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Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605 nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Forster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1A. Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was logY=3.65786+0.43863·logX (R=0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0.4 ng mL(-1). By assaying test samples against the standard curve, the coefficient of variations was

Purpose: Recently, greater attention has been paid to interactions between biomolecules particularly at the single-molecular level. Due to their novel properties, integrated lab-on-a-chip (LOC) devices and fluorescence correlation spectroscopy (FCS) are in high demand. Methods: The LOC was manufactured using the technique of proton beam writing. The biomolecule fluorescein was used to probe flow profiles in the micro/nanochannels on the LOC by FCS. The FCS optical system was based on a confocal microscopy setup. At different locations on the LOC, the numbers and traveling time of the fluorescein fluidic solution were investigated. Results: From calibrations, ω 0 and τ D were 217 nm and 2.2 × 10−5 s, respectively. Particle number and duration in passing through the detect volume, τ F , were investigated. Results indicated that particle number was proportional to the size of micro/nanochannels in the LOC. The particle number distribution and speed of the flow were mirror-symmetric in the two parallel (inlet/outlet) microchannels. The distribution of the fluidic particles remained stable and the speed of the flow was nearly symmetrical when being transferred in the nanochannels. Conclusions: The results were realistic and in line with the hydromechanics, indicating that in multidisciplinary areas measurements of flow profiles by FCS are possible inside the LOC channels. This study paves the way to investigate biomolecular interactions at the single-molecular level.

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.

Time-resolved fluorometry of lanthanide chelates is one of the most useful non-isotopic detection techniques and has been used in numerous applications in biomedical science. We developed a time-resolved fluoroimmunoassay (TRFIA) to quantify α-fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg) in human serum. Based on a two-site sandwich protocol, monoclonal antibodies (McAbs) against AFP and HBsAg were co-coated in 96 microtitration wells and tracer McAbs against HBsAg and AFP were labeled with europium (Eu) and samarium (Sm) chelates, respectively. After application of diluted serum samples, Eu3+- and Sm3+-McAbs were added and fluorescence signals of Sm3+ and Eu3+ tracers were collected. Detection limits of AFP and HBsAg were 0.09 mIU/L and 0.01 µg/L, respectively. Measurement ranges of AFP-TRFIA and HBsAg-TRFIA were 1–1000 mIU/L and 0.2-150 µg/L, respectively. Intra- and inter-assay coefficients of variation of AFP-TRFIA were 3.3-4.1% and 5.7-7.2% and for HBsAg-TRFIA were 2.9-3.9% and 4.9-6.8%, respectively. Linear correlation of TRFIA and chemiluminescence immunoassay measurements resulted in a correlation coefficient of 0.9949 for AFP and 0.9940 for HBsAg. For the endurance test, Eu-labeled McAbs were stable for at least one year at −20°C and the results of the TRFIA with the same reagents were also reproducible after one year. The availability of a highly sensitive, reliable and convenient AFP/HBsAg TRFIA will allow the quantification of both AFP and HBsAg, thereby providing diagnostic value in various clinical conditions and could be applied for clinical use. Copyright © 2012 John Wiley & Sons, Ltd.

Human ubiquitin-conjugating enzyme E2, also known as UbcH10, is defined as a cyclin-selective ubiquitin carrier protein and is essential for selective degradation of many short-lived proteins in eukaryotic cells. Recently more and more data show that UbcH10 could be a potential cancer biomarker. In this study, we have developed a monoclonal antibody (MAb) against UbcH10 using an expression recombinant protein. Hybridomas F001, F007, and F008 with high affinities belong to IgG1 subclass with κ light and are highly specific for UbcH10. Further experimentation showed that MAbs F001, F007, and F008 are suitable for the development of immunoassay core agents with sufficient sensitivity and specificity in vitro by Western-blot, immunofluorescence, and immunohistochemistry. These MAbs can be used as a tool for further investigation on functions related to the role of UbcH10 in tumorigenesis and development.

We developed a luminescent terbium sensor (LTS) based on energy resonance transfer for homogeneous bioassays. The effect of temperature on photoluminescence and time-resolved fluorescence of the LTS was investigated. When the temperature was increased from 277 K to 369 K, the photoluminescence quantum yield decreased by up to 25 %, time-resolved fluorescence decreased by up to 54 %, and the lifetime shortened dramatically. Studies showed that both photoluminescence and time-resolved fluorescence quantum yields were largely recovered after samples were heated from 298 to 310, 333 or 369 K and subsequently cooled to 298 K. These results indicate that the homogeneous bioassay with LTS is sensitive to temperature and should be conducted at a constant temperature to ensure the temperature effect does not influence data and to increase the accuracy of the results. The results of this study are important for LTS applications in homogeneous bioassays. Copyright © 2012 John Wiley & Sons, Ltd.

