Sensitive electrochemical immunosensor array for the simultaneous detection of multiple tumor markers

A novel electrochemical immunosensor array for the simultaneous detection of multiple tumor markers was developed by incorporating electrochemically addressing immobilization and one signal antibody strategy. As a proof-of-principle, an eight-electrode array including six carbon screen-printed working electrodes was used as a base array for the analysis of two important tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) and a horseradish peroxidase-labeled antibody was employed as a signal antibody. The immunosensor in the array was fabricated in sequence by covalently coupling the capture antibody onto the surface of the desired working electrode, which was firstly electrochemically addressably grafted with an aminophenyl group by reduction of in situ generated aminophenyl diazonium cation generated from p-phenylenediamine, using glutaraldehyde as cross-linker. This allowed the selective immobilization of the capture antibody at the desired position on a single array via an electrochemical operation. The immunoassay in sandwich mode was performed by specifically binding the targets, second antibodies and one signal antibody to the immunosensor array. The result showed that the steady current density was directly proportional to the concentration of target CEA/AFP in the range from 0.10 to 50 ng mL(-1) with a detection limit of 0.03 ng mL(-1) for CEA and 0.05 ng mL(-1) for AFP (S/N = 3), respectively. This work demonstrates that the employment of an electrochemically addressing method for the fabrication of an immunosensor array and one signal antibody is a promising approach for the determination of multiple tumor markers in clinical samples.     

Determination of urine melamine by validated isotopic ultra‐performance liquid chromatography/tandem mass spectrometry

Little is known about melamine (MEL) analysis in children's urine. In this study, an isotopic ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and systematically validated for the analysis of MEL in urine. The method is easily performed and comprises acidification, solid‐phase extraction (SPE) and UPLC/MS/MS analysis. ¹³C₃N₃(¹⁵NH₂)₃ was used as the internal standard (IS) for calibration. Transition ions m/z 127 > 85 of MEL and m/z 133 > 89 of the IS were used for quantification and m/z 127 > 68 of MEL was used for quantitative confirmation. Recovery and precision were assessed to guarantee the applicability of the method. The limit of quantification (LOQ) was 0.01 µg/mL while the calculated method detection limit was 0.006 µg/mL. The mean recoveries ranged from 96–99%. The method was then applied to analyze urine samples from children who had potentially consumed MEL‐tainted dairy products during screening in Taiwan. Ten nephrolithiasis cases and 20 age‐ and gender‐matched controls were selected for this study. Three out of the 10 nephrolithiasis cases had elevated levels of MEL. Comparatively, twenty age‐ and gender‐matched non‐nephrolithiasis controls consuming Taiwan brand milk powder all showed MEL levels lower than the detection limit except for two children with background levels of 0.02 µg/mL. The background level in these children urine samples was established by UPLC/MS/MS analysis. Positive results of urine MEL tests might be associated with nephrolithiasis in these candidates. Measurement of urine MEL concentration can be helpful in confirming MEL‐related nephrolithiasis, but its clinical application needs further clarification. Copyright © 2009 John Wiley & Sons, Ltd.     

Docking of Antibodies into the Cavities of DNA Origami Structures

Immobilized antibodies are extensively employed for medical diagnostics, such as in enzyme‐linked immunosorbent assays. Despite their widespread use, the ability to control the orientation of immobilized antibodies on surfaces is very limited. Herein, we report a method for the covalent and orientation‐selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA)‐modified strands are inserted into the cavity to form NTA–metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysine residues. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) confirmed the efficient immobilization of the antibodies in the origami structures. This increased control over the orientation of antibodies in nanostructures and on surfaces has the potential to direct the interactions between antibodies and targets and to provide more regular surface assemblies of antibodies.     

