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Establishing personalized medication plans for patients to maximize therapeutic efficacy and minimize the toxicity of vancomycin (VAN) requires rapid, simple, and accurate monitoring of VAN concentration in body fluid. In this study, we have developed a simple and rapid analytical method by integrating Eu (III) chelate nanoparticles (CN-EUs) and lateral flow immunoassay (LFIA) to achieve the real-time monitoring of VAN concentration in serum within 15 min. This approach was performed on nitrocellulose (NC) membrane assembled LFIA strips via indirect competitive immunoassay and exhibited a wide linear range of detection (0.1–80 μg*ml−1) with a low limit of detection (69.2 ng*ml−1). The coefficients of variation (CV) of the intra- and inter-assay in the detection of VAN were 7.12–8.53% and 8.46–11.82%, respectively. The dilution test and specificity indicated this method had a stability that was not affected by the serum matrix and some other antibiotics. Furthermore, the applicability of the proposed method was assessed by comparing the determined results with those measured by LC-MS/MS, showing a satisfactory correlation (R2 = 0.9713). The proposed CN-EUs-based LFIA manifested promising analytical performance, which showed potential value in the real-time monitoring of VAN and could help optimize the clinical use of more antibiotics.

Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker for diagnosing acute kidney injury (AKI). Currently, there are few assays for determining NGAL and they are complex, time-consuming or expensive. We aimed to establish an efficient immunoassay to measure NGAL in human urine simply and rapidly. A novel immunoassay for NGAL determination was established by combining a dissociation-enhanced-free time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a “sandwich”-type immunoassay format, analytes in samples were captured by a pair of monoclonal antibodies (mAb) in which one mAb was coated in magnetic beads and the other mAb was labeled with europium(III) chelate microparticles (CM-EUs) as “fluorescent reporters”. NGAL concentrations were determined in a linear range (10–1500 ng mL−1) with a limit of detection of 0.32 ng mL−1. The reproducibility, recovery, and specificity of our TRFIA were acceptable. Our method was compared with that of a chemiluminescence immunoassay (CMIA) using 115 urine samples, and the results showed good correlation (R2 = 0.8677). We expect our novel method to be useful for the early diagnosis of AKI.

Development of sensitive homogeneous assays is a high-priority research target for clinical diagnostics. Quantum dots (QDs) present favorable photophysical properties, which implies their potential as an exceptional dye in fluorescence detection. QDs-based biosensors have been described in the literature; however, few of them have truly progressed to widespread clinical usage. In this work, a chemiluminescent homogeneous detecting biosensor is fabricated using QDs-doped polystyrene nanospheres to sensitively detect biomarkers in low-volume serum samples. Phthalocyanine-dyed and QDs-encapsulated carboxylate-functionalized polystyrene nanospheres with surface carboxyl groups (PPs and QPs, respectively) were fabricated and served as triggers and fluorescent probes, respectively, in this biosensing system. In this sandwich-format immunoassay, the PPs produced singlet oxygen once sensitized by 680 nm diode lasers, and the QPs, conjugated with antibodies, and then reacted with the singlet oxygen in the presence of specific antigens and emitted anti-Stokes fluorescence with wavelengths around 605 nm, as a result of fluorescence resonance energy transfer (FRET) within the QPs. We demonstrated the determination of carcinoembryonic antigen as a model protein target in 25 μL of serum samples with an unprecedented detection limit of 2.56 × 10-13 M (46 pg/mL) using this biosensor. Furthermore, excellent correlations ( R2 = 0.99718, n = 107) were obtained between utilizing this biosensor and commercialized chemiluminescence immunoassay kits in clinical serum detection. These results demonstrate that our flexible and reliable biosensor is suitable for direct integration into clinical diagnostics, and it is expected to be a promising diagnostic tool for early detection and screening tests as well as prognosis evaluation for patients.