Background Valid assays measuring free thyroxine (FT4) must perform without bias despite large variations in the concentrations and affinities of serum thyroxine-binding proteins in the population. We developed a new, rapid one-step labelled-antibody time-resolved fluoroimmunoassay (TRFIA) for FT4. Methods Based on the heterologous combination of anti-T4 monoclonal antibody and triiodothyronine–immunoglobulin G conjugate, a one-step TRFIA for FT4 detection was established and compared with the two-step DELFIA® Free Thyroxine Assay. Matrix interference caused by endogenous binders and exogenous non-esterified fatty acids (NEFA) was also accessed in the proposed assay. Results The developed method generally took only one hour, had a detection limit of 0.6 pmol/L and a large linear range of 2.5–120 pmol/L. The inter- and intra-assay coefficients of variation were 3.5–6.6% and 4.4–9.8%, respectively. Results from 110 specimens showed apparent agreement with that from the DELFIA® FT4 Assay with the square of the correlation coefficient of 0.975. This assay indicated that there was no significant dependence on endogenous binders and displayed potential interference by exogenous NEFA up to 5 mmol/L. Conclusions The proposed one-step heterologous TRFIA FT4 assay possesses simplicity, accuracy, high sensitivity and exhibits great potential for FT4 measurement. The combination of heterologous immunoassay with TRFIA may be advantageous for FT4 immunoassay development.

The aim of this study was to produce monoclonal and polyclonal antibodies against procalcitonin (PCT), a valid post-mortem marker of sepsis marker. Monoclonal antibodies (MAbs) were made from hyperimmune Balb/c mice by injecting 50 microg of purified antigen. These mice were immunized four times and given a final boost, and their spleen cells were collected and fused with SP2/0 myeloma cells under the presence of PEG 1450. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using purified protein. Lastly, five MAbs were obtained and designated successfully and were characterized by ELISA and Western immunoblotting. By Western immunoblotting, MAbs were found reactive with the patient's serum specifically. The results showed that the monoclonal antibodies could be used for various pathophysiological analyses in further investigations of procalcitonin and in preparing diagnostic kits.

Different diameter CdSe/ZnS semiconductor nanocrystals (average diameter: from approximately 3.5 to approximately 20 nm), quantum dots (QDs) were synthesized by changing the nucleation time, using organometallic reagents. These quantum dots possess narrow and symmetrical fluorescent emissions. The emission wavelengths of these composite dots span most of the visible spectrum from 500 through 700 nm. Furthermore, it is found that the quantum dots with an emission at approximately 590 nm, tend to have a good quantum yield (such as Phi(590)=43.5%). While the emission wavelength of prepared CdSe/ZnS QDs shifts toward blue or red from 590 nm, the quantum yield tends to decrease. After that, optical encoding of microbeads with these quantum dots was carried out, and the spectra of encoded beads were identified. The result indicates that, to identify the encoded beads with different emission wavelengths and emission intensities, it is needed to acquire and differentiate the spectra of beads. After immobilized with human IgG, the encoded beads were used to detect the corresponding antibody in solution. The result indicates that the encoded beads can detect the antibody signal effectively. And the effective detection range of the antibody is about 2-15 microM.

Quantum dots had been covalently attached to human IgG by activating agents. The formation of QDs bioconjugates was confirmed by fluorescence spectrum and gel chromatography. Results of circular dichroism showed that the conjugates still maintained biological activity. When goat anti-human IgG labeled with fluorescin isothiocyanate (FITC) was added, the immunocomplex formed and the mutual affinity of the antigen and antibody brought the FITC and QDs close enough. The existing of FRET between FITC (donor) and QDs (acceptor) was further confirmed by observing the changes of their spectra. This investigation provides a new trail of thought for the fluorescence resonance energy transfer (FRET) research based on QDs.

Background WAP four-disulfide core domain protein 2 (WFDC2) shows a tumor-restricted upregulated pattern of expression in ovarian cancer. Methods We investigated the role of estradiol (E2) on cell growth in estrogen-sensitive or estrogen-insensitive ovarian cancer cell lines. Real-time (RT)-PCR and western blotting were used to examine the expression of WFDC2 at RNA and protein levels. Growth traits of cells transfected with WFDC2-shRNA or blank control were assessed using MMT arrays. Cell apoptosis was analyzed using annexin V-FITC/PI and flow cytometry. Estrogen receptor expression was evaluated using RT-PCR and flow cytometry. Apoptosis-related proteins induced by E2 directly and indirectly were determined using an antibody array comparing cells transfected with WFDC2- shRNA or a blank control. Results High-dose (625 ng/ml) E2 increased the expression of WFDC2 in HO8910 cells at both the mRNA and protein levels. However, E2 had no effect on WFDC2 expression in estrogen-insensitive SKOV3 cells. Of interest, knockdown of WFDC2 enabled a considerable estrogen response in SKOV3 cells in terms of proliferation, similar to estrogen-responsive HO8910 cells. This transformation of SKOV3 cells into an estrogen-responsive phenotype was accompanied by upregulation of estrogen receptor beta (ERß) and an effect on cell apoptosis under E2 treatment by regulating genes related to cell proliferation and apoptosis. Conclusions We postulate that increased WFDC2 expression plays an important role in altering the estrogen pathway in ovarian cancer, and the identification of WFDC2 as a new player in endocrine-related cancer encourages further studies on the significance of this gene in cancer development and therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13048-015-0210-y) contains supplementary material, which is available to authorized users.

