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1.
An innovative scheme for signal amplification using random tetramer-modified gold nanoparticles, termed “nanoamplicons,” has
been developed for hybridization assay without PCR. Large numbers of nanoamplicons could be integrated onto one target, providing
much greater amplification than the larger nanoparticles usually adopted. Using M13mp18 single-strand DNA as a target, this
concept is shown to be a feasible approach to detecting 0.17 amol L−1 DNA without target amplification, based on microgravimetric detection of the adsorption of the probe–target–nanoamplicons
complex via thiol–gold binding. To our knowledge, this method has a sensitivity that is close to that of PCR and superior
to those of nanoparticle-based methods reported previously. Additionally, this novel nanoamplicon could be prepared in the
same way and used for all diagnostic tests; such universality would make the nanoamplicons highly advantageous for the generalization
and standardization of bioassays, and when applying this new technology in clinical laboratories.
Figure A novel signal amplification method for DNA detection with subattomolarsensitivity has been developed using random tetramer-modified
gold nanoparticlesas nanoamplicons, which are easily prepared with high uniformity and can be universally adaptedto any sequences 相似文献
2.
Lillian Roth Jutta Zagon Anke Ehlers Lothar W. Kroh Hermann Broll 《Analytical and bioanalytical chemistry》2009,394(2):529-537
A new approach for the detection of DNA target molecules is described, using capture probes and subsequent signal enhancement
by a uniform polymerase chain reaction (PCR). Peptide nucleic acid probes were immobilized in real-time PCR-compatible microtiter
plates. After hybridization of biotinylated DNA targets, detection was performed by real-time immuno-PCR, a method formerly
used for protein detection. We demonstrate the feasibility of this strategy for the qualitative detection of DNA oligonucleotides
with a detection limit (LOD) of 6 attomol. Furthermore, the method was applied to PCR-amplified samples from genetically modified
maize DNA (Mon810). A 483-bp DNA fragment was detected in mixture with 99.9% of noncomplementary DNA with a sensitivity down
to the level of attomole.
Figure 相似文献
3.
Digital bioanalysis 总被引:3,自引:1,他引:2
Digital microfluidics has recently emerged as a new paradigm in the world of lab-on-a-chip technology. A wide variety of bioanalyses
have been successfully implemented in this format. This paper reviews the various techniques that have been adapted to digital
microfluidic systems, and the current state of the field.
Figure A multiplexed digital microfluidic device. Six analytical platforms are wired in series, allowing multiple independent analyses
to be performed simultaneously from a single set of controls. 相似文献
4.
An aptamer-based label-free approach to hemin recognition and DNA assay using capillary electrophoresis with chemiluminescence
detection is introduced here. Two guanine-rich DNA aptamers were used as the recognition element and target DNA, respectively.
In the presence of potassium ions, the two aptamers folded into the G-quartet structures, binding hemin with high specificity
and affinity. Based on the G-quartet–hemin interactions, the ligand molecule was specifically recognized with a K
d ≈ 73 nM, and the target DNA could be detected at 0.1 μM. In phosphate buffer of pH 11.0, hemin catalyzed the H2O2-mediated oxidation of luminol to generate strong chemiluminescence signal; thus the target molecule itself served as an indicator
for the molecule–aptamer interaction, which made the labeling and/or modification of aptamers or target molecules unnecessary.
This label-free method for molecular recognition and DNA detection is therefore simple, easy, and effective.
Figure A label-free approach to aptamer-based hemin recognition and DNA detection is introduced, which gives great potential for
using a small molecule itself as the indicator for molecular recognition and DNA detection thereby avoiding any labeling or
modification step 相似文献
5.
A chemiluminescent (CL) detection method has been developed for DNA hybridization. The assay relies on a sandwich-type DNA
hybridization in which gold nanoparticles modified with alkylthiol-capped oligonucleotide strands are used as probes to monitor
the presence of the specific target DNA. The , which is the dissolving product of the gold nanoparticles anchored on the DNA hybrids, serves as an analyte in the H2O2–luminol– CL reaction for the indirect measurement of the target DNA. The combination of the remarkable sensitivity of the CL analysis
with the large number of released from each DNA hybrid allows a detection limit at levels as low as 0.1 pM of the target DNA. Moreover, with a further
silver amplification step, the detection limit will be pushed down to the femtomolar domain.
相似文献
6.
Rapid amplification of genetically modified organisms using a circular ferrofluid-driven PCR microchip 总被引:1,自引:0,他引:1
Yi Sun Yien-Chian Kwok Peter Foo-Peng Lee Nam-Trung Nguyen 《Analytical and bioanalytical chemistry》2009,394(5):1505-1508
The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. Polymerase
chain reaction (PCR) technology is extensively used for the detection of GMOs in food products in order to verify compliance
with labeling requirements. In this paper, we present a novel close-loop ferrofluid-driven PCR microchip for rapid amplification
of GMOs. The microchip was fabricated in polymethyl methacrylate by CO2 laser ablation and was integrated with three temperature zones. PCR solution was contained in a circular closed microchannel
and was driven by magnetic force generated by an external magnet through a small oil-based ferrofluid plug. Successful amplification
of genetically modified soya and maize were achieved in less than 13 min. This PCR microchip combines advantages of cycling
flexibility and quick temperature transitions associated with two existing microchip PCR techniques, and it provides a cost
saving and less time-consuming way to conduct preliminary screening of GMOs.
Figure Schematic of the circular ferrofluid-driven PCR microchip 相似文献
7.
Yokokawa R Miwa J Tarhan MC Fujita H Kasahara M 《Analytical and bioanalytical chemistry》2008,391(8):2735-2743
Massively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled
molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were
digested by a restriction enzyme, XhoI, for DNA analysis. One end of the λDNA was modified with biotin and the other end was modified with digoxin molecules by
fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin
antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was
attached to a biotinylated microtubule via avidin–biotin biding and the DNA was stretched by a kinesin-based gliding assay.
A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was
supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method
of performing molecular surgery at the single-molecule scale.
Figure DNA molecule manipulation by motor proteins for analysis at the single-molecule level 相似文献
8.
Chen XW Xu ZR Qu BY Wu YF Zhou J Zhang HD Fang J Wang JH 《Analytical and bioanalytical chemistry》2007,388(1):157-163
Bead injection in a lab-on-valve (LOV) system was adopted for DNA purification via micro solid-phase extraction (SPE) with
a renewable silica microcolumn packed in a channel of the LOV unit. The complex matrix components in human whole blood, including
proteins, were well eliminated by choosing properly the sample loading and elution media. The DNA purification process was
monitored on-line by using laser-induced fluorescence in a demountable side part of the LOV unit incorporating optical fibers.
The practical applicability of the entire system was demonstrated by separation/purification of λ-DNA in a simulated matrix
and human blood genetic DNA by performing SPE, in situ monitoring of the purified products, and postcolumn PCR amplification.
When DNAs in a simulated matrix (10.0 ng μl−1 λ-DNA, 50 ng μl−1 bovine serum albumin, 1.0% Triton X-100) were processed in the present system and laser-induced fluorescence was monitored
at 610 nm, an overall extraction/collection efficiency of 70% was achieved by employing identical sample loading and an elution
flow rate of 0.5 μl s−1, along with a precision of 3.8% relative standard deviation. DNA separation and purification from human whole-blood samples
were performed under similar conditions.
Figure Lab-on-valve mesofluidic system employed for DNA separation and purification integrating a demountable fluorescence flow cell
for in-situ laser induced fluorescence detection 相似文献
9.
Addition of gold nanoparticles to real-time PCR: effect on PCR profile and SYBR Green I fluorescence
Haber AL Griffiths KR Jamting AK Emslie KR 《Analytical and bioanalytical chemistry》2008,392(5):887-896
Real-time quantitative polymerase chain reaction (qPCR) is the industry standard technique for the quantitative analysis of
nucleic acids due to its unmatched sensitivity and specificity. Optimisation and improvements of this fundamental technique
over the past decade have largely consisted of attempts to allow faster and more accurate ramping between critical temperatures
by improving assay reagents and the thermal geometry of the PCR chamber. Small gold nanoparticles (Au-NPs) have been reported
to improve PCR yield under fast cycling conditions. In this study, we investigated the effect of Au-NPs on optimised real-time
qPCR assays by amplifying DNA sequences from genetically modified canola in the presence and absence of 0.9 nM Au-NPs of diameter
12 ± 2nm. Contrary to expectations, we found that Au-NPs altered the PCR amplification profile when using a SYBR Green I detection
system due to fluorescence quenching; furthermore, high-resolution melt (HRM) analysis demonstrated that Au-NPs destabilised
the double-stranded PCR product. The results indicate that effects on the assay detection system must be carefully evaluated
before Au-NPs are included in any qPCR assay.
Figure Raw amplification profiles in the presence and absence of gold nanoparticles 相似文献
10.
We screened a series of RNA and DNA aptamers for their ability to serve in the dye displacement assays in which analytes compete
with TO dye. We conclude that, while the performance of the TO dye displacement approach is not always predictable, it is
still a simple and sensitive assay to detect binding between RNA aptamers and small molecules. In particular, we describe
efficient assays for tobramycin and theophylline, with up to 90% displacement of TO observed, and we describe the first aptameric
assay for cAMP.
Figure An RNA or DNA aptamer against a molecule (circle) binds TO dye, resulting in a fluorescent complex. Presence of free molecule in solution results in the displacement of TO
from the complex and a reduction in fluorescence
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
11.
Takahashi K Kishine K Matsuyama S Saito T Kato H Kinugasa S 《Analytical and bioanalytical chemistry》2008,391(6):2079-2087
Poly(ethylene glycol) (PEG) is a useful water-soluble polymer that has attracted considerable interest in medical and biological
science applications as well as in polymer physics. Through the use of a well-calibrated evaporative light-scattering detector
coupled with high performance supercritical fluid chromatography, we are able to determine exactly not only the average mass
but also all of the molecular mass fractions of PEG samples needed for certified reference materials issued by the National
Metrology Institute of Japan. In addition, experimental uncertainty was determined in accordance with the Guide to the expression of uncertainty in measurement (GUM). This reference material can be used to calibrate measuring instruments, to control measurement precision, and to confirm
the validity of measurement methods when determining molecular mass distributions and average molecular masses. Especially,
it is suitable for calibration against both masses and intensities for matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry.
Figure Comparison between the molecular mass fractions of PEG 1000 before calibration (si) (○) and after calibration (wi) (⧫). The error bar shows the expanded uncertainty of k = 2 of each mass fraction 相似文献
12.
一种可绝对定量核酸的数字PCR微流控芯片 总被引:2,自引:0,他引:2
构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究. 相似文献
13.
McCarthy EL Egeler TJ Bickerstaff LE Pereira da Cunha M Millard PJ 《Analytical and bioanalytical chemistry》2006,386(7-8):1975-1984
Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia
virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and
rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained
from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary
tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers,
which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock
probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal
cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial,
and protozoan pathogens within localized regions of microcapillary tubes.
相似文献
14.
Bonanni A Esplandiu MJ Pividori MI Alegret S del Valle M 《Analytical and bioanalytical chemistry》2006,385(7):1195-1201
Impedance spectroscopy is proposed as the transduction principle for detecting the hybridization of DNA complementary strands.
In our experiments, different DNA oligonucleotides were used as model gene substances. The gene probe is first immobilized
on a graphite-epoxy composite working electrode based genosensor. Detection principle is based on changes of impedance spectra
of a redox marker, the ferro/ferricyanide couple, after hybridization with target DNA. Resistance offered to the electrochemical
reaction serves as the working signal, allowing for an unlabelled gene assay.
相似文献
15.
Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids 总被引:1,自引:0,他引:1
Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination
with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms
(SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids
and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive
indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface.
When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached
to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal
from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very
effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic
digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring
the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the
target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism
(GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect
SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and
pharmaceutical applications.
Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant
structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively
choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
16.
Stratis-Cullum DN Griffin GD Mobley J Vo-Dinh T 《Analytical and bioanalytical chemistry》2008,391(5):1655-1660
This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this
system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system.
