Direct detection of DNA conformation in hybridization processes

DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a "discrete molecule binding" approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or "Y-shaped". Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays.     

Parallel single nucleotide polymorphism genotyping by surface invasive cleavage with universal detection

Large-scale investigations of sequence variation within the human species will provide information about the basis of heritable variation in disease susceptibility and human migration. The surface invader assay (an adaptation of the invasive cleavage reaction to an array format) is capable of exquisitely sensitive and specific detection of genetic variation. It is shown here that this genotyping technology can be multiplexed in a DNA array format, permitting the parallel analysis of a panel of single nucleotide polymorphisms (SNPs) directly from an unamplified genomic DNA target. In addition, a "universal" mode of detection was developed that makes use of a mixture of degenerate templates for DNA ligation to the surface-bound cleaved oligonucleotides and thereby makes this strategy amenable to any desired SNP site or combination of SNP sites, without regard to their particular DNA sequences. This approach was demonstrated on a proof-of-principle scale using small DNA arrays to genotype 6 SNP markers in the PTPN1 gene and 10 mutations in the cystic fibrosis transmembrane conductance regulator gene. This ability to analyze many different genetic variations in parallel, directly from unamplified human genomic DNA samples, lays the groundwork for the development of high-density arrays able to analyze hundreds of thousands or even millions of SNPs.     

Accurate DNA fragment sizing by capillary electrophoresis with laser-induced fluorescence array for detection of sequence specificity of DNA damage

Cancer has been linked to mutations within specific codons in genes that code for critical biomolecules such as tumor suppressor proteins (e.g., p53). Activated metabolites like benzo[a]pyrenediol epoxide act on preferred nucleotide sequences of DNA, and such mutations have been identified in cancers. DNA reaction site identification depends on accurate analysis of oligonucleotide fragment sizes produced by strand breakage at the damaged sites. Herein, we report a new method for DNA fragment sizing using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Absolute sizing accuracy and speed are achieved by utilizing a CE-LIF array with two-color fluorescence detection. Accuracy depends on correcting results with commercial standards by referring them to primary standards with the same sequences and identical labels as sample fragments. The method is demonstrated by detection of a [...GGCGCGCAG...] G reaction site for styrene oxide on an oligonucleotide representing the CYP1B1 gene. This approach avoids the need for radioactive isotopes and is less labor intensive and faster than the alternative PAGE with (32)P end labeling.     

DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.     

Ultrasensitive PCR and real-time detection from human genomic samples using a bidirectional flow microreactor

In this paper we present a reliable bidirectional flow DNA amplification microreactor for processing real-world genomic samples. This system shares the low-power thermal responsiveness of a continuous flow reactor with the low surface area to volume ratio character of stationary reactors for reducing surface inhibitory effects. Silanization with dimethyldichlorosilane in combination with dynamic surface passivation was used to enhance PCR compatibility and enable efficient amplification. For real-time fragment amplification monitoring we have implemented an epimodal fluorescent detection capability. The passivated bidirectional flow system was ultrasensitive, achieving an RNase P gene detection limit of 24 human genome copies with a reaction efficiency of 77%. This starts to rival the performance of a conventional real-time PCR instrument with a reaction efficiency of 93% and revitalizes flow-through PCR as a viable component of lab on a chip DNA analysis formats.     

Optical Detection of Human Papillomavirus Type 16 and Type 18 by Sequence Sandwich Hybridization With Oligonucleotide-Functionalized Au Nanoparticles

The importance of detecting and subtyping human papillomaviruses (HPVs) in clinical and epidemiological studies has been well addressed. In detecting the most common types of HPV, type 16 (HPV-16) and type 18 (HPV-18), in the cervical mucous of patients in a simple and rapid manner, the assay of a label-free colorimetric DNA sensing method based on sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles (AuNPs) was fabricated in this study. Specific oligonucleotide probes were designed for the sequence detection within the L1 gene of HPV-16 and HPV-18, and the probes were capped onto AuNPs, as AuNP probes. The target HPV sequences in clinical specimens were obtained by an asymmetric polymerase chain reaction (PCR) with universal primers, which can amplify the target sequences from several HPV serotypes, including HPV-16 and HPV-18. The DNA sandwich hybridization between the target sequences and the specific AuNP probes was performed at a temperature closer to the theoretical melting temperature of the DNA hybridization. Next, the procedure of increasing salt concentration and cooling the hybridizing solution was immediately utilized to discriminate the target sequences of HPV-16 or HPV-18. If the target sequences were not complementary to sequences of AuNP probes, the AuNPs would aggregate because no duplex DNA formation occurred such that the color of the reaction solution changed from red to purple. If the AuNP probes were a perfect match to the target sequences and a full DNA sandwich hybridization occurred, the reaction solution maintained its red color. A total of 70 mucous specimens from patients with cervical intraepithelial neoplasia were tested by the AuNP probes sandwich hybridization.      

