An enzymatic determination of hydrogen peroxide by using spectrophotometry.

A new spectrophotometric method for determining low hydrogen peroxide concentrations by using horseradish peroxidase in the presence of NADH at pH 7.5 has been described. Both total NADH consumption and initial reaction rate may be used for the determination. Using the NADH consumption, a linear response with respect to hydrogen peroxide was observed in the concentration range 7 x 10(-8)-2.5 x 10(-6) M. Due to the presence of superoxide dismutase, hydrogen peroxide is partly regenerated and an amplification of the signal results, which explains the sensitivity.     

A ratiometric and two-photon fluorescent probe for imaging hydrogen peroxide in living cells

As an important reactive oxygen species (ROS), hydrogen peroxide plays a significant role in the life activity system, and its abnormal levels are closely related to many diseases. Developing effective fluorescent probes for detecting hydrogen peroxide is very urgent. Therefore, we constructed a probe Z that can detect hydrogen peroxide in ratio. It has naphthimide as the fluorophore and phenylboronic acid pinacol esters as the recognition group. It shows higher sensitivity, lower detection limit, higher selectivity, and broad pH applicability. Moreover, probe Z has low cytotoxicity that can be used to detect exogenous hydrogen peroxide in HeLa cells and might be a potential tool for studying hydrogen peroxide in physiological activities.     

Quadruplex priming amplification combined with nicking enzyme for diagnostics

Quadruplex priming amplification (QPA) is a straightforward assay that allows isothermal amplification of DNA and possesses an intrinsic real-time detection mechanism. QPA can be employed as a diagnostic tool for both linear and exponential signal amplification. The linear QPA, which is less prone to background activity characteristics of exponential systems, suffers from low sensitivity. To increase the sensitivity, here we introduce specific probe molecules that are designed for combined activities of Bst 2.0 polymerase and Nt.BstNBI nicking enzyme. The current assay, which is suitable for single-tube isothermal signal amplification, has increased sensitivity of plain linear QPA by three orders of magnitude to levels of low femtomolar concentration of target molecules.     

DNA hybridization detection in a microfluidic channel using two fluorescently labelled nucleic acid probes

A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5'-CTGAT TAGAG AGAGAA-TAMRA-3' and 5'-TET-ATGTC TGAGC TGCAGG-3') and target DNA (3'-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5') were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 x 10(-6) to 1.0 x 10(-7)M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase.     

Novel signal amplification technology with applications in DNA and protein detection systems.

A non-enzymatic approach to signal amplification has practical advantages over conventional target amplification methods. We have designed a simple, cost-efficient signal amplification system that can be used to enhance the detection of nucleic acids or protein. The signal amplification process requires initial capture of analyte by a specific probe, which, depending on the analyte, can be an oligomer or an antibody. Once the analyte is captured, amplification moieties are applied to significantly enhance the sensitivity of analyte detection. Nucleic acid amplification is typically greater than 1000-fold, increasing the sensitivity of target detection to less than 1 amol/100 microL. This amplification strategy presents a very flexible system with components that are easily altered to accommodate diverse assay requirements.     

The effect of avidin-biotin interactions in detection systems for in situ hybridization.

The effect of avidin-biotin interactions in several detection systems for the non-radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems.     

Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry.

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.     

A near‐infrared fluorescent probe based on boric acid hydrolysis for hydrogen peroxide detection and imaging in HeLa cells

Using the characteristics of hydrogen peroxide that are able to cleave phenyl‐boric acid selectively and efficiently, we here report a dicyanoisophorone‐boric acid ( DCP‐BA )‐based near‐infrared (NIR) fluorescent probe for detection of hydrogen peroxide. This probe shows a rapid, highly selective, and sensitive detection process for hydrogen peroxide with a significant NIR fluorescent turn‐on response that has been successfully applied to detect exogenous hydrogen peroxide in HeLa cells.     

Oligonucleotide probes applied for sensitive enzyme-amplified electrochemical assay of mercury(II) ions

We developed a novel electrochemical sensor for Hg(2+) detection using two mercury-specific oligonucleotide probes and streptavidin-horseradish peroxidase (HRP) enzymatic signal amplification. The two mercury-specific oligonucleotide probes comprised a thiolated capture probe and a biotinated signal probe. The thiolated capture probe was immobilized on a gold electrode. In the presence of Hg(2+), the thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction between the mismatched T-T base pairs directed the biotinated signal probe hybridizing to the capture probe and yielded a biotin-functioned electrode surface. HRP was then immobilized on the biotin-modified substrate via biotin-streptavidin interaction. The immobilized HRP catalyzed the oxidation of hydroquinone (H(2)Q) to benzoquinone (BQ) by hydrogen peroxide (H(2)O(2)) and the generated BQ was further electrochemically reduced at the modified gold electrode, producing a readout signal for quantitative detection of Hg(2+). The results showed that the enzyme-amplified electrochemical sensor system was highly sensitive to Hg(2+) in the concentration of 0.5 nM to 1 μM with a detection limit of 0.3 nM, and it also demonstrated excellent selectivity against other interferential metal ions.     

