Precise characterization method of antibody‐conjugated magnetic nanoparticles for pathogen detection using stuffer‐free multiplex ligation‐dependent probe amplification

Antibody‐conjugated magnetic nanoparticles (Ab‐MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab‐MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab‐MNPs as a pathogen detection method requires accurate characterization of Ab‐MNP capture ability and standardization of all handling processes. In this study, we used high‐resolution CE‐single strand conformational polymorphism coupled with a stuffer‐free multiplex ligation‐dependent probe amplification system to characterize Ab‐MNPs. The capture ability of Ab‐MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab‐MNPs was also assessed. The results showed that the stuffer‐free multiplex ligation‐dependent probe amplification system has the potential to serve as a standard characterization method for Ab‐MNPs. Moreover, the precise characterization of Ab‐MNPs facilitated robust pathogen detection in various applications.     

Quantitative determination of amphetamine in plasma using negative ion chemical ionization GC‐MS of o‐(pentafluorobenzyl‐oxycarbonyl)‐2,3,4,5‐tetrafluorobenzoyl derivatives

Quantitative determination of amphetamine in plasma by the use of a novel electrophoric derivatization reagent, o‐(pentafluorobenzyloxycarbonyl)‐2,3,4,5‐tetrafluorobenzoyl chloride is described. Amphetamine can be quantitatively measured down to 49 pg/mL plasma using only 250 μL of sample due to the extraordinary sensitivity of the derivatives under negative ion chemical ionization MS. Plasma samples were made alkaline with carbonate buffer and treated with n‐hexane and reagent solution for 20 min, which, after concentration was measured by negative ion chemical ionization GC‐MS. The method is rapid as extraction and derivatization occur in one single step. [²H₅]‐Amphetamine was used as an internal standard. Validation data are given to demonstrate the usefulness of the assay, including specificity, linearity, accuracy and precision, benchtop stability, freeze–thaw stability, autosampler stability, aliquot analysis, and prospective analytical batch size accuracy.     

Metal Enhanced Electrochemical Cyclooxygenase‐2 (COX‐2) Sensor for Biological Applications

Pain measurement is commonly required in biomedical and other emergency situations, yet there has been no pain biosensor reported in literature. Conventional approaches for pain measurement relies on Wong‐Baker face diagrams, which are grossly inadequate for situations involving children or unconscious people. We report a label‐free immunosensor for monitoring the pain biomarker cylooxygenase‐2 (COX‐2) in blood. The sensor is based on the concept of metal‐enhanced detection (MED). MED relies on the idea that the immobilization of underpotential deposition (upd) metallic films deposited either as a monolayer or electrostatically held onto a solid gold substrate could significantly amplify bimolecular recognition such as involving antigen‐antibody (Ab‐Ag) interactions. The surface bound Ab‐Ag complex insulates the electrode; causing a decrease in concentration‐dependent redox signals. A linear detection range of (3.64–3640.00)×10^?4 ng/mL was recorded with a detection limit of 0.25×10^?4 ng/mL, which was 4 orders of magnitude lower than that reported for ELISA for the same biomarker. The immunosensor exhibited selectivity of less than 6 % for potential interferents.     

A novel freeze‐dried storage and preparation method for the determination of mycophenolic acid in plasma by high‐performance liquid chromatography

Plasma samples were conventionally stored at freezing conditions until the time of detection. Such a technique, when carried out over an extended period, is energy consuming; in addition, preparation and transportation of stored samples is inconvenient. In this study, a freeze‐dried storage and preparation method was proposed to determine the presence of mycophenolic acid (MPA) in plasma. Fresh plasma samples were freeze‐dried using a device, and then stored at ambient temperature. After the stored samples were soaked with methanol spiked with the internal standard, high‐performance liquid chromatography was conducted to detect MPA. The proposed method was demonstrated to be precise and accurate over the linear range of 0.5–50 μg mL⁻¹, with both intra‐ and inter‐day precision being <7% and biases <10%. The freeze‐dried samples were stable at ambient temperature for at least 40 days. This method was also successfully applied to the pharmacokinetic study of MPA in healthy volunteers. Pharmacokinetic parameters, such as maximum plasma concentration, time point of maximum plasma concentration and elimination half‐life, among others, were consistent with the results in the published study. This proposed technique was proved to be simple, reproducible and energy saving. This approach could also simplify the storage and analysis of samples in clinical and scientific drug research.     

Two‐step signal amplification for high‐sensitivity detection of biomarkers using gold nanoparticle–based conjugates

The measurement of biomarkers in bodily fluids is extremely important for diagnosing disease, monitoring disease progression, and evaluating treatment efficacy. In this paper, we present a highly sensitive and compatible gold nanoparticle (AuNP)‐based, two‐step signal amplification system for biomarker detection. First, AuNPs were coated onto the surfaces of 96‐well plates to generate rough surfaces, which enable immobilization of many more capture antibodies than a smooth substrate. As a result, detection sensitivity was enhanced significantly. Second, the horseradish peroxidase (HRP)‐conjugated detection antibodies were labeled on large‐size AuNPs, which increase the localized amounts of HRP and thus further lower the detection limit. Based on the consecutive signal amplification system, a high‐sensitivity assay was achieved, with a LOD of 0.07 ng/mL for prostate‐specific antigen (PSA). This assay was allowed to detect the PSA levels in clinical samples without changing the current standard immunoassay setups, showing great potential in many settings where immunoassays are needed.     

Enhancement of sensitivity and specificity of the fluoroimmunoassay of Hepatitis B virus surface antigen through “flexible” coupling between quantum dots and antibody

Quantum dots (QDs) are widely used in the immune detection. Yet, the sensitivity and specificity of the immune detection are not satisfactory because the binding sites of QDs onto antibody (Ab) are often arbitrary and the influence of the large surface electronic potential energy of QDs on the directly conjugated Ab is nonnegligible. In this work, we provide a “flexible” coupling method, in which protein G (PG) is selected as the flexible bridge between the QDs and the Hepatitis B virus surface antibody (HBsAb), to improve the sensitivity and specificity of the fluoroimmunoassay compared to the directly covalent conjugation. Successful coupling of the HBsAb to our highly luminescent CdTe/CdS core/shell QDs is proven with Gel electrophoresis and atomic force microscopy (AFM). The assay results, based on the microelisa well plate as matrix to immobilize the sandwich structure, show that both sensitivity and specificity can be improved greatly through the flexible coupled QDs-PG-Ab conjugates.     

Design of Sensitive Biocompatible Quantum‐Dots Embedded in Mesoporous Silica Microspheres for the Quantitative Immunoassay of Human Immunodeficiency Virus‐1 Antibodies

A novel sandwich‐type electrochemiluminescence (ECL) immunosensor was developed to enable the sensitive detection of HIV‐1 antibodies. This system incorporated mesoporous silica (mSiO₂) complexed with quantum dots (QDs) and nano‐gold particles, which were assembled to enhance signal detection. Magnetic beads were used by immobilizing the secondary anti‐IgG antibody. This was first employed to capture HIV‐1 antibody (Ab) to form a Fe₃O₄/anti‐IgG/Ab complex. A high loading and signal‐enhanced nanocomposite (hereafter referred to as Au‐mSiO₂‐CdTe) was used as a HIV‐1 antigen label. The Au‐mSiO₂‐CdTe nanocomposite was conjugated with the Fe₃O₄/anti‐IgG/Ab complex to form an immunocomplex (hereafter referred to as Fe₃O₄/anti‐IgG/Ab/HIV‐1/CdTe‐mSiO₂‐Au). This complex could be further separated by an external magnetic field to produce ECL signals. Due to the large specific surface area and pore volume of mSiO₂, the loading of the CdTe QDs was markedly increased. Thus, the loaded QDs released a powerful chemiluminescent signal with a concordantly increased sensitivity of the immunosensor. The immunosensor was highly sensitive, and displayed a linear range of responses for HIV‐1 antibody across a dilution range of 1 : 1500 through 1 : 50 with the detection limit of 1 : 4500. The immunoassay can be a promising candidate in early diagnosis of HIV infection.     

