Quantum dots-based multifunctional dendritic superstructure for amplified electrochemiluminescence detection of ATP

A novel multifunctional dendrimeric CdSe-CdS-Quantum dots (QDs) hybrid superstructure with highly intense electrochemiluminescence (ECL), fluorescence and excellent magnetic property is prepared for the first time, and successfully applied to amplified ECL assays of ATP using DNA cycle amplification technique. The magnetic nanoparticles (MNPs) were firstly assembled with unique dendrimer nanoclusters (NCs), then large numbers of QDs were labeled onto the dendrimer NCs, the superstructure exhibits highly enhanced ECL and fluorescence than the pure QDs. Remarkable ECL quenching of the nanocomposites by gold nanoparticles (GNPs) was observed, based on which a novel strategy for highly sensitive ATP detection was developed by cycle amplification technique. Furthermore, the nanocomposites with excellent magnetic properties can be easily labeled, separated and immobilized onto a magnetic electrode. In particular, all the procedures such as linking GNPs, sensing target and DNA cycle amplification were directly accomplished on the nanocomposites, which is more rapid, convenient, complete and has better reproducibility than the conventional methods on electrode. To the best of our knowledge, this is the first report on the multifunctional QDs superstructure with highly intense ECL, fluorescence, excellent magnetism and its ECL biosensing, which opens a new pathway for developing QD-based nanocomposites for broad applications in ECL bioassays and optical imaging.     

Nano-magnetic primer based electrochemiluminescence-polymerase chain reaction (NMPE-PCR) assay

Here we have developed a novel nano-magnetic primer based electrochemiluminescence-polymerase chain reaction (NMPE-PCR) strategy for detection of genome. The key idea of this method is integrating the two in situ processes: PCR on the surface of magnetic nanoparticles (MNPs) and magnetic beads based ECL readout platform, to avoid some laborious manual operations and achieve rapid yet sensitive detection. At first, the approach employs a pair of functional primers for amplification: one is tris-(2,2'-bipyridyl) ruthenium (TBR) labeled primer; the other one is nano-magnetic primer which is prepared by attaching the primer to the surfaces of MNPs. With the presence of DNA analyte and PCR mixture, the TBR labeled products are directly loaded and enriched on the surface of MNPs during PCR cycling. Then the MNPs-TBR complexes can be analyzed by a magnetic ECL platform without any post-modification or post-incubation. Finally, we used Listeria monocytogenes as the target to examine these desirable properties of this assay, reaching a detection limit of 500 fg/μL for genome in 1 h. The proposed study has provided the evidence as a proof-of-concept, thus having potential for development of automatic mode for detection of specific gene.     

Quantitative electrochemiluminescence detection of proteins: Avidin-based sensor and tris(2,2'-bipyridine) ruthenium(II) label

Quantitative electrochemiluminescence (ECL) detection of a model protein, bovine serum albumin (BSA) was achieved via biotin–avidin interaction using an avidin-based sensor and a well-developed ECL system of tris(2,2′-bipyridine) ruthenium(II) derivative as label and tri-n-propylamine (TPA) as coreactant. To detect the protein, avidin was linked to the glassy carbon electrode through passive adsorptions and covalent interaction with carboxylate-terminated carbon nanotubes that was used as binder to immobilize avidin onto the electrode. Then, biotinylated BSA tagged with tris(2,2′-bipyridine) ruthenium(II) label was attached to the prepared avidin surface. After binding of BSA labeled with tris(2,2′-bipyridine) ruthenium(II) derivative to the surface-immobilized avidin through biotin, ECL response was generated when the self-assembled modified electrode was immersed in a TPA-containing electrolyte solution. Such double protein labeling protocol with a biotin label for biorecognition and ruthenium label for ECL detection facilitated the detection of protein compared to the classical double antibody sandwich format. The ECL intensity was linearly proportional to the feed concentration of BSA over two orders of magnitude in the range of 15 nM to 7.5 μM. The detection limit was estimated to be 1.5 nM. Further application to the lysozyme analysis was carried out to validate the present approach for an effective and favorable protocol for the quantitative detection of proteins. The dynamic range of lysozyme was from 0.001 g L⁻¹ to 0.1 g L⁻¹ and the detection limit was 0.1 mg L⁻¹. Electrochemical impedance and cyclic voltammetric measurements along with some necessary control experiments were conducted to characterize the successful formation of self-assembled modified electrodes and to grant the whole detection process.     

Fluorescent bio-barcode DNA assay for the detection of Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis is one of the most frequently reported causes of foodborne illness. It is a major threat to the food safety chain and public health. A highly amplified bio-barcode DNA assay for the rapid detection of the insertion element (Iel) gene of Salmonella Enteritidis is reported in this paper. The biosensor transducer is composed of two nanoparticles: gold nanoparticles (Au-NPs) and magnetic nanoparticles (MNPs). The Au-NPs are coated with the target-specific DNA probe which can recognize the target gene, and fluorescein-labeled barcode DNA in a 1:100 probe-to-barcode ratio. The MNPs are coated with the 2nd target-specific DNA probe. After mixing the nanoparticles with the 1st target DNA, the sandwich structure (MNPs-2nd DNA probe/Target DNA/1st DNA probe-Au-NPs-barcode DNA) is formed. A magnetic field is applied to separate the sandwich from the unreacted materials. Then the bio-barcode DNA is released from the Au-NPs. Because the Au-NPs have a large number of barcode DNA per DNA probe binding event, there is substantial amplification. The released barcode DNA is measured by fluorescence. Using this technique, the detection limit of this bio-barcode DNA assay is as low as 2.15 x 10(-16)mol (or 1 ng/mL).     

Sensitive and isothermal electrochemiluminescence gene-sensing of Listeria monocytogenes with hyperbranching rolling circle amplification technology

Listeria monocytogenes (L. monocytogenes) is one of the most problematic human pathogens, as it is mainly transmitted through the food chain and cause listeriosis. Thus, specific and sensitive detection of L. monocytogenes is required to ensure food safety. In this study, we proposed a method using hyperbranching rolling circle amplification (HRCA) combined with magnetic beads based electrochemiluