Label‐Free Homogeneous Immunosensor Based on FRET for the Detection of Virus Antibody in Serum

Abstract

As a member of the polyomaviridae family, simian vacuolating virus 40 (SV40), a non-enveloped small tumorigenic virus with a circular double-stranded DNA genome, was first identified in 1960 in cultures of rhesus monkey kidney cells. Its capsid with a diameter of about 45 nm is formed by 72 pentameric capsomers composed of three capsid proteins, VP1, VP2, and VP3. [1] The occurrence of nucleic acid sequences identical to those of SV40 has been reported in certain types of human tumors, including osteosarcoma, mesothelioma, choroid plexus tumors, and ependymomas. [2] Especially, SV40 is closely related to the human polyomaviruses BK virus (BKV) and JC virus (JCV), for the SV40 genome has about 70 % nucleotide sequence homology with the BKV and JCV genomes. [3] Besides, it has been suggested that antibody to the SV40 in low levels was found in human sera. [4] Thus, owing to the potential human health risk, the detection of antibody to SV40 in serum is expected to be a main approach to human cancer diagnosis relating to SV40. So far, the acknowledged detection method of antibodies to SV40 is serological. Levels of antibodies to SV40 can be measured by plaque inhibition neutralization assay, but the outcomes may take one to two weeks. Another widely used method is enzyme immunoassay (EIA) technology, which is the preferred method for measurement of antiviral antibodies, because it provides greater sensitivity and precision than tissue-culture-based assays. [5] However, the assay is inconvenient and unsuitable for the rapid and simple determination of antibody, due to laboratory operation and the need for skilled technicians and special facilities. [6] To overcome these drawbacks, a homogeneous immunoassay without any separation step could allow the detection of targets by ligand binding interactions under physiological conditions. [7] Besides, fluorescence has been selected for signal output due to its rapidness, simplicity, sensitivity, and reproducibility. [8] Fortunately, it has been found that the SV40 VP1 alone has the capacity to package the viral minichromosome [9] and then efficiently self-assembles to form SV40 virus-like particles (SVLP) in vitro, and that it can be used to encapsulate quantum dots through molecular self-assembly to form SLVP-QDs chimeric particles. [10] SVLP and SVLP-QDs chimeric particles both can stably exist in the presence of appropriate salt concentration and Ca 2 + over a pH range from 5 to 8. [11] Given a hint by that, we report herein a label-free homogenous fluorescence immunosensor for anti-SV40 antibody in serum for the first time. The immunosensor is based on fluorescence resonance energy transfer (FRET) where SVLP-QDs and graphene oxide (GO) are the energy donor and acceptor, respectively. QDs have always been a popular choice of energy donor [12] in FRET because of a variety of distinguished optical features, including high quantum yield, broad excitation spectra, narrow emission spectra, and photobleaching resistance. [13] However, the labeling the surface of a virus with QDs may affect the viral infectivity and the immunogenicity. [14] SVLP-QDs can solve these problems excellently and keep the strong fluorescence of QDs. But as a coin has the other side, SVLP-QDs may increase the distance between donor and acceptor, which will interfere with the efficiency of FRET. Theoretical calculations have indicated that graphene could act as a superquencher of organic dyes, as a result of nonradiative transfer of electronic excitation energy from dye excited states to the p system of graphene. The rate of this long-range resonance energy transfer was suggested to have a d 4 dependence on distance d, in sharp contrast to traditional FRET, for which the rate has a d 6 dependence. [15] Graphene and GO were widely used as acceptors and exhibited high efficiency in quenching the donor fluorescence emission. [16] Thus, GO was selected as the acceptor, and a FRET-based immunosensor was developed. We revealed the energy transfer from SVLP-QDs to GO and constructed an immunosensing platform which could be used for rapid, inexpensive, and easy-to-perform detection of anti-SV40 antibody directly in serum.