Simple multiplex genotyping by surface-enhanced resonance Raman scattering

The accurate detection of DNA sequences is essential for a variety of post human genome projects including detection of specific gene variants for medical diagnostics and pharmacogenomics. A specific DNA sequence detection assay based on surface-enhanced resonance Raman scattering (SERRS) and an amplification refractory mutation system (ARMS) is reported. Initially, generation of PCR products was achieved by using specifically designed allele-specific SERRS active primers. Detection by SERRS of the PCR products confirmed the presence of the sequence tested for by the allele-specific oligonucleotides. This lead directly to the multiplex genotyping of human DNA samples for the deltaF508 mutational status of the cystic fibrosis transmembrane conductance regulator gene using SERRS active primers in an ARMS assay. Removal of the unincorporated primers allowed fast and accurate analysis of the three genotypes possible in this system in a multiplex format without any separation of amplicons. The results indicate that SERRS can be used in modern genetic analysis and offers an opportunity for the development of novel assays. This is the first demonstration of the use of SERRS in multiplex genotyping and shows potential advantages over fluorescence as a detection technique with considerable promise for future development.     

Gold nanoparticles with asymmetric polymerase chain reaction for colorimetric detection of DNA sequence

We developed a novel strategy for rapid colorimetric analysis of a specific DNA sequence by combining gold nanoparticles (AuNPs) with an asymmetric polymerase chain reaction (As-PCR). In the presence of the correct DNA template, the bound oligonucleotides on the surface of AuNPs selectively hybridized to form complementary sequences of single-stranded DNA (ssDNA) target generated from As-PCR. DNA hybridization resulted in self-assembly and aggregation of AuNPs, and a concomitant color change from ruby red to blue-purple occurred. This approach is simpler than previous methods, as it requires a simple mixture of the asymmetric PCR product with gold colloid conjugates. Thus, it is a convenient colorimetric method for specific nucleic acid sequence analysis with high specificity and sensitivity. Most importantly, the marked color change occurs at a picogram detection level after standing for several minutes at room temperature. Linear amplification minimizes the potential risk of PCR product cross-contamination. The efficiency to detect Bacillus anthracis in clinical samples clearly indicates the practical applicability of this approach.     

Integrated portable genetic analysis microsystem for pathogen/infectious disease detection

An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance.     

Chip-based electrical detection of DNA

A variety of methods have been developed for the detection of the binding of the complementary strand of DNA to a gene chip using electrical rather than the established optical signal techniques. Chip-based DNA sensors offer sensitivity, specificity, parallelisation and miniaturisation for the detection of selected DNA sequences or mutated genes associated with human diseases. Problems associated with the established fluorescence-based optical detection technique include the high equipment costs and the need to use sophisticated numerical algorithms to interpret the data. These problems generally limit its use to research laboratories and make it hard to adapt this detection scheme for on-site or point-of-care use. An electrical readout might be a solution to these problems. A review of a number of different approaches to achieve an electrical readout for a DNA chip is presented. The review covers various methods that are based on the use of metal nanoparticles as labels and also electrochemical methods that use polymer-modified electrodes, DNA-specific redox reporters, and DNA-mediated charge transport techniques.     

Single-mismatch position-sensitive detection of DNA based on a bifunctional ruthenium complex

A ruthenium complex, pentaamine ruthenium [3-(2-phenanthren-9-yl-vinyl)-pyridine] (which we refer to as RuL in the text) generated in situ has been used as a sensitive and selective electrochemical indicator in DNA sensing. The complex incorporates dual functionalities with the Ru center providing a redox probe and the ligand (L) providing a fluorescent tag. The presence of the aromatic groups in the ligand endows the complex with an intercalative character and makes it capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Combining spectroscopic and electrochemical techniques, we have elucidated the nature of the interactions. From these data we conclude that the binding mode is fundamentally intercalative. The ligand-based fluorescence allows characterization of the complex formation as well as for melting experiments to be carried out. The metal-based redox center is employed as an electrochemical indicator to detect the hybridization event in a DNA biosensor. The biosensor has been developed by immobilization of a thiolated capture probe sequence from Helicobacter pylori onto gold electrodes. With the use of this approach, complementary target sequences of Helicobacter can be quantified over the range of 106 to 708 pmol with a detection limit of 92+/-0.4 pmol and a linear correlation coefficient of 0.995. In addition, this approach allows the detection, without the need for a hybridization suppressor in solution, such as formamide, of not only a single mismatch but also its position in a specific sequence of H. pylori, due to the selective interaction of this bifunctional ruthenium complex with dsDNA.     