Simultaneous determination of triptolide, tripdiolide and tripterine in human urine by high-performance liquid chromatography coupled with ion trap atmospheric-pressure chemical ionization mass spectrometry

An accurate and selective method for the simultaneous determination of triptolide, tripdiolide and tripterine in human urine using hydrocortisone as an internal standard (IS) by high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization mass spectrometry in negative ion mode has been developed. After triptolide, tripdiolide and tripterine in human urine were extracted with ethyl acetate and cleaned by solid-phase extraction with C(18) cartridges, a satisfactory separation was achieved on an XDB C(18) short column (30 x 2.1 mm i.d., 3 microm) using the mobile phase of acetic acid-ammonium acetate (5 mmol/L, pH = 4.5)-acetonitrile-methanol in gradient elution. Detection was operated by APCI in selected ion monitoring mode. The target ions m/z 359, m/z 375, m/z 449 and m/z 419 were selected for the quantification of triptolide, tripdiolide, tripterine and IS, respectively. The linear range was 1.0-100.0 ng mL(-1), and the limits of quantification in human urine were found to be 0.1-0.5 ng mL(-1) for the three compounds. The precisions (CV%) and accuracies were 6.6-12.9 and 85.1-97.0%, respectively. The developed method could be applied to the determination of triptolide, tripdiolide and tripterine in human urine for diagnosis of the intoxication and for forensic purposes.     

Label-free capacitive immunosensors for ultra-trace detection based on the increase of immobilized antibodies on silver nanoparticles

Detection of ultra-trace amounts of antigens by label-free capacitive immunosensors was investigated using electrodes modified with silver nanoparticles (AgNPs) that allows for an increase in the amount of immobilized antibodies. The optimal amount of AgNPs that provided the highest immobilization yield was 48 pmol (in 2.0 mL). The performances of immunosensor electrodes for human serum albumin prepared with AgNPs, were compared to electrodes prepared with gold nanoparticles. The two systems provided the same linear range (1.0 × 10⁻¹⁸ to 1.0 × 10⁻¹⁰ M) and detection limit (1.0 × 10⁻¹⁸ M). The system with AgNPs was used to analyze albumin in urine samples and the results agreed well with the immunoturbidimetric assay (P > 0.05). Electrodes modified with AgNPs and appropriate antibodies were tested for their performances to detect analytes of different sizes. For a macromolecule (human serum albumin) the incorporation of AgNPs improved the detection limit from 100 to 1 aM. For small molecules, microcystin-LR and penicillin G, the detection limits were lowered from 100 and 10 fM to 10 and 0.7 fM, respectively. The high sensitivity and very low detection limits are potentially useful for the analysis of toxins or residues present in samples at ultra-trace levels and this method could easily be applied to other affinity pairs.     

Biosensor Based on TTF@ SiO2 Nanoparticles as Electron Transfer Mediator for the Determination of Glucose

IntroductionBecause of the high demand for blood glucose mo-nitoring, significant research and development effortshave been devoted to produce reliable glucose sensorsforin vitroorin vivoapplications[1,2]. With the appli-cations of this device, many relev…     

Determination and pharmacokinetic study of norcantharidin in human serum by HPLC-MS/MS method

A sensitive, simple and selective high-performance liquid chromatography-tandem mass spectrometry method was developed and applied to the determination of norcantharidin concentration in human serum. Norcantharidin (NCTD) and cyclophosphamide (IS) in serum were extracted with acetone, separated on a C18 reversed-phase column, gradiently eluted with a mobile phase of acetonitrile-water containing 2 mm ammonium acetate and 0.1% formic acid (pH 3), ionized by positive ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor-->product ions of m/z 169.3-->123.1 for NCTD and 261.2-->140.2 for IS, respectively. The linear range of the calibration curve for NCTD was 2.5-50 ng/mL, with a lowest limit of quantification of 2.5 ng/mL, and the intra/inter-day RSD was less than 10%. The method was suitable for determination of low NCTD concentration in human serum after therapeutic oral doses, and has been successfully used for pharmacokinetic studies in healthy Chinese volunteers.     

Highly sensitive method for the determination of JI-101, a multi-kinase inhibitor in human plasma and urine by LC-MS/MS-ESI: method validation and application to a clinical pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of JI-101 in human plasma and urine using LC-MS/MS-ESI in the positive-ion mode. The assay procedure involves extraction of JI-101 and alfuzosin (internal standard, IS) from human plasma/urine with a solid-phase extraction process. Chromatographic resolution was achieved on two Zorbax SB-C(18) columns connected in series with a PEEK coupler using an isocratic mobile phase comprising acetonitrile-0.1% formic acid in water (70:30, v/v). The total run time was 2.0 min. The MS/MS ion transitions monitored were 466.20 → 265.10 for JI-101 and 390.40 → 156.10 for IS. The method was subjected to rigorous validation procedures to cover the following: selectivity, sensitivity, matrix effect, recovery, precision, accuracy, stability and dilution effect. In both matrices the lower limit of quantitation was 10.0 ng/mL and the linearity range extended from ~10.0 to 1508 ng/mL in plasma or urine. The intra- and inter-day precisions were in the ranges 1.57-14.5 and 6.02-12.4% in plasma and 0.97-15.7 and 8.66-10.2% in urine. This method has been successfully applied for the characterization of JI-101 pharmacokinetics in cancer patients.     

