Development of an automatic multi-channel ink-jet ejection chemiluminescence system and its application to the determination of horseradish peroxidase

In this work, an automatic multi-channel ink-jet for chemiluminescence (CL) analysis was developed. The four-channel ink-jet device was controlled by a home-made circuit. Differing from the classic flow injection CL, the whole procedure for CL analysis was automatically completed on a hydrophobic glass side. CL reaction of luminal and hydrogen peroxide for the determination of horseradish peroxidase (HRP) was selected as an application to automatic CL analysis platform. All solutions delivered by different channels were precisely ejected to the same position of the glass slide for the CL analysis. The consumption of reaction solution was reduced to nanoliter level. The whole CL analysis could be completed in less than 4 min, which was benefited from the prompt solution mixing in small size of droplet. The CL intensity increased linearly with HRP concentration in the range from 0.01 to 0.5 μg mL⁻¹. The limit of detection (LOD) (S/N = 3) was 0.005 μg mL⁻¹. Finally, the automatic CL system could also be used for the detection of HRP in HRP–protein conjugates, which showed its practical application in immunoassay.     

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.     

Flow injection chemiluminescence determination of the total phenolics levels in plant-derived beverages using soluble manganese(IV)

This study established a flow injection (FI) methodology for the determination of the total phenolic content in plant-derived beverages based on soluble manganese(IV) chemiluminescence (CL) detection. It was found that mixing polyphenols with acidic soluble manganese(IV) in the presence of formaldehyde evoked chemiluminescence. Based on this finding, a new FI-CL method was developed for the estimation of the total content of phenolic compounds (expressed as milligrams of gallic acid equivalent per litre of drink) in a variety of wine, tea and fruit juice samples. The proposed method is sensitive with a detection limit of 0.02 ng mL⁻¹ (gallic acid), offers a wide linear dynamic range (0.5-400 ng mL⁻¹) and high sample throughput (247 samples h⁻¹). The relative standard deviation for 15 measurements was 3.8% for 2 ng mL⁻¹ and 0.45% for 10 ng mL⁻¹ of gallic acid. Analysis of 36 different samples showed that the results obtained by the proposed FI-CL method correlate highly with those obtained by spectrophotometric methods commonly used for the evaluation of the total phenolic/antioxidant level. However, the FI-CL method was found to be far simpler, more rapid and selective, with almost no interference from non-phenolic components of the samples examined.     

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.     

Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum

A novel immunoassay for the determination of tumor markers in human serum was established by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a sandwich-type immunoassay format, analytes in samples were captured by magnetic beads coated with one monoclonal antibody and “sandwiched” by another monoclonal antibody labeled with europium chelates. The immunocomplex was separated and washed by exposure to a magnetic field and treatment with enhancement solution; fluorescence was then measured according to the number of europium ions dissociated. Levels of the model analyte, carcinoembryonic antigen (CEA), were determined in a linear range (1–1000 ng mL⁻¹) with a limit of detection of 0.5 ng mL⁻¹ under optimal conditions. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. To evaluate this novel assay for clinical applications, 239 serum samples were evaluated. Compared with the conventional TRFIA and chemiluminescence immunoassay (CLIA), the correlation coefficients of the developed immunoassay were 0.985 and 0.975, respectively. These results showed good correlation and confirmed that our method is feasible and could be used for the clinical determination of CEA (or other tumor antigens) in human 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.     

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 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 high-throughput homogeneous immunoassay based on Förster resonance energy transfer between quantum dots and gold nanoparticles

A novel homogeneous immunoassay based on Förster resonance energy transfer for sensitive detection of tumor, e.g., marker with carcinoembryonic antigen (CEA), was proposed. The assay was consisted of polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor. In presence of CEA, the bio-affinity between antigen and antibody made the QDs and AuNPs close enough, thus the photoluminescence (PL) quenching of CdTe QDs occurred. The PL properties could be transformed into the fluorometric variation, corresponding to the target antigen concentration, and could be easily monitored and analyzed with the home-made image analysis software. The fluorometric results indicated a linear detection range of 1–110 ng mL⁻¹ for CEA, with a detection limit of 0.3 ng mL⁻¹. The proposed assay configuration was attractive for carcinoma screening or single sample in point-of-care testing, and even field use. In spite of the limit of available model analyte, this approach could be easily extended to detection of a wide range of biomarkers.     

