A one-step highly sensitive method for DNA detection using dynamic light scattering

A one-step homogeneous DNA detection method with high sensitivity was developed using gold nanoparticles (AuNPs) coupled with dynamic light scattering (DLS) measurement. Citrate-protected AuNPs with a diameter of 30 nm were first functionalized with two sets of single-stranded DNA probes and then used as optical probes for DNA detection. In the presence of target DNA, the hybridization between target DNA and the two nanoparticle probes caused the formation of nanoparticle dimers, trimers, and oligomers. As a result, the nanoparticle aggregation increased the average diameter of the whole nanoparticle population, which can be monitored simply by DLS measurement. A quantitative correlation can be established between the average diameter of the nanoparticles and the target DNA concentration. This DLS-based assay is extremely easy to conduct and requires no additional separation and amplification steps. The detection limit is around 1 pM, which is 4 orders of magnitude better than that of light-absorption-based methods. Single base pair mismatched DNAs can be readily discriminated from perfectly matched target DNAs using this assay.     

基于纳米金胶标记DNA探针的电化学DNA传感器研究

以纳米金胶为标记物,将其标记于人工合成的5-端巯基修饰的寡聚核苷酸片段上,制成了具有电化学活性的金胶标记DNA电化学探针;在一定条件下,使其与固定在玻碳电极表面的靶序列进行杂交反应,利用ssDNA与其互补链杂交的高度序列选择性和极强的分子识别能力,以及纳米金胶的电化学活性,实现对特定序列DNA片段的电化学检测以及对DNA碱基突变的识别.     

A thermodynamic investigation into the binding properties of DNA functionalized gold nanoparticle probes and molecular fluorophore probes

We report the first quantitative analysis of the oligonucleotide binding thermodynamics for DNA functionalized gold nanoparticle probes and compare our findings to molecular fluorophore probes on a sequence-for-sequence basis. With proper design, nanoparticle probes show significantly increased binding over molecular fluorophore probes under identical conditions. This is significant because probe binding strength directly influences detection sensitivity limits.     

DNA detection method based on the two-dimensional aggregation and selective desorption of nanoparticle probes

A label-free two-dimensional colorimetric DNA sensor is reported. This sensor is based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes induced by the molecular hybridization of single-stranded oligonucleotide probes and their complementary single-stranded DNA targets. To detect the aggregation, we have developed a new detection method based on the selective desorption of nonaggregated nanoparticles. We will show here that this detection method is highly specific and allows the quantification of the DNA targets.     

Direct visual detection of DNA based on the light scattering of silica nanoparticles on a human papillomavirus DNA chip

A detection system for a human papillomavirus (HPV) DNA chip based on the light scattering of aggregated silica nanoparticle probes is presented. In the assay, a target HPV DNA is sandwiched between the capture DNA immobilized on the chip and the probe DNA immobilized on the plain silica nanoparticle. The spot where the sandwich reaction occurs appears bright white and is readily distinguishable to the naked eye. Scanning electron microscopy images clearly show the aggregation of the silica nanoparticle probes. When three different sized (55 nm, 137 nm, 286 nm) plain silica nanoparticles were compared, probes of the larger silica nanoparticles showed a higher scattering intensity. Using 286-nm silica nanoparticles, the spots obtained with 200 pM of target DNA were visually detectable. The demonstrated capability to detect a disease related target DNA with direct visualization without using a complex detection instrument provides the prerequisite for the development of portable testing kits for genotyping.     