The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with
the intensified biochip device. This system was capable of detecting approximately 1 × 105
Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact
system that does not require laser excitation and is readily adaptable to field use.
Figure Schematic diagram of the miniature biochip detection system 相似文献
17.
Pérez Pavón JL García Pinto C Guerrero Peña A Moreno Cordero B 《Analytical and bioanalytical chemistry》2008,391(2):599-607
In the present work we report the results obtained with a methodology based on direct coupling of a headspace generator to
a mass spectrometer for the identification of different types of petroleum crudes in polluted soils. With no prior treatment,
the samples are subjected to the headspace generation process and the volatiles generated are introduced directly into the
mass spectrometer, thereby obtaining a fingerprint of volatiles in the sample analysed. The mass spectrum corresponding to
the mass/charge ratios (m/z) contains the information related to the composition of the headspace and is used as the analytical signal for the characterization
of the samples. The signals obtained for the different samples were treated by chemometric techniques to obtain the desired
information. The main advantage of the proposed methodology is that no prior chromatographic separation and no sample manipulation
are required. The method is rapid, simple and, in view of the results, highly promising for the implementation of a new approach
for oil spill identification in soils.
Figure PCA score plots illustrate clear discrimination of types of crude oil in polluted soil samples (e.g. results are shown for
vertisol) 相似文献
18.
Towards biochips using microstructured optical fiber sensors 总被引:2,自引:0,他引:2
Rindorf L Høiby PE Jensen JB Pedersen LH Bang O Geschke O 《Analytical and bioanalytical chemistry》2006,385(8):1370-1375
In this paper we present the first incorporation of a microstructured optical fiber (MOF) into biochip applications. A 16-mm-long
piece of MOF is incorporated into an optic-fluidic coupler chip, which is fabricated in PMMA polymer using a CO2 laser. The developed chip configuration allows the continuous control of liquid flow through the MOF and simultaneous optical
characterization. While integrated in the chip, the MOF is functionalized towards the capture of a specific single-stranded
DNA string by immobilizing a sensing layer on the microstructured internal surfaces of the fiber. The sensing layer contains
the DNA string complementary to the target DNA sequence and thus operates through the highly selective DNA hybridization process.
Optical detection of the captured DNA was carried out using the evanescent-wave-sensing principle. Owing to the small size
of the chip, the presented technique allows for analysis of sample volumes down to 300 nL and the fabrication of miniaturized
portable devices.
相似文献
19.
Harir M Frommberger M Gaspar A Martens D Kettrup A El Azzouzi M Schmitt-Kopplin P 《Analytical and bioanalytical chemistry》2007,389(5):1459-1467
The photodecomposition of imazamox, a herbicide of the imidazolinone family, was investigated in pure water. The main photoproducts
from the photolysis were followed over time by liquid chromatography mass spectrometry and structures were proposed from exact
mass determinations obtained by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The method
comprised exact mass determination with better than 0.2 ppm mass accuracy and a corresponding structural visualization taking
care of respective isotopes with an adapted van Krevelen diagram that enabled a systematic approach to the characterisation
of the elementary composition of each photoproduct. By taking advantage of the high resolving power of FT-ICR MS to make precise
formula assignments, the derived 2D van Krevelen diagram (O/C; H/C; m/z) enabled one to structurally differentiate the formed photoproducts and to propose a degradation pathway for imazamox.
Figure Overview of applied method to analyse the photolysis process of imazamox herbicide 相似文献
20.
Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. It is isolated from bacterial media as a mixture of two epimers, which readily equilibrate in most solvents. Experiments based on high-performance liquid chromatography/electrospray ionization mass spectrometry are reported here, allowing the investigation of the different Fe(III)-chelating properties of pyochelin diastereomers in solution without the need for labourious isolation. It is demonstrated in this study that only one of the two pyochelin diastereomers is able to chelate Fe(III); no Fe(III) complexes of the other diastereomer could be detected. The Fe(III)–pyochelin complex exhibited a 1:1 metal-to-siderophore ratio and no evidence for other stoichiometries was found.
相似文献