Highly sensitive multianalyte immunochromatographic test strip for rapid chemiluminescent detection of ractopamine and salbutamol

A novel immunochromatographic assay (ICA) was proposed for rapid and multiple assay of β₂-agonists, by utilizing ractopamine (RAC) and salbutamol (SAL) as the models. Owing to the introduction of chemiluminescent (CL) approach, the proposed protocol shows much higher sensitivity. In this work, the described ICA was based on a competitive format, and horseradish peroxidase-tagged antibodies were used as highly sensitive CL probes. Quantitative analysis of β₂-agonists was achieved by recording the CL signals of the probes captured on the two test zones of the nitrocellulose membrane. Under the optimum conditions, RAC and SAL could be detected within the linear ranges of 0.50–40 and 0.10–50 ng mL⁻¹, with the detection limits of 0.20 and 0.040 ng mL⁻¹ (S/N = 3), respectively. The whole process for multianalyte immunoassay of RAC and SAL can be completed within 20 min. Furthermore, the test strip was validated with spiked swine urine samples and the results showed that this method was reliable in measuring β₂-agonists in swine urine. This CL-based multianalyte test strip shows a series of advantages such as high sensitivity, ideal selectivity, simple manipulation, high assay efficiency and low cost. Thus, it opens up new pathway for rapid screening and field analysis, and shows a promising prospect in food safety.     

A sensitive and semi-quantitative method for determination of multi-drug residues in animal body fluids using multiplex dipstick immunoassay

The objective of this research was to develop a multiplex dipstick immunoassay method for the simultaneous determination of multi-veterinary drug residues, such as β-agonists, sulfonamides, and tetracyclines in milk, urine, and serum. The multiplex dipstick assay format was based on an indirect competitive approach: Three test lines (different antigens) and one control line (goat anti-mouse IgG) were located on the strip membrane. Labeled antibodies were freeze-dried in microwells. Samples did not require pretreatment and could be directly analyzed within 10 min. Threshold levels in different sample matrices were visually estimated at 0.3–0.45 ng mL⁻¹ for clenbuterol; 3–4 ng mL⁻¹ for sulfadiazine; and 4.5–6 ng mL⁻¹ for tetracycline, respectively. The linear relationship between the concentrations of veterinary drug residues and the Au nanoparticles plasmon absorbance allowed quantitative determination of these veterinary drug residues. The recoveries of clenbuterol, sulfadiazine and tetracycline in spiked samples ranged from 78.4% to 112.6%, and the relative standard deviations were below 11.2%. Analysis of animal samples suggested that the proposed multiplex dipstick assay method was consistent with the LC-MS/MS method. The percentage of false results was less than or equal to 5%. Thus, the proposed multiplex dipstick assay is inexpensive, easy-to-use, and suitable for the purposes of rapid and comprehensive screening of 3 families of β-agonists, sulfonamides and tetracyclines including 26 drugs in animal body fluids.     

Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk

A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC₅₀) of cephalexin and cefadroxil were obtained at 1.5 ng mL⁻¹; IC₅₀ of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ng mL⁻¹, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ng mL⁻¹). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ng mL⁻¹ in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk.     

Surface plasmon resonance-based immunoassay for procalcitonin

A surface plasmon resonance (SPR) biosensor has been developed for rapid immunoassay of procalcitonin (PCT) with high detection sensitivity and reproducibility. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A (PrA), diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES) was dispensed on a KOH-treated Au-coated SPR chip, resulting in the covalent binding of PrA in 30 min. This “single-step” PrA immobilization strategy led to the oriented binding of the anti-PCT antibody (Ab) on a PrA-functionalized gold (Au) chip. The leach-proof immobilization procedure is five-fold faster than conventional counterparts, enabling high detection specificity and reproducibility. The IA detects 4–324 ng mL⁻¹ of PCT with a limit of detection (LOD) and a limit of quantification (LOQ) of 4.2 ng mL⁻¹ and 9.2 ng mL⁻¹, respectively. It was capable of detecting PCT in real sample matrices and patient samples with high precision. The Ab-bound SPR chips were stable for more than five weeks.     

Colloidal gold nanoparticle probe-based immunochromatographic assay for the rapid detection of chromium ions in water and serum samples

An immunochromatographic assay (ICA) using gold nanoparticles coated with monoclonal antibody (McAb) for the detection of chromium ions (Cr) in water and serum samples was developed, optimized and validated. Gold nanoparticles coated with affinity-purified monoclonal antibodies against isothiocyanobenzyl-EDTA (iEDTA)-chelated Cr³⁺ were used as the detecting reagent in this completive immunoassay-based one-step test strip. The ICA was investigated to measure chromium speciation (Cr³⁺ and Cr⁶⁺ ions) in water samples. Chromium standard samples of 0–80 ng mL⁻¹ in water were determined by the test strips. The results showed that the visual lowest detection limit (LDL) of the test strip was 50.0 ng mL⁻¹. A portable colorimetric lateral flow reader was used for the quantification of Cr. The results indicated that the linear range of the ICA with colorimetric detection was 5–80 ng mL⁻¹. The ICA was also validated for the detection of chromium ions in serum samples. The test trips showed high stability in that they could be stored at 37 °C for at least 12 weeks without significant loss of activity. The test strip also showed good selectivity for Cr detection with negligible interference from other heavy metals. Because of its low cost and short testing time (within 5 min), the test strip is especially suitable for on-site large-scale screening of Cr-polluted water samples, biomonitoring of Cr exposure, and many other field applications.     

Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal

A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB₁ was developed. The anti-idiotypic nanobody–alkaline phosphatase (Ab2β−Nb−AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB₁ were 2.69 and 0.35 ng mL⁻¹, respectively, with a linear range of 0.93–7.73 ng mL⁻¹. The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL⁻¹, and the IC₅₀ was 0.89 ± 0.09 ng mL⁻¹ with a linear range of 0.29–2.68 ng mL⁻¹. Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β−Nb−AP fusion protein based one-step competitive immunoassay for monitoring FB₁ contamination in cereals. The Ab2β−Nb−AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems.     

Improved porous silicon microarray based prostate specific antigen immunoassay by optimized surface density of the capture antibody

Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA – prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5 ng mL⁻¹, 80 pg mL⁻¹, and 800 fg mL⁻¹ when arraying the PSA antibody, H117 at the concentration 15 μg mL⁻¹, 35 μg mL⁻¹, and 154 μg mL⁻¹, respectively. We further investigated PSA spiked into human female serum in the range of 800 fg mL⁻¹ to 500 ng mL⁻¹. The microarray showed a LOD of 800 fg mL⁻¹ and a dynamic range of 800 fg mL⁻¹ to 80 ng mL⁻¹ in serum spiked samples.     

A naked-eye based strategy for semiquantitative immunochromatographic assay

It is critical to develop a cost-effective quantitative/semiquantitative assay for rapid diagnosis and on-site detection of toxic or harmful substances. Here, a naked-eye based semiquantitative immunochromatographic strip (NSI-strip) was developed, on which three test lines (TLs, TL-I, TL-II and TL-III) were dispensed on a nitrocellulose membrane to form the test zone. Similar as the traditional strip assay for small molecule, the NSI-strip assay was also based on the competitive theory, difference was that the analyte competed three times with the capture reagent for the limited number of antibody binding sites. After the assay, the number of TLs developed in the test zone was inversely proportional to the analyte concentration, thus analyte content levels could be determined by observing the appeared number of TLs. Taking aflatoxin B₁ as the model analyte, visual detection limit of the NSI-strip was 0.06 ng mL⁻¹ and threshold concentrations for TL-I–III were 0.125, 0.5, and 2.0 ng mL⁻¹, respectively. Therefore, according to the appeared number of TLs, the following concentration ranges would be detectable by visual examination: 0–0.06 ng mL⁻¹ (negative samples), and 0.06–0.125 ng mL⁻¹, 0.125–0.5 ng mL⁻¹, 0.5–2.0 ng mL⁻¹ and >2.0 ng mL⁻¹ (positive samples). That was to say, compared to traditional strips the NSI-strip could offer more parameter information of the target analyte content. In this way, the NSI-strip improved the qualitative presence/absence detection of traditional strips by measuring the content (range) of target analytes semiquantitatively.     

