Electrochemical aptamer-based assays coupled to isothermal nucleic acid amplification techniques: New tools for cancer diagnosis

Highly sensitive detection of cancer biomarkers in blood is key not only to find cancer at an early stage but also to help clinicians to decide the best treatment plan and to find how well treatment is working. To quantify the small changes in clinically validated biomarkers associated with carcinogenesis both selective receptors and signal amplification strategies of the recognition event between the receptor and the biomarker are highly in demand. This report covers the most recent developments in the integration of aptamer-based recognition of blood-circulating cancer biomarkers and isothermal nucleic acid amplification platforms with electrochemical readout, highlighting the potential of these novel tools, and the challenges to translate these assays to the clinical practice.     

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.     

Antibiotics as inhibitors of nucleic acid and protein synthesis

The antibiotics of the rifamycin, actinomycin, chromomycin, and anthracycline groups have been found to be specific inhibitors for the DNA-controlled synthesis of RNA in vitro. Streptomycin, chloramphenicol, and puromycin can specifically suppress certain steps in the biosynthesis of proteins. The investigation of the mode of action of such substances may help us to gain a better insight into the transmission of hereditary information.     

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。     

An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification

A compact hand-held heated fluorometric instrument for performing real-time isothermal nucleic acid amplification and detection is described. The optoelectronic instrument combines a Printed Circuit Board/Micro Electro Mechanical Systems (PCB/MEMS) reaction detection/chamber containing an integrated resistive heater with attached miniature LED light source and photo-detector and a disposable glass waveguide capillary to enable a mini-fluorometer. The fluorometer is fabricated and assembled in planar geometry, rolled into a tubular format and packaged with custom control electronics to form the hand-held reactor. Positive or negative results for each reaction are displayed to the user using an LED interface. Reaction data is stored in FLASH memory for retrieval via an in-built USB connection. Operating on one disposable 3 V lithium battery >12, 60 min reactions can be performed. Maximum dimensions of the system are 150 mm (h) × 48 mm (d) × 40 mm (w), the total instrument weight (with battery) is 140 g. The system produces comparable results to laboratory instrumentation when performing a real-time nucleic acid sequence-based amplification (NASBA) reaction, and also displayed comparable precision, accuracy and resolution to laboratory-based real-time nucleic acid amplification instrumentation. A good linear response (R² = 0.948) to fluorescein gradients ranging from 0.5 to 10 μM was also obtained from the instrument indicating that it may be utilized for other fluorometric assays. This instrument enables an inexpensive, compact approach to in-field genetic screening, providing results comparable to laboratory equipment with rapid user feedback as to the status of the reaction.     

Sequence-specific detection of nucleic acids utilizing isothermal enrichment of G-quadruplex DNAzymes

G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Various chemical sensors and biosensors have been developed, based on such DNAzymes. Here we report a novel, specific nucleic acid detection method utilizing the isothermal amplification strategy of G-quadruplex DNAzymes. In this method, an unlabeled oligonucleotide probe was used. The probing sequence of the oligonucleotide was in the form of a stem-loop structure. A G-rich sequence, containing three GGG repeats, was linked to the 5′-end of the stem-loop structure. In the presence of target, the probing sequence hybridized to the target, and a G_n (n ≥ 2) repeat was extended from its 3′-end. This G_n repeat, together with the three GGG repeats at the 5′-end, folded into a G-quadruplex, and displayed enhanced peroxidase acitivity upon hemin binding. Utilizing the dynamic binding interaction between the probe and its target, the enrichment of G-quadruplex DNAzymes was achieved. Using this method, simple, rapid and cost-effective nucleic acid detection could be achieved. This method displayed high target-length tolerance and good detection specificity; one-base mismatch could be judged easily, even by visual inspection. This method may be used as an auxiliary tool for amplified detection of specific DNA targets in some situations, in which isothermal detection is desirable.     

Bioanalytical applications of aptamer and molecular-beacon probes in fluorescence-affinity assays

This critical review summarizes recent developments in highly sensitive, specific assays using nucleic-acid (NA)-affinity probes and fluorescence detection. We emphasize two groups of DNA and RNA probes (i.e. aptamers and molecular beacons) because of the increase in their bioanalytical applications. The affinity and the specificity of these NA probes combined with the diverse detection capability of fluorescence measurements (e.g., intensity, polarization, resonance-energy transfer and decay life-time) enable ultrasensitive assays for proteins, gene mutations, single-nucleotide polymorphisms and molecular-binding events.     

Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids

Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain DNA polymerases in which long single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA template by using a single DNA primer. A method traditionally used for ultrasensitive DNA detection in areas of genomics and diagnostics, RCA has been used more recently to generate large-scale DNA templates for the creation of periodic nanoassemblies. Various RCA strategies have also been developed for the production of repetitive sequences of DNA aptamers and DNAzymes as detection platforms for small molecules and proteins. In this way, RCA is rapidly becoming a highly versatile DNA amplification tool with wide-ranging applications in genomics, proteomics, diagnosis, biosensing, drug discovery, and nanotechnology.     

单细胞核酸编码扩增成像分析

单细胞成像可在单细胞水平观测目标物位置、 确定目标物含量, 在生命科学与临床医学研究领域应用广泛. 核酸编码扩增技术利用特定分子反应将待测目标识别转化为核酸条码的扩增, 具有探针种类多、 易编程、 反应条件温和及信号放大效率高等特点, 在单细胞低丰度、 高灵敏、 多目标物成像中优势显著, 为理解细胞状态、 探索生命过程提供了新思路. 本文综合评述了核酸编码扩增在单细胞荧光成像领域的研究进展, 以目标物的编码方式为分类依据, 系统阐述了固定细胞原位成像和活细胞成像中不同目标物编码与扩增成像方式的区别, 并对活细胞成像中多重检测面临的问题以及未来发展前景进行了展望.     

A label-free fluorescent probe based on DNA-templated silver nanoclusters and exonuclease III-assisted recycling amplification detection of nucleic acid

A number of specific nucleic acids are closely related with many serious diseases, in the current research, a platform taking advantage of exonuclease III (Exo III) to realize double recycling amplification and label-free fluorescent DNA-templated silver nanoclusters (DNA-AgNCs) for detecting of nucleic acid had been developed. In this method, a molecular beacon (MB) with 3′-protruding termini and a single-stranded cytosine-rich (C-rich) probe were designed that coexist stably with Exo III. Once the target DNA appeared, portion of the MB could hybridize with target DNA and was digested by Exo III, which allowed the release of target DNA and a residual sequence. Subsequently, the residual sequence could trigger the Exo III to digest C-rich probe, and the DNA-AgNCs was not able to be synthesized because of the C-rich probe was destroyed; finally the fluorescent of solution was quenched. This assay enables to monitor human hemochromatosis gene (as a model) with high sensitivity, the detection limit is as low as 120 pM compared with other fluorescence DNA-AgNCs methods, this assay also exhibits superior specificity even against single base mismatch. The strategy is applied to detect human hemochromatosis gene in real human serum samples successfully.     

负压驱动皮升级等温核酸精确定量微流控芯片

精准医疗急需更加精确、灵敏、方便快捷的核酸定量方法。设计开发了一种结合双荧光等温扩增的高密度皮升级核酸定量微流控芯片。芯片采用多层软光刻技术制成,有3个反应通道,每通道有40 000个反应小室,共120 576个皮升级反应小室,反应小室的密度达7 000/cm~2。芯片利用负压驱动样品精确分配,热凝固油相封闭小室,无需阀门及其他仪器辅助。芯片中嵌入的氟硅烷纳米涂层能有效地阻止反应过程中水蒸气的散失。以乙肝病毒(hepatitis B virus,HBV)质粒作为模板,进行等温多底物自配引发扩增(isothermal multiple self-matchinginitiated amplification,IMSA),测试芯片定量性能。芯片检测模板的动态范围达6个数量级,可检测的最大模板量为36μL中1.13×10~6拷贝数核酸。该装置具有定量精确、灵敏、快捷、操作简单等优势,可用于精准检测。     

Fluorimetric determination of nucleic acids with terbium and lutetium

In this paper, fluorescence-enhancement of Tb-nucleic acids [fish sperm DNA (fsDNA) and yeast RNA (yRNA)] by Lu³⁺ is studied in detail and is applied to determine nucleic acids. The experiments indicated that under the optimum conditions, a linear relationship was obtained between the fluorescence intensity (I_f) and the concentration of nucleic acids. The linear range is 1.2×10⁻⁸-1.0×10⁻⁴ g/ml for DNA and 3.0×10⁻⁸-8.0×10⁻⁴ g/ml for RNA. The detection limits (signal/noise=3) for DNA and RNA were 4.8×10⁻⁹ and 7.0×10⁻⁹ g/ml, respectively. The mechanism of the co-luminescence effect is also discussed.     

