Sensitivity by combination: immuno-PCR and related technologies

The versatility of immunoassays for the detection of antigens can be combined with the signal amplification power of nucleic acid amplification techniques in a broad range of innovative detection strategies. This review summarizes the spectrum of both, DNA-modification techniques used for assay enhancement and the resulting key applications. In particular, it focuses on the highly sensitive immuno-PCR (IPCR) method. This technique is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR or related techniques for signal generation and read-out. Various strategies for the combination of antigen detection and nucleic acid amplification are discussed with regard to their laboratory analytic performance, including novel approaches to the conjugation of antibodies with DNA, and alternative pathways for signal amplification and detection. A critical assessment of advantages and drawbacks of these methods for a number of applications in clinical diagnostics and research is conducted. The examples include the detection of viral and bacterial antigens, tumor markers, toxins, pathogens, cytokines and other targets in different biological sample materials.     

Colloidal gold-polystyrene bead hybrid for chemiluminescent detection of sequence-specific DNA

A sensitive chemiluminescent (CL) detection of sequence-specific DNA has been developed by taking advantage of a magnetic separation/mixing process and the amplification feature of colloidal gold labels. In this protocol, the target oligonucleotides are hybridized with magnetic bead-linked capture probes, followed by the hybridization of the biotin-terminated amplifying DNA probes and the binding of streptavidin-coated gold nanoparticles; the nanometer-sized gold tags are then dissolved and quantified by a simple and sensitive luminol CL reaction. The proposed CL protocol is evaluated for a 30-base model DNA sequence, and the amount as low as 0.01 pmol of DNA is determined, which exhibits a 150 x enhancement in sensitivity over previous gold dissolution-based electrochemical formats and an enhancement of 20 x over the ICPMS detection. Further signal amplification is achieved by the assembly of biotinylated colloidal gold onto the surface of streptavidin-coated polystyrene beads. Such amplified CL transduction allows detection of DNA targets down to the 100 amol level, and offers great promise for ultrasensitive detection of other biorecognition events.     

Sensor Array: Impedimetric Label‐Free Sensing of DNA Hybridization in Real Time for Rapid,PCR‐Based Detection of Microorganisms

A novel, impedance‐based electronic sensor format was used for label‐free, real‐time detection of microbial DNA on oligonucleotide probe arrays. Detection limits of 5–10 nM were achieved for single‐stranded, PCR‐amplified DNA targets. Hybridization selectivity yielded 9‐ to 12‐fold signal increases for specific targets, and the sensor arrays were re‐used multiple times without significant signal degradation. These and other features of the SHARP Laboratories of America (SLA) sensor array, such as its ability to acquire continuous measurements of DNA as it accumulates on the array surface, make it an attractive biosensor platform for field detection and monitoring of sentinel and/or pathogenic microorganisms.     

Label-free electrical detection of pyrophosphate generated from DNA polymerase reactions on field-effect devices

We introduce a label-free approach for sensing polymerase reactions on deoxyribonucleic acid (DNA) using a chelator-modified silicon-on-insulator field-effect transistor (SOI-FET) that exhibits selective and reversible electrical response to pyrophosphate anions. The chemical modification of the sensor surface was designed to include rolling-circle amplification (RCA) DNA colonies for locally enhanced pyrophosphate (PPi) signal generation and sensors with immobilized chelators for capture and surface-sensitive detection of diffusible reaction by-products. While detecting arrays of enzymatic base incorporation reactions is typically accomplished using optical fluorescence or chemiluminescence techniques, our results suggest that it is possible to develop scalable and portable PPi-specific sensors and platforms for broad biomedical applications such as DNA sequencing and microbe detection using surface-sensitive electrical readout techniques.     

基于聚苯乙烯微球放大的凝血酶化学发光检测新技术

本文以羧基96孔板为分离载体,核酸适配体作为分子特异性识别元件,聚苯乙烯微球作为放大载体,辣根过氧化物酶为标记物,构建了化学发光（CL）高灵敏度凝血酶检测新技术.实验结果表明：该放大技术不但灵敏度高,且抗干扰能力强,其他蛋白质如IgG、IgM、IgA、IgE、IFN均无明显干扰.聚苯乙烯微球放大体系中凝血酶的线性范围为7.8～250pmol/L,最低检测浓度可达3.9pmol/L;而不放大检测技术的线性范围为0.94～30nmol/L,最低检测浓度为0.46nmol/L,放大体系将检测灵敏度提高100多倍.综合而言,基于适配体识别和聚苯乙烯微球放大的凝血酶CL检测新技术具有通量大、简单快速和灵敏度高的特点,有望在凝血酶高通量检测领域获得应用.     