Objective: To understand the role of WFDC2 in metastasis of ovarian cancer. Methods: By knockdown or overexpression of WFDC2, we demonstrated the role of WFDC2 in epithelial-mesenchymal transition (EMT). Results: We demonstrated that stable knockdown of WFDC2 suppressed EMT along with the upregulation of E-cadherin and the downregulation of Vimentin. In addition, WFDC2 knockdown decreases matrix metalloproteinase-2 (MMP-2) expression in in vitro cell model and in in vivo nude mice xenografts. The correlation of WFDC2 and MMP-2 expression in the clinical sample confirmed that WFDC2 was tightly correlated with the development of tumor. More importantly, the EMT phenotype and cell invasion induced by WFDC2 overexpressing can be reversed by the siMMP-2 and P13K/AKT signaling inhibitor. Conclusion: WFDC2 contributed to ovarian cancer metastasis and EMT as a positive regulator by activating AKT signaling pathway and inducing MMP-2 expression.

Currently, quality control of glycoprotein in the human rabies vaccine is based on enzyme-linked immunosorbent assay (ELISA). However, ELISA does not match the needs of a modernised quality control system. For a long time, human rabies virus vaccine manufacturers have been devoted to seeking a detection platform that is sensitive, accurate, automatic, and feasible for practical applications. Therefore, our team invested major efforts into establishing a fully automated micromagnetic particle (MMP)-based chemiluminescence immunoassay (CLIA) platform. For vaccine quality control, MMP-coupled rabies virus glycoprotein monoclonal antibodies (S037) were used to capture the rabies virus. Another rabies virus glycoprotein antibody (S053) labelled with acridinium ester was added as a signal tracer. After pretreating the vaccine sample, the entire analysis was performed using a fully automated machine, which had a limited detection time (only 30 min) and eliminated manual error. Multiple experiments have identified the optimal conditions allowing valid and reliable assessment of vaccine potency. The CLIA platform has exhibited merits in terms of speed, robustness, high sensitivity (with a minimum detection value of 0.45 mIU/mL), considerable accuracy, and a wide linear range of detection (9.4-1200 mIU/mL). Furthermore, the results showed that the CLIA platform is consistent with the National Institutes of Health test and time-resolved fluorescent immunoassay (TRFIA) in quantitative analysis, and had a better analytic performance than TRFIA. Therefore, the CLIA platform presented here may be important for application in modern vaccine quality control.

Nano-radiosensitizers provide a powerful tool for cancer radiation therapy. However, their limited tumor retention/penetration and the inherent or adaptive radiation resistance of tumor cells hamper the clinical success of radiation therapy. Herein, we report a synergistic strategy for potentiated cancer radiation/gene therapy based on transformable gold nanocluster aggregates loaded with antisense oligonucleotide-targeting survivin mRNA (named AuNC-ASON). AuNC-ASON exhibited acidic pH-triggered structure splitting from a gold nanocluster aggregate (around 80 nm) to gold nanocluster (

Ameliorated therapy based on the tumor microenvironment is becoming increasingly popular, yet only a few methods have achieved wide recognition. Herein, targeting multifunctional hydrophilic nanomicelles, AgBiS2@DSPE-PEG2000-FA (ABS-FA), were obtained and employed for tumor treatment. In a cascade amplification mode, ABS-FA exhibited favorable properties of actively enhancing computed tomography/infrared (CT/IR) imaging and gently relieving ambient oxygen concentration by cooperative photothermal and sonodynamic therapy. Compared with traditional Bi2S3 nanoparticles, the CT imaging capability of the probe was augmented (43.21%), and the photothermal conversion efficiency was increased (33.1%). Furthermore, remarkable ultrasonic dynamic features of ABS-FA were observed, with increased generation of reactive oxygen species (24.3%) being obtained compared to Ce6, a commonly used sonosensitizer. Furthermore, ABS-FA exhibited obvious inhibitory effects on HeLa cell migration at 6 μg/mL, which to some extent, demonstrated its suppressive effect on tumor growth. A lower dose, laser and ultrasonic power, and shorter processing time endowed ABS-FA with excellent photothermal and sonodynamic effects. By mild cascade mode, the hypoxic condition of the tumor site was largely improved, and a suitable oxygen-rich environment was provided, thereby endowing ABS-FA with a superior synergistically enhanced treatment effect compared with the single-mode approach, which ultimately realized the purpose of "one injection, multiple treatment". Moreover, our data showed that ABS-FA was given with a biological safety profile while harnessing in vivo. Taken together, as a synergistically enhanced medical diagnosis and treatment method, the one-for-all nanoplatform will pave a new avenue for further clinical applications.