Photopolymerization as an innovative detection technique for low-density microarrays

One limitation that accounts in part for the scarcity of commercially available diagnostic microarrays is the expense associated with fluorescence detection. Here we present a colorimetric method based on photopolymerization as an "on-chip" signal amplification technique. Proof of principle experiments are detailed and followed by the use of a simple influenza microarray to demonstrate the technique for the first time with clinical samples. The advantages of this new technique include rapid (<5 min) signal amplification ( approximately 105) in ambient conditions for both DNA and protein microarrays, low reagent cost (<$1 per assay), visual or inexpensive detection, and signal preservation for at least two years under ambient conditions.     

Femtomol SPR detection of DNA-PNA hybridization with the assistance of DNA-guided polyaniline deposition

The inability of surface plasmon resonance (SPR) spectroscopy to detect extremely small refractive index changes has hindered its applications in ultrasensitive DNA analysis. In this study we report a signal amplification strategy that uses DNA-templated polyaniline deposition, suitable for DNA hybridization analysis with charge neutral peptide nucleic acid (PNA) being probes. Under acidic conditions, protonated aniline monomers are adsorbed on DNA backbones through electrostatic interaction. The microenvironment provided by the DNA facilitates oxidative aniline polymerization initialized by H₂O₂ in the presence of horseradish peroxide. Under optimal conditions, the detection limit is lowered from 5 nM for conventional SPR detection to 0.1 pM. The significant sensitivity improvement is attributed to the in-situ polymer chain growth along DNA strands, which introduces drastic refractive index increases. This signal amplification approach does not involve secondary hybridization processes. The detection sensitivity obtained is much better than that of gold nanoparticle-based amplification involving a secondary hybridization process and labeled DNA detection probes.     

Competitive reporter monitored amplification (CMA)--quantification of molecular targets by real time monitoring of competitive reporter hybridization

Background

State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests.

Methodology and Principal Findings

The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique.

Conclusions and Significance

The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.     

Using liquid crystals for the label-free detection of catalase at aqueous-LC interfaces

In this study, we developed a simple label-free method for the detection of catalase (CAT) using liquid crystals (LCs). The optical appearance of LCs changed from bright to dark when hydrogen peroxide was in contact with the dodecanal-doped nematic LC, 4-cyano-4′-pentylbiphenyl (5CB). Since hydrogen peroxide can oxidize aldehyde into carboxylic acid, an orientational transition of the LC from the planar to homeotropic state was induced by the self-assembled carboxylate monolayer formed at the aqueous/LC interface. The optical response of LCs exhibited a higher sensitivity to the presence of hydrogen peroxide in an alkaline solution. A new type of LC-based sensor was developed to monitor the presence of CAT in the aqueous phase. Due to the enzymatically catalytic hydrolysis of hydrogen peroxide, the bright-to-dark shift in the optical signal did not take place in the aqueous mixture of hydrogen peroxide and catalase. In contrast, the optical response changed from bright to dark when the mixture in the optical cell was replaced with an aqueous solution of hydrogen peroxide. Considering the optical response of LCs related to the absence and presence of hydrogen peroxide, the aldehyde-doped 5CB might have potential utility in real-time recognition and detection of chemical and biological events associated with hydrogen peroxide.     

Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Molecular beacons represent a new family of fluorescent probes for nucleic acids, and have found broad applications in recent years due to their unique advantages over traditional probes. Detection of nucleic acids using molecular beacons has been based on hybridization between target molecules and molecular beacons in a 1:1 stoichiometric ratio. The stoichiometric hybridization, however, puts an intrinsic limitation on detection sensitivity, because one target molecule converts only one beacon molecule to its fluorescent form. To increase the detection sensitivity, a conventional strategy has been target amplification through polymerase chain reaction. Instead of target amplification, here we introduce a scheme of signal amplification, nicking enzyme signal amplification, to increase the detection sensitivity of molecular beacons. The mechanism of the signal amplification lies in target-dependent cleavage of molecular beacons by a DNA nicking enzyme, through which one target DNA can open many beacon molecules, giving rise to amplification of fluorescent signal. Our results indicate that one target DNA leads to cleavage of hundreds of beacon molecules, increasing detection sensitivity by nearly three orders of magnitude. We designed two versions of signal amplification. The basic version, though simple, requires that nicking enzyme recognition sequence be present in the target DNA. The extended version allows detection of target of any sequence by incorporating rolling circle amplification. Moreover, the extended version provides one additional level of signal amplification, bringing the detection limit down to tens of femtomolar, nearly five orders of magnitude lower than that of conventional hybridization assay.     

综述与专论: 基于核酸适配体传感器的即时检测（POCT）技术

即时检测（point-of-care testing，POCT）是一种检测成本低、检测速度快、准确度高、能自我采样获得临床诊断结果的新型诊断技术。该技术在临床诊断、病情监控与疫情防控等领域发挥了重要作用。核酸适配体是一种能够特异性识别多种靶标的分子探针，具有易合成、批间差异小、易实现信号放大等突出优势，是生物医学传感器中重要的分子识别元件。本文概述了核酸适配体探针的现有筛选方法和进展，总结了核酸适配体POCT传感器信号放大策略，着重介绍了各类核酸适配体传感器在POCT领域的应用现状，并对核酸适配体POCT传感器的发展前景进行了展望。     

Field-effect amperometric immuno-detection of protein biomarker

The field-effect enzymatic detection technique has been applied to the amperometric immunoassay of the cancer biomarker, carcinoma antigen 125 (CA 125). The detection adopted a reagentless approach, in which the analyte, CA 125, was immobilized on the detecting electrode, which was modified using carbon nanotubes, and the detection signal was obtained by measuring the reduction peak current of the enzyme that was used to label the antibody. A gating voltage was applied to the detecting electrode, inducing increase in the signal current and therefore providing amplification of the detection signal. The voltage-controlled signal amplification of the detection system has increased the sensitivity and lowered the detection limit of the system. A detection limit of 0.9U/ml was obtained in the work.     

Fluorescent resonance energy transfer (FRET) based detection of a multiplex ligation-dependent probe amplification assay (MLPA) product

A fluorescent resonance energy transfer (FRET)-based hybridization assay for detecting multiplex ligation-dependent probe amplification (MLPA) products has been developed, extending the diagnostic power of the technique and demonstrating the possibility of combining MLPA with microarrays for the detection of multiple mutations. FRET is one of the most commonly used detection techniques for hybridization assays. To investigate the applicability of FRET based detection of MLPA products, a sandwich assay was designed to detect gene copy number by exploiting an immobilized probe labeled with an acceptor dye, Alexa Fluor 555, which hybridises to specific PCR amplicons, followed by hybridization of a second probe labeled with the donor dye, Alexa Fluor 488. Following excitation of the Alexa Fluor 488, a FRET signal was produced only if a DNA sequence specific to the BRCA1 exon 13 was present in the test sample. We have verified this assay on a DNA sample of a patient carrying a heterozygous BRCA1 exon 13 deletion using male genomic DNA as control. Here we demonstrate that the DNA sample containing the heterozygous deletion generated a considerably reduced FRET signal as compared to the control male human DNA. Our results show that the FRET design presented in this study can differentiate between reduced copy numbers any genomic DNA sequence after MLPA analysis, and the reported format is applicable to multiplex detection of MLPA products, using microarrays, or optical biosensor arrays, and future work will focus on the demonstration of this.     

Real-time monitoring of the strand displacement amplification (SDA) of human cytomegalovirus by a new SDA-piezoelectric DNA sensor system

Nucleic acid amplification has long been used in biosensor technologies, such as DNA sensors, DNA chips and microarrays, due to its advantage of high sensitivity in detecting target DNA. However, dynamic monitoring of nucleic acid amplifications with traditional DNA sensors in real-time is difficult since a constant temperature must be maintained during detection. Thus, the piezoelectric sensor, one type of traditional DNA sensor, is not applicable in real-time monitoring PCR due to the dramatic change in temperature that occurs during reaction. In this study, we introduced strand displacement amplification (SDA), an well-developed nucleic acid amplification technique that can work under conditions of constant temperature, into the development of a novel piezoelectric sensor. Using the new SDA-piezoelectric DNA sensor, we designed a stable system for liquid-phase detection, in which the crystal oscillator plate was fixed by an easily adjustable screw-threaded clamping mechanism and successfully applied the new sensor system to real-time SDA monitoring of human cytomegalovirus (HCMV). This new technique overcomes the shortcomings of traditional DNA sensors in real-time monitoring of nucleic acid amplification. The technique has proved to be a markedly simplified procedure with a number of advantages, such as higher sensitivity, better time efficiency, and the ability of dynamic real-time detection.