BACKGROUND: Because of the life‐consuming treatment and severe consequences associated with thalassemia, it is more effective to prevent than cure thalassemia. Rapid and sensitive detection is critical for controlling thalassemia. In this study, we developed a rapid and accurate test to genotype nondeletional α‐ and β‐thalassemia mutations by an electrochemical DNA sensor. METHODS: Screen‐printed electrodes were used as electrochemical transducers for the sensor, in which the capture probe DNA was attached to the golden surface of the working electrode via an S–Au covalent bond, which is highly suitable for immobilizing the biological element. In addition, two types of ferrocene with varying redox potentials for modified signal probe DNA were adopted. The hybridization signal is detected by alternating current voltammetry when the capture probe and signal probe hybridize with the target DNA. RESULTS: With this technique, 12 types of nondeletional α‐ and β‐thalassemia mutations were detected, which constitute more than 90% of all the nondeletional types of thalassemia mutation determinants found in China, including the CD142 (TAA>CAA) Constand spring, CD125 (CTG>CCG) Quonsze, CD122 (CAC>CAG) Weastmead, −28 (A>G), Cap+1 (A>C), initiation codon (ATG>AGG), CD17 (AAG>TAG), CD26 (GAG>AAG), CD31(‐C), CD41‐42 (‐CTTT), CD71‐72 (+A), and IVS‐II‐654 (C>T) mutations. Concordance levels were 100% within the 20 blood samples of homozygous wild‐type individuals and 238 blood samples of heterozygous mutant individuals. CONCLUSIONS: The electrochemical DNA sensor developed here can be applied for rapid genotyping of thalassemia or other clinical genotyping applications and is useful for early screening of thalassemia in high‐risk groups by minimizing the time and investment cost.

The Epstein-Barr virus (EBV) transactivator protein (ZEBRA) is an immediate-early protein that plays an important role in the switch from latency to productive cycle in EBV virus. ZEBRA is an important marker of EBV reactivation. In order to diagnose EBV infection status correctly and timely, a novel immunoassay was developed based on an indirect time-resolved fluoroimmunoassay (TRFIA) for Zta IgA, which used recombinant Zta antigen as solid-phase antigen and Eu(3+)-labeled mouse antihuman IgA as corresponding probe. The precision, sensitivity, specificity test, and stability of the TRFIA kit were evaluated, and comparison with the traditional enzyme-linked immunosorbent assay (ELISA) was also investigated. The cutoff value for the TRFIA was 2.5. Intra- and interassay coefficients of variation for the TRFIA were 2.45-3.30% and 3.38-4.61% respectively. There was no cross-reactivity with the antibodies of cytomegalovirus (CMV) or herpes simplex virus (HSV) types 1 and 2, or other potential interferences. The established assay kit also behaved better in sensitivity and stability than the ELISA one. Additionally, the results in 382 serum samples using two analytical methods showed there was good agreement between the TRFIA and commercial ELISA kit. In the current study, the results demonstrated that the TRFIA that was developed for Zta IgA detection was more sensitive and reliable for the diagnosis of EBV infection and had potential value in automation and high-throughput screening.

Enzyme-linked immunosorbent assay (ELISAs) specific for Epstein-Barr virus nuclear antigen 1 (EBNA1)-immunoglobulin A (IgA) are most commonly used in the clinical diagnosis of EBV infection. But they have a low sensitivity and the enzyme-labeled antibodies are unstable. In this study, a novel immunoassay based on an indirect time-resolved fluoroimmunoassay (TRFIA) was developed. Microtiter plates were coated with recombinant EBNA1. We used Eu(3) (+)-labeled anti-human IgA as probe. The precision, sensitivity, specificity, and stability were evaluated, and comparison with traditional and commercially available ELISAs was also made. The cut-off value for our TRFIA was 2.7. Intra- and inter-assay coefficients of variation for the TRFIA were 1.56-4.99% and 3.92-6.95%, respectively; whereas those for the ELISA were 4.54-8.16% and 7.07-10.52%, respectively. Sensitivity was obviously better than traditional ELISA when diluted positive samples serially. Additionally, stability, specificity test and comparison of sensitivity and specificity between the TRFIA and commercial ELISAs all proved satisfactory. In conclusion, the results demonstrated that EBNA1 IgA TRFIA was a sensitive immunoassay and had potential value in large-scale screening of human serum samples in developing countries.