The current protein interaction method is time consuming and cumbersome or the instrument is expensive. A new method that is convenient, fast, and high throughput needs to be studied urgently. The purpose of this study was to establish a homogeneous immunoassay to detect the interaction between insulin-like growth factor-1 receptor-β (IGF1R-β) and suppressor of cytokine signaling 1 (SOCS1). The recombinant vectors IGF1R-β/pENTER and SOCS1/pENTER were constructed and transfected into 293T cells. Based on homogeneous immunoassay technology, we established a suitable method. The signal intensity in the 293T lysate that overexpressed IGF1R-β and SOCS1, respectively, was compared with the signal intensity in the simultaneous expression of IGF1R-β and SOCS1. The interaction between IGF1R-β and SOCS1 was verified in vitro. The detection system for the interaction between IGF1R-β and SOCS1 was established. Compared with other methods, homogeneous immunoassay has the advantages of being rapid and sensitive, having higher sensitivity, and easy to operate. The interaction between IGF1R-β and SOCS1 was tested to verify the feasibility of this method and prove its practicability and sensitivity. This new method can be used as a high-throughput platform for protein-protein interaction, with the advantages of trace detection, short detective time, and high detective sensitivity.

Objective To evaluate the analytical performance of our previously developed chemiluminescence immunoassay (CLIA) kit for the detection of procalcitonin (PCT) and compare with the results obtained using the Vidas B.R.A.H.M.S. PCT™ test (PCT-V). Design and methods Our laboratory previously designed a novel CLIA kit and supporting instrument (AE-180) for the detection of PCT. We analyzed the clinical performance of this system, including the imprecision, limit of detection, and linearity of analyses of 305 serum specimens. The results were compared with measurements of the same serum samples obtained with PCT-V. Results The limit of detection and blank of our kit were 0.0075 and 0.0039 ng/mL, respectively. The intra- and inter-assay coefficient of variation of the kit were both between 0.8% and 3.9%. The equation of linearity was found to be y = 1.03× + 0.06 (r = 0.99) for concentrations in the range of 0.01–110 ng/mL. The correlation coefficient with the results of PCT-V was 0.995, and the equation obtained for Passing and Bablok regression analysis was 1.061 for our CLIA PCT kit and − 0.003 for PCT-V. Our kit slightly overestimated the concentration according to comparison with PCT-V results. Conclusion The kit that was previously developed in our laboratory for the measurement of serum PCT concentration using CLIA technology shows excellent performance, just that the functional sensitivity is not as good as the PCT-V; therefore, we suggest that this kit is suitable for clinical use.

Janus-activated kinase-1 (JAK1) plays an important role in many signaling pathways, including the JAK–STAT and SOCS pathways. The activation of the JAK1–STAT3 pathway is a key role in tumor inflammation and immunity by promoting inflammation. The detection of its interacting proteins is of great significance in many diseases. Although luminescent oxygen channeling is commonly used in immunoassay technologies, its use in the detection of JAK1 and interacting proteins has not been reported to date. We constructed eukaryotic overexpression plasmids pENTER–JAK1-His and pENTER–STAT3-His and, following single transfection and co-transfection, measured their expression by western blotting. Co-immunoprecipitation and luminescent oxygen channeling were then used to identify the protein interactions. JAK1 and STAT3 proteins were shown to be successfully overexpressed, and co-immunoprecipitation identified their interaction in vitro. We also detected protein interactions between JAK1 and interleukin 4 receptor (IL4R), JAK1 and growth hormone receptor (GHR). In conclusion, we verified that JAK1 and STAT3 interact in vitro using co-immunoprecipitation, and demonstrated for the first time the potential application of luminescent oxygen channeling for detecting JAK1 and its interacting proteins. Also we think this assay could help other researchers find unreported proteins that also have interactions in vitro.