Direct detection of point mutations in nonamplified human genomic DNA

Ultrasensitive detection protocols not requiring polymerase chain reaction (PCR)-mediated target DNA amplification are expected to significantly improve our possibilities in several research and diagnostic applications for which minute cell quantities are available. For this reason we have tested a nanoparticle-enhanced surface plasmon resonance imaging (SPRI) sensing strategy to detect point mutations in nonamplified genomic DNA. We have used genomic DNAs, not subject to costly, time-consuming, and prone to contamination PCR-based amplification procedures, obtained from both healthy individuals and homozygous or heterozygous patients affected by β-thalassemia, in order to demonstrate the specificity and the sensitivity of the described sensing strategy. The assay we describe is ultrasensitive and convenient. Attomolar concentrations of target genomic DNA are detected, DNAs from healthy individuals and homozygous or heterozygous patients affected by β-thalassemia are discriminated, and only simple manipulations of the genetic samples are required before the analysis. The proposed ultrasensitive detection of DNA point mutations involved in genomic disorders possibly represents an important advantage in several biomedical applications.     

Colorimetric‐based detection of Ureaplasma urealyticum using gold nanoparticles

Ureaplasma urealyticum (uu) is one of the most common agents of urogenital infections and is associated with complications such as infertility, spontaneous abortion and other sexually transmitted diseases. Here, a DNA sensor based on oligonucleotide target‐specific gold nanoparticles (AuNPs) was developed, in which the dispersed and aggregated states of oligonucleotide‐functionalised AuNPs were optimised for the colorimetric detection of a polymerase chain reaction (PCR) amplicon of U. urealyticum DNA. A non‐cross‐linking approach utilising a single Au‐nanoprobe specific of the urease gene was utilised and the effect of a PCR product concentration gradient evaluated. Results from both visual and spectral analyses showed that target–Au‐nanoprobe hybrids were stable against aggregation after adding the inducer. Furthermore, when a non‐target PCR product was used, the peak position shifted and salt‐induced aggregation occurred. The assay''s limit of detection of the assay was 10 ng with a dynamic range of 10–60 ng. This procedure provides a rapid, facile and low‐cost detection format, compared to methods currently used for the identification of U. urealyticum.Inspec keywords: patient diagnosis, diseases, enzymes, nanosensors, microorganisms, molecular biophysics, DNA, nanoparticles, aggregation, cellular biophysics, colorimetry, genetics, gold, nanomedicineOther keywords: urogenital infections, infertility, spontaneous abortion, sexually transmitted diseases, DNA sensor, oligonucleotide target‐specific gold nanoparticles, oligonucleotide‐functionalised AuNPs, colorimetric detection, polymerase chain reaction amplicon, noncross‐linking approach, single Au‐nanoprobe specific, urease gene, visual analyses, spectral analyses, target–Au‐nanoprobe hybrids, nontarget PCR product, salt‐induced aggregation, rapid cost detection format, facile cost detection format, low‐cost detection format, PCR product concentration, Ureaplasma urealyticum DNA, Au     

Designed biointerface using near-infrared quantum dots for ultrasensitive surface plasmon resonance imaging biosensors

The surface plasmon resonance imaging chip biointerface is fully designed using near-infrared (NIR) quantum dots (QDs) for the enhancement of surface plasmon resonance imaging (SPRi) signals in order to extend their application for medical diagnostics. The measured SPRi detection signal following the QD binding to the surface was amplified 25-fold for a 1 nM concentration of single-stranded DNA (ssDNA) and 50-fold for a 1 μg/mL concentration of prostate-specific antigen (PSA), a cancer biomarker, thus substantiating their wide potential to study interactions of a diverse set of small biomolecules. This significant enhancement is attributed to the QD's mass-loading effect and spontaneous emission coupling with propagating surface plasmons, which allowed the SPRi limit of detection to be reduced to 100 fM and 100 pg/mL for ssDNA and PSA, respectively. Furthermore, this study illustrates the potential of SPRi to be easily integrated with fluorescent imaging for advanced correlative surface-interaction analysis.     

An immunosensor based on the glucose oxidase label and optical oxygen detection

A novel optical immunosensor setup is described which uses glucose oxidase enzyme as a label in conjunction with a luminescence lifetime-based oxygen sensor and phase measurements. The oxygen sensor membranes prepared on microporous filters were used as a solid phase on which the immunoassay was carried out. These sensing materials in combination with a new measurement setup provided high sensitivity for the detection of oxidase enzymes, being at nanogram per milliliter level, i.e., 10(-11)-10(-12) M, with respect to glucose oxidase and its conjugates. Experimental data on the sensitivity were validated using theoretical equations and calculations. Using the new measurement setup and IgG-anti-IgG as a model, a number of different sensing materials were studied aimed to optimize the immunosensor and evaluate its performance. This approach was then applied to a practical system for the detection of human lactate dehydrogenase isoenzymes. It provided similar sensitivity of approximately 1 ng/mL, which is comparable to that of standard ELISA. The attributes of the new immunosensor approach are discussed with respect to performance and versitility.     

