A Novel Homogeneous Time-Resolved Fluoroimmunoassay for Carcinoembryonic Antigen Based on Water-Soluble Quantum Dots

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 Förster distance (R0) was calculated as 61.9 Å 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 Å. 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.

[1]  C. Kuo,et al.  Carcinoembryonic antigen in monitoring of response to cetuximab plus FOLFIRI or FOLFOX-4 in patients with metastatic colorectal cancer. , 2008, The International journal of biological markers.

[2]  S. Ashley,et al.  A Novel Role for Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6 as a Determinant of Gemcitabine Chemoresistance in Pancreatic Adenocarcinoma Cells , 2004, Cancer Research.

[3]  T. Roux,et al.  Homogeneous time-resolved fluorescence-based assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a. , 2011, Analytical biochemistry.

[4]  Jiang Xia,et al.  Fast self-assembly kinetics of quantum dots and a dendrimeric peptide ligand. , 2012, Langmuir : the ACS journal of surfaces and colloids.

[5]  Tian-Cai Liu,et al.  Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum. , 2012, Analytica chimica acta.

[6]  P. Selvin,et al.  Amine-reactive forms of a luminescent diethylenetriaminepentaacetic acid chelate of terbium and europium: attachment to DNA and energy transfer measurements. , 1997, Bioconjugate chemistry.

[7]  N. Hildebrandt,et al.  Semiconductor quantum dots as FRET acceptors for multiplexed diagnostics and molecular ruler application. , 2012, Advances in experimental medicine and biology.

[8]  T. M. Chu,et al.  Demonstration of Carcinoembryonic Antigen in Normal Human Plasma , 1972, Nature.

[9]  Hans-Gerd Löhmannsröben,et al.  Lanthanides to quantum dots resonance energy transfer in time-resolved fluoro-immunoassays and luminescence microscopy. , 2006, Journal of the American Chemical Society.

[10]  Jin-Ming Lin,et al.  Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen , 2007, Analytical and bioanalytical chemistry.

[11]  J E Hearst,et al.  Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[12]  T. Mitsudomi,et al.  Serum carcinoembryonic antigen level as a surrogate marker for the evaluation of tumor response to chemotherapy in nonsmall cell lung cancer. , 2010, Annals of thoracic and cardiovascular surgery : official journal of the Association of Thoracic and Cardiovascular Surgeons of Asia.

[13]  Niko Hildebrandt,et al.  Lanthanide Complexes and Quantum Dots: A Bright Wedding for Resonance Energy Transfer , 2008 .

[14]  Xiaoyan Yang,et al.  Luminol/antibody labeled gold nanoparticles for chemiluminescence immunoassay of carcinoembryonic antigen. , 2010, Analytica chimica acta.

[15]  I. Hemmilä,et al.  Development of luminescent lanthanide chelate labels for diagnostic assays , 1997 .

[16]  D. Balding,et al.  HLA Sequence Polymorphism and the Origin of Humans , 2006 .

[17]  W. Hsu,et al.  Preoperative serum carcinoembryonic antigen level is a prognostic factor in women with early non-small-cell lung cancer. , 2007, The Annals of thoracic surgery.

[18]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[19]  I. Willner,et al.  Amplified multiplexed analysis of DNA by the exonuclease III-catalyzed regeneration of the target DNA in the presence of functionalized semiconductor quantum dots. , 2011, Nano letters.

[20]  K. Chaouachi,et al.  Hookah smoking and cancer: carcinoembryonic antigen (CEA) levels in exclusive/ever hookah smokers , 2008, Harm reduction journal.

[21]  Frank Morgner,et al.  Terbium to Quantum Dot FRET Bioconjugates for Clinical Diagnostics: Influence of Human Plasma on Optical and Assembly Properties , 2011, Sensors.

[22]  E. Donaldson,et al.  The clinical significance of carcinoembryonic antigen in the plasma and tumors of patients with gynecologic malignancies , 1978, Cancer.

[23]  T. Lövgren,et al.  Europium as a label in time-resolved immunofluorometric assays. , 1984, Analytical biochemistry.

[24]  Shimon Weiss,et al.  Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated CdSe/ZnS Semiconductor Quantum Dots† , 2001 .

[25]  G. Mathis,et al.  Homogeneous immunoassays using rare earth cryptates and time resolved fluorescence: principles and specific advantages for tumor markers. , 1997, Anticancer research.

[26]  B. Valeur,et al.  Molecular Fluorescence: Principles and Applications , 2001 .

[27]  S. Nie,et al.  Quantum dot bioconjugates for ultrasensitive nonisotopic detection. , 1998, Science.

[28]  L. Kricka,et al.  An enhanced chemiluminescent enzyme immunoassay for serum carcinoembryonic antigen based on a modification of a commercial kit. , 1986, Journal of bioluminescence and chemiluminescence (Print).

[29]  S. Schulman.  Fluorescence and phosphorescence spectroscopy : physicochemical principles and practice , 1977 .

[30]  Phil Gold,et al.  SPECIFIC CARCINOEMBRYONIC ANTIGENS OF THE HUMAN DIGESTIVE SYSTEM , 1965, The Journal of experimental medicine.

[31]  Zhen-Li Huang,et al.  A Feasible and Quantitative Encoding Method for Microbeads with Multicolor Quantum Dots , 2007, Journal of Fluorescence.

[32]  M. S. Miranda,et al.  CdSe quantum dots capped PAMAM dendrimer nanocomposites for sensing nitroaromatic compounds. , 2011, Talanta.

[33]  R. Stockley,et al.  Effect of cigarette smoking, pulmonary inflammation, and lung disease on concentrations of carcinoembryonic antigen in serum and secretions. , 1986, Thorax.

[34]  Z. Wang,et al.  Effect of temperature on the photoproperties of luminescent terbium sensors for homogeneous bioassays. , 2013, Luminescence : the journal of biological and chemical luminescence.

[35]  V. Yang,et al.  Development and Validation of an Enzyme Linked Immunosorbent Assay for the Quantification of Carcinoembryonic Antigen in Mouse Plasma , 2009, Journal of immunoassay & immunochemistry.

[36]  Xiaogang Peng,et al.  Experimental Determination of the Extinction Coefficient of CdTe, CdSe, and CdS Nanocrystals , 2003 .

[37]  M. Kuroki,et al.  Carcinoembryonic Antigen–Targeted Selective Gene Therapy for Gastric Cancer through FZ33 Fiber-Modified Adenovirus Vectors , 2006, Clinical Cancer Research.

[38]  L. Kricka.  Selected strategies for improving sensitivity and reliability of immunoassays. , 1994, Clinical chemistry.

[39]  Qingming Luo,et al.  Temperature-dependent photoluminescence of water-soluble quantum dots for a bioprobe , 2006 .

[40]  A. de Diego,et al.  Usefulness of carcinoembryonic antigen determination in bronchoalveolar lavage fluid. A comparative study among patients with peripheral lung cancer, pneumonia, and healthy individuals. , 1991, Chest.

[41]  A. Fletcher,et al.  Fluorescence quantum yields of some rhodamine dyes , 1982 .

[42]  Frank Morgner,et al.  A quantum-dot-based molecular ruler for multiplexed optical analysis. , 2010, Angewandte Chemie.

[43]  E. Diamandis,et al.  Ultrasensitive bioanalytical assays using time-resolved fluorescence detection. , 1995, Pharmacology & therapeutics.

[44]  W. Law,et al.  Aqueous-phase synthesis of highly luminescent CdTe/ZnTe core/shell quantum dots optimized for targeted bioimaging. , 2009, Small.

[45]  Phil Gold,et al.  DEMONSTRATION OF TUMOR-SPECIFIC ANTIGENS IN HUMAN COLONIC CARCINOMATA BY IMMUNOLOGICAL TOLERANCE AND ABSORPTION TECHNIQUES , 1965, The Journal of experimental medicine.

[46]  K. Shin,et al.  The clinical values of squamous cell carcinoma antigen and carcinoembryonic antigen in patients with cervical cancer treated with concurrent chemoradiotherapy , 2006, International Journal of Gynecologic Cancer.

[47]  D. Bravo,et al.  Biomimetic, Mild Chemical Synthesis of CdTe-GSH Quantum Dots with Improved Biocompatibility , 2012, PloS one.

[48]  S. Nie,et al.  Luminescent quantum dots for multiplexed biological detection and imaging. , 2002, Current opinion in biotechnology.

[49]  M. Kuroki,et al.  Establishment and Evaluation of a New Chemiluminescent Enzyme Immunoassay for Carcinoembryonic Antigen Adapted to the Fully Automated ACCESS ® System , 1996, European journal of clinical chemistry and clinical biochemistry : journal of the Forum of European Clinical Chemistry Societies.

[50]  Steven C Kazmierczak,et al.  Nanodiagnostics: a new frontier for clinical laboratory medicine. , 2006, Clinical chemistry.

[51]  L. Leong,et al.  A Phase I Study of a Combination of Yttrium-90–Labeled Anti–Carcinoembryonic Antigen (CEA) Antibody and Gemcitabine in Patients with CEA-Producing Advanced Malignancies , 2009, Clinical Cancer Research.

[52]  S. Nie,et al.  Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules , 2001, Nature Biotechnology.

[53]  I. Mackay,et al.  Increased carcinoembryonic antigen in heavy cigarette smokers. , 1973, Lancet.

[54]  P. de Paepe,et al.  Carcinoembryonic antigen elevation in agnogenic myeloid metaplasia , 2000, British journal of haematology.