Bioelectronic DNA detection of human papillomaviruses using eSensor™: a model system for detection of multiple pathogens

Research article Open Access Open Peer Review Bioelectronic DNA detection of human papillomaviruses using eSensor™: a model system for detection of multiple pathogens Suzanne D Vernon1Email author, Daniel H Farkas2, 3, Elizabeth R Unger1, Vivian Chan2, 4, Donna L Miller1, Yin-Peng Chen2, Gary F Blackburn2 and William C Reeves1 BMC Infectious Diseases20033:12 https://doi.org/10.1186/1471-2334-3-12 ©  Vernon et al; licensee BioMed Central Ltd. 2003 Received: 06 January 2003Accepted: 19 June 2003Published: 19 June 2003 Open Peer Review reports Abstract Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor™) in identifying multiple pathogens. Methods Two chips were spotted with capture probes consisting of DNA oligonucleotide sequences specific for HPV types. Electrically conductive signal probes were synthesized to be complementary to a distinct region of the amplified HPV target DNA. A portion of the HPV L1 region that was amplified by using consensus primers served as target DNA. The amplified target was mixed with a cocktail of signal probes and added to a cartridge containing a DNA chip to allow for hybridization with complementary capture probes. Results Two bioelectric chips were designed and successfully detected 86% of the HPV types contained in clinical samples. Conclusions This model system demonstrates the potential of the eSensor platform for rapid and integrated detection of multiple pathogens. Keywords FerroceneSignal ProbeCapture ProbeReverse Line BlotMultiple Pathogen Background Global emergence of pathogenic infectious diseases by both natural and intentional means presents a formidable challenge to infectious disease surveillance and response, namely timely and efficient pathogen detection. Many laboratory methods exist for identifying pathogens, but most require exquisite care in sample handling and processing prior to characterization of a pathogen. In addition, costly and perishable reagents, equipment, and supplies are required for sensitive and specific detection. The ideal detection system would integrate sample processing and pathogen characterization into a single automated device that would eliminate laborious, and time consuming sample processing and costly detection. Bioelectronic detection of nucleic acids on a miniature solid support is one of the first steps toward development of such an integrated detection device. Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [1–4]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Current eSensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample. Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5, 6] and viruses [7, 8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need. Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers [9]. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample. Methods eSensor™ DNA Chip Design We used previously described printed circuit board technology to manufacture eSensor chips with 16 gold electrodes, one reference electrode, and one auxiliary electrode [2–4]. Each electrode was wired to a connector at the edge of the chip. Capture probes were synthesized using an alkane chain of 16 residues terminated at the 3'-end with a disulfide which was spotted onto the gold electrode on the chips to form a self-assembled monolayer [10]. The chips were glued into a plastic housing with a port for sample introduction. Hybsimulator (RNAture, Inc., Irvine, CA) was used to design the HPV target mimics, and signal and capture probes. For these experiments, we designed capture (Table 1) and signal (Table 2) probes to hybridize specifically to the exon sequence of β-globin and to HPV sequences within the 450-bp L1 region amplified by the PGMY primer set. Signal probes (Table 2) tagged with eight ferrocenes were synthesized with a modified adenine residue (N6) containing a ferrocene substitution on the ribose ring [11]. No signal probes were synthesized for HPV types 33, 39, 55, and 73. HPV target mimic oligonucleotides were synthesized for types 6, 11, 16, 18, 26, 31, 35, 40, 42, 45, 51, 52, 54, 56, 58, 59, 66, 68, 82, 83, and 84 (Table 3). Table 1 Human papillomavirus (HPV) capture probe and PCR primers sequences. 6 capture CAG AAT TGG TGT ATG TGG AAG A(N152) 11 capture TAA TCT GAA TTA GTG TAT GTA GCA GAT TTA GAC A(N152) 16 capture GTA GTT TCT GAA GTA GAT ATG G(N152) 18 capture TGG TAG CAT CAT ATT GCC CAG G(N152) 26 capture ATC AGA TGG TTT AAA TGG AGT GGA TGC(N152) 31 capture TAC TAC TTT TAA ATG TAG TAT CAC(N152) 35 capture ACT GTC ACT AGA AGA CAC AGC AGA ACA CA(N152) 40 capture GGG GGA CTG TGT GGC ACC A(N152) 42 capture AGC AGC TGT ATA TGT ATC ACC AGA TGT TGC AGT GGC TCA(N152) 45 capture CTT AGT AGG GTC ATA TGT ACT TGG C(N152) 51 capture TTG GGG AAA CCG CAG CAG TGG CAG GGC TA(N152) 52 capture TAT GTG CTT TCC TTT TTA ACC T(N152) 54 capture GTC AGA ATT ATT AAA GCT ATC CTG CG(N152) 56 capture TTT TCG TGC ATC ATA TTT ACT TA(N152) 58 capture GTA CCT TCC TTA GTT ACT TCA G(N152) 59 capture CTG GTA GGT GTG TAT ACA TTA G(N152) 66 capture CAC GGG CAT CAT ATT TAG TTA A(N152) 68 capture TTA AAT T (...truncated)