Electrochemical quantification of single-nucleotide polymorphisms using nanoparticle probes

We report a new approach for electrochemical quantification of single-nucleotide polymorphisms (SNPs) using nanoparticle probes. The principle is based on DNA polymerase I (Klenow fragment)-induced coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA under the Watson-Crick base pairing rule. After liquid hybridization events occurred among biotinylated DNA probes, mutant DNA, and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a biotin-avidin affinity reaction and magnetic separation. A cadmium phosphate-loaded apoferritin nanoparticle probe, which is modified with nucleotides and is complementary to the mutant site, is coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Subsequent electrochemical stripping analysis of the cadmium component of coupled nanoparticle probes provides a means to quantify the concentration of mutant DNA. The method is sensitive enough to detect 21.5 attomol of mutant DNA, which will enable the quantitative analysis of nucleic acid without polymerase chain reaction preamplification. The approach was challenged with constructed samples containing mutant and complementary DNA. The results indicated that it was possible to accurately determine SNPs with frequencies as low 0.01. The proposed approach has a great potential for realizing an accurate, sensitive, rapid, and low-cost method of SNP detection.     

Electric detection of DNA with PDMS microgap electrodes and silver nanoparticles

In this work a novel microdevice sensor has been developed by plating gold on the PDMS surface to generate a sandwich-type gap electrode for DNA detection. The microdevice utilizes a gold band electrode-PDMS-gold band electrode configuration and the minimum detectable volume could be as low as 5 μL. The 20 μm PDMS-based gap was chemically modified with DNA capture probes and DNA sandwich hybrids were formed with the addition of DNA target and silver nanoparticle probes. To increase detection sensitivity, parallel detection zones have been developed in which the relevant resistances decrease substantially upon hybridyzation. By measuring the change in electrical conductivity, the DNA target in the concentration range of 1000-0.1 nM can be assayed and the limit of lowest detectable concentration was achieved at 0.01 nM.     

基于纳米金探针和基因芯片的DNA检测新方法

运用荧光纳米金探针和基因芯片杂交建立一种新的DNA检测方法. 荧光纳米金探针表面标记有两种DNA探针: 一种为带有Cy5荧光分子的信号探针BP1, 起信号放大作用; 另一种为与靶DNA一部分互补的检测探针P532, 两种探针比例为5∶1. 当靶DNA存在时, 芯片上捕捉探针(与靶DNA的另一部分互补)通过碱基互补配对结合靶DNA, 将靶DNA固定于芯片上; 荧光纳米金探针通过检测探针与靶DNA及芯片结合, 在芯片上形成“三明治”复合结构, 最后通过检测信号探针上荧光分子的信号强度来确定靶DNA的量. 新方法检测灵敏度高, 可以检测浓度为1 pmol/L的靶DNA, 操作简单, 检测时间短. 通过改进纳米金探针的标记和优化杂交条件, 可进一步提高核酸检测的灵敏度, 这将在核酸检测方面具有重要的应用价值.     

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.     

Electroanalysis of single-nucleotide polymorphism by hairpin DNA architectures

Genetic analysis of infectious and genetic diseases and cancer diagnostics require the development of efficient tools for fast and reliable analysis of single-nucleotide polymorphism (SNP) in targeted DNA and RNA sequences often responsible for signalling disease onset. Here, we highlight the main trends in the development of electrochemical genosensors for sensitive and selective detection of SNP that are based on hairpin DNA architectures exhibiting better SNP recognition properties compared with linear DNA probes. SNP detection by electrochemical hairpin DNA beacons is discussed, and comparative analysis of the existing SNP sensing strategies based on enzymatic and nanoparticle signal amplification schemes is presented.     

A two-color-change, nanoparticle-based method for DNA detection

Herein, we describe the detailed synthesis of Ag/Au core-shell nanoparticles, the surface-functionalization of these particles with thiolated oligonucleotides, and their subsequent use as probes for DNA detection. The Ag/Au core-shell nanoparticles retain the optical properties of the silver core and are easily functionalized with thiolated oligonucleotides due to the presence of the gold shell. As such, the Ag/Au core-shell nanoparticles have optical properties different from their pure gold counterparts and provide another “color” option for target DNA-directed colorimetric detection. Size-matched Ag/Au core-shell and pure gold nanoparticles perform nearly identically in DNA detection and melting experiments, but with distinct optical signatures. Based on this observation, we report the development of a two-color-change method for the detection and simultaneous validation of single-nucleotide polymorphisms in a DNA target using Ag/Au core-shell and pure gold nanoparticle probes.     