Multiplex detection of tumor markers with photonic suspension array

A novel photonic suspension array was developed for multiplex immunoassay. The carries of this array were silica colloidal crystal beads (SCCBs). The codes of these carriers are the characteristic reflection peak originated from their structural periodicity, and therefore they do not suffer from fading, bleaching, quenching, and chemical instability. In addition, because no dyes or materials related with fluorescence are included, the fluorescence background of SCCBs is very low. With a sandwich format, the proposed suspension array was used for simultaneous multiplex detection of tumor markers in one test tube. The results showed that the four tumor markers, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125) and carcinoma antigen 19-9 (CA 19-9) could be assayed in the ranges of 1.0-500 ng mL⁻¹, 1.0-500 ng mL⁻¹, 1.0-500 U mL⁻¹ and 3.0-500 U mL⁻¹ with limits of detection of 0.68 ng mL⁻¹, 0.95 ng mL⁻¹, 0.99 U mL⁻¹ and 2.30 U mL⁻¹ at 3σ, respectively. The proposed array showed acceptable accuracy, detection reproducibility, storage stability and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for low cost, automated, and simultaneous multiplex immunoassay.     

Immunochemical method for sulfasalazine determination in human plasma

Sulfasalazine is an antibiotic used in the treatment of inflammatory bowel diseases. For the assessment of sulfasalazine in several biological matrices, an Enzyme-Linked Immunosorbent Assay (ELISA) method based on polyclonal antibodies was developed and characterized.The immunoassay showed a high sensitivity (IC₅₀ = 0.51 ng mL⁻¹) and specificity, a detection limit of 0.02 ng mL⁻¹ and a dynamic range of 0.06-3.75 ng mL⁻¹ (80-20% inhibition). The immunoassay performed well when it was applied to spiked plasma samples (from 0.5 to 2.0 ng mL⁻¹) previously cleaned up by protein precipitation with methanol. Recoveries ranged from 83 to 119%, with a mean value of 99% (CV = 13%).Since sulfasalazine remaining of a treatment reaches the systemic circulation in unchanged form, the immunoassay can be applied to the determination of this pharmaceutical in human plasma in order to facilitate the control of the patients through the application of personal doses.     

Electro membrane extraction of sodium diclofenac as an acidic compound from wastewater,urine, bovine milk,and plasma samples and quantification by high-performance liquid chromatography

Electro membrane extraction (EME) as a new microextraction method was applied for extraction of sodium diclofenac (SDF) as an acidic compound from wastewater, urine, bovine milk and plasma samples. Under applied potential of 20 V during the extraction, SDF migrated from a 2.1 mL of sample solution (1 mM NaOH), through a supported liquid membrane (SLM), into a 30 μL acceptor solution (10 mM NaOH), exist inside the lumen of the hollow fiber. The negative electrode was placed in the donor solution, and the positive electrode was placed in the acceptor solution. 1-octanol was immobilized in the pores of a porous hollow fiber of polypropylene as SLM. Then the extract was analyzed by means of high-performance liquid chromatography (HPLC) with UV-detection for quantification of SDF. Best results were obtained using a phosphate running electrolyte (10 mM, pH 2.5). The ranges of quantitation for different samples were 8–500 ng mL⁻¹. Intra- and inter-day RSDs were less than 14.5%. Under the optimized conditions, the preconcentration factors were between 31 and 66 and also the limit of detections (LODs) ranged from 2.7 ng mL⁻¹ to 5 ng mL⁻¹ in different samples. This procedure was applied to determine SDF in wastewater, bovine milk, urine and plasma samples (spiked and real samples). Extraction recoveries for different samples were between 44–95% after 5 min of extraction.     

Multiplex immunodetection of tumor markers with a suspension array built upon core-shell structured functional fluorescence-encoded microspheres

A new suspension array built upon laboratory-prepared functional fluorescence-encoded polystyrene beads (FFPBs) was developed for multiplex immunodetection of tumor markers. The FFPBs were synthesized by copolymerizing rhodamine 6G (R6G) and carboxyl function groups on the surface of the seed beads forming a core-shell structure. The fabrication process was facile and the encoding fluorescence intensity of the beads can be precisely controlled by adjusting the quantity of R6G. In present work, we demonstrated that the quantity variation of impregnated R6G had negligible effect on the coupling efficiency of biomolecules onto the surface of the FFPBs. The R6G encoding fluorescence remained good monodispersity upon capture probe coupling and immunocomplex formation. No fluorescence resonance energy transfer was observed between the R6G doped in the bead shell and fluorophore used for antibody labeling. Under the optimal conditions, the proposed suspension array allowed simultaneous detection of α-fetoprotein, carcinoembryonic antigen, and prostate specific antigen in the ranges of 0.07-500 ng mL⁻¹, 1-2000 ng mL⁻¹, and 0.5-500 ng mL⁻¹, respectively, with detection limits of 0.0626 ng mL⁻¹, 0.554 ng mL⁻¹, and 0.250 ng mL⁻¹. Test on clinical serum samples demonstrated that the results obtained with suspension array were in good agreement with those of the reference electrochemiluminescence immunoassay method. We conclude that the laboratory-made FFPBs are sufficient as the microcarrier for the construction of suspension array in clinical diagnosis.     

