immobilize antibody biomolecules (anti-AFP). Enhanced sensitivity was obtained by using bioconjugates featuring HRP labeled (HRP-CNTs), which had lager specific surface area and good electronic catalysis (current response signal) compared to carbon nanotubes. Under optimized conditions, the linear ranges were from 0.2 to 200 ng mL⁻¹ with a detection limit of 0.067 ng mL⁻¹ (at an S/N of 3). The proposed immunosenor showed good precision, acceptable stability and reproducibility and could be used for the detection AFP in normal human serum, which provided a potential alternative tool for the detection of protein in clinical diagnosis.     

Label-free electrochemical immunosensor for sensitive detection of kanamycin

A novel label-free electrochemical immunosensor for sensitive detection of kanamycin based on water-soluble graphene sheet (WGS)/prussian blue-chitosan (PB-CTS)/nanoporous gold (NPG) composited film has been reported. PB was selected as an electron transfer mediator, and was modified onto the electrode together with WGS through electrostatic adsorption. Then NPG was immobilized onto the as-prepared film for biomolecules anchoring. The electroactivity of PB was greatly enhanced in the presence of WGS and NPG. It could mainly be ascribed to the fact that the good conductivity of WGS and NPG promoted electron transfer and enhanced the sensitivity. kanamycin antibody, as a model, was immobilized onto the composite film for the detection of kanamycin. Under optimum conditions, the amperometric signal of PB decreased linearly with kanamycin concentration (0.02-14 ng mL⁻¹), a linear calibration plot (y = 1.3817 + 4.7544x, r = 0.9993), resulting in a low limit of detection (6.31 pg mL⁻¹). The novel immunosensor for the detection of kanamycin in real sample with satisfactory results has been proved. In addition, this method would be easily adapted for the detection of other residual antibiotics in animal derived foods.     

A CO2 sensor based upon a continuous-wave thermoelectrically-cooled quantum cascade laser

A CO₂ sensor based upon a continuous-wave thermoelectrically-cooled distributed feedback quantum cascade laser operating between 2305 and 2310 cm⁻¹ and a 54.2 cm long optical cell has been developed. Two approaches for direct absorption spectroscopy have been evaluated and applied for monitoring of the CO₂ concentration in gas lines and ambient laboratory air. In the first approach optical transmittance was derived from the single channel laser intensity, whilst in the second approach a ratio of signal and reference laser intensities (balanced detection) was used. The optimum residual absorption standard deviation was estimated to be 1.9 × 10⁻⁴ for 100 averages of 1 ms duration and 0.1 cm⁻¹ scans over the P(46) CO₂ absorption line of the ν₃ vibrational band at 2306.926 cm⁻¹. A CO₂ detection limit (1 standard deviation) of 36 ppb was estimated for 0.1 s average and balanced detection.     

Screen-printed carbon electrode for choline based on MnO2 nanoparticles and choline oxidase/polyelectrolyte layers

This paper presents the amperometric biosensor that determines choline and cholinesterase activity using a screen printed graphite electrode. In order to detect H₂O₂ we have blanket modified the electrode material with manganese dioxide nanoparticles layer. Using layer-by-layer technique on the developed hydrogen peroxide sensitive electrode surface choline oxidase was incorporated into the interpolyelectrolyte nanofilm. Its ability to serve as a detector of choline in bulk analysis and cholinesterase assay was investigated. We examined the interferences from red-ox species and heavy metals in the blood and in the environmental sample matrixes. The sensor exhibited a linear increase of the amperometric signal at the concentration of choline ranging from 1.3 × 10⁻⁷ to 1.0 × 10⁻⁴ M, with a detection limit (evaluated as 3σ) of 130 nM and a sensitivity of 103 mA M⁻¹ cm⁻² under optimized potential applied (480 mV vs. Ag/AgCl). The biosensor retained its activity for more than 10 consecutive measurements and kept 75% of initial activity for three weeks of storage at 4 °C. The R.S.D. was determined as 1.9% for a choline concentration of 10⁻⁴ M (n = 10) with a typical response time of about 10 s. The developed choline biosensor was applied for butyrylcholinesterase assay showing a detection limit of 5 pM (3σ). We used the biosensor to develop the cholinesterase inhibitor assay. Detection limit for chlorpyrifos was estimated as 50 pM.     

