A microfluidic biosensor based on nucleic acid sequence recognition

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome–target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol L^–1 (10 pmol L^–1) . Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.     

A generic sandwich-type biosensor with nanomolar detection limits

A quantitative and highly sensitive, yet simple and rapid, biosensor system was developed for the detection of nucleic acid sequences that can also be adapted to the detection of antigens. A dipstick-type biosensor with liposome amplification, based on a sandwich assay format with optical detection, was combined with a simple coupling reaction that allows the transformation of the generic biosensor components to target specific ones by a mere incubation step. This biosensor platform system was developed and optimized, and its principle was proven using DNA oligonucleotides that provided a nucleic acid biosensor for the specific detection of RNA and DNA sequences. However, the coupling reaction principle chosen can also be used for the immobilization of antibodies or receptor molecules, and therefore for the development of immunosensors and receptor-based biosensors. The generic biosensor consists of liposomes entrapping sulforhodamine B that are coated with streptavidin on the outside, and polyethersulfone membranes with anti-fluorescein antibodies immobilized in the detection zone. In order to transform the generic biosensor into a specific DNA/RNA biosensor, two oligonucleotides that are able to hybridize to the target sequence were labeled with a biotin and a fluorescein molecule, respectively. By simultaneously incubating the liposomes, both oligonucleotides, and the target sequence in a hybridization buffer for 20–30 min at 42 ^°C, a sandwich complex was formed. The mixture was applied to the polyethersulfone membrane. The complex was captured in the detection zone and quantified using a handheld reflectometer. The system was tested using RNA sequences from B. anthracis, C. parvum and E. coli. Quantitation of concentrations between 10 fmol and 1000 fmol (10–1000 nM) was possible without altering any biosensor assay conditions. In addition, no changes to hybridization conditions were required when using authentic nucleic acid sequence-based amplified RNA sequences, and the generic biosensor compared favorably with those previously developed specifically for the RNA sequences. Therefore, the universal biosensor described is an excellent tool, for use in laboratories or at test sites, for rapidly investigating and quantifying any nucleic acid sequence of interest, as well as potentially any antigen of interest that can be bound by two antibodies simultaneously.     

Biosensor for the specific detection of a single viable B. anthracis spore

A simple membrane strip-based biosensor for the detection of viable B. anthracis spores was developed and combined with a spore germination procedure as well as a nucleic acid amplification reaction to identify as little as one viable B. anthracis spore in less than 12 h. The biosensor is based on identification of a unique mRNA sequence from the anthrax toxin activator (atxA) gene encoded on the toxin plasmid, pXO1. Preliminary work relied on plasmid vectors in both E. coli and B. thuringiensis expressing the atxA gene. Once the principle was firmly established, the vaccine strain of B. anthracis was used. After inducing germination and outgrowth of spores of B. anthracis (Sterne strain), RNA was extracted from lysed cells, amplified using nucleic acid sequence-based amplification (NASBA), and rapidly identified by the biosensor. While the biosensor assay requires only 15-min assay time, the overall process takes 12 h for the detection of as little as one viable B. anthracis spore, and is shortened significantly, if larger amounts of spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized oligonucleotide probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second oligonucleotide probe that has been coupled to dye-encapsulating liposomes. The dye in the liposomes then provides a signal that can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1.5 fmol of target mRNA. Specificity analysis revealed no crossreactivity with closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis etc.     

滚环扩增型压电石英晶体传感器检测乙型肝炎病毒

通过捕获探针与纳米金膜之间的共价连接, 保证了滚环扩增(RCA)产物始终结合于金膜表面, Phi29 DNA聚合酶的高效扩增和Escherichia coli DNA链接酶的高度精确性使检测达到单碱基识别, 检测灵敏度达到10⁴ copies/mL. 实验结果表明, 与单碱基错配序列相比, RCA可明显增强检测的灵敏度. 该RCA基因传感器操作简单, 灵敏度和特异性较高, 在乙型肝炎病毒的快速检测方面具有一定的开发潜力.     

Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification

Nucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed.     

Detection of MicroRNAs using target-guided formation of conducting polymer nanowires in nanogaps

A nanogapped microelectrode-based biosensor array is fabricated for ultrasensitive electrical detection of microRNAs (miRNAs). After peptide nucleic acid (PNA) capture probes were immobilized in nanogaps of a pair of interdigitated microelectrodes and hybridization was performed with their complementary target miRNA, the deposition of conducting polymer nanowires, polyaniline (PAn) nanowires, is carried out by an enzymatically catalyzed method, where the electrostatic interaction between anionic phosphate groups in miRNA and cationic aniline molecules is exploited to guide the formation of the PAn nanowires onto the hybridized target miRNA. The conductance of the deposited PAn nanowires correlates directly to the amount of the hybridized miRNA. Under optimized conditions, the target miRNA can be quantified in a range from 10 fM to 20 pM with a detection limit of 5.0 fM. The biosensor array is applied to the direct detection of miRNA in total RNA extracted from cancer cell lines.     