In order to early screen and detect suspected biomarkers from pathogens and the human body itself, tracers or reaction strategies that can act as signal enhancers have been proposed forth at purpose. In this paper, we discussed the applicability of magnetic microparticles-assisted time-resolved fluoroimmunoassay (MMPs-TRFIA) for sensitive determination of potential analytes. Hepatitis B e antigen, antibody to hepatitis B surface antigen and free triiodothyronine were used as biomarker models to explore the reliability of the method. By coupling with bioprobes, MMPs were used as immunoassay carriers to capture target molecules. Under optimal condition, assay performance, including accuracy, precision and specificity, was outstanding and demonstrated satisfactory. To further evaluate the performance of the MMPs-TRFIA in patients, a total of 728 serum samples from hospital were analyzed for three biomarkers in parallel with the proposed method and chemiluminescence immunoassay kit commercially available. Fairly good agreements are obtained between the two methods via data analysis. Not only that but the reliability of MMPs-TRFIA has also been illustrated by three different reaction models. It is confirmed that the novel method modified with MMPs has been established and showed great potential applications in both biological detection and clinical diagnosis, including big molecule protein and low molecular weight haptens.

Water-soluble quantum dots (QDs) were used to label goat anti-human immunoglobulin antibodies (Abs), and the labeling process was characterized by column purification. The QDs obtained in organic solvent were modified with mercaptoacetic acid (MAA) and became water-soluble. These water-soluble QDs were linked to the antibodies using the coupling reagents ethyl-3-(dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The linking process was shown to be effective by ultra-filter centrifugation and column purification. After comparing the quantities of Abs and water-soluble QDs involved in the linking reaction via column purification, it was found that a molar Abs:QD ratio of >1.2 resulted in most of the water-soluble QDs becoming covalently linked to the Abs. The circular dichroism (CD) spectra of Abs and QD-Ab conjugates were very similar to each other, indicating that the secondary structure of Abs remained largely intact after the conjugation. Finally, antigen (Ag)-antibody (Ab) recognition reactions perfomed on the surface of a glass slide showed that the conjugate retained the activity of Abs. This work lends support to the idea of linking biomolecules to QDs, and thus should aid the application of QDs to the life sciences.

Novel –COOH modified polystyrene beads were prepared by sulfonation grafting, and the surface area and pore volume are greatly improved in comparison with the swelling-treated beads. The optimization coating time is 4 h, and the corresponding –COOH content is approximately 2.1 mmol/g. The scanning electron microscope results show that the silica particles deposited on the beads and formed a silica shell that decreases the leakage of quantum dots (QDs) preferably and improves the bar code stability greatly. The anti-photobleaching of silica-coated beads was studied systemically, and the results show that the half-decay time (t1/2) of the coated beads increases to 537 s—seven times longer than that of the uncoated ones. Further DNA probe hybridization experiments indicated that the coding signal and target signal can be detected simultaneously and that the assays based on these probe-conjugated silica/QD/polystyrene beads have good specificity and sensitivity that can detect a concentration as low as 0.01 μg/ml target DNA in denatured calf thymus DNA solution, indicating that it is feasible to use this kind of bead for multiplex analysis.

It was reported that a novel detection method, continuous wave (CW)-based multiphoton excitation (MPE) fluorescence detection with diode laser (DL), has been firstly proposed for capillary electrophoresis (CE). Special design of end-column detection configuration proved to be superior to on-column type, considering the detection sensitivity. Three different kinds of fluorescent tags that were widely used as molecular label in bio-analysis, such as small-molecule dye, fluorescent protein and nano particle or also referred to as quantum dot (QD), have been evaluated as samples for the constructed detection scheme. Quantitative analyses were also performed using rhodamine species as tests, which revealed dynamic linear range over two orders of magnitude, with detection limit down to zeptomole-level. Simultaneous detection of fluorescent dyestuffs with divergent excitation and emission wavelengths in a broad range showed advantage of this scheme over conventional laser-induced fluorescence (LIF) detection. Further investigations on CW-MPE fluorescence detection with diode laser for capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations of fluorescein isothiocyanate (FITC) labeled amino acids indicated good prospect of this detection approach in various micro or nano-column liquid phase separation technologies.