Simultaneous Determination of β-Artemether and its Metabolite Dihydroartemisinin in Human Plasma and Urine by a High-Performance Liquid Chromatography-Mass Spectrometry Assay Using Electrospray Ionisation

A sensitive and selective method is described for the determination of β-artemether (AM) and its metabolite dihydroartemisinin (DHA) in human plasma and urine using artemisinin (IS) as internal standard. The method consists of a liquid-liquid extraction using 2,2,4-trimethylpentane – ethyl acetate (7:3 v/v) with subsequent evaporation of the supernatant to dryness followed by the analysis of the reconstituted sample by liquid chromatography – mass spectrometry (LC-MS) using positive electrospray ionisation (ESI). The acquisition was performed using a mass range scan and the ions (MH+?CH3OH) m/z 267.2, (MH+?H2O) m/z 267.2 and (MH+) m/z 283.2 for AM, DHA and IS respectively were used for compound quantifications. Chromatography was performed on a C18 reversed-phase column using a gradient of acetonitrile – ammonium acetate 10 mM, glacial acetic acid 0.1% as a mobile phase. The method was validated over a concentration range of 10–1000 ng mL^?1 using 1 mL of human plasma per assay and over a concentration range of 5–500 ng mL^?1 using 2 mL of human urine per assay. The method was applied to the quantitation of β-artemether and dihydroartemisinin in human plasma and urine of volunteers participating in a drug pharmacokinetic study.     

Multivalent binding of small guest molecules and proteins to molecular printboards inside microchannels

Beta-Cyclodextrin (beta-CD) monolayers have been immobilized in microchannels. The host-guest interactions on the beta-CD monolayers inside the channels were comparable to the interactions on beta-CD monolayers on planar surfaces, and a divalent fluorescent guest attached with a comparable binding strength. Proteins were attached to these monolayers inside microchannels in a selective manner by employing a strategy that uses streptavidin and orthogonal linker molecules. The design of the chip, which involved a large channel that splits into four smaller channels, allowed the channels to be addressed separately and led to the selective immobilization of antibodies. Experiments with labeled antibodies showed the selective immobilization of these antibodies in the separate channels.     

Simultaneous determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using ultra-performance liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive, and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for the determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using sildenafil as an internal standard (IS) was developed and validated. Udenafil, DA-8164 and IS from a 100 microL aliquot of biological samples were extracted by protein precipitation using acetonitrile. Chromatographic separation was carried on an Acquity UPLC BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with an isocratic mobile phase consisting of acetonitrile and containing 0.1% formic acid (75:25, v/v) at flow rate of 0.4 mL/min, and total run time was within 1 min. Detection and quantification was performed by the mass spectrometer using multiple reaction-monitoring mode at m/z 517 --> 283 for udenafil, m/z 406 --> 364 for DA-8164 and m/z 475 --> 100 for IS. The assay was linear over a concentration range of 1-600 ng/mL with a lower limit of quantification of 1 ng/mL in both human plasma and urine. The coefficient of variation of this assay precision was less than 13.7%, and the accuracy exceeded 92.0%. This method was successfully applied for pharmacokinetic study after oral administration of udenafil 100 mg to healthy Korean male volunteers.     