A bare-eye based one-step signal amplified semiquantitative immunochromatographic assay for the detection of imidacloprid in Chinese cabbage samples

A novel bare-eye based one-step signal amplified semi-quantitative immunochromatographic assay (SAS-ICA) was developed for detection of the pesticide imidacloprid. This method was based on competitive immunoreactions. Signal amplification was achieved by dual labeling of the test lines (TLs) on the strip using high affinity nanogold-biotinylated anti-imidacloprid mAb (BAb) and nanogold-streptavidin (Sa) probes. The relative color intensities of three TLs (TL-I, TL-II and TL-III) on a nitrocellulose (NC) membrane were used for direct visual analysis of the SAS-ICA strips, and could be used for semi-quantitation of analyte concentrations by observing what TLs disappeared in the amplification zone. Under optimized conditions, the following imidacloprid concentration ranges would be detected by visual examination of the SAS-ICA strip: 0–5 ng mL⁻¹ (negative samples), and 5–25 ng mL⁻¹, 25–250 ng mL⁻¹, 250–1000 ng mL⁻¹ and >1000 ng mL⁻¹ (positive samples). The sensitivity (the visual detection limit (VDL) of TL-III) and semi-quantitative analytical capacity (when TL-III disappeared completely) of the SAS-ICA strip were 10-fold and 160-fold higher than those of traditional ICA, respectively. The developed SAS-ICA strip was applied to the analysis of spiked and authentic contaminated Chinese cabbage samples in the laboratory and under field conditions, and the results were validated by high-performance liquid chromatography (HPLC). This process could be adopted as a potential generous technique for all ICA-based detection methods.     

Determination of beta-conglycinin in soybean and soybean products using a sandwich enzyme-linked immunosorbent assay

Soybean protein has long been recognized as a source of dietary allergens for humans and animals with β-conglycinin being the major allergen. This paper presents a sandwich enzyme-linked immunosorbent assay (ELISA) that allows for the detection of trace amount of β-conglycinin in soybean and soybean products. In the sandwich ELISA, mouse anti-β-conglycinin monoclonal antibody (Mab 5C5) was used as coating antibody, and rabbit anti-β-conglycinin polyclonal antibody (Pab) was used as secondary antibody. The assay showed high specificity for β-conglycinin with minimum cross-reactions with other soy proteins. The practical working range for the determination of β-conglycinin using the developed assay was 3–100 ng mL⁻¹ and the limit of determination (LOD) was 1.63 ng mL⁻¹. The recoveries of β-conglycinin in spiked soybean samples were between 88.1% and 106.6% with relative standard deviation less than 8.9% (intra-day) and 13.1% (inter-day). The developed method was used to analyze 469 soybean seed samples from different sources as well as five soybean products treated with different processing techniques. The data showed that the concentration of β-conglycinin decreased significantly after processing, especially for soybean protein isolation, where the concentration of β-conglycinin dropped to nearly zero. The assay provides a specific and sensitive method for the screening of β-conglycinin and allows for further investigation into hypersensitive mechanisms of soybean proteins and development of soybean processing techniques to reduce their negative effects.     

Direct hapten coated immunoassay format for the detection of atrazine and 2,4-dichlorophenoxyacetic acid herbicides

A novel immunoassay format employing direct coating of small molecular hapten on microtiter plates is reported for the detection of atrazine and 2,4-dichlorophenoxyacetic (2,4-D). In this assay, the polystyrene surface of microtiter plates was first treated with an acid to generate -NO₂ groups on the surface. Acid treated plates were further treated with 3-aminoprpyltriethoxysilane (APTES) to functionalize the plate surface with amino groups for covalent linkage to small molecular hapten with carboxyl groups. The modified plates showed significantly high antibody binding in comparison to plates coated with hapten-carrier protein conjugates and presented excellent stability as a function of the buffer pH and reaction time. The developed assay employing direct hapten coated plates and using affinity purified atrazine and 2,4-D antibodies demonstrated very high sensitivity, IC₅₀ values for atrazine and 2,4-D equal to 0.8 ng mL⁻¹ and 7 ng mL⁻¹, respectively. The assay could detect atrazine and 2,4-D levels in standard water samples even at a very low concentration upto 0.02 and 0.7 ng mL⁻¹ respectively in the optimum working range between 0.01 and 1000 ng mL⁻¹ with good signal reproducibility (p values: 0.091 and 0.224 for atrazine and 2,4-D, respectively). The developed immunoassay format could be used as convenient quantitative tool for the sensitive screening of pesticides in samples.     

Nanogold-penetrated poly(amidoamine) dendrimer for enzyme-free electrochemical immunoassay of cardiac biomarker using cathodic stripping voltammetric method

Methods based on immunoassays have been developed for cardiac biomarkers, but most involve the low sensitivity and are unsuitable for early disease diagnosis. Herein we design an electrochemical immunoassay for sensitive detection of myoglobin (a cardiac biomarker for acute myocardial infarction) by using nanogold-penetrated poly(amidoamine) dendrimer (AuNP-PAMAM) for signal amplification without the need of natural enzymes. The assay was carried out on the monoclonal mouse anti-myoglobin (capture) antibody-anchored glassy carbon electrode using polyclonal rabbit anti-myoglobin (detection) antibody-labeled AuNP-PAMAM as the signal tag. In the presence of target myoglobin, the sandwiched immunocomplex could be formed between capture antibody and detection antibody. Accompanying AuNP-PAMAM, the carried gold nanoparticles could be directly determined via stripping voltammetric method under acidic conditions. Under optimal conditions, the detectable electrochemical signal increased with the increasing target myoglobin in the sample within a dynamic working range from 0.01 to 500 ng mL⁻¹ with a detection limit of 3.8 pg mL⁻¹. The electrochemical immunoassay also exhibited high specificity and good precision toward target myoglobin. Importantly, our strategy could be applied for quantitative monitoring of myoglobin in human serum specimens, giving well matched results with those obtained from commercialized enzyme-linked immunosorbent assay (ELISA) method.     

A disposable chronocoulometric sensor for heavy metal ions using a diaminoterthiophene-modified electrode doped with graphene oxide

The rapid simultaneous determination of cadmium, lead, copper, and mercury ions is performed by employing a disposable sensor modified with graphene oxide (GO) doped diaminoterthiophene (GO/DTT) for chronocoulometry (CC). The performances of CC with and without pre-deposition in two opposite potential step directions were compared with square wave anodic stripping voltammetry (SWASV) under various conditions. The surface of the GO/DTT modified screen print carbon electrode (SPCE) was characterized by SEM, EDXS, and electrochemical impedance spectroscopy (EIS). Experimental variables that affect the response signal such as the pH, deposition time, type of supporting electrolyte, concentration of DTT, content ratio of GO to DTT, and Nafion content were optimized. Interference effects due to other heavy metal ions were also investigated. The dynamic ranges of SWASV and CC were between 1 ng mL⁻¹ and 2.5 μg mL⁻¹ and between 1 ng mL⁻¹ and 10 μg mL⁻¹, respectively. The detection limits for Cd²⁺, Pb²⁺, Cu²⁺, Hg²⁺ ions were 1.9 ± 0.4 ng mL⁻¹, 2.8 ± 0.6 ng mL⁻¹, 0.8 ± 0.2 ng mL⁻¹, and 2.6 ± 0.9 ng mL⁻¹ for the CC stripping method; 2.6 ± 0.2 ng mL⁻¹, 0.5 ± 0.1 ng mL⁻¹, 1.8 ± 0.3 ng mL⁻¹, and 3.2 ± 0.3 ng mL⁻¹ for the CC deposition method; and 7.1 ± 0.9, 1.9 ± 0.3, 0.4 ± 0.1, and 0.7 ± 0.1 ng mL⁻¹ for SWASV. The reliability of the method for point-of-analysis was evaluated by analyzing a urine standard reference material and some water samples.     

Magnetic-particle-based,ultrasensitive chemiluminescence enzyme immunoassay for free prostate-specific antigen

We report a magnetic-particle (MMP)-based chemiluminescence enzyme immunoassay (CLEIA) for free prostate-specific antigen (f-PSA) in human serum. In this method, the f-PSA is sandwiched between the anti-PSA antibody coated MMPs and alkaline phosphatase (ALP)-labeled anti-f-PSA antibody. The signal produced by the emitted photons from the chemiluminescent substrate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2′-adamantane)) is directly proportional to the amount of f-PSA in a sample. The present MMP-based assay can detect f-PSA in the range of 0.1–30 ng mL⁻¹ with the detection limit of 0.1 ng mL⁻¹. The linear detection range could match the concentration range within the “diagnostic gray zone” of serum f-PSA levels (4–10 ng mL⁻¹). The detection limit was sufficient for measuring clinically relevant f-PSA levels (>4 ng mL⁻¹). Furthermore, the method was highly selective; it was unaffected by cross-reaction with human glandular kallikrein-2, a kallikrein-like serine protease that is 80% similar to f-PSA. The proposed method was finally applied to determine f-PSA in 40 samples of human sera. Results obtained using the method showed high correlation with those obtained using a commercially available microplate CLEIA kit (correlation coefficient, 0.9821). This strategy shows great potential application in the fabrication of diagnostic kits for determining f-PSA in serum.     

An ultrasensitive streptavidin-functionalized carbon nanotubes platform for chemiluminescent immunoassay

An ultrasensitive chemiluminescent (CL) immunoassay system was developed for the detection of tumor marker. This sandwich CL assay method was for the first time designed based on a highly efficient streptavidin-functionalized multi-walled carbon nanotubes (MWCNTs) platform. The glass slide was firstly silylanized with 3-gycidoxypropyltrimethoxysilane (GPTMS) to generate surface epoxy group functionality. Subsequently, the MWCNTs/chitosan solution was mixed with streptavidin solution, and a certain amount of the resulting suspension was dropped on the surface of the epoxy-activated glass substrate to form a firm streptavidin-functionalized MWCNTs platform. The biofunctionalized-MWCNTs platform shows large reactive surface area and excellent biocompatibility. The capture antibody can be efficiently immobilized on the biosensing platform surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as model analyte, the proposed method exhibits wide linear range of 0.001–0.1 ng mL⁻¹ with a low detection limit down to 0.52 pg mL⁻¹. The CL immunoassay system displays 7.9-fold increase in the detection sensitivity compared to the immunosensor without using MWCNTs. Moreover, the resulting immunosensor demonstrates excellent specificity, good reproducibility, and acceptable stability. This streptavidin-functionalized MWCNTs platform opens a novel and promising avenue for fabricating ultrasensitive CL immunoassay system.     

Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork

A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V_H and V_L) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V_H and V_L genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 ± 0.03 and 0.02 ± 0.004 ng mL⁻¹, respectively, and the linear response range extended from 0.05 to 1.45 ng mL⁻¹. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS). The results showed a good correlation between the data of dc-CLEIA and HPLC–MS (R² > 0.99), indicating that the assay was an efficient analytical method for monitoring food safety.     

Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810)

The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development.The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL⁻¹ with a decision limit (CCα) of 1.5 ng mL⁻¹ and detection capability (CCβ) of 2.3 ng mL⁻¹. Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma.In total, 20 plasma samples from cows (n = 7) fed non-transgenic maize and 24 samples from cows (n = 8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL⁻¹; CCα). No plasma sample was positive for the presence of the Cry1Ab protein at CCα and CCβ of the assay.     

Analysis of β-agonists and β-blockers in urine using hollow fibre-protected liquid-phase microextraction with in situ derivatization followed by gas chromatography/mass spectrometry

A method using hollow fibre-protected liquid-phase microextraction (HF-LPME) with in situ derivatization followed by gas chromatography/mass spectrometry (GC/MS) was established for the analysis of β-agonists and β-blockers in urine. Because it can simultaneously extract and derivatize compounds of interest by methylbenzol and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) in HF-LPME, the approach overcomes the drawbacks of considerable time-consuming and tedious operation, meanwhile improves enrichment multiple. The optimized conditions were extraction for 20 min at 35 °C with 5.0 μL of mixed extraction solvent (methylbenzol/MSTFA = 1:1, v/v) with stirring speed of 925 rpm in 5.0 mL sample under pH 12.0 and 14% (w/v) NaCl. The method provided very wide linear ranges (0.25–400 ng mL⁻¹) and low detection limits in the range of 0.08–0.10 ng mL⁻¹ for clenbuterol, metoprolol and propranolol while enrichment factors reached up to 256. The analytes could be determined in spiked urine by the method with high extraction efficacy (93.79–109.04% recoveries) and precision (<9.70% RSD). It has a satisfactory result for metoprolol in practical human urine samples for a single-dose administration of 50 mg after 36 h. The proposed method only needs few microliters of organic solvent and derivatizing agent; the operation is simple, convenient and rapid for the trace analysis of β-agonists and β-blockers in biological fluids; it can be readily generalized for high sample throughput. So, it is hopeful that the study will facilitate the monitoring of β-agonists and β-blockers in the competition sports.     

Amorphous carbon nanoparticle used as novel resonance energy transfer acceptor for chemiluminescent immunoassay of transferrin

Amorphous carbon nanoparticles (ACNPs) showing highly efficient quenching of chemiluminescence (CL) were prepared from candle soot with a very simple protocol. The prepared ACNP was employed as the novel energy acceptor for a chemiluminescence resonance energy transfer (CRET)-based immunoassay. In this work, ACNP was linked with transferrin (TRF), and horseradish peroxidase (HRP) was conjugated to TRF antibody (HRP–anti-TRF). The immunoreaction rendered the distance between the ACNP acceptor and the HRP-catalyzed CL emitter to be short enough for CRET occurring. In the presence of TRF, this antigen competed with ACNP–TRF for HRP–anti-TRF, thus led to the decreased occurrence of CRET. A linear range of 20–400 ng mL⁻¹ and a limit of detection of 20 ng mL⁻¹ were obtained in this immunoassay. The proposed method was successfully applied for detection of TRF levels in human sera, and the results were in good agreement with ELISA method. Moreover, the ACNPs show higher energy transfer efficiency than other conventional nano-scaled energy acceptors such as graphene oxide in CRET assay. It is anticipated that this approach can be developed for determination of other analytes with low cost, simple manipulation and high specificity.     

Application of direct-injection detector integrated with the multi-pumping flow system to chemiluminescence determination of the total polyphenol index

In this work, we present a novel chemiluminescence (CL) method based on direct-injection detector (DID) integrated with the multi-pumping flow system (MPFS) to chemiluminescence determination of the total polyphenol index. In this flow system, the sample and the reagents are injected directly into the cone-shaped detection cell placed in front of the photomultiplier window. Such construction of the detection chamber allows for fast measurement of the CL signal in stopped-flow conditions immediately after mixing the reagents. The proposed DID-CL-MPFS method is based on the chemiluminescence of nanocolloidal manganese(IV)-hexametaphosphate-ethanol system. The application of ethanol as a sensitizer, eliminated the use of carcinogenic formaldehyde. Under the optimized experimental conditions, the chemiluminescence intensities are proportional to the concentration of gallic acid in the range from 5 to 350 ng mL⁻¹. The DID-CL-MPFS method offers a number of advantages, including low limit of detection (0.80 ng mL⁻¹), high precision (RSD = 3.3%) and high sample throughput (144 samples h⁻¹) as well as low consumption of reagents, energy and low waste generation. The proposed method has been successfully applied to determine the total polyphenol index (expressed as gallic acid equivalent) in a variety of plant-derived food samples (wine, tea, coffee, fruit and vegetable juices, herbs, spices).