Fluorescent polymeric transducer for the rapid, simple, and specific detection of nucleic acids at the zeptomole level

We report the specific detection of a few hundred molecules of genetic material using a fluorescent polythiophene biosensor. Such recognition is based on simple electrostatic interactions between a cationic polymeric optical transducer and the negatively charged nucleic acid target and can be done in less than 1 h, simply and affordably, and without any chemical reaction. This simple system is versatile enough to detect nucleic acids of various lengths, including a segment from the RNA genome of the Influenza virus.     

新型超支状液晶核酸传感器用于p53突变基因的检测

基于核酸杂交链式反应影响液晶取向的原理, 构建了一种新型的超支状液晶核酸传感器用于检测p53突变基因. 本文突破传统构建超支状分子的方式, 采用杂交链式反应方法, 以目标序列p53突变基因作为引发剂, 3种不同的发卡探针Hairpin A, Hairpin B和Hairpin C为单体, 在温和的条件下, 通过改变单体的浓度和反应时间自发杂交组装形成尺寸和分子量可控的超支状DNA(branched-like DNA, bDNA). 借助捕获探针将该超支状DNA连接到液晶传感基底表面, 观察液晶分子取向改变前后的光学信号, 实现了p53基因含249密码子突变序列的快速检测. 本方法有望为核酸诊断的发展提供一种新的方法和思路.     

PNA探针与DNA探针的系统比较

肽核酸（Peptide Nucleic Acid，PNA）是近十几年发展起来的以中性酰胺键为骨架的脱氧核糖核酸（Deoxyribonucleic Acid，DNA）类似物，其结构介于多肽和DNA之间。由于PNA能够与DNA和RNA特异性地结合，可以制备PNA探针。与DNA探针相比，其杂交的稳定性和特异性增加且能在低盐浓度下进行杂交。本文从DNA和PNA的分子结构和性质、DNA探针和PNA探针的设计制备、杂交亲和性、杂交动力学以及在生物传感器上的应用等方面进行了系统比较。     

Direct acoustic profiling of DNA hybridisation using HSV type 1 viral sequences

We describe the detection of specific, conserved DNA sequences of herpes simplex virus (HSV) type 1 by means of a novel, high sensitivity acoustic biosensor. Repeated assays on planar and polymeric carboxylic acid- and biotin-presenting surface chemistries enabled statistical comparison of assay specificity and sensitivity and evaluation of assay Z-factor scores. Using a three minute hybridisation with NeutrAvidin capture for signal enhancement, it was possible to detect HSV viral nucleic acids at 5.2 x 10(-11) M concentration.     

Quantitative detection of well-based DNA array using switchable lanthanide luminescence

In this report a novel wash-free method for multiplexed DNA detection is demonstrated employing target specific probe pairs and switchable lanthanide luminescence technology on a solid-phase array. Four oligonucleotide capture probes, conjugated at 3′ to non-luminescent lanthanide ion carrier chelate, were immobilized as a small array on the bottom of a microtiter plate well onto which a mix of corresponding detection probes, conjugated at 5′ to a light absorbing antenna ligand, were added. In the presence of complementary target nucleic acid both the spotted capture probe and the liquid-phase detection probe hybridize adjacently on the target. Consequently the two non-luminescent label molecules self-assemble and form a luminescent mixed lanthanide chelate complex. Lanthanide luminescence is thereafter measured without a wash step from the spots by scanning in time-resolved mode. The homogeneous solid-phase array-based method resulted in quantitative detection of synthetic target oligonucleotides with 0.32 nM and 0.60 nM detection limits in a single target and multiplexed assay, respectively, corresponding to 3× SD of the background. Also qualitative detection of PCR-amplified target from Escherichia coli is described.     

