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Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5′- and 3′- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1 × 10 −19 mol in mass and 2.9 pM in concentration. [ABSTRACT FROM AUTHOR]

Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5′- and 3′- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1 × 10 −19 mol in mass and 2.9 pM in concentration. [ABSTRACT FROM AUTHOR]

Human DNA is exposed to a variety of endogenous and environmental agents that may induce a wide range of damage. The critical role of DNA damage in cancer development makes it essential to develop highly sensitive and specific assays for DNA lesions. We describe here ultrasensitive assays for DNA damage, which incorporate immuno-affinity with capillary electrophoresis (CE) separation and laser induced fluorescence (LIF) detection. Both competitive and non-competitive assays using CE/LIF were developed for the determination of DNA adducts of benzo[a]pyrene diol epoxide (BPDE). A fluorescently labeled oligonucleotide containing a single BPDE adduct was synthesized and used as a fluorescent probe for competitive assay. Binding between this synthetic oligonucleotide and a monoclonal antibody (MAb) showed both 1:1 and 1:2 complexes between the MAb and the oligonucleotide. The 1:1 and 1:2 complexes were separated by CE and detected with LIF, revealing binding stoichiometry information consistent with the bidentate nature of the immunoglobulin G antibody. For non-competitive assay, a fluorescently labeled secondary antibody fragment F(ab′)2 was used as an affinity probe to recognize a primary antibody that was specific for the BPDE-DNA adducts. The ternary complex of BPDE-DNA adducts with the bound antibodies was separated from the unbound antibodies using CE and detected with LIF for quantitation of the DNA adducts. The assay was used for the determination of trace levels of BPDE-DNA adducts in human cells. Analysis of cellular DNA from A549 human lung carcinoma cells that were incubated with low doses of BPDE (32 nM–1 μM) showed a clear dose–response relationship. BPDE is a potent environmental carcinogen, and the ultrasensitive assays for BPDE-DNA adducts are potentially useful for monitoring human exposure to this carcinogen and for studying cellular repair of DNA damage. [Copyright &y& Elsevier]

We described an aptamer based and Mg2+ mediated free zone capillary electrophoresis-laser induced fluorescence (CE-LIF) assay for aflatoxin B1 (AFB1) detection. This CE-LIF assay applied an anti-AFB1 aptamer with a single fluorescein (FAM) label at 5′ end and a short complementary DNA (cDNA). In the absence of AFB1, the cDNA hybridized with the aptamer probe and formed a duplex DNA. The use of running buffer containing MgCl 2 allowed good isolation of the duplex DNA from the single stranded DNA in CE. We found introducing a biotin label on the cDNA further improved the isolation. When AFB1 existed in sample solution, the aptamer probe bound with AFB1, dissociating from the duplex DNA. Thus, the duplex DNA peak decreased, while the aptamer probe peak increased during CE-LIF analysis. We achieved detection of AFB1 by measuring the aptamer probe peak. The length of cDNA, the ratio of aptamer to cDNA, and the concentration of MgCl 2 in sample buffer and separation buffer had great effect on the aptamer based CE-LIF assay. Under optimized conditions, the detection limit of AFB1 was 0.2 nM, and the dynamic range was from 0.2 nM to 500 nM. Limit of quantitation was 0.5 nM. This CE-LIF assay enabled detection of AFB1 spiked in diluted human serum, diluted human urine, and corn flour samples. This assay exhibits potential for wide application as it integrates the rapidity, high sensitivity, low sample consumption of CE-LIF analysis and the strengths of aptamer. An aptamer free zone capillary electrophoresis coupled with laser induced fluorescence assay (CE-LIF) was developed for detection of aflatoxin B1 (AFB1). AFB1 competes with a short complementary DNA (cDNA) in the binding to a fluorescein (FAM)-labeled aptamer probe. The duplex DNA is well isolated from the FAM-labeled aptamer in CE with separation buffer containing MgCl 2. The presence of AFB1 caused decrease of the aptamer-cDNA duplex peak and increase of aptamer probe peak during CE-LIF analysis. Image 1 • Aptamer free zone capillary electrophoresis (CE) assay for aflatoxin B1 (AFB1). • Complementary DNA (cDNA) and fluorescein-labeled aptamer probe are used. • MgCl 2 mediated CE allows separation of aptamer probe and aptamer-cDNA duplex. • AFB1 binding causes decrease of duplex peak and increase of aptamer probe peak. • AFB1 is detected by peaks changes in CE coupled with laser induced fluorescence. [ABSTRACT FROM AUTHOR]

Aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) combines the advantages of affinity aptamer, rapid CE separation, and high sensitivity detection. Here we reported an affinity CE-LIF assay for thrombin by using a fluorophore-labeled RNA aptamer containing 2′-fluoro modification in sugar rings of pyrimidine nucleotides (C and U) as affinity ligand. This RNA aptamer has high binding affinity, specificity and biostability. Thrombin at 0.2 nM was successfully detected. This RNA aptamer allowed for the detection of thrombin spiked in diluted human serum sample due to the nuclease resistance. The RNA aptamer has comparable binding affinity to a 29-mer DNA aptamer for thrombin, and the binding site of the RNA aptamer on thrombin partially overlaps with the binding site of the 29-mer DNA aptamer on thrombin. It shows the nuclease-resistant RNA aptamers are promising in assays for thrombin. [ABSTRACT FROM AUTHOR]

Abstract: Assessment of the underlying molecular events leading to xenobiotic toxicity is challenging especially when techniques are applied in isolation. We examined transcriptional and metabolic changes in Lumbriculus variegatus exposed to benzo(a)pyrene (B(a)P), cadmium (Cd) or pentachlorophenol (PCP) by DNA microarrays (7422 ESTs) and gas chromatography–mass spectrometry (GC–MS), respectively. In addition, the DNA damage response of worms exposed to B(a)P was assessed by a capillary electrophoresis laser induced fluorescence (CE-LIF) immunoassay. We found elevated expression of oxidative stress responsive genes, which correlated positively with the changes in antioxidant vitamin precursors including alpha-tocopherol and cholecalciferol. Other genes with strong differential expressions were mostly involved in actin related processes and proteolysis, despite an apparent delayed Cd response. Phosphates, sugars and fatty acids were effectively reduced and suggested that chemical treatments may have interfered with energy metabolism. The increased amount of B(a)P diol-epoxide (BPDE)–DNA adducts in exposed worms appeared to correlate with the variability in uridine, inosine and xanthine, which are key components of nucleoside metabolism. This suggests that DNA damage was imminent or peaked within 6h. The results conformed to transcriptional changes in B(a)P exposed worms and compliment other approaches to elucidate underlying molecular changes. [Copyright &y& Elsevier]

Abstract: The stereospecific binding of monoclonal antibody (mAb) 8E11 to anti-benzo(a)pyrene diol epoxide (BPDE)-dG adducts in single nucleoside, long oligonucleotide, and genomic DNA were quantitatively evaluated using noncompetitive and competitive capillary electrophoresis (CE) immunoassays. Two single-stranded TMR-BPDE-90mers containing a single anti-BPDE-dG adduct with defined stereochemistry and a fluorescent label at 5′-end were used as fluorescent probes for competitive CE immunoassay. To quantitatively evaluate the binding affinity through competitive CE immunoassays, a series of equations were derived according to the binding stoichiometry. The binding of mAb 8E11 to trans-(+)-anti-BPDE-dG displays strongest affinity (K b: 3.57×108 M−1) among all four investigated anti-BPDE-dG mononucleoside adducts, and the cis-(−)-anti-BPDE-dG displays lowest affinity (K b: 1.14 ×107 M−1). The binding of monoclonal antibody (mAb) 8E11 to BPDE-dG adducts in long DNA (90mer) preferentially forms the complex with a stoichiometry of 1:1, and that mAb 8E11 displays a slightly higher affinity with trans-(+)-anti-BPDE-90mers (K b: 6.36±0.54×108 M−1) than trans-(−)-anti-BPDE-90mers (K b: 4.52±0.52×108 M−1). The mAb 8E11 also displays high affinity with BPDE-dG adducts in genomic DNA (K b: 3.74×108 M−1), indicating its promising applications for sensitive immuno-detection of BPDE-DNA adducts in genomic DNA. [Copyright &y& Elsevier]

Abstract: The cellular mechanism based on P-glycoprotein (PGP) for its drug pump function has become very important in multidrug resistance (MDR) research. A method has been established to characterize PGP on single K562 cell by coupling capillary electrophoresis with laser induced fluorescence detection. A permeable intact cell after the immunoassay binding with fluorescence labeling antibody was injected into the capillary and directly separated without lysis. It was found that once 5–10 optional cells were detected in batch, the PGP amount on this cell line could be outlined and calculated clearly. The PGP amount on K562 MDR cell line is 3.88 times higher than that on K562 sensitive cell line. These two cell lines with immunoassay binding were also analyzed by injection of multi-cells in order to improve the throughput. A resistance factor so called multidrug resistance multiple (MRM) was introduced to evaluate the MDR difference between cell lines. The MRM values of the cell line K562 measured by single cell analysis are well correlated with those by flow cytometry, which also prove the validity of our method in single cell analysis for the possibility of cancer diagnosis, pharmacokinetics and drug screening in future. [Copyright &y& Elsevier]