Micropeptides (≤100 amino acids) are essential regulators of physiological and pathological processes, which can be encoded by small open reading frames (smORFs) derived from long non-coding RNAs (lncRNAs). Recently, lncRNA-encoded micropeptides have been shown to have essential roles in tumorigenesis. Since translated smORF identification remains technically challenging, little is known of their pathological functions in cancer. Therefore, we created classifiers to identify translated smORFs derived from lncRNAs based on ribosome-protected fragment sequencing and machine learning methods. In total, 537 putative translated smORFs were identified and the coding potential of five smORFs was experimentally validated via green fluorescent protein-tagged protein generation and mass spectrometry. After analyzing 11 lncRNA expression profiles of seven cancer types, we identified one validated translated lncRNA, ZFAS1, which was significantly up-regulated in hepatocellular carcinoma (HCC). Functional studies revealed that ZFAS1 can promote cancer cell migration by elevating intracellular reactive oxygen species production by inhibiting nicotinamide adenine dinucleotide dehydrogenase expression, indicating that translated ZFAS1 may be an essential oncogene in the progression of HCC. In this study, we systematically identified translated smORFs derived from lncRNAs and explored their potential pathological functions in cancer to improve our comprehensive understanding of the building blocks of living systems.

In rheumatoid arthritis(RA) pathogenesis, activated RA fibroblast-like synoviocytes (RA-FLSs) exhibit similar proliferative features as tumor cells and subsequent erosion to cartilage will eventually lead to joint destruction. Therefore, it is imperative to search for compounds, which can effectively inhibit the abnormal activation of RA-FLSs, and retard RA progression.3'3-Diindolylmethane (DIM), the major product of the acid-catalyzed oligomerization of indole-3-carbinol from cruciferous vegetables, has been reported to be functionally relevant to inhibition of migration, invasion and carcinogenesis in some solid tumors. In this study, we explored the anti-proliferation, anti-metastasis and anti-inflammation effects of DIM on RA-FLSs as well as the underlying molecular mechanisms. To do this, primary RA-FLSs were isolated from RA patients and an animal model. Cell proliferation, migration and invasion were measured using CCK-8, scratch, and Transwell assays, respectively. The effects of DIM on Matrix metalloproteinases (MMPs) and some inflammatory factors mRNA and key molecules such as some inflammatory factors and those involved in aberrantly-activated signaling pathway in response to tumor necrosis factor α(TNF-α), a typical characteristic mediator in RA-FLS, were quantitatively measured by real-time PCR and western blotting. Moreover, the effect of DIM on adjuvant induced arthritis(AIA) models was evaluated with C57BL/6 mice in vivo. The results showed that DIM inhibited proliferation, migration and invasion of RA-FLS in vitro. Meanwhile, DIM dramatically suppressed TNF-α-induced increases in the mRNA levels of MMP-2, MMP-3, MMP-8, and MMP-9; as well as the proinflammatory factors IL-6, IL-8, and IL-1β. Mechanistic studies revealed that DIM is able to suppress phosphorylated activation not only of p38, JNK in MAPK pathway but of AKT, mTOR and downstream molecules in the AKT/mTOR pathway. Moreover, DIM treatment decreased expression levels of proinflammatory cytokines in the serum and alleviated arthritis severity in the knee joints of AIA mice. Taken together, our findings demonstrate that DIM could inhibit proliferation, migration and invasion of RA-FLSs and reduce proinflammatory factors induced by TNF-α in vitro by blocking MAPK and AKT/mTOR pathway and prevent inflammation and knee joint destruction in vivo, which suggests that DIM might have therapeutic potential for RA.

Single nucleotide polymorphism (SNP) was associated with many human diseases, therefore, SNP detection was important for early diagnosis and clinical prognosis. Herein, a simple and accurate method for visual detection SNP sites (A/A, G/G, A/G) in CYP1A1 gene related to cancers based on colloidal gold nucleic acid strip biosensor and primer-specific polymerase chain reaction (PCR) was established. This method could directly distinguish SNP sites on strip biosensor by introducing twice PCR amplifications. The second PCR (primer-specific PCR) was performed using specific product of the first PCR as template, thus this twice PCR could reduce non-specific amplification greatly and obtain target product. In addition, single-strand or double-strand DNA (ssDNA or dsDNA) was accurately produced by introducing mismatched base at the 3’ end of forward primers in primer-specific PCR. The designed strip biosensor could only combine with the ssDNA, thus visual detection of SNP could be achieved within 10 min by color difference of a pair of strips. 61 human blood samples by this method were identical with those of pyrosequencing. This method had the advantages of rapid, visual and low-cost and was expected to be applied in medical diagnosis.