The photoluminescence of water-soluble CdSe/ZnS core/shell quantum dots is found to be temperature-dependent: as temperature arising from 280 K to 351 K, the photoluminescence declines with emission peak shifting towards the red at a rate of ∼0.11 nm K −1 . And the studies show that the photoluminescence of water-soluble CdSe/ZnS quantum dots with core capped by a thinner ZnS shell is more sensitive to temperature than that of ones with core capped by a thicker one. That is, with 50% decrement of the quantum yield the temperature of the former need to arise from 280 K to 295 K, while the latter requires much higher temperature (315.6 K), which means that the integrality of shell coverage is a very important factor on temperature-sensitivity to for the photoluminescence of water-soluble CdSe/ZnS quantum dots. Moreover, it is found that the water-soluble CdSe quantum dots with different core sizes, whose cores are capped by thicker ZnS shells, possess almost the same sensitivity to the temperature. All of the studies about photoluminescence temperature-dependence of water-soluble CdSe/ZnS core/shell quantum dots show an indispensable proof for their applications in life science.

A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions was demonstrated using quantum dots (QDs) as a fluorescence label coupled with protein. 1,3-Dipolar cycloaddition between azide and alkyne was exploited to attach α-D-glucopyranoside to a C 1 4 hydrocarbon chain that noncovalently binds to the microtiter well surface, and the product formation was detected by both electrospray ionization-mass spectrometry (ESI-MS) and QD- (or fluorescein isothiocyanate (FITC))-conjugated lectin binding. It indicated that the peak intensity of the fluorescence emission was proportional to the initial concanavalin A (Con A) concentration in the range of 2 × 10 - 3 μmol/L to 2 x 10 - 2 n-imol/L with a detection limit at least 100 times lower than that of the FITC-based method.

Viral capsid antigen (VCA) IgA is one of the most commonly tested antibodies for Epstein-Barr virus (EBV) in the clinic and is a proven biomarker to predict the risk of nasopharyngeal carcinoma (NPC) and other diseases. At present, a VCA-IgA antibody is used for clinical diagnosis by enzyme-linked immunosorbent assay (ELISA), which can detect samples only qualitatively or semi-quantitatively, with unsatisfactory sensitivity and specificity. In this study, an indirect time-resolved fluoroimmunoassay (TRFIA) using Eu(3+) labeled mouse anti-human IgA monoclonal antibodies as a tracer was developed. This method produced a linear range of 0-30 AU/mL, with a limit of detection of 0.018 AU/mL. The intra- and inter-assay precisions were 1.62-4.30% and 3.56-7.57%, respectively. TRFIA showed no cross-reactivity against potentially interfering substances and a better sensitivity and specificity compared with commercial ELISA. This study confirmed that an indirect TRFIA meets the requirement for clinical testing and could be an alternative to detect VCA-IgA levels in human serum in the clinic.

A sensitive, rapid and novel measurement method for cytokeratin 19 fragment (CYFRA 21–1) in human serum by magnetic particle-based time-resolved fluoroimmunoassay (TRFIA) is described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating onto the surface of magnetic beads and “sandwiched” by another monoclonal antibody labeled with europium chelates. The coefficient variations of the method were lower than 7 %, and the recoveries were in the range of 90–110 % for serum samples. The lower limit of quantitation of the present method for CYFRA 21–1 was 0.78 ng/ml. The correlation coefficient of CYFRA 21–1 values obtained by our novel TRFIA and CLIA was 0.980. The present novel TRFIA demonstrated high sensitivity, wider effective detection range and excellent reproducibility for determination of CYFRA 21–1 can be useful for early screening and prognosis evaluation of patients with non-small cell lung cancer.

Background Hepatitis B virus (HBV) poses a serious risk to human health and the hepatitis B surface antigen (HBsAg) is a popular biomarker in the diagnosis of HBV infection. A quantitative method with a high degree of accuracy, sensitivity and throughput is needed. Methods A novel amplified luminescent proximity homogeneous assay (AlphaLISA) was developed for HBsAg determination. A set of monoclonal antibodies was screened against the main subtypes of HBsAg (adr, ay) to confirm the assay's sensitivity to mutants. Technological processes and reaction conditions were optimized and the assay performance was evaluated. Results HBsAg concentrations were determined within a linear range of 0.04 to 100 IU ml− 1. The detection sensitivity was established as 0.01 IU ml− 1. Assay sensitivity to mutant HBsAg was achieved through antibody screening. The results demonstrate that the reproducibility, recovery, and specificity of this assay for HBsAg were better than acceptable. Compared with the commercial light-initiated chemiluminescence assay, the correlation coefficient of the novel assay was established as 0.921. Conclusions The novel AlphaLISA developed in this study has shorter incubation time and easier protocol than the ones of conventional ELISA. It could be used for the clinical determination of HBsAg in human serum. We established a platform for further development of other biomarkers.

Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02-700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7-8.7% and 3.8-7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X - 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus.