Preparation and evaluation of an immunoaffinity sorbent with Fab′ antibody fragments for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry

An immunoaffinity (IA) sorbent with antibody fragments was prepared for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry (IA-SPE-CE–MS). The antibody fragmentation was evaluated by MALDI-TOF-MS. Fab′ fragments obtained from a polyclonal IgG antibody against Endomorphins 1 and 2 (End1 and End2) were covalently attached to succinimidyl silica particles to prepare the IA sorbent. An IA-SPE-CE–MS methodology was established analyzing standard solutions of End1 and End2 and acceptable repeatability, linearity ranges and LODs (0.5 and 5 ng mL⁻¹, respectively) were obtained. The LOD of End1 was slightly better than that previously obtained using an IA sorbent with intact antibodies (1 ng mL⁻¹). In human plasma samples, End1 and End2 could be detected at 1 and 50 ng mL⁻¹, respectively, which meant an improvement of 100 and 2-fold with regard to the LODs using an IA sorbent with intact antibodies (100 ng mL⁻¹).     

Ultrasound-assisted surfactant-enhanced emulsification microextraction for the determination of carbamate pesticides in water samples by high performance liquid chromatography

A novel ultrasound-assisted surfactant-enhanced emulsification microextraction (UASEME) coupled with high performance liquid chromatography-diode array detection has been developed for the extraction and determination of six carbamate pesticides (metolcarb, carbofuran, carbaryl, pirimicarb, isoprocarb and diethofencarb) in water samples. In the UASEME technique, Tween 20 was used as emulsifier, and chlorobenzene and chloroform were used as dual extraction solvent without using any organic dispersive solvent that is normally required in the previously described common dispersive liquid–liquid microextraction method. Parameters that affect the extraction efficiency, such as the kind and volume of the extraction solvent, the type and concentration of the surfactant, ultrasound emulsification time and salt addition, were investigated and optimized for the method. Under the optimum conditions, the enrichment factors were in the range between 170 and 246. The limits of detection of the method were 0.1–0.3 ng mL⁻¹ and the limits of quantification were between 0.3 and 0.9 ng mL⁻¹, depending on the compounds. The linearity of the method was obtained in the range of 0.3–200 ng mL⁻¹ for metolcarb, carbaryl, pirimicarb, and diethofencarb, 0.6–200 ng mL⁻¹ for carbofuran, and 0.9–200 ng mL⁻¹ for isoprocarb, with the correlation coefficients (r) ranging from 0.9982 to 0.9998. The relative standard deviations varied from 3.2 to 4.8% (n = 5). The recoveries of the method for the six carbamates from water samples at spiking levels of 1.0, 10.0, 50.0 and 100.0 ng mL⁻¹ were ranged from 81.0 to 97.5%. The proposed UASEME technique has demonstrated to be simple, practical and environmentally friendly for the determination of carbamates residues in river, reservoir and well water samples.     

Orthogonal array optimization of ultrasound-assisted emulsification–microextraction for the determination of chlorinated phenoxyacetic acids in river water