A sensitive, rapid and homogeneous reaction measurement method for quantitation of human epididymis protein 4 (HE4) in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) was described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one biotinylated monoclonal antibody combining on the surface of streptavidin coated donor beads, and "sandwiched" by another monoclonal antibody coated on acceptor beads. The coefficient variations of the method were lower than 10%, and the recoveries were in the range of 90-110% for serum samples. A value of 0.88pmol/l was identified as the minimum detectable dose of the present method for HE4. Compared with the results from electrochemiluminescence immunoassay kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. The present assay demonstrated high sensitivity, wider effective detection range and excellent reproducibility for quantitation of HE4 can be useful for early screening and prognosis evaluation of patients with ovarian cancer.

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 isoenzyme creatine kinase MB is very important for diagnosis of acute myocardial infarction (AMI). Some CK-MB immunoassays are sensitive, accurate and available for clinical application, but they are expensive and time-consuming procedures. Furthermore, conventional fluorescence immunochromatographic assays (FL-ICAs) have suffered from background fluorescence interference and low analytical sensitivity. A rapid and simple FL-ICA with Eu (III) chelate polystyrene microparticles was developed to determine CK-MB in 50uL serum samples using a portable test strip reader by measuring the fluorescence peak heights of the test line (HT) and the control line (HC) in 12 min. The assay was reliable with a good correlation coefficient between HT/HC ratio and CK-MB concentration in samples. A linear range was 0.85-100.29 ng/mL for CK-MB, and the LOD was 0.029 ng/mL. The intra- and inter-assay coefficients of variation (CV) were both

Alpha-fetoprotein (AFP), a primary marker for many diseases including various cancers, is important in clinical tumor diagnosis and antenatal screening. Most immunoassays provide high sensitivity and accuracy for determining AFP, but they are expensive, often complex, time-consuming procedures. A simple and rapid point-of-care system that integrates Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) has been developed to determine AFP in serum with an assay time of 15 min. The approach is based on a sandwich immunoassay performed on lateral flow test strips. A fluorescence strip reader was used to measure the fluorescence peak heights of the test line (HT) and the control line (HC); the HT/HC ratio was used for quantitation. The Eu (III) chelate microparticles-based LFIA assay exhibited a wide linear range (1.0-1000 IU mL(-1)) for AFP with a low limit of detection (0.1 IU mL(-1)) based on 5ul of serum. Satisfactory specificity and accuracy were demonstrated and the intra- and inter-assay coefficients of variation (CV) for AFP were both

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 Eu3+-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. J. Med. Virol. 87:1940–1945, 2015. © 2015 Wiley Periodicals, Inc.

Quantitative hepatitis B core antigen (anti-HBc) measurements could play an important role in evaluating therapeutic outcomes and optimizing the antiviral therapy of chronic hepatitis B infection. In this study, we have developed a simple and rapid fluorescence point-of-care test based on a lateral flow immunoassay (LFIA) method integrated with Eu (III) chelate microparticles to quantitatively determine anti-HBc concentrations in serum. This assay is based on a direct competitive immunoassay performed on lateral flow test strips with an assay time of 15 min. The Eu (III) chelate microparticle-based LFIA assay could quantitatively detect anti-HBc levels with a limit of detection of 0.31 IU mL−1, and exhibited a wide linear range (0.63–640 IU mL−1). The intra- and inter-assay coefficients of variation for anti-HBc were both less than 10% and a satisfactory dilution test and accuracy were demonstrated. There were no statistically significant differences in sensitivity or specificity in serum samples between the Eu (III) chelate microparticle-based LFIA strips and the Abbott Architect kit. A simple, rapid and effective quantitative detection of anti-HBc was possible using the Eu (III) chelate microparticle-based LFIA strips. The strips will provide diagnostic value for clinical application.