A Simple and Highly Sensitive Electrochemical Biosensor for microRNA Detection Using Target‐Assisted Isothermal Exponential Amplification Reaction

A simple and highly sensitive electrochemical biosensor for microRNA (miRNA) detection was successfully developed by integrating a target‐assisted isothermal exponential amplification reaction (EXPAR) with enzyme‐amplified electrochemical readout. The binding of target miRNA with the immobilized linear DNA template generated a part duplex and triggered primer extension reaction to form a double‐stranded DNA. Then one of the DNA strands was cleaved by nicking endonuclease and extended again. The short fragments with the same sequence as the target miRNA except for the replacement of uridines and ribonucleotides with thymines and deoxyribonucleotides could be displaced and released. Hybridization of these released DNA fragments with other amplification templates and their extension on the templates led to target exponential amplification. Integrating with enzyme‐amplified electrochemical readout, the electrochemical signal decreases with the increasing target microRNA concentration. The method could detect miRNA down to 98.9 fM with a linear range from 100 fM to 10 nM. The fabrication and binding processes were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The specificity of the method allowed single‐nucleotide difference between miRNA family members to be discriminated. The established biosensor displayed excellent analytical performance toward miRNA detection and might present a powerful and convenient tool for biomedical research and clinic diagnostic application.     

Multiple sample amplification and genotyping integrated on a single electronic microarray

We report a novel method that allows simultaneous in situ amplification and then genotyping of single nucleotide polymorphism (SNP) for multiple samples on a single electronic microarray. The locus coding for one of the common inherited thrombosis risk factors, Factor V Leiden (FVL), was chosen as a model system for SNP analysis. This method combines strand displacement amplification (SDA) with electrophoretic movement and concentration of DNA on electronic microarrays to provide a single platform for DNA amplification and analysis. The method includes: electronic anchoring of allele-specific SDA amplifiable primers (APs) and a nonamplifiable primer (NAP) to different electrodes, electronic hybridization of genomic DNA from different samples to those primers, in situ amplification of target DNA, and genotyping of FVL. Compared to previous anchored SDA methods, the addition of a NAP improves detection signals by at least 20-fold. The sensitivity of this method is dependent on the amplification time. Using this method, nine different genomic DNA samples with known FVL genotypes were amplified and correctly genotyped on a single electronic microarray without any contamination between samples. The present method could streamline development of nucleic acid-based assays in applications of molecular diagnostic, point-of-care testing, and forensic detection, which often require the capability to analyze multiple samples efficiently.     

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.     

Amplified analysis of DNA by the autonomous assembly of polymers consisting of DNAzyme wires

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10(-14) M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene.     

Ultrasensitive assays for proteins

Proteins are essential components of organisms and are involved in a wide range of biological functions. There are increasing demands for ultra-sensitive protein detection, because many important protein biomarkers are present at ultra-low levels, especially during the early stages of disease. Measuring proteins at low levels is also crucial for investigations of the protein synthesis and functions in biological systems. In this review, we summarize the recent developments of novel technology enabling ultrasensitive protein detection. We focus on two groups of techniques that involve either polymerase amplification of affinity DNA probes or signal amplification by the use of nano-/micro-materials. The polymerase-based amplification of affinity DNA probes indirectly improves the sensitivity of protein detection by increasing the number of detection molecules. The use of nano-/micro-materials conjugated to affinity probes enhances the measurement signals by using the unique electrical, optical, and catalytic properties of these novel materials. This review describes the basic principles, performances, applications, merits, and limitations of these techniques.     

Express and sensitive detection of multiple miRNAs via DNA cascade reactors functionalized photonic crystal array

Array based detection techniques with fluorescence signal reading is a powerful tool for multiple targets analysis. However,when applied fluorescence array for micro RNA detection, time-consuming multi-steps surface signal amplification is usually required due to the low abundance of micro RNA in total RNA expressions, which impairs detection efficiency and limits its application in point of care test(POCT) manner. Herein, DNA cascade reactors(DCRs) functionalized photonic crystal(PC)array was fabricated for express and sensitive detections of mi RNA-21 and mi RNA-155. DCRs were assembled by interval conjugation of self-quenched hairpin DNA probes to single strand DNA nanowire synthesized by rolling circle amplification,which generated cascade DNA hybridization reactions in response to target mi RNAwith instant fluorescence recovery signal. PC array patterns with multi-structure colors further amplified fluorescence with their respective photonic bandgaps(PBGs)matching with the emission peaks of fluorescence molecules labelled on DCRs. The as-prepared DCRs functionalized PC array demonstrated express and sensitive simultaneous detections of mi RNA-21 and mi RNA-155 with hundreds f M detection limits only in 15 min, and was successfully applied in fast quantifications of low abundance mi RNAs from cell lysates and spiked mi RNAs from human serum, which would hold great potential for disease diagnosis and therapeutic effect monitoring with a POCT manner.     

Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses

The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z.     

Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.)

A species-specific endogenous reference gene system was developed for polymerase chain reaction (PCR)-based analysis in common wheat (Triticum aestivum L.) by targeting the ALMT1 gene, an aluminium-activated malate transporter. The primers and probe were elaborated for real-time PCR-based qualitative and quantitative assay. The size of amplified product is 95 base pairs. The specificity was assessed on 17 monocot and dicot plant species. The established real-time PCR assay amplified only T. aestivum-derived DNA; no amplification occurred on other phylogenetically related species, including durum wheat (T. durum). The robustness of the system was tested on the DNA of 15 common wheat cultivars using 20 000 genomic copies per PCR the mean cycle threshold (Ct) values of 24.02 +/- 0.251 were obtained. The absolute limits of detection and quantification of the real-time PCR assay were estimated to 2 and 20 haploid genome copies of common wheat, respectively. The linearity was experimentally validated on 2-fold serial dilutions of DNA from 650 to 20 000 haploid genome copies. All these results show that the real-time PCR assay developed on the ALMT1 gene is suitable to be used as an endogenous reference gene for PCR-based specific detection and quantification of T. aestivum-derived DNA in various applications, in particular for the detection and quantification of genetically modified materials in common wheat.     

Enhancing the fluorescence intensity of DNA microarrays by using cationic surfactants

DNA microarrays have been used as powerful tools in genomics studies and single nucleotide polymorphisms analysis. However, the fluorescence detection used in most conventional DNA microarrays is still limited by its sensitivity. The aim of this study is to use a cationic surfactant, cetyl trimethylammonium bromide (CTAB), to enhance the fluorescence intensity of 6-carboxy-fluorescene (FAM)-labeled DNA probes immobilized on a DNA microarray. We show that in the presence of CTAB the immobilized FAM-labeled DNA probes is 11-fold brighter than that without exposure to CTAB. Similarly, when we hybridize FAM-labeled DNA targets to a DNA microarray and treat the surface with CTAB solution, the fluorescence intensity shows a 26-fold increase for perfect-match DNA targets. More importantly, the contrast between perfect-match and 1-mismatch DNA is also increased from 1.3-fold to 15-fold. This method offers a simple and efficient technique to enhance the detection limit of DNA microarrays.     

Amplified detection of single-base mismatches in DNA using microgravimetric quartz-crystal-microbalance transduction

Three different methods for the amplified detection of a single-base mismatch in DNA are described using microgravimetric quartz-crystal-microbalance as transduction means. All methods involve the primary incorporation of a biotinylated base complementary to the mutation site in the analyzed double-stranded primer/DNA assembly. The double-stranded assembly is formed between 25 complementary bases of the probe DNA assembled on the Au-quartz crystal and the target DNA. One method of amplification includes the association of avidin- and biotin-labeled liposomes to the sensing interface. The second method of amplified detection of the base mismatch includes the association of an Au-nanoparticle-avidin conjugate to the sensing interface, and the secondary Au-nanoparticle-catalyzed deposition of gold on the particles. The third amplification route includes the binding of the avidin-alkaline phosphatase biocatalytic conjugate to the double-stranded surface followed by the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate to the insoluble product indigo derivative that precipitates on the transducer. Comparison of the three amplification routes reveals that the catalytic deposition of gold on the Au-nanoparticle/avidin conjugate is the most sensitive method, and the single-base mismatch in the analyzed DNA is detected with a sensitivity that corresponds to 3x10(-16) M.