A simple 96 well microfluidic chip combined with visual and densitometry detection for resource-poor point of care testing

There is a well-recognized need for low cost biodetection technologies for resource-poor settings with minimal medical infrastructure. Lab-on-a-chip (LOC) technology has the ability to perform biological assays in such settings. The aim of this work is to develop a low cost, high-throughput detection system for the analysis of 96 samples simultaneously outside the laboratory setting. To achieve this aim, several biosensing elements were combined: a syringe operated ELISA lab-on-a-chip (ELISA-LOC) which integrates fluid delivery system into a miniature 96-well plate; a simplified non-enzymatic reporter and detection approach using a gold nanoparticle-antibody conjugate as a secondary antibody and silver enhancement of the visual signal; and carbon nanotubes (CNT) to increase primary antibody immobilization and improve assay sensitivity. Combined, these elements obviate the need for an ELISA washer, electrical power for operation and a sophisticated detector. We demonstrate the use of the device for detection of Staphylococcal enterotoxin B, a major foodborne toxin using three modes of detection, visual detection, CCD camera and document scanner. With visual detection or using a document scanner to measure the signal, the limit of detection (LOD) was 0.5 ng/ml. In addition to visual detection, for precise quantitation of signal using densitometry and a CCD camera, the LOD was 0.1 ng/ml for the CCD analysis and 0.5 ng/ml for the document scanner. The observed sensitivity is in the same range as laboratory-based ELISA testing. The point of care device can analyze 96 samples simultaneously, permitting high throughput diagnostics in the field and in resource poor areas without ready access to laboratory facilities or electricity.     

Electrochemical behaviors and determination of isoniazid at ordered mesoporous carbon modified electrode

In this work, an electrochemical sensor based on ordered mesoporous carbon (OMC) for the amperometric detection of isoniazid was developed. OMC was dispersed in a solution of Nafion, and the suspension was modified onto the surface of glassy carbon (GC) electrode. Cyclic voltammetry and amperometry were used to investigate the electrochemical behaviors of isoniazid on Nafion-OMC modified electrode (Nafion-OMC/GC). The results indicate that OMC can facilitate the electrochemical oxidation of isoniazid with a great decrease of overpotential in pH 7.0 phosphate buffer solution. The proposed biosensor provides excellent performance towards the determination of isoniazid with a high sensitivity of 0.031 μA/μM, a low detection limit of 8.4 × 10⁻⁸ M and wide linear range from 1.0 × 10⁻⁷ M to 3.7 × 10⁻⁴ M at +0.20 V vs. Ag/AgCl. The method was successfully applied to the determination of isoniazid tablets with satisfying results. All the results suggest that Nafion-OMC/GC electrode is a potential candidate for a stable and efficient electrochemical sensor to detect isoniazid.     

Multifunctional mesoporous silica nanoparticles as sensitive labels for immunoassay of human chorionic gonadotropin

A novel method to construct amperometric immunosensor for human serum chorionic gonadotrophin (hCG) has been described. In this study, horseradish peroxidase (HRP), Pt nanoparticles and secondary antibody (Ab₂) modified MSN (Pt@MSN/HRP/Ab₂) was synthesized and the multifunctional MSN was used as label for the preparation of immunosensor. With the hCG primary antibody immobilized onto thionine/graphene modified glassy carbon electrode (GCE) via crosslinking with glutaraldehyde, the electrochemical immunosensor was able to realize a reliable determination of hCG in the range of 0.01-12 ng mL⁻¹ with a detection limit of 7.50 pg mL⁻¹. This immunoassay system has many desirable merits including high sensitivity, accuracy, and little instrumentation requirement. Significantly, the new method may be quite promising, with potentially broad applications for clinical immunoassays.     

Development of a new fluorescent sensor based on a triazolo-thiadiazin derivative immobilized in polyvinyl chloride membrane for sensitive detection of lead(II) ions

A new sensor membrane based on a novel triazolo-thiadiazin derivative immobilized in polyvinyl chloride has been developed for the determination of Pb(II) ions that displays excellent performance. The parameters involved in the preparation of the optode and determination of Pb(II) were optimized. Under the optimal conditions, the proposed sensor displays a calibration response for Pb(II) over a wide concentration range of 5.0 × 10⁻⁸ to 3.8 × 10⁻⁴ M with the detection limit of 2.2 × 10⁻⁸ M. In addition to high reproducibility and reversibility of the fluorescence signal, the sensor also exhibits good selectivity over common metal ions. The optode membrane developed is easily prepared, stable, rapid, and simple for the determination of Pb(II). The accuracy of the proposed sensor was confirmed by analyzing standard reference materials of natural water and surface water. The sensor was successfully used for the determination of Pb(II) ions in water samples with satisfactory results.     