Oriented immobilization of antibodies and their fragments on modified silicon for the production of nanosensors

Different methods for the covalent immobilization of specific antibodies and their fragments on a silicon surface with the subsequent formation of immune complexes that consist of an immobilized monoclonal antibody, an antigen molecule, and a molecule of a second monoclonal antibody labeled with gold nanoparticles have been studied. Prostate-specific antigen (PSA), which is a molecular biomarker for prostate cancer, was used as an antigen. A covalent conjugate of the fragments of PSA-specific antibodies with gold nanoparticles has been obtained using the thiol groups of the antibodies. Scanning electron microscopy (SEM) was used for the registration of immune complexes on the surface. The high resolution of the method made it possible to detect individual immune complexes by the presence of gold nanoparticles and to calculate their number. A new method for the chemical modification of silicon by 3-aminopropyltrimetoxysilane (APTMS) and a bifunctional reagent 1,4-phenylene diisothiocyanate (PDITC) has been developed. This method provides a uniform distribution of antigen-binding centers and their availability for the formation of immune complexes. The developed immobilization method is promising for the formation of a biospecific biosensor layer based on silicon nanowires.     

Histamine determination in human urine using sub-2 μm C18 column with fluorescence and mass spectrometric detection

A fast, sensitive, and selective method for the determination of histamine in human urine samples by ultrahigh pressure liquid chromatography (LC) with fluorescence and mass spectrometry (MS) detection is investigated. A fluorescent reagent, 4-(1-pyrene) butyric acid N-hydroxysuccinimide ester was conjugated to the primary and secondary amino moieties of histamine. The structure of dipyrene-labeled histamine in human urine was determined by quadrupole time-of-flight MS with electospray ionization interface. The determination of the dipyrene derivative of histamine in urine samples was achieved within 3.9 min on an ultrahigh pressure LC Eclipse Zorbax XDB-C(18) column with 1.8 μm particle diameter. In this work, histamine separation was achieved significantly faster (3.9 min) with improved detection limit (signal-to-noise = 3) of 0.04 nM than 19.5 min with a detection limit of 0.183 nM as reported in a previous method.     

Modification of a Capillary for Electrophoresis by Electrostatic Self‐Assembly of an Enzyme for Selective Determination of the Enzyme Substrate

The integration of a separation capillary for capillary electrophoresis (CE) with an on‐column enzyme reaction for selective determination of the enzyme substrate is described. Enzyme immobilization is achieved by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the GOx produces hydrogen peroxide which migrates the length of the capillary and is detected amperometrically at the capillary outlet. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre‐ or post‐separation chemical modification of the analyte. The enzyme reactor is found to have an optimal response to glucose when a 5 : 1 mixture of PSS:GOx is used. Under these conditions the limit of detection for glucose is found to be between 5.0×10^?4 and 1.3×10^?3 M, dependent upon the inner‐diameter of the capillary. The apparent Michaelis‐Menten constant for the enzyme reaction was determined to be 0.047 (±0.001) M and 0.0037 (±0.0007) M for a 50 and 10 μm inner‐diameter capillaries, respectively. These results indicate that the enzyme reaction is efficient, having enzyme kinetics similar to that of a reaction occurring in solution. This enzyme immobilization method was also applied to another enzyme, glutamate oxidase, yielding similar results.     

Determination of 3-hydroxy pterocarpan, a novel osteogenic compound in rat plasma by liquid chromatography-tandem mass spectrometry: application to pharmacokinetics study

A rapid, sensitive and selective LC‐MS/MS method for the quantitative analysis of 3‐hydroxy pterocarpan (S006‐1709) in female rat plasma has been developed and validated. A Discovery RP₁₈ column was used for the chromatographic elution using acetonitrile and 0.1% acetic acid in water as mobile phase (80:20 v/v) at the flow rate of 0.5 mL/min. MS/MS analysis was performed using a triple quadrupole mass spectrometer with electrospray ionization in negative ion mode using biochanin as an internal standard (IS). Extraction of S006‐1709 and IS from rat plasma was done by liquid–liquid extraction method using diethyl ether. The LC‐MS/MS method was sensitive with 1.95 ng/mL as the limit of detection and 3.9 ng/mL as the lower limit of quantification. The method was linear in the concentration range of 3.9–1000 ng/mL. The percentage bias for intraday and interday accuracy was not greater than 4.2 and the %RSD for intraday and interday precision was not greater than 13.2. The recoveries of S006‐1709 and IS were 73.9–79.3 and 85.7%, respectively. S006‐1709 was found to be stable in various stability studies. The validated LC‐MS/MS method was successfully applied for the oral pharmacokinetics study of S006‐1709 at 10 mg/kg in female Sprague–Dawley rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of ethylenethiourea as a biomarker in human urine using liquid chromatography/triple quadrupole mass spectrometry