Replacement of the in vivo rabies vaccine potency test (NIH test) by in vitro methods had been discussed by several researcher including WHO expert working groups. In this paper, a time-resolved fluoroimmunoassay (TRFIA) for the assay of rabies virus glycoprotein in rabies vaccine was first established to estimate the rabies vaccine potency by using specific monoclonal antibody that only recognized the native, trimeric and immunogenic form of rabies virus glycoprotein. Potency of the rabies virus glycoprotein was assayed with satisfactory performance under optimal conditions, and the method demonstrated satisfactory results when applied in practical samples. The correlation coefficient of potency values obtained from the present TRFIA and ELISA was 0.912, and 0.903 for those from the present TRFIA and NIH test. These preliminary results confirmed that this TRFIA can replace ELISA with higher performance, and could be a promising replacement of the NIH test. Based upon these results, the present TRFIA seemed to be a convenient tool for evaluating rabies vaccine potency and its products at different stages accordingly.

Enzyme-linked immunosorbent assays (ELISA) specific for anti-HSV glycoprotein G (gG) are most commonly used in the clinical diagnosis of HSV infection. But most of them are qualitative and with narrow detection ranges. A novel time-resolved fluoroimmunoassay (TRFIA) methodology was developed for the quantitative determination of HSV IgG in human serum. The assay was based on an indirect immunoassay format, and performed in 96-well microtiter plates. HSV-1 and HSV-2 were used as the coating antigens. Eu3+-labeled goat anti-(human IgG) polyclonal antibodies were used as tracers. The fluorescence intensity of each well was measured and serum HSV IgG levels quantified against a calibration curve. The detection range of the novel TRFIA was between 5 and 500 AU/mL. Assay sensitivity was 0.568 AU/mL. The intra- and inter-assay coefficients of variation were 0.59–3.63% and 3.65–6.81%, respectively. Analytical recovery, dilution tests and serum panel tests were performed using TRFIA and the results proved satisfactory. There were no statistically significant differences in sensitivity and specificity between the TRFIA and commercial ELISAs. An effective, sensitive and accurate quantitative HSV type 1 and type 2 IgG TRFIA was successfully developed and provided diagnostic value in clinical use. Copyright © 2014 John Wiley & Sons, Ltd.

Objectives Diabetes mellitus is a chronic disease affecting millions of people globally and resulting in significant death rates each year. A fast, inexpensive alternative to traditional testing and monitoring techniques is desirable, since secretion of insulin and C-peptide is impaired in diabetes mellitus. Design and methods A highly sensitive immunoassay was developed for the simultaneous measurement of C-peptide and insulin levels in human serum, utilizing dual-label time-resolved fluoroimmunoassay (TRFIA) and magnetic particle technologies. This assay was characteristic for a single-step sandwich-type immunoassay, wherein antibody-coated magnetic particles were used as the solid phase and Eu 3 + and Sm 3 + chelate labels were used for detection. Results Antibody-coated magnetic particles in a TRFIA format performed well in addressing a number of quantitative needs. Conclusions The results of this assay correlated well with commercial chemiluminescence assays and provided a number a advantages, including reduced sample volume, reduced reagent and personnel costs and reduced assay time, while maintaining the required clinical sensitivity.

A highly sensitive, rapid and novel simultaneous measurement method for cytokeratin 19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA) in human serum by magnetic nanoparticle-based dual-label time-resolved fluoroimmunoassay was developed. On the basis of a sandwich-type immunoassay format, analytes in the samples were captured by antibodies coated onto the surface of the magnetic beads and sandwiched by other antibodies labeled with europium and samarium chelates. The lower limit of quantitation of the present method for CYFRA 21-1 was 0.77 ng mL−1 and CEA was 0.85 ng mL−1. The coefficient variations of the method were less than 7%, and the recoveries were in the range of 90–110% for serum samples. The concentrations of CYFRA 21-1 and CEA serum samples determined by the present method were compared with those obtained by the chemiluminescence immunoassay. A good correlation was obtained with the correlation coefficients of 0.961 for CYFRA 21-1 and 0.938 for CEA. This novel method demonstrated high sensitivity, wide effective detection range and excellent reproducibility for the simultaneous determination of CYFRA 21-1 and CEA, which can be useful for the early screening and prognosis evaluation of patients with lung cancer.