Quantum dots are not widely used in clinical diagnosis. However, the homogeneous time-resolved fluorescence assay possesses many advantages over current methods for the detection of carcinoembryonic antigen (CEA), a primary marker for many cancers and diseases. Therefore, a novel luminescent terbium chelates- (LTCs) and quantum dots-based homogeneous time-resolved fluorescence assay was developed to detect CEA. Glutathione-capped quantum dots (QDs) were prepared from oil-soluble QDs with a 565 nm emission peak. Conjugates (QDs-6 F11) were prepared with QDs and anti-CEA monoclonal antibody. LTCs were prepared and conjugates (LTCs-S001) were prepared with another anti-CEA monoclonal antibody. The fluorescence lifetime of QDs was optimized for sequential analysis. The Forster distance (R0) was calculated as 61.9 A based on the overlap of the spectra of QDs-6 F11 and LTCs-S001. Using a double-antibody sandwich approach, the above antibody conjugates were used as energy acceptor and donor, respectively. The signals from QDs were collected in time-resolved mode and analyzed for the detection of CEA. The results show that the QDs were suitable for time-resolved fluoroassays. The spatial distance of the donor-acceptor pair was calculated to be 61.9 A. The signals from QDs were proportional to CEA concentration. The standard curve was LogY = 2.75566 + 0.94457 LogX (R = 0.998) using the fluorescence counts (Y) of QDs and the concentrations of CEA (X). The calculated sensitivity was 0.4 ng/mL. The results indicate that water-soluble QDs are suitable for the homogenous immunoassay. This work has expanded future applications of QDs in homogeneous clinical bioassays. Furthermore, a QDs-based homogeneous multiplex immunoassay will be investigated as a biomarker for infectious diseases in future research.

Time-resolved fluorometry of lanthanide chelates is one of the most useful non-isotopic detection techniques and has been used in numerous applications in biomedical science. We developed a time-resolved fluoroimmunoassay (TRFIA) to quantify α-fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg) in human serum. Based on a two-site sandwich protocol, monoclonal antibodies (McAbs) against AFP and HBsAg were co-coated in 96 microtitration wells and tracer McAbs against HBsAg and AFP were labeled with europium (Eu) and samarium (Sm) chelates, respectively. After application of diluted serum samples, Eu(3+)- and Sm(3+)-McAbs were added and fluorescence signals of Sm(3+) and Eu(3+) tracers were collected. Detection limits of AFP and HBsAg were 0.09 mIU/L and 0.01 µg/L, respectively. Measurement ranges of AFP-TRFIA and HBsAg-TRFIA were 1-1000 mIU/L and 0.2-150 µg/L, respectively. Intra- and inter-assay coefficients of variation of AFP-TRFIA were 3.3-4.1% and 5.7-7.2% and for HBsAg-TRFIA were 2.9-3.9% and 4.9-6.8%, respectively. Linear correlation of TRFIA and chemiluminescence immunoassay measurements resulted in a correlation coefficient of 0.9949 for AFP and 0.9940 for HBsAg. For the endurance test, Eu-labeled McAbs were stable for at least one year at -20°C and the results of the TRFIA with the same reagents were also reproducible after one year. The availability of a highly sensitive, reliable and convenient AFP/HBsAg TRFIA will allow the quantification of both AFP and HBsAg, thereby providing diagnostic value in various clinical conditions and could be applied for clinical use.

To develop an amplified luminescent proximity homogeneous immunoassay (AlphaLISA) kit for the detection of human hepatitis B virus e antibody (HBeAb).The neutralizing and competitive inhibition method was used to develop the AlphaLISA kit for detection of serum HBeAb.The working range of the kit was 0.003-16 NCU/ml with a sensitivity up to 0.003 NCU/ml. The intra- and inter-assay coefficient of variation was 5.3% and 6.8%, respectively. The kit showed no cross-reaction with HBcAb, and comparison of the detection results with those of a commercially available Elecsys HBeAb kit (Roche) for 136 samples showed a correlation coefficient of 0.961.The AlphaLISA kit for HBeAb detection meets the clinical requirements for detection HBeAb in human serum.

Transferrin and mouse anti-human CD71 monoclonal antibody were respectively conjugated covalently to the core/shell CdSe/ZnS quantum dots with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydrocylsulfo-succinimide (Sulfo-NHS). The conjugation worked well and the bioactivities of these macromolecules still remained, which was verified by column filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis, absorption spectra, fluorescence spectra, and circular dichroism spectrometry. Thus, these two kinds of quantum dot conjugates were used to recognize the tumor cells involved. In case of pseudo positivity, FITC-labeling secondary antibody IgG was used, and the results showed that as-prepared fluorescent quantum dot bioprobes were highly specific to tumor cells.