Characterization of solid standards prepared by freeze‐drying

Solid standards prepared by freeze‐drying consistently showed a high degree of homogeneity. The freezing process, completed in fractions of a second, preserves the original homogeneous distribution of the dopants, and the subsequent sublimation step would minimize any disturbance. Compared to those prepared by conventional methods such as blending and spiking, freeze‐dried standards exhibited superior lateral distribution and better uniform depth distribution. There is, however, a concentration constraint for achieving homogeneity. At 5% doping, segregation was observed in both lateral and depth distribution. Many tungsten standards doped with 10–28 elements ranging from 10 to 200 ppm were successfully prepared and used as controls for a number of analytical techniques including glow discharge mass spectrometry (GDMS) and d.c.‐arc optical emission spectroscopy (OES). Copyright © 2009 John Wiley & Sons, Ltd.     

Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze‐dried powder

A simple and rapid liquid chromatography–mass spectrometry (LC‐MS) method was developed and validated for analysis of ginsenoside Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁, icariin and epimedin A, B, C in rat target tissues (spinal cord, brain, muscle and sciatic nerve) after intravenous administration of Jiweiling freeze‐dried powder using genistein as an internal standard (IS). The tissue samples were treated by protein precipitation with methanol prior to HPLC and chromatographic separation was performed on a C₁₈ column utilizing a gradient elution program with acetonitrile and 0.1% formic acid aqueous. Electrospray ionization (ESI) source was employed and the 11 analytes and IS were detected by multiple reaction monitoring (MRM) scanning under the negative ionization mode. Higher sensitivity was achieved and the optimized mass transition ion‐pairs (m/z) for quantitation were selected. The calibration curves were linear over the investigated concentration ranges with correlation coefficients higher than 0.995. The intra‐ and inter‐day RSDs were all less than 10% with the relative error (RE) within ±9.3%. The mean extraction recoveries for all compounds were between 93.3 and 106%. The proposed method was successfully applied to investigate the target tissue distribution of the 11 compounds in rat after intravenous administration of Jiweiling freeze‐dried powder. Copyright © 2011 John Wiley & Sons, Ltd.     

Differential scanning calorimetry study on thermal behaviors of freeze‐dried poly(L‐lactide) from dilute solutions

Glass transition, cold crystallization, and melting of freeze‐dried poly(L‐lactide) (PLLA) prepared from dilute 1,4‐dioxane solutions were investigated by differential scanning calorimetry (DSC). Conventional DSC measurements of heating scans revealed that freeze‐dried PLLA prepared from a 0.07 wt % solution undergoes a two‐step cold crystallization (or reorganization) with a lower exotherm appearing at about 78 °C and with a higher broad exotherm between 110–155 °C. The peak temperature of the former exotherm is about 50 K lower than that observed for a reference bulk sample. Step‐scan mode DSC, which provides information essentially equivalent to that obtained from the temperature‐modulated DSC, revealed that the glass‐transition temperature is about 6 K lower than that of the reference bulk. These findings suggest enhanced chain mobility for freeze‐dried PLLA. Freeze‐dried PLLA that crystallized at 80 °C for 40 min was revealed to contain a rather large amount of rigid amorphous material (42%). © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 115–124, 2005     

Biomolecule‐Doped Organic/Inorganic Hybrid Nanocomposite Film for Label‐Free Electrochemical Immunoassay of α‐1‐Fetoprotein