A novel biosensor for detecting toxicity in water using sulfur-oxidizing bacteria

A novel toxicity detection methodology based on sulfur-oxidizing bacteria (SOB) has been developed for the rapid and reliable detection of toxic chemicals in water. The methodology exploits the ability of SOB to oxidize sulfur particles in the presence of oxygen to produce sulfuric acid according to the following equation: S + H₂O + 1.5O₂ → SO₄²⁻ + 2H⁺, ΔG°′ = −587.1 kJ/reaction. The reaction results in an increase in electrical conductivity (EC) and a decrease in pH as SOB convert insoluble sulfur particles to sulfate and protons. The proposed technique is validated using EC and pH data. Using a synthetic stream water (EC = 0.12 mS/cm and pH 7.2), the baseline steady-state EC and pH values were ∼1.0 mS/cm and ∼2.5 over 30 days of testing when hexavalent chromium (Cr⁶⁺) was not added to the system. When Cr⁶⁺ was added to the system, the effluent EC decreased and the pH increased due to inhibition of SOB. We found that the system can detect Cr⁶⁺ at a concentration of 5 ppb which is lower than any method to date.     

Study of an amperometric glucose sensor based on Pd-Ni/SiNW electrode

An amperometric glucose sensor based on Pd-Ni/SiNW electrode has been investigated. The silicon nanowire (SiNW) electrodes were first fabricated by chemical etching, and then nickel and palladium particles were deposited onto the surfaces of SiNWs via electroless co-plating technique followed by annealing in nitrogen atmosphere at 350 °C for 300 s. The morphology of Pd-Ni/SiNW electrode was characterized by scanning electron microscope (SEM) and X-ray diffraction (XRD). The sensor performance was characterized by cyclic voltammetry (CV) and fixed potential amperometry techniques. In 0.1 M KOH alkaline medium with different glucose concentrations, the sensor shows an excellent sensitivity of 190.72 μA mM⁻¹ cm⁻² with the detection limit (S/N ratio = 3) of 2.88 μM. And it also exhibits superior anti-interference properties to the species including ascorbic acid (AA), uric acid (UA) and 4-acetamidophenol (AP). All results demonstrate that this Pd-Ni/SiNW electrode is a candidate with great potential for glucose detection.     

A sensitive hydrazine electrochemical sensor based on electrodeposition of gold nanoparticles on choline film modified glassy carbon electrode

A highly sensitive hydrazine sensor was developed based on the electrodeposition of gold nanoparticles onto the choline film modified glassy carbon electrode (GNPs/Ch/GCE). The electrochemical experiments showed that the GNPs/Ch film exhibited a distinctly higher activity for the electro-oxidation of hydrazine than GNPs with 3.4-fold enhancement of peak current. The kinetic parameters such as the electron transfer coefficient (α) and the rate of electron exchange (k) for the oxidation of hydrazine were determined. The diffusion coefficient (D) of hydrazine in solution was also calculated by chronoamperometry. The sensor exhibited two wide linear ranges of 5.0 × 10⁻⁷-5.0 × 10⁻⁴ and 5.0 × 10⁻⁴-9.3 × 10⁻³ M with the detection limit of 1.0 × 10⁻⁷ M (s/n = 3). The proposed electrode presented excellent operational and storage stability for the determination of hydrazine. Moreover, the sensor showed outstanding sensitivity, selectivity and reproducibility properties. All the results indicated a good potential application of this sensor in the detection of hydrazine.     

Optical fiber chemical sensing of Hg(II) ions in aqueous samples using a microfluidic device containing a selective tripodal chromoionophore-PVC film

A novel methodology for the determination of Hg(II) ions was developed based on optical fiber chemical sensing in a microfluidic device containing a selective tripodal chromoionophore (i.e. tris[2-(4-phenyldiazenyl)phenylamino)ethoxy]cyclotriveratrylene/TPPECTV)-PVC film. Absorbance detection was performed by incorporating a single optical fiber on the top and the bottom of the detection zone of the microfluidic device. In this micro-sensing system, the intensity of the absorption maximum at 495 nm of the TPPECTV-Hg(II) complex linearly increases as a function of the Hg(II) ion concentration in the range 1.0 × 10⁻⁶ to 2.5 × 10⁻⁴ M, with a detection limit of 0.5 μM. Interference from other heavy metal ions was not observed at significant levels. The absorbance results of the detection of Hg(II) ions in environmental water samples (river water) are in good agreement with those obtained by a macro-scale system (cold vapor atomic absorption spectrometry/CVAAS).