The luminescent silica nanoparticle has attracted the researchers' concentration in bioanalysis recently. Its extensive application is based on the immobilization of various biomolecules such as deoxyribonucleic acid, antibody, and so forth onto the surface. By comparing different introduction methods of amine groups, it was confirmed that the “two-step” route is more preferable by adding tetraethyl orthosilicate and 3-aminopropyl-(triethoxyl)silane in sequence, to attain ideal amine-modified silica nanoparticles. On this basis, carboxyl groups were derived from amine groups on the nanoparticle surface and then were activated by 1-ethyl-3-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy-succinimide. Finally, mouse monoclonal antihuman CD71 antibody (McAb CD71) and transferrin were effectively linked with the carboxyl groups and successfully labeled the receptors in the membrane of fibroblast cells, respectively. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2007

Multicolor encoded beads were achieved by incorporating two color core-shell quantum dots (QDs) (CdSe/ZnS) to commercial polystyrene (PS) beads. By controlling the concentration ratios of the two quantum dots (QDs) in doping solutions, a series of codes with different intensity ratios were obtained. Based on the multiple encoded carboxylic modified polystyrene beads, fluorescent dyes labeled antibodies were distinguished successfully on the beads’ surface. It suggests that the encoded beads from this method have the practicability in biological applications and chemical analysis.

Ferrite/polymer composites were prepared by mixing ferrite powders with epoxy resin. The powders are W-type barium ferrites BaCoxZn2-xFe16O27 (x=1.0, 1.3 and 1.5) doped with V2O5 of 0-1.5 wt%. X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), extended X-ray absorption fine structure spectroscopy (EXAFS), scanning electron microscope (SEM), electric resistivity, static and high-frequency magnetic measurements were performed to study the microstructure, static and high-frequency properties of the composites. The experimental results show that the doping of V2O5 can effectively modify the shape of particles, greatly decrease the value of real permittivity, and significantly enhance the complex permeability. The composites, therefore, are potential candidates for EM attenuation materials with low reflectivity and broad bandwidth.

A flow cytometric detecting technology based on quantum dots (QDs)-encoded beads has been described. Using this technology, several QDs-encoded beads with different code were identified effectively, and the target molecule (DNA sequence) in solution was also detected accurately by coupling to its complementary sequence probed on QDs-encoded beads through DNA hybridization assay. The resolution of this technology for encoded beads is resulted from two longer wavelength fluorescence identification signals (yellow and red fluorescent signals of QDs), and the third shorter wavelength fluorescence signal (green reporting signal of fluorescein isothiocyanate (FITC)) for the determination of reaction between probe and target. In experiment, because of QDs’ unique optical character, only one excitation light source was needed to excite the QDs and probe dye FITC synchronously comparing with other flow cytometric assay technology. The results show that this technology has present excellent repeatability and good accuracy. It will become a promising multiple assay platform in various application fields after further improvement.

Microbeads with embedded encode characteristics are of considerable interest due to their potential use in multiplexed bioassays, high-throughput screening and combinatorial chemistry. Lots of encoding strategies fortagging or labeling microbeads have arisen in the past couple of years. Compared with the organic dye counterparts,the ideal optical properties of quantum dots (QDs) (e.g. characteristic narrow and symmetric spectra, size-tunableemission and simultaneous excitation) make it possible to tag microbeads in a quantitative way. In this paper,quantitative doping of commercial polystyrene microbeads with single color quantum dots was reported. A detailedanalysis of the optical characteristics of the QD-tagged microbeads was presented based on the combination offluorescent spectroscopy and microscope imaging.Keywords: Quantum dots, microbeads, fluorescence, imaging 1. INTRODUCTION Microbeads encoded with optical signatures are of considerable interest in analytical chemistry and bioengineeringdue to their potential applications in multiplexed bioassays, medical diagnostics, drug screening, and combinatorial

A novel green synthesis of semiconductor nanoparticles was introduced and it was used as optical probes for bio-imaging. Quantum dots are known as

The (1-->3)-beta-D-pentaglucoside was synthesized as its (R)-2,3-epoxypropyl glycoside via 2+3 strategy. The disaccharide donor 8 was obtained by 3-selective coupling of 2 with 4, followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor 12 was prepared by coupling of 10 with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside. The results of these bioassays demonstrated that the (1-->3)-beta-D-glucanase was obviously inactivated by 15 with k(app)=3.79 x 10(-4) min(-1). At the same time, we found that the 15 was more active as compared to the laminaripentaose in eliciting phytoalexin accumulation in tobacco cotyledon tissue, and it could be kept longer time than laminaripentaose, which indicated it is much more stable than laminaripentaose.