A novel detection system for the genetically modified canola (Brassica rapa) line RT73

The herbicide-tolerant genetically modified Roundup Ready canola (Brassica napus) line RT73 has been approved worldwide for use in animal feed and human food. However, RT73 Brassica rapa lines derived from interspecific crosses with RT73 B. napus have not been approved in Japan. Here, we report on a novel system using individual kernel analyses for the qualitative detection of RT73 B. rapa in canola grain samples. We developed a duplex real-time polymerase chain reaction (PCR) method to discriminate B. napus and B. rapa DNA using scatter plots of the end-point analyses; this method was able to discriminate a group comprising B. rapa and Brassica juncea from a group comprising B. napus, Brassica carinata, and Brassica oleracea. We also developed a duplex real-time PCR method for the simultaneous detection of an RT73-specific sequence and an endogenous FatA gene. Additionally, a DNA-extraction method using 96-well silica-membrane plates was developed and optimized for use with individual canola kernels. Our detection system could identify RT73 B. rapa kernels in canola grain samples enabling the accurate and reliable monitoring of RT73 B. rapa contamination in canola, thus playing a role in its governmental regulation in Japan.     

Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions

A sensitive method for the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA that utilizes nanoparticle-enhanced surface plasmon resonance imaging (SPRI) measurements of surface enzymatic ligation reactions on DNA microarrays is demonstrated. SNP identification was achieved by using sequence-specific surface reactions of the enzyme Taq DNA ligase, and the presence of ligation products on the DNA microarray elements was detected using SPRI through the hybridization adsorption of complementary oligonucleotides attached to gold nanoparticles. The use of gold nanoparticles increases the sensitivity of the SPRI so that single bases in oligonucleotides can be successfully identified at a concentration of 1 pM. This sensitivity is amply sufficient for performing multiplexed SNP genotyping by using multiple PCR amplicons and should also allow for the direct detection and identification of SNP sequences from 1 pM unamplified genomic DNA samples with this array-based and label-free SPRI methodology. As a first example of SNP genotyping, three different human genomic DNA samples were screened for a possible point mutation in the BRCA1 gene that is associated with breast cancer.     

One-pot polymerase chain reaction with gold nanoparticles for rapid and ultrasensitive DNA detection

We report a novel method for rapid, colorimetric detection of a specific deoxyribonucleic acid (DNA) sequence by carrying out a polymerase chain reaction in the presence of gold nanoparticles functionalized with two primers. Extension of the primers when the target DNA is present as a template during the polymerase chain reaction process affords the complementary sequences on the gold nanoparticle surfaces and results in the formation of gold nanoparticle aggregates with a concomitant color change from red to pinkish/purple. This method provides a convenient and straightforward solution for ultrasensitive DNA detection without any further post-treatment of the polymerase chain reaction products being necessary, and is a promising tool for rapid disease diagnostics and gene sequencing.     

Label-free electrochemical hybridization genosensor for the detection of hepatitis B virus genotype on the development of Lamivudine resistance

The resistance analysis related to the hepatitis B virus (HBV) genotyping and treatment procured key information for the study of infected patients. The aim of this study was to develop a novel assay for the voltammetric detection of DNA sequences related to the HBV genotype on the development of lamuvidine resistance by monitoring the oxidation signal of guanine. This new technique not only provides a rapid, cost-effective, simple analysis but also gives information concerning both genotyping and lamivudine resistance. Synthetic single-stranded oligonucleotides ("probe") including YMDD (HBV wild type) YVDD, or YIDD (mutations in the YMDD) variants have been immobilized onto pencil graphite electrodes with the adsorption at a controlled potential. The probes were hybridized with different concentrations of their complementary ("target") sequences such as synthetic complementary sequences, clonned PCR products, or real PCR samples. The formed synthetic hybrids on the electrode surface were evaluated by a differential pulse voltammetry technique using a label-free detection method. The oxidation signal of guanine was observed as a result of the specific hybridization between the probes and their synthetic targets and specific PCR products. The response of the hybridization of the probes with their single-base mismatch oligonucleotides at PGE was also detected. Control experiments using the noncomplementary oligonucleotides were performed to determine whether the DNA genosensor responds selectively. Numerous factors, affecting the probe immobilization, target hybridization, and nonspecific binding events, were optimized to maximize the sensitivity and reduce the assay time. Under the optimum conditions, 457 fmol/mL was found as the detection limit for target DNA. With the help of the appearance of the guanine signal, the new protocol is based on the electrochemical detection of HBV genotype for the development of lamuvidine resistance for the first time. Features of this protocol are discussed and optimized.     

Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection

An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification. The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation. Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors. In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions. After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation. An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence. Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences. The limit of detection is about six copies of target DNA.