Ethylenebisdithiocarbamates (EBDCs) are widely used fungicides. Ethylenethiourea (ETU), the main metabolite and also a contaminant in the commercially available products, is of major toxicological concern. In this study, a method using liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) is described for the analysis of ETU in human urine after a single-step extractive derivatization using pentafluorobenzyl bromide (PFBBr). Analysis was carried out using selected reaction monitoring (SRM) in the positive ion mode. Quantification of ETU was performed using [(2)H(4)]-labeled ETU as internal standard (IS). The limit of detection (LOD) was determined to 0.05 ng/mL. The method was linear in the range 0.1-54 ng/mL urine and had a within-run precision of 3-5%. The between-run precision was determined at an average urine level of 2 and 10 ng/mL urine and found to be 9%. The inter-batch precision was 6%. To validate ETU as a biomarker of exposure, the method was applied in a human experimental oral exposure to the commercial fungicide Ridomil Gold, containing 64% mancozeb and 4.5% ETU. Two healthy volunteers received 8.9 microg/kg body weight (b.w.) Ridomil Gold in a single oral dose followed by urine sampling for 104 h post-exposure. The elimination half-life of ETU was estimated to 17-23 h.     

Quantitation of the novel anticonvulsant remacemide in rat and dog plasma and urine: application of the plasma methodology to measure the plasma protein binding of remacemide.

Sensitive and selective methods have been developed for quantitation of the novel anticonvulsant remacemide in rat and dog plasma and urine. The methods employed liquid-liquid extraction (urine) or ion-exchange solid-phase extraction (plasma), with an internal standard, followed by high-performance liquid chromatography with ultraviolet detection. The detection limit for both methods was 10 ng/ml. Overall accuracy was 0.00% for plasma and -1.4% for urine with a precision of 6.04 and 3.87% for plasma and urine, respectively. The standard curves were linear for both plasma and urine over a wide concentration range (9.96-2490 ng/ml). The plasma method was also applied to measurement of in vitro plasma protein binding of remacemide in rat, dog and human plasma.     

Validation of a sensitive LC/MS/MS method for the determination of zidovudine and lamivudine in human plasma

A sensitive LC/MS/MS assay for determining zidovudine (ZDV) and lamivudine (3TC) in human plasma was validated to support antiretroviral pharmacology research programs. After addition of stable labeled isotopic zidovudine (ZDV‐IS) and lamivudine (3TC‐IS) as internal standard, a solid‐phase extraction was performed with an Oasis HLB 1 cm³ cartridge, with recoveries of 92.3% for ZDV and 93.9% for 3TC. A Phenomonex Synergi Hydro‐RP (2.0 × 150 mm) reversed‐phase analytical column was utilized for chromatographic separation. The mobile phase consisted of an aqueous solution of 15% acetonitrile and 0.1% acetic acid. Detection was accomplished by ESI/MS/MS in the positive ion mode, monitoring 268/127, 271/130, 230/112 and 233/115 transitions, for ZDV, ZDV‐IS, 3TC and 3TC‐IS, respectively. The method was linear from 1 to 3000 ng/mL with a minimum quantifiable limit of 1 ng/mL when 100 μL of plasma was analyzed. Validation results demonstrated high accuracy (≤8.3% deviation) and high precision (≤10% CV) for the quality control samples. The method was also shown to be specific and reproducible. The value of the high sensitivity was demonstrated by quantitation of approximately 100 existing samples that had ZDV below the limit of quantitation using a previously validated, less sensitive HPLC‐UV method utilized in the laboratory. Copyright © 2011 John Wiley & Sons, Ltd.     

蛋白质微阵列芯片技术及其在抗体筛选中的应用

以兔IgG为模式蛋白质,对其在醛基修饰玻片表面的固定浓度、固定时间和温度等条件进行了优化,结果表明：在室温下,当固定蛋白质的浓度为1g／L、固定时间为4h时,可获得理想的蛋白质固定效果;蛋白质的定量检测范围为1μg／L～10mg／L。按优化的蛋白质微阵列芯片制作条件将规模化制备的抗体制作成抗体微阵列芯片,通过与荧光标记的人球蛋白和人白蛋白的相互作用,实现了对不同抗体株抗球蛋白和抗白蛋白活性的快速筛选与比较。