Universal liposomes: preparation and usage for the detection of mRNA

Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L⁻¹ and an assay range spanning four orders of magnitude (5 pmol L⁻¹−50 nmol L⁻¹) with a coefficient of variation ≤5.8% was found for synthetic DNA. For synthetic RNA the LOQ was half that of synthetic DNA. A comparison was made to alkaline phosphatase-conjugated streptavidin for detection which yielded a limit of quantitation approximately 80 times higher than that for liposomes in the same system. Thus, liposomes and the optimized sandwich hybridization method are well suited for detecting single-stranded nucleic acid sequences and compares favorably to other sandwich hybridization schemes recently described in the literature. The assay was then used successfully for the clear detection of mRNA amplified by nucleic acid sequence-based amplification (NASBA) isolated from as little as one Cryptosporidium parvum oocyst. The detection of mRNA from oocysts isolated from various water sample types using immunomagnetic separation was also assessed. Finally, to prove the wider applicability and sensitivity of this universal method, RNA amplified from the atxA gene of Bacillus anthracis was detected when the input to the preceding NASBA reaction was as low as 1.2 pg. This highly sensitive liposome-based microtiter plate assay is therefore a platform technology allowing for high throughput and wide availability for routine clinical and environmental laboratory applications.     

Nucleic acid biosensors for environmental pollution monitoring

Nucleic acid-based biosensors are finding increasing use for the detection of environmental pollution and toxicity. A biosensor is defined as a compact analytical device incorporating a biological or biologically-derived sensing element either integrated within or intimately associated with a physicochemical transducer. A nucleic acid-based biosensor employs as the sensing element an oligonucleotide, with a known sequence of bases, or a complex structure of DNA or RNA. Nucleic acid biosensors can be used to detect DNA/RNA fragments or either biological or chemical species. In the first application, DNA/RNA is the analyte and it is detected through the hybridization reaction (this kind of biosensor is also called a genosensor). In the second application, DNA/RNA plays the role of the receptor of specific biological and/or chemical species, such as target proteins, pollutants or drugs. Recent advances in the development and applications of nucleic acid-based biosensors for environmental application are reviewed in this article with special emphasis on functional nucleic acid elements (aptamers, DNAzymes, aptazymes) and lab-on-a-chip technology.     

Quantum dot-based isothermal chain elongation for fluorescence detection of specific DNA sequences via template-dependent surface-hybridization

A new quantum dot-based method to detect specific sequences of DNA is proposed. The capture and reporter probes do not hybridize to each other, but in the presence of a template they can anneal to each other via the formation of a stable ternary complex. Because of the specific design of the capture and reporter probes, the 5' end of the template target DNA remains free to hybridize with another reporter. In this way, each capture DNA is an initiator strand that triggers a cascade of hybridization events between the target DNA and the reporter probe. This forms a superstructure, enhances base stacking, and produces a strong fluorescent signal. The introduction of T4 DNA ligase further stabilizes the superstructure and greatly increases the fluorescence intensity, and the detection limit is as low as 10 fM. This fluorescence method is advantageous over conventional techniques because of its excellent ability to discriminate single base-pair mismatches and single nucleotide gap or flap. This simple technique is promising for improving medical diagnosis and treatment.     

Simultaneous detection of small molecules,proteins and microRNAs using single molecule arrays

Biological samples such as blood, urine, cerebrospinal fluid and saliva contain a large variety of proteins, nucleic acids, and small molecules. These molecules can serve as potential biomarkers of disease and therefore, it is desirable to simultaneously detect multiple biomarkers in one sample. Current detection techniques suffer from various limitations including low analytical sensitivity and complex sample processing. In this work, we present an ultrasensitive method for simultaneous detection of small molecules, proteins and microRNAs using single molecule arrays (Simoa). Dye-encoded beads modified with specific capture probes were used to quantify each analyte. Multiplex competitive Simoa assays were established for simultaneous detection of cortisol and prostaglandin E2. In addition, competitive and sandwich immunoassays were combined with a direct nucleic acid hybridization assay for simultaneous detection of cortisol, interleukin 6 and microRNA 141. The multi-analyte Simoa assay shows high sensitivity and specificity, which provides a powerful tool for the analysis of many different samples.

The first example of multiplexed detection of proteins, nucleic acids, and small molecules using single molecule measurement methodology.     