Orthogonal array design (OAD) was utilized for the first time to optimize the experimental conditions of ultrasound-assisted emulsification–microextraction (USAEME) for determining chlorinated phenoxyacetic acids (CPAs) in river water samples. The use of ultrasound facilitates the mass transfer of CPAs from an aqueous phase into a water-immiscible organic extraction solvent (dichloromethane, DCM) without adding dispersive solvent to form numerous microdroplets. The water-immiscible extractant was collected by centrifugation, dried under low pressure, reconstituted in methanol–water mixture (1:1), and injected into a HPLC system for the determination of CPAs. The linear range was 2–1000 ng mL⁻¹ (2, 5, 10, 50, 200, 500 and 1000 ng mL⁻¹) for each analyte and the relative standard deviations of CPAs among the seven different concentrations were in the range of 1.5–17.0% (n = 3). The detection limits (signal-to-noise ratio of 3) of CPAs ranged from 0.67 to 1.50 ng mL⁻¹. The ranges of intra-day precision (n = 3) for CPAs at the levels of 5 and 200 ng mL⁻¹ were 3.6–11.9% and 5.3–9.5%, respectively. The range of inter-day precision (n = 3) at 5 and 200 ng mL⁻¹ were 1.4–7.7% and 8.5–12.2%, respectively. The applicability of USAEME for environmental analysis was demonstrated by determining CPAs in river water. The recoveries of CPAs from five-spiked river water samples at 10 and 200 ng mL⁻¹ were 96.3–112.5% and 94.8–109.4%, respectively. The maximum contaminant level (MCL) of 2,4-D in drinking water and the tolerance of residues in food for p-CPA are 70 and 200 μg L⁻¹, respectively, according to the US EPA regulations. These contaminant levels fall in the linear range investigated in this study. In addition, this USAEME method provided detection limits lower than their contaminant levels, which made USAEME an effective sample preparation method for determining organic environmental contaminants, such as CPAs, in river water samples with little consumption of organic solvent.     

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.     

Palladium@gold bimetallic nanostructures as peroxidase mimic for development of sensitive fluoroimmunoassay

In this paper, gold nanoparticles coated with palladium dots (Pd@Au) bimetallic nanostructures have been reported to have a peroxidase like activity which is not found in their monometallic counterparts. Based on this finding, we have developed an immunoassay in which antibody-modified Pd@Au nanostructure catalyzes the dimerization of a fluorogenic substrate for peroxidase, 3-(4-dihydroxy phenyl) propionic acid (HPPA), to generate high fluorescence signal. Specific antibodies against bensulfuron-methyl were generated by using a well characterized bensulfuron–protein conjugate as an immunogen, and the assay was performed in a competitive immunoassay format where Pd@Au nanostructure was bound to secondary antibody to show the peroxidase like activity. The developed immunoassay exhibited an excellent sensitivity showing a dynamic response range from 0.001 to 100 ng mL⁻¹ for herbicide bensulfuron-methyl with a detection limit of 0.01 ng mL⁻¹ (n = 3). The newly synthesized bimetallic nanostructure shows the advantages of low cost, easy synthesis and tunable catalytic activity, making it a promising substitution of enzyme peroxidase in different applications.     

Enzyme-catalyzed silver deposition on irregular-shaped gold nanoparticles for electrochemical immunoassay of alpha-fetoprotein

A new and disposable electrochemical immunosensor was designed for detection of alpha-fetoprotein (AFP), as a model analyte, with sensitivity enhancement based on enzyme-catalyzed silver deposition onto irregular-shaped gold nanoparticles (ISGNPs). The assay was carried out with a sandwich-type immunoassay protocol by using ISGNP-labeled anti-AFP antibodies conjugated with alkaline phosphatase (ALP–Ab₂) as detection antibodies. The enzymatically catalytic deposition of silver on the electrode could be measured by stripping analysis in KCl solution due to the Ag/AgCl solid-state voltammetric process. Several labeling protocols including spherical gold nanoparticle-labeled ALP–Ab₂ and ISGNP-labeled ALP–Ab₂ were investigated for determination of AFP, and improved analytical properties were achieved with the ISGNP labeling. With the ISGNP labeling method, the effects of incubation time and incubation temperature for antigen-antibody reaction, and deposition time of silver on the current responses of the electrochemical immunosensors were also monitored. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range from 0.01 ng mL⁻¹ to 200 ng mL⁻¹ with a detection limit of 5.0 pg mL⁻¹ AFP. The immunosensor displayed a good stability and acceptable reproducibility and accuracy. No significant differences at the 95% confidence level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of AFP.     

Light emitting diode induced chemiluminescence and its application as a detector for high performance liquid chromatography

Some categories of compounds, including quinones, coumarins, flavins, and xanthene dyes, were found to produce strong chemiluminescence (CL) signals with luminol in sample solution under the irradiation of light emitting diodes (LED) with proper wavelengths. Based on this phenomenon, a compact photochemical reactor was constructed to develop a novel LED induced CL detector for high performance liquid chromatography (HPLC). The effects of related parameters including LED wavelength, luminol concentration, flow rate, pH, and eluents of HPLC were investigated in detail. Under the optimized conditions, the limits of detections (LODs) were in the range of 0.2–80 ng mL⁻¹. The applications and accuracy of the proposed method were validated by analyzing food samples such as milk powder, beer, candy and beverage with satisfactory results.