DNA detection on lateral flow test strips: enhanced signal sensitivity using LNA-conjugated gold nanoparticles

A lateral flow test strip assay, enabling sensitive detection of DNA specific to the foodborne pathogen E. coli O157:H7, is described. The use of LNA-conjugated gold nanoparticle probes, along with signal amplification protocols, results in minimum detectable concentrations of ~0.4 nM.     

Simultaneous electrochemical determination of two analytes based on nuclease-assisted target recycling amplification

In the present study, a method for simultaneous determination of two different DNAs is developed based on nuclease-assisted target recycling and nanoparticle amplification. The target recycling process is accomplished by taking advantage of the cleavage property of nicking endonuclease (NEase) for specific nucleotide sequences in duplex. In the presence of target DNA, the linker DNA in our detection system can hybridize with the target and be cleaved to form short fragments. Thus the target DNA is released and recognized by another linker DNA, activating the next round of cleavage reaction. On the other hand, two bio-barcode probes, a PbS nanoparticles (NPs)-DNA probe and a CdS NPs-DNA probe, are used for tracing two target DNAs to further amplify the detection signals. Based on a sensitive differential pulse anodic stripping voltammetry (DPASV) method for the simultaneous detection of Pb²⁺ and Cd²⁺ obtained by dissolving two probes, two different target DNAs are determined with high sensitivity and single-base mismatch selectivity.     

Stimuli responsive release of metalic nanoparticles on semiconductor substrates

Optically active metal nanoparticles have been of recent and broad interest for applications to biomarker detection because of their ability to enable high sensitivity enhancements in various optical detection techniques. Here, we report stimuli responsive release of metallic nanoparticles on a semiconductor thin film array structure based on pH change. The metallic nanoparticles are obtained by a simple redox procedure on the semiconductor surface. This approach allows controlling nanoparticle surface coatings in situ for biomolecule conjugation, such as DNA probes on nanoparticles, and rapid stimuli responsive release of these nanoparticles upon pH change.     

Surface-enhanced Raman nanoparticle beacons based on bioconjugated gold nanocrystals and long range plasmonic coupling

We have developed a new class of surface-enhanced Raman scattering beacons (SERS beacons) that can be turned on and off by long-range plasmonic coupling, induced by biomolecular recognition and binding events. The beacons are based on colloidal gold nanocrystals in two sizes (40 and 60 nm) and are prepared by spectral encoding with a Raman reporter molecule, functionalized with thiolated DNA probes, and stabilized and protected by low molecular weight poly(ethylene glycol)s (PEGs). The results show the SERS signal intensities increase by 40-200-fold when the nanoparticle beacons are activated by plasmonic coupling, much higher than the bright-to-dark intensity ratios reported for traditional molecular beacons. Multivalent gold nanoparticles also have exquisite specificity and are able to recognize single-base mismatches or mutations. This class of SERS nanoparticle beacons has novel mechanisms for molecular detection and signal amplification, and its long-range coupling nature raises new opportunities in developing plasmonic probes to detect proteins, cells, and intact viruses.     

Applications of aptamers in cancer cell biology

Identifying cells associated with specific disease states is critically important for the early detection and diagnosis of cancer. To facilitate this task, molecular probes, which bind biomarkers that are either specifically or differentially expressed in diseased cells relative to healthy cells, provide a simple and effective method. This review focuses on the use of DNA aptamers as molecular probes for cancer cells. These aptamers are created by means of the cell-based Systematic Evolution of Ligands by EXponential enrichment (SELEX) process, which uses whole disease cells as targets. We describe at length the steps of the cell-SELEX process and discuss several applications for the aptamers, including profiling leukemia patient samples and discovering cell-surface cancer biomarkers. We conclude with a discussion of an aptamer-conjugated nanoparticle enrichment and detection scheme.     

Dynamic-light-scattering-based sequence-specific recognition of double-stranded DNA with oligonucleotide-functionalized gold nanoparticles

An ultrasensitive and simple dynamic-light-scattering (DLS) assay for the sequence-specific recognition of double-stranded DNA (dsDNA) was developed based on detection of the average diameter change of Au nanoparticle (AuNP) probes modified with oligonucleotides 5'-TTTCTCTTCCTT- CTCTTC-(T)(12)-SH-3' (Oligo 1) and 5'-TTCTTTCTTTTCTTTTTC-(T)(12)- SH-3' (Oligo 2). The target dsDNA was composed of two complementary oligonucleotides: 5'-AAAGAGAAGGAAGAGAAGAAGAAAGAAAAGAAAAAG-3' (Oligo 3) and 3'-TTTCTCTTCCTTCTCTTCTTCTTTCTTTTCTTTTTC-5' (Oligo 4). Hybridization of the two AuNPs-Oligo probes with the target dsDNA induced aggregation of the target dsDNA by forming triplex DNA, which accordingly increased the average diameter. This diameter change could then be detected by DLS. The average diameter was proportional to the target dsDNA concentration over the range from 593 fM to 40 pM, with a detection limit of 593 fM. Moreover, the assay had good sequence specificity for the target dsDNA.     

The design and application of fluorophore-gold nanoparticle activatable probes

Fluorescence-based assays and detection techniques are among the most highly sensitive and popular biological tests for researchers. To match the needs of research and the clinic, detection limits and specificities need to improve, however. One mechanism is to decrease non-specific background signals, which is most efficiently done by increasing fluorescence quenching abilities. Reports in the literature of theoretical and experimental work have shown that metallic gold surfaces and nanoparticles are ultra-efficient fluorescence quenchers. Based on these findings, subsequent reports have described gold nanoparticle fluorescence-based activatable probes that were designed to increase fluorescence intensity based on a range of stimuli. In this way, these probes can detect and signify assorted biomarkers and changes in environmental conditions. In this review, we explore the various factors and theoretical models that affect gold nanoparticle fluorescence quenching, explore current uses of activatable probes, and propose an engineering approach for future development of fluorescence based gold nanoparticle activatable probes.     

Sequence-specific detection of femtomolar DNA via a chronocoulometric DNA sensor (CDS): effects of nanoparticle-mediated amplification and nanoscale control of DNA assembly at electrodes

We herein report a novel nanoparticle-based electrochemical DNA detection approach. This DNA sensor is based on a "sandwich" detection strategy, which involves capture probe DNA immobilized on gold electrodes and reporter probe DNA labeled with gold nanoparticles that flank the target DNA sequence. Electrochemical signals are generated by chronocoulometric interrogation of [Ru(NH(3))(6)](3+) that quantitatively binds to surface-confined capture probe DNA via electrostatic interactions. We demonstrated that the incorporation of a gold nanoparticle in this sensor design significantly enhanced the sensitivity and the selectivity. Nanoscale control of the self-assembly process of DNA probes at gold electrodes further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar (zeptomoles) DNA targets and exhibited excellent selectivity against even a single-base mismatch. In addition, this novel DNA sensor showed fairly good reproducibility, stability, and reusability.     

Rolling‐Circle Amplification Detection of Thrombin Using Surface‐Enhanced Raman Spectroscopy with Core‐Shell Nanoparticle Probe

An ultrasensitive surface‐enhanced Raman spectroscopy (SERS) sensor based on rolling‐circle amplification (RCA)‐increased “hot‐spot” was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO₂ core‐shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer‐by‐layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of thrombin, the aptamer sequence was released; this allowed the remaining single‐stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0×10^?12 to 1.0×10^?8 M and a detection limit of 4.2×10^?13 M , and showed good performance in real serum samples.