Lateral flow devices for nucleic acid analysis exploiting quantum dots as reporters

There is a growing interest in the development of biosensors in the form of simple lateral flow devices that enable visual detection of nucleic acid sequences while eliminating several steps required for pipetting, incubation and washing out the excess of reactants. In this work, we present the first dipstick-type nucleic acid biosensors based on quantum dots (QDs) as reporters. The biosensors enable sequence confirmation of the target DNA by hybridization and simple visual detection of the emitted fluorescence under a UV lamp. The ‘diagnostic’ membrane of the biosensor contains a test zone (TZ) and a control zone (CZ). The CZ always fluoresces in order to confirm the proper function of the biosensor. Fluorescence is emitted from the TZ, only when the specific nucleic acid sequence is present. We have developed two general types of QD-based nucleic acid biosensors, namely, Type I and Type II, in which the TZ consists of either immobilized streptavidin (Type I) or immobilized oligodeoxynucleotides (Type II). The control zone consists of immobilized biotinylated albumin. No purification steps are required prior to the application of the DNA sample on the strip. The QD-based nucleic acid biosensors performed accurately and reproducibly when applied to (a) the visual detection of PCR amplification products and (b) visual genotyping of single nucleotide polymorphisms (SNPs) in human genomic DNA from clinical samples. As low as 1.5 fmol of double-stranded DNA were clearly detected by naked eye and the dynamic range extended to 200 fmol. The %CV were estimated to be 4.3–8.2.     

核酸等温扩增技术在电化学生物传感器中的应用

生物分子检测在临床诊断、基因治疗、基因突变分析等方面变得日益重要,因而,建立简单、快速、灵敏的检测方法具有重要意义。近年,电化学生物传感器因其简单、便携、易操作、成本低等优势在生物分子检测的研究中备受关注。为了提高检测方法的灵敏度,不同的核酸等温扩增技术被应用于电化学生物传感器的构建中。本文简单介绍了电化学生物传感器的工作原理,着重综述了几种主要应用于电化学传感器中的核酸等温扩增技术,同时比较了各方法的优缺点。     

A Single Electrochemical Probe Used for Analysis of Multiple Nucleic Acid Sequences

Electrochemical hybridization sensors have been explored extensively for analysis of specific nucleic acids. However, commercialization of the platform is hindered by the need for attachment of separate oligonucleotide probes complementary to a RNA or DNA target to an electrode's surface. Here we demonstrate that a single probe can be used to analyze several nucleic acid targets with high selectivity and low cost. The universal electrochemical four‐way junction (4J)‐forming (UE4J) sensor consists of a universal DNA stem‐loop (USL) probe attached to the electrode's surface and two adaptor strands (m and f) which hybridize to the USL probe and the analyte to form a 4J associate. The m adaptor strand was conjugated with a methylene blue redox marker for signal ON sensing and monitored using square wave voltammetry. We demonstrated that a single sensor can be used for detection of several different DNA/RNA sequences and can be regenerated in 30 seconds by a simple water rinse. The UE4J sensor enables a high selectivity by recognition of a single base substitution, even at room temperature. The UE4J sensor opens a venue for a re‐useable universal platform that can be adopted at low cost for the analysis of DNA or RNA targets.     

Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels

This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro-channel delivers fresh analyte solution to the reaction site which maintains a high concentration gradient differential to enhance mass transport. Based on the dual signal amplification strategy, the developed microfluidic bead-based nucleic acid sensor could discriminate as low as 5 fM (signal-to-noise (S/N) 3) of synthesized carcinoembryonic antigen (CEA) gene fragments and showed a 1000-fold increase in detection limit compared to the off-chip test. In addition, using spiked colorectal cancer cell lines (HT29) in the blood as a model system, the detection limit of this chip-based approach was found to be as low as 1 HT29 in 1 mL blood sample. This microfluidic bead-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence.     

Electrocatalytic assay of mercury(II) ions using a bifunctional oligonucleotide signal probe

Engineered nucleic acid probes containing recognition and signaling functions find growing interest in biosensor design. In this paper, we developed a novel electrochemical biosensor for sensitive and selective detecting of Hg²⁺ based on a bifunctional oligonucleotide signal probe combining a mercury-specific sequence and a G-quadruplex (G4) sequence. For constructing the electrochemical Hg²⁺ biosensor, a thiolated, mercury-specific oligonucleotide capture probe was first immobilized on gold electrode surface. In the presence of Hg²⁺, a bifunctional oligonucleotide signal probe was hybridized with the immobilized capture probe through thymine–mercury(II)–thymine interaction-mediated surface hybridization. The further interaction between G4 sequence of the signal probe and hemin generated a G4–hemin complex, which catalyzed the electrochemical reduction of hydrogen peroxide, producing amplified readout signals for Hg²⁺ interaction events. This electrochemical Hg²⁺ biosensor was highly sensitive and selective to Hg²⁺ in the concentration of 1.0 nM to 1 μM with a detection limit of 0.5 nM. The new design of bifunctional oligonucleotide signal probes also provides a potential alternative for developing simple and effective electrochemical biosensors capable of detecting other metal ions specific to natural or artificial bases.