In this paper, we present the optical properties and the platforms on fluorescent quantum dots for biological labeling, biomedical engineering and biosensor in molecular imaging. Quantum dots possess several properties that make them very attractive for fluorescent tagging: broad excitation spectrum, narrow emission spectrum, precise tunability of their emission peak, longer fluorescence lifetime than organic fluorophores and negligible photobleaching. We describe how to take such advantages of quantum dots to develop the technology and employ it to build assay platforms. Finally, ultrasensitivity, multicolor, and multiplexing of the technology of semiconductor quantum dots open up promising and interesting possibilities for bioassay platform.

A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions was demonstrated using quantum dots (QDs) as a fluorescence label coupled with protein. 1,3-Dipolar cycloaddition between azide and alkyne was exploited to attach alpha-d-glucopyranoside to a C(14) hydrocarbon chain that noncovalently binds to the microtiter well surface, and the product formation was detected by both electrospray ionization-mass spectrometry (ESI-MS) and QD- (or fluorescein isothiocyanate (FITC))-conjugated lectin binding. It indicated that the peak intensity of the fluorescence emission was proportional to the initial concanavalin A (Con A) concentration in the range of 2 x 10(-3) micromol/L to 2 x 10(-2)mmol/L with a detection limit at least 100 times lower than that of the FITC-based method.

The (1-->3)-beta-D-pentaglucoside was synthesized as its (R)-2,3-epoxypropyl glycoside via 2+3 strategy. The disaccharide donor 8 was obtained by 3-selective coupling of 2 with 4, followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor 12 was prepared by coupling of 10 with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside. The results of these bioassays demonstrated that the (1-->3)-beta-D-glucanase was obviously inactivated by 15 with k(app)=3.79 x 10(-4) min(-1). At the same time, we found that the 15 was more active as compared to the laminaripentaose in eliciting phytoalexin accumulation in tobacco cotyledon tissue, and it could be kept longer time than laminaripentaose, which indicated it is much more stable than laminaripentaose.

M Xie, H Zhang, Y Xu, T Liu, S Chen, J Wang, T ZhangDepartment of Otorhinolaryngology Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, People's Republic of ChinaAbstract: Immunohistochemistry and an immunofluorescence technique was used to detect folate receptor expression in tissue samples and cell lines of head and neck squamous carcinoma, including 20 tissue samples of nasopharyngeal carcinoma, 16 tissue samples of laryngeal carcinoma, and HNE-1, HNE-2, CNE-1, CNE-2, SUNE-1, 5-8F, and Hep-2 cell lines. Iron staining, electron microscopy, and magnetic resonance imaging were used to observe endocytosis of folate-conjugated cisplatin-loaded magnetic nanoparticles (CDDP-FA-ASA-MNP) in cultured cells and transplanted tumors. As shown by immunohistochemistry, 83.3% (30/36) of the head and neck squamous carcinomas expressed the folate receptor versus none in the control group (0/24). Only the HNE-1 and Hep-2 cell lines expressed the folate receptor, and the other five cell lines did not. Endocytosis of CDDP-FA-ASA-MNP was seen in HNE-1 and Hep-2 cells by iron staining and electron microscopy. A similar result was seen in transplanted tumors in nude mice. Magnetic resonance imaging showed low signal intensity of HNE-1 cells and HNE-1 transplanted tumors on T2-weighted images after uptake of CDDP-FA-ASA-MNP, and this was not seen in CNE-2 transplanted tumors. In conclusion, head and neck squamous carcinoma cell strongly expressed the folate receptor, while normal tissue did not. The folate receptor can mediate endocytosis of folate-conjugated anticancer nanomedicines, and lays the foundation for molecular targeted treatment of cancer.Keywords: nasopharyngeal carcinoma, laryngeal carcinoma, folate receptor, molecular targeting, cisplatin, nanomedicine

In this paper, a novel time-resolved fluoroimmunoassay (TRFIA) protocol using magnetic particles for the simultaneous determination of α-fetoprotein (AFP) and the free β-subunit of human chorionic gonadotropin (free β-hCG) in human serum is described. The new approach uses magnetic particles as an immobilization matrix and means of separation, while the luminescent europium and samarium chelates are used as probes. The proposed method was evaluated via a single-step, sandwich-type TRFIA immunoassay of AFP and free β-hCG as model analytes in serum. With the advantages of magnetic particles, the TRFIA immunoassay exhibited a wide dynamic range for AFP of 0.1-750 ng mL(-1), with a lower detection limit of 0.05 ng mL(-1). The dynamic range for free β-hCG was 0.16-450 ng mL(-1), with a lower detection limit of 0.08 ng mL(-1). Satisfactory specificity, reproducibility, and recovery of the immunoassay were demonstrated. Good correlations were obtained in the analysis of 446 human serum samples between the proposed method and a commercial TRFIA kit. These results demonstrate the feasibility and potential of the new method as a rapid and highly sensitive immunoassay that could be developed into a platform for multi-analyte determinations in clinical practice.