Novel biosensor chip for simultaneous detection of DNA-carcinogen adducts with low-temperature fluorescence.
A monoclonal antibody (MAb)-gold biosensor chip with low-temperature laser-induced fluorescence detection for analysis of DNA-carcinogen adducts is described. Optimization of the detection limit, dynamic range, and biosensing applicability of the MAb-gold biosensor chip was achieved by: (1) using dithiobis(succinimidyl propionate (DSP)) as a protein linker and (2) employing recombinant protein A to provide oriented immobilization of the MAbs. The use of DSP, which has a short methylene chain length, led to faster protein binding kinetics and higher protein surface density than a longer dithiobis(succinimidyl undecanoate) (DSU) linker. The incorporation of recombinant protein A increased the distance between the oriented MAb-bound analytes and the gold surface. The increased distance minimized fluorescence quenching, resulting in about a 10-fold increase in the fluorescence signal in comparison with a chip without protein A. The improved chip architecture was used to demonstrate that biosensing of two structurally similar benzo[a]pyrene (BP)-derived DNA adducts, BP-6-N7Gua and BP-diolepoxide-10-N2dG, bound to two specific MAbs immobilized from a mixture at the same address on the chip, is feasible. These mutagenic adducts are formed by one-electron oxidation and monooxygenation pathways, and are depurinating and stable DNA adducts, respectively. It is shown that the DNA adducts can be easily identified at the same address using time-resolved, low-temperature laser-based fluorescence spectroscopy. The current limit of detection is in the low femtomole range. These results indicate that a single biosensor chip consisting of a Au/DSP/protein A/MAb nano-assembly, with analyte-specific MAbs and low-temperature fluorescence detection should be suitable for simultaneous detection and quantitation of the above adducts, as well as the luminescent antigens for which selective MAbs exist.
Grubor
Nenad M
NM
Ames Laboratory--USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Shinar
Ruth
R
Jankowiak
Ryszard
R
Porter
Marc D
MD
Small
Gerald J
GJ
D001564:Benzo(a)pyrene / Q000032:analysis* / Q000737:chemistry; D001580:Benzopyrenes / Q000032:analysis / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002273:Carcinogens / Q000032:analysis* / Q000737:chemistry; D003080:Cold Temperature; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015200:Fluoroimmunoassay / Q000295:instrumentation* / Q000379:methods; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
C102824:7-(benzo(a)pyren-6-yl)guanine; D001580:Benzopyrenes; D002273:Carcinogens; D018736:DNA Adducts; D001564:Benzo(a)pyrene; D006147:Guanine