A new electrochemical immunosensor for the detection of α‐1‐fetoprotien (AFP) was developed based on AFP antibody (anti‐AFP)‐functionalized organic/inorganic hybrid nanocomposite membrane. To fabricate such a hybrid composite membrane, 3,4,9,10‐perylenetetracarboxylic acid‐bound thionine molecules (PTCTH) were initially doped into titania colloids (TiO₂), and then gold nanoparticles and anti‐AFP were immobilized onto the composite film in turn. Comparison with the electrode fabricated only with thionine not 3,4,9,10‐perylenetetracarboxylic acid, the immunosensor with PTCTH exhibited high sensitivity and fast electron transfer. The presence of gold nanoparticles provided a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The modified process was characterized by scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The surface topography of the membrane was investigated by scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 2.5 to 200.0 ng/mL towards AFP with a detection limit of 0.5 ng/mL (S/N=3). The stability, reproducibility and precision of the immunosensor were acceptable. Comparison with the conventional enzyme‐linked immunosorbent assay (ELISA), the present method did not require more labeled procedures and washing steps. Significantly, the detection methodology provides a promising approach for other proteins or biosecurities.     

Double‐Layer Nanogold and Double‐Strand DNA‐Modified Electrode for Electrochemical Immunoassay of Cancer Antigen 15‐3

Various sensor‐based immunoassay methods have been extensively developed for the detection of cancer antigen 15‐3 (CA 15‐3), but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. The aim of this work is to develop a simple and sensitive electrochemical immunoassay for CA 15‐3 in human serum by using nanogold and DNA‐modified immunosensors. Prussian blue (PB), as a good mediator, was initially electrodeposited on a gold electrode surface, then double‐layer nanogold particles and double‐strand DNA (dsDNA) with the sandwich‐type architecture were constructed on the PB‐modified surface in turn, and then anti‐CA 15‐3 antibodies were adsorbed onto the surface of nanogold particles. The double‐layer nanogold particles provided a good microenvironment for the immobilization of biomolecules. The presence of dsDNA enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The performance and factors influencing the performance of the immunosensor were evaluated. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 1.0 to 240 ng/mL with a relatively low detection limit of 0.6 ng/mL (S/N=3) towards CA 15‐3. The stability, reproducibility and precision of the as‐prepared immunosensor were acceptable. 57 serum specimens were assayed by the developed immunosensor and standard enzyme‐linked immunosorbent assay (ELISA), respectively, and the results obtained were almost consistent. More importantly, the proposed methodology could be further developed for the immobilization of other proteins and biocompounds.     

Liquid chromatography quadrupole time‐of‐flight mass spectrometry identification and determination of tri‐ and hexaaryl chloro imidazoles in sewage sludge

The identification and further quantification of 2‐chloro‐triarylimidazole (o‐Cl‐TAI) and its dimer (o‐DCl‐HABI) in sludge from a sewage treatment plant (STP) is reported for the first time. Liquid chromatography (LC) quadrupole time‐of‐flight (QTOF) mass spectrometry (MS) was used as analytical technique during screening and determination steps. Pollutants were identified following a post‐run search strategy, applying the chlorine mass filter, and characterized by their accurate MS and product ion scan spectra. Finally, their identities were confirmed with authentic standards. The species (o‐Cl‐TAI) has been rated as potentially genotoxic and carcinogenic for mice and rats. Effects of sample preparation in the stability and the extraction efficiency of both compounds are discussed. Under final conditions, they were extracted from freeze‐dried samples (0.5 g of sludge or biosolids dispersed with 2 g of C₁₈ and packed into a polypropylene syringe) with 20 ml of methanol, which also flowed through a clean‐up layer of Florisil and PSA sorbents (0.5 g each). This method attained quantitative extraction yields and limits of quantification between 4 and 10 ng/g. The pollutants o‐Cl‐TAI and o‐DCl‐HABI were ubiquitous in sludge and biosolids obtained in consecutive years from the investigated STP. Their concentrations varied from 0.02 to more than 13 μg/g (freeze‐dried sample). Copyright © 2016 John Wiley & Sons, Ltd.     