Study on the interaction between nucleic acid and Eu3+-oxolinic acid and the determination of nucleic acid using the resonance light scattering technique

At pH 9.75, the resonance light scattering (RLS) intensity of OA–Eu³⁺ system is greatly enhanced by nucleic acid. Based on this phenomenon, a new quantitative method for nucleic acid in aqueous solution has been developed. Under the optimum condition, the enhanced RLS is proportional to the concentration of nucleic acid in the range of 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ g/ml for herring sperm DNA, 8.0 × 10⁻¹⁰ to 1.0 × 10⁻⁶ g/ml for calf thymus DNA and 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ g/ml for yeast RNA, and their detection limits are 0.020, 0.011 and 0.010 ng/ml, respectively. Synthetic samples and actual samples were satisfactorily determined. In addition, the interaction mechanism between nucleic acid and OA–Eu³⁺ is also investigated.     

Fluorimetric determination of nucleic acid using the enhancement of terbium-gadolinium-nucleic acid-cetylpyridine bromide system

It is found that the fluorescence intensity of Tb-cetylpyridine bromide (CPB)-nucleic acid system can be enhanced by La³⁺, Gd³⁺, Lu³⁺, Sc³⁺ and Y³⁺, of which Gd³⁺ has the greatest enhancement. The experiments indicate that under the optimum condition, the fluorescence intensity of the system is in proportion to the concentration of nucleic acids in the range from 9×10⁻⁸ to 1×10⁻⁵ g ml⁻¹ for yeast RNA, from 1×10⁻⁷ to 1×10⁻⁵ g ml⁻¹ for fish sperm DNA. The detection limits are 3.2 and 4.1 ng ml⁻¹, respectively. This method has been used satisfactorily for the determination of both synthetic and actual samples. In comparison with most fluorescence method for the determination of nucleic acids, this method has higher sensitivity and stability.     

Determination of nucleic acids based on the fluorescence quenching of Hoechst 33258 at pH 4.5

It was found the strong fluorescence emitted by the bis-benzimidazole derivative Hoechst 33258 at 490 nm could be efficiently quenched in pH 4.5 buffer when nucleic acids were added. Analysis of fluorescence intensity showed that the procedure was a static quenching dominated one, which was also demonstrated by the electron absorption spectra and lifetime of the excited state. The binding constant and numbers of binding sites were obtained from the Scatchard plot. The decreased fluorescence intensity was in proportion to the concentration of nucleic acids in the range 40-1800 ng ml⁻¹ for dsDNA and 26-1700 ng ml⁻¹ for ssDNA. The limits of detection were 12 and 8 ng ml⁻¹, respectively. The sensitivity of the method was about 3.4 times higher for dsDNA detection and 5.4 times higher for ssDNA detection compared with the widely used fluorescence enhancement method using the same dye. Application results to synthetic samples showed simplicity, rapidity and satisfactory reproducibility of the presented method. Measurement of real samples extracted from leaves of Crassula argentea and E. coli genome also gave satisfactory results, which were in good agreement with those obtained using spectrophotometric method.     

Fluorescence-based nucleic acid detection and microarrays

Recent analytical innovations for nucleic acid detection have revolutionized the biological sciences. Single nucleic acid sequence detection methods have been expanded to incorporate multiplexed detection strategies. A variety of nucleic acid detection formats are now available that can address high throughput genomic interrogation. Many of these parallel detection platforms or arrays, employ fluorescence as the signaling method. Fluorescence-based assays offer many advantages, including increased sensitivity, safety and multiplexing capabilities, as well as the ability to measure multiple fluorescence properties. Multiplexed microarray platforms provide parallel detection capabilities capable of measuring thousands of simultaneous responses. This review will discuss both single target detection and microarray applications with a focus on gene expression and pathogenic microorganism (PM) detection.     

Practical physical aspects of interfacial nucleic acid oligomer hybridisation for biosensor design

A significant amount of research concerning rapid, selective biomolecular analysis has focused on development of analytical methods that make use of nucleic acid hybridisation as the basis for selective recognition. The development of biosensors based on nucleic acid hybridisation requires consideration of the thermodynamics of hybrid formation at a solid interface, because the relative thermodynamic stability can dictate the selectivity of hybridisation. Careful control of hybridisation conditions such as the density of oligonucleotides, as well as the temperature, pH, and ionic strength, may therefore enhance the selectivity, sensitivity and speed of a nucleic acid hybridisation assay that is located at an interface.     

Micropacked capillary nucleic acid analysis

Nucleic acid compositional analysis is discussed, followed by a brief review of the current state of the technology applied in which the benefits and limitations of current methodology are enumerated. An alternative method is presented, namely the use of a micropacked capillary ion exchange column to separate nucleoside monophosphates via an anion exchange mechanism. The separated nucleotides are then quantitated by means of post-column photodiode-array detection. The use of a photodiode array also enables the verification of peak identity and purity by acquisition of UV spectra at any point in the separation. The technique has applications in the compositional analysis of both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).