Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.     

Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes

The aim of this study was to develop new strategies for analyzing molecular signatures of disease states approaching real-time using single pair fluorescence resonance energy transfer (spFRET) to rapidly detect point mutations in unamplified genomic DNA. In addition, the detection process was required to discriminate between normal and mutant (minority) DNAs in heterogeneous populations. The discrimination was carried out using allele-specific primers, which flanked the point mutation in the target gene and were ligated using a thermostable ligase enzyme only when the genomic DNA carried this mutation. The allele-specific primers also carried complementary stem structures with end-labels (donor/acceptor fluorescent dyes, Cy5/Cy5.5, respectively), which formed a molecular beacon following ligation. We coupled ligase detection reaction (LDR) with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the molecular beacon probes formed upon ligation. LDR-spFRET provided the necessary specificity and sensitivity to detect single-point mutations in as little as 600 copies of human genomic DNA directly without PCR at a level of 1 mutant per 1000 wild type sequences using 20 LDR thermal cycles. We also demonstrate the ability to rapidly discriminate single base differences in the K-ras gene in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA (600 copies). Real-time LDR-spFRET detection of point mutations in the K-ras gene was accomplished in PMMA microfluidic devices using sheath flows.     

Electrochemical biosensors for detection of point mutation based on surface ligation reaction and oligonucleotides modified gold nanoparticles

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.     

A Fluorescence Assay Based on GoldMag-CS Nanoparticles for Hepatitis B Virus DNA

A sensitive fluorescence assay for hepatitis B virus (HBV) DNA was developed based on the dissociation of bio-bar-code DNA probes from GoldMag-CS nanoparticles (NPs) and magnetic separation. In this method, the target sequence (HBV DNA) was recognized through sandwich hybridization by the catching probes and the detection probes. Catching probes were modified with biotins, and were specifically bound on streptavidin-coated 96-well microplates; detection probes were all attached on the GoldMag-CS nanoparticles, which also bound bio-bar-code strands with fluorescent tags. Bio-bar-codes were dissociated from the NPs by dithiothreitol (DTT) after DNA target recognition and magnetic separation, and then quantified. Streptavidin-coated 96-well microplates diminished the nonspecific binding of DNA-conjugated GoldMag-CS nanoparticles, thus lowering the background; and GoldMag-CS nanoparticles provided easy separation and significant signal amplification. Together, these two effects brought about the detection limit as low as 7.52 fM.     

Aptamers-based sandwich assay for silver-enhanced fluorescence multiplex detection

In this work, aptamers-modified silver nanoparticles (AgNPs) were prepared as capture substrate, and fluorescent dyes-modified aptamers were synthesized as detection probes. The sandwich assay was based on dual aptamers, which was aimed to accomplish the highly sensitive detection of single protein and multiplex detection of proteins on one-spot. We found that aptamers-modified AgNPs based microarray was much superior to the aptamer based microarray in fluorescence detection of proteins. The result shows that the detection limit of the sandwich assay using AgNPs probes for thrombin or platelet-derived growth factor-BB (PDGF-BB) is 80 or 8 times lower than that of aptamers used directly. For multiplex detection of proteins, the detection limit was 625 pM for PDGF-BB and 21 pM for thrombin respectively. The sandwich assay based on dual aptamers and AgNPs was sensitive and specific.     

吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒的制备及其性质研究

以蛋白质或多肽修饰的吲哚类菁染料Cy3为内核, 采用实验条件简单的油包水反相微乳液方法成核, 通过正硅酸乙酯水解形成的网状二氧化硅包壳的方法制备吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒. 考察了以不同等电点的蛋白质和多肽修饰的Cy3为内核材料对吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒制备的影响. 结果表明, 分别采用人免疫球蛋白(IgG)或多聚赖氨酸修饰的Cy3为内核材料, 都能制备荧光强度高、荧光稳定性强和染料泄漏极少的Cy3嵌入的核壳荧光纳米颗粒. 进一步对Cy3嵌入的核壳荧光纳米颗粒进行了表征, 并将基于这一新型的荧光纳米颗粒建立起来的生物标记方法初步应用于流感病毒DNA的检测, 其检测线性范围为3.18×10-10～1.27×10-9 mol/L, 检测下限为3.51×10-10 mol/L, 相关系数r为0.986 5.     

Amplification free detection of herpes simplex virus DNA

Amplification-free detection of nucleic acids in complex biological samples is an important technology for clinical diagnostics, especially in the case where the detection is quantitative and highly sensitive. Here we present the detection of a synthetic DNA sequence from Herpes Simplex Virus-1 within swine cerebrospinal fluid (CSF), using a sandwich-like, magnetic nanoparticle pull-down assay. Magnetic nanoparticles and fluorescent polystyrene nanoparticles were both modified with DNA probes, able to hybridise either end of the target DNA, forming the sandwich-like complex which can be captured magnetically and detected by fluorescence. The concentration of the target DNA was determined by counting individual and aggregated fluorescent nanoparticles on a planar glass surface within a fluidic chamber. DNA probe coupling for both nanoparticles was optimized. Polystyrene reporter nanoparticles that had been modified with amine terminated DNA probes were also treated with amine terminated polyethylene glycol, in order to reduce non-specific aggregation and target independent adhesion to the magnetic particles. This way, a limit of detection for the target DNA of 0.8 pM and 1 pM could be achieved for hybridisation buffer and CSF respectively, corresponding to 0.072 and 0.090 femtomoles of target DNA, in a volume of 0.090 mL.     

An ultrasensitive immunosensor array for determination of staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB) is a potent gastrointestinal toxin and is heat resistant. SEB is also a potential bioterrorism agent. The ability to measure accurately very low amounts of staphylococcal enterotoxin B in food and other samples is very important. A highly sensitive and stable sandwich fluorescence immunoassay based on a pair of monoclonal antibodies against SEB which were produced by us was developed. Classical sandwich immunoassay was adopted and the glass slides were used as the base of the immunologic reaction. The functionalized fluorescent core-shell silica nanoparticles were used as labels. The fluorescence issued from the labels was detected by a laser-induced fluorescence millimeter sensor array detection platform. The fluorescence intensity has a linear relationship with the amount of SEB in the range of 50 pg/mL-5 ng/mL, and the detection limit of SEB was 20 pg/mL (the absolute detection limit was 0.02 pg). The relative standard deviation (RSD) for 5 parallel measurements of SEB (1 ng/mL) was 9.2%.     

Comparative studies of thiol-sensitive fluorogenic probes for HAT assays

Histone acetyltransferases (HATs) catalyze the acetylation of specific lysine residues in histone and nonhistone proteins. Recent studies showed that acetylation is widely distributed among cellular proteins, suggestive of diverse functions of HATs in cellular pathways. Nevertheless, currently available assays for HAT activity study are still quite limited. Here, we evaluated a series of thiol-sensitive fluorogenic compounds for the detection of the enzymatic activities of different HAT proteins. Upon conjugation to the thiol group of HSCoA, these molecules gain enhanced quantum yields and strong fluorescence, permitting facile quantitation of HAT activities. We investigated and compared the assay performances of these fluorogenic compounds for their capability as HAT activity reporters, including kinetics of reaction with HSCoA, influence on HAT activity, and fluorescence amplification factors. Our data suggest that CPM and coumarin maleic acid ester are excellent HAT probes owing to their fast reaction kinetics and dramatic fluorescence enhancement during the HAT reaction. Further, the microtiter plate measurements show that this fluorescent approach is robust and well suited for adaption to high-throughput screening of small molecule inhibitors of HATs, highlighting the value of this assay strategy in new drug discovery.     

D-Proline capped gold nanoclusters for turn-on detection of serum Raltitrexed

Using D-proline (D-Pro) as the reducing agent and capper, D-Pro@AuNCs was rapidly constructed. Its fluorescence could be quenched by AuNPs. Due to the electrostatic interaction between anticancer drug Raltitrexed (RTX) and AuNPs induced fluorescence "turn-on" principle, the resultant fluorescent probe exhibited good selectivity and sensitivity for detecting RTX in rat serums.     

d-Proline capped gold nanoclusters for turn-on detection of serum Raltitrexed

With d-proline as the reducing and capping agent, fluorescent gold nanoclusters were rapidly prepared (d-Pro@AuNCs) within 10 min at 100 °C. In the present of gold nanoparticles, the fluorescence of d-Pro@AuNCs was remarkably quenched. Interestingly, based on the electrostatic interaction between anticancer drug Raltitrexed and gold nanoparticles induced fluorescence “turn-on” principle, a high selective assay for detection of Raltitrexed was established with the probe associating the fluorescence emission at 435 nm. The fluorescence intensity of d-Pro@AuNCs linearly correlated with the concentration of Raltitrexed in the range from 5.0 μmol/L to 40.0 μmol/L (R² = 0.999) and the limit of detection was 1.9 μmol/L. Further, after Raltitrexed was abdominal injected in rats, a metabolic approach was constructed with the prepared fluorescent probe. It showed great potential of AuNCs-based sensing probes for application in analysis of serum anticancer drugs.     

Design and synthesis of intramolecular charge transfer-based fluorescent reagents for the highly-sensitive detection of proteins

Novel fluorescent molecular probes possessing both a hydroxystyryl and a cyanopyranyl moieties were designed and synthesized to detect the proteins via noncovalent bonding. These fluorescent probes indicated very weak fluorescence emission in the absence of protein. On the other hand, the fluorescence spectra of these probes showed a large Stokes shift and dramatic increase of fluorescence intensity, and red emission was observed after addition of BSA. These fluorescence spectral changes upon binding proteins were caused by the ICT process. Fluorescence intensities of the probes were plotted as a function of protein concentrations. A good linear relationship was observed up to 1000 microg/mL of protein, and the detection limit was found to be 100 ng/mL at the given assay conditions. Similar results were observed for the measurements of not only BSA but also other proteins (BGG, etc.). The responses of these probes to various nonprotein substances (inorganic salts, chelating agents, etc.) were observed, the fluorescence intensity did not change before and after the addition of foreign substances, and correct protein monitoring was successful using these fluorescent probes. To demonstrate the application of these probes, proteins after the separation using SDS-PAGE were stained in the medium containing 1, and the imaging of the proteins in the gel was successful. The experimental results clearly showed that these probes are good protein indicators for easy and highly sensitive detection.     