Development of Electrochemical Paper‐based Glucose Sensor Using Cellulose‐4‐aminophenylboronic Acid‐modified Screen‐printed Carbon Electrode

The present work describes the fabrication of paper‐based analytical devices (μPADs) by immobilization of glucose oxidase onto the screen printed carbon electrodes (SPCEs) for the electrochemical glucose detection. The sensitivity towards glucose was improved by using a SPCE prepared from homemade carbon ink mixed with cellulose acetate. In addition, 4‐aminophenylboronic acid (4‐APBA) was used as a redox mediator giving a lower detection potential for improvement selectivity. Under optimized condition, the detection limit was 0.86 mM. The proposed device was applied in real samples. This μPAD has many advantages including low sample consumption, rapid analysis method, and low device cost.     

Amperometric Immunosensor for the Determination of α‐1‐Fetoprotein Based on Core‐Shell‐Shell Prussian Blue‐BSA‐Nanogold Functionalized Interface

In the present work, a newly functional nanoparticle has been prepared to immobilize the protein for the detection of α‐1‐fetoprotein (AFP). Prussian blue (PB) nanoparticle was initially synthesized under ultrasonic condition, then bovine serum albumin (BSA) was used to coat the PB nanoparticle to improve the stability of the PB nanoparticle as well as functionalize the surface of PB nanoparticle, and then gold colloids were loaded on the BSA‐coated PB nanoparticle to construct a core‐shell‐shell nanostructure via the conjunction of thiolate linkages or alkylamines of the BSA. Finally, a convenient, effective and sensitivity amperometric immunosensor for the detection of α‐1‐fetoprotein (AFP) was constructed by the employment of these functional core‐shell‐shell microspheres. The preparation of the nanoparticle (Au‐BSA‐PB NPs) was characterized by transmission electron microscopy (TEM), and the assembly of the biosensor was characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The dynamic range of the resulted immunosensor for the detection of AFP is from 0.02 ng/mL to 200.0 ng/mL with a detection limit of 0.006 ng/mL (S/N=3). Moreover, this biosensor displays good selectivity, stability and reproducibility.     

Sensitive Electrochemical Detection of Horseradish Peroxidase at Disposable Screen‐Printed Carbon Electrode

A rapid, simple and sensitive electrochemical assay of horseradish peroxidase (HRP) performed on disposable screen‐printed carbon electrode was developed. HRP activities were monitored by square‐wave voltammetric (SWV) measuring the electroactive enzymatic product in the presence of o‐aminophenol and hydrogen peroxide substrate solution. SWV analysis demonstrated a greater sensitivity and shorter analysis time than the widely used amperometric and differential‐pulsed voltammetric methods. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were optimized. Under optimized conditions, a linear response for HRP from 0.003 to 0.1 U/mL and a detection limit of 0.002 U/mL (1.25×10^?15 mol in 25 μL) were obtained with a good precision (RSD=8%; n=6). This rapid and sensitive HRP assay with microliter‐assay volume could be readily integrated to portable devices and point‐of‐care (POC) diagnosis applications.     

Anchoring Iron‐EDTA Complex on Graphene toward the Synthesis of Highly Efficient Fe‐N‐C Oxygen Reduction Electrocatalyst for Fuel Cells

Developing nonprecious carbon electrocatalysts as alternatives to platinum for cathodic oxygen reduction reaction in fuel cells is of significance. Herein, an efficient precursor‐controlled synthesis strategy based on extremely rapid nucleation and deposition process assisted by the liquid nitrogen freeze drying method is explored to anchor cheap iron‐EDTA complex evenly dispersed on graphene to realize microstructural homogeneity of the derived Fe‐N‐C oxygen reduction electrocatalyst. The prepared electrocatalyst possesses excellent performance including high activity with more positive onset and half‐wave potential, such long‐term stability, and anti‐poisoning effect compared to commercial Pt/C. The activity correlates well with the unique sheet‐shaped morphology, high surface area, hierarchical porous structure, and the introduction of Fe‐Nx/C species. Especially, both the assembled practical alkaline and acid fuel cells based on the synthesized cathode catalysts reveal excellent performance with high open‐circuit voltage and power density.     