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.

Background Cytokeratin 19 fragment antigen (CYFRA 21–1) is used to diagnose and monitor neoplasms. However, the main disadvantages of the currently available CYFRA 21–1 assays include heterogenous technology, being time-consuming, and having low through-put with low insensitivity. This study investigated the use of amplified luminescent proximity homogeneous immunoassay (AlphaLISA) for the quantization of CYFRA 21–1 in human serum. Methods The AlphaLISA kit was developed based on AlphaScreen detection technology with two different anti-CYFRA 21–1 monoclonal antibodies. One was coated on AlphaLISA acceptor beads and the other was biotinylated. Donor beads were coated with streptavidin. The test conditions were optimized and analytical performance was studied. Results The measurement range of AlphaLISA CYFRA 21–1 kit was 0.08–500 ng/ml. Assay detection limit was 0.08 ng/ml. The intra- and interassay coefficients of variation were 3.00–9.00% and 4.00–10.00%, respectively. There was no cross-reaction to alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 19–9 (CA19–9), cytokeratins 8 (CK8), and cytokeratins 18 (CK18). The correlation coefficient of blood samples involved was 0.974 between CYFRA 21–1-AlphaLISA assay and a commercial electrochemiluminescence immunoassay (ECLIA) CYFRA 21–1 kit (Roche). Conclusions The AlphaLISA CYFRA 21–1 kit developed in this study had favorable performance characteristics for clinical application with acceptable analytical sensitivity, specificity, and accuracy.

A novel quantum dot-doped polystyrene nanoparticles-based lateral flow test strips (QPs-LFTS) system was developed to simultaneously detect a cytokeratin-19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA) in human serum to aid the diagnosis and prognosis of lung cancer. Quantum dot-doped carboxylate-functionalized polystyrene nanoparticles (QPs) were prepared and introduced as fluorescent reporters in QPs-LFTS. The detection was based on a sandwich immunoassay and performed on lateral flow test strips, with an assay time of 15 min. The strips were read by a fluorescence strip reader to obtain the fluorescence peak heights of the test lines (HT) and the control line (HC). The ratio of HT/HC was used for quantitation. The QPs showed excellent photoproperties and good performance. Under optimal conditions, the QPs-LFTS system exhibited a wide linear range for CYFRA 21-1 (1.3–480 ng/mL) and CEA (2.8–680 ng/mL). The detection limits for CYFRA 21-1 and CEA were 0.16 and 0.35 ng/mL, respectively. The recovery and reproducibility of the method were satisfactory. Furthermore, excellent correlations (n =120, R2 =0.9862, P

In this paper, a novel time-resolved fluorescence immunoassay (TRFIA) is described that allows the simultaneous quantitative detection of hepatitis B virus surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in human serum to aid the diagnosis and monitoring of hepatitis B virus infection. The proposed method was developed based on a two-step sandwich immunoassay protocol in which monoclonal antibodies against HBsAg and HBeAg were co-coated in 96 microtitration wells, then tracer polyclonal antibodies against HBsAg labeled with samarium and tracer monoclonal antibodies against HBeAg labeled with europium chelates were used for detection. The detection range was 0.1–150 IU/mL for HBsAg and 0.5–160 PEIU/mL for HBeAg, and the detection limits were 0.03 IU/L and 0.09 PEIU/ml, respectively. The intra- and inter-assay coefficients of variation were below 8 % for both virus antigens. The dilution linearity and accuracy of the assay were satisfactory. No statistically significant differences were observed in sensitivity or specificity for the serum samples between the dual-label TRFIA and a commercial single-label TRFIA. These results demonstrate that an effective, reliable and convenient HBsAg/HBeAg dual-label TRFIA was successfully developed that may be clinically applicable for blood screening to monitor the course of hepatitis B virus infection and predict treatment responses.

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

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.

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.

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.

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.

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.

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.

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

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.