Preparation of highly fluorescent magnetic nanoparticles for analytes-enrichment and subsequent biodetection

Bifunctional nanoparticles with highly fluorescence and decent magnetic properties have been widely used in biomedical application. In this study, highly fluorescent magnetic nanoparticles (FMNPs) with uniform size of ca. 40 nm are prepared by encapsulation of both magnetic nanoparticles (MNPs) and shell/core quantum dots (QDs) with well-designed shell structure/compositions into silica matrix via a one-pot reverse microemulsion approach. The spectral analysis shows that the FMNPs hold high fluorescent quantum yield (QY). The QYs and saturation magnetization of the FMNPs can be regulated by varying the ratio of the encapsulated QDs to MNPs. Moreover, the surface of the FMNPs can be modified to offer chemical groups for antibody conjugation for following use in target-enrichment and subsequent fluorescent detection. The in vitro immunofluorescence assay and flow cytometric analysis indicate that the bifunctional FMNPs-antibody bioconjugates are capable of target-enrichment, magnetic separation and can also be used as alternative fluorescent probes on flow cytometry for biodetection.     

Fluorescence-labelled DNA probes to detect complementary sequences in homogeneous media

Sensitive, safe and easy-to-use probes for the detection of nucleic acids are urgently called for. To this end we are in the process of developing a fluorescence-based technique to work in homogeneous assay media. We have examined pyrene and fluorescein as fluorescent labels for natural DNA probes. A fraction of the cytosine residues of a single-stranded cDNA was randomly labelled with either pyrene or fluorescein using the bisulfite-catalyzed diamine reaction. Both fluorophores showed fluorescence quenching when the labelled probe was hybridized with its complementary strand and we describe the changes in steady-state fluorescence intensity that occurred upon hybridization. Our results demonstrate that pyrene quenching is more efficient than fluorescein quenching and thus pyrene-labelled probes are more sensitive for detecting and quantifying DNA from natural sources.     

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.     

基于线性荧光探针对T4 DNA连接酶的活性分析

利用线性荧光探针作为核酸连接反应的模板和信号分子, 通过实时监测荧光信号的降低来表征连接产物的生成过程, 从而建立了一种连续、简单且特异性高的T4 DNA连接酶活性分析的新方法, 检出限可达1.2 U/mL; 同时, 该方法还可用于快速考察金属离子和化学药物对酶促反应的影响. 实验结果表明, 该法不仅为灵敏、实时监测核酸连接反应提供了一种简便快捷的非同位素分析方法, 也为开展核酸连接酶活性分析、反应动力学机制探讨和药物快速筛选提供了一种新技术.     

High-throughput ribozyme-based assays for detection of viral nucleic acids

Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.     

A versatile platform for highly sensitive detection of protein: DNA enriching magnetic nanoparticles based rolling circle amplification immunoassay

A novel rolling circle amplification (RCA) immunoassay based on DNA enriching magnetic nanoparticles and assembled fluorescent DNA nanotags, magnetic nanoparticles-RCA immunoassay, is developed as a versatile fluorescence assay platform for highly sensitive proteins detection.     

Fluorescent pH probes for alkaline pH range based on perylene tetra-(alkoxycarbonyl) derivatives

The pH detection in the alkaline range is particularly important in many fields such as leather processing, waste water treatment, paper industry, and metal mining and finishing. Compared with traditional analysis methods such as colorimetric sensors and electrochemical sensors, the fluorescence and colorimetric probes for pH measurements have attracted much more attention due to their advantages of high sensitivity, excellent selectivity, noninvasiveness, low cost, fast response time, the possibility of continuously measurement, etc. However, there are few fluorescent probes fiting for alkaline pH monitoring. Acturally, the design and synthesis of them were more significant for new probes producing. In this study, the design, synthesis, and practical application of two novel fluorescent pH probes for alkaline pH assay were discussed. Both of the two probes were derived from perylene tetra-(alkoxycarbonyl). The red or blue shift of the absorption/fluorescence spectrum was caused by the introduction of electron donor amino or oxygen ring in the bay region. Due to electronic separation of the OH group from the electron-withdrawing core, the probes have high pKa values and cover the pH range from 8 to 12. They exist in either fluorescent acid form or non-fluorescent basic form. It was investigated that the amino substituent at bay region had a higher pKa value than O-heterocyclic annulated perylene, which showed that the adjustable pKa value could be achieved by the modification of electron withdrawing groups. The probes would have a wide use for testing strips measurements and monitoring pH changes in concrete.