Humic acid‐derived hierarchical porous carbon preparation using vacuum freeze‐drying for electric double layer capacitors

Electric double layer capacitors (EDLCs) preserve charge by reversible physisorption of electrolyte ions on the surface of porous active materials. Therefore, engineering a reasonable pore structure and reducing oxygen‐containing groups of carbon materials are efficient approaches to enable rapid ion diffusion pathways and long life span, respectively. Here, humic acid (HA)‐derived hierarchical porous carbon was fabricated by vacuum freeze‐drying, KOH activation, and subsequent annealing. The macropores were generated from the vacancies where the ice crystals in the HA–KOH gels initially occupied during vacuum‐freeze drying, while abundant micropores were created from homogeneous KOH activation. In addition, subsequent annealing further reduced the oxygen‐containing groups. When used as EDLC electrodes in 1 mol/L TEABF₄/PC organic electrolyte, they could give a high capacitance of 150 F/g at 0.05 A/g and excellent rate performance of 81% (with capacitance of 121.46 F/g at 10 A/g). More importantly, the hierarchical porous carbon displays superior capacity retention of 85.6% after 10,000 cycles at 1 A/g at 2.7 V.     

High‐throughput polymer tip‐electrospray ionization mass spectrometry for enhanced detection of neopterin and biopterin in clinical urine samples

Urinary biopterin (Bio) and neopterin (Neo) are important markers for clinical diagnosis of hyperphenylalaninemia. Herein, we developed a high‐throughput analysis method based on electrospray ionization mass spectrometry (ESI‐MS) with polymer tips for the rapid quantitative detection of Bio and Neo in clinical urine samples. Different polymer tips were investigated. It is found that the best detection sensitivity was achieved with hydrophobic polymer tip, ie, polyethylene tips. The high‐throughput polymer tip‐ESI‐MS method allowed a rapid analysis speed at ~40 seconds per sample. The limits of quantification (LOQ) (S/N ≥ 10) for the detection of Bio and Neo were improved to be 5.0 ng/mL. Acceptable relative standard deviation (RSD) values for Neo and Bio were measured to be 12.2% and 13.4% for direct measurement of Bio and Neo in raw urine samples, respectively. Furthermore, Bio and Neo were directly quantified from 18 clinical urine samples by presented method. The ratios of urinary Bio‐to‐Neo were analyzed for diagnosis of hyperphenylalaninemia. The results demonstrated that the present polymer tip‐ESI‐MS method is a promising strategy for the rapid analysis of clinical samples.     

Constructing Mie‐Scattering Porous Interface‐Fused Perovskite Films to Synergistically Boost Light Harvesting and Carrier Transport

Light harvesting (LH) and carrier transport abilities of a photoactive layer, which are both crucial for optoelectronic devices such as solar cells and photodetectors (PDs), are typically hard to be synergistically improved. Taking perovskite as an example, a freeze‐drying recrystallization method is used to construct porous films with improvements of both LH and carrier transport ability. During the freeze‐drying casting process, the rapid solvent evaporation produces massive pores, the sizes of which can be adjusted to exploit the Mie scattering for enhancement of the LH ability. Meanwhile, owing to the strong iconicity, the interface between perovskite nanocrystals fused during recrystallization, which favors carrier transport. Subsequently, PDs based on these Mie porous and interface‐fused films show a high on/off ratio of more than 10⁴ and an external quantum efficiency value of 658 % under 9 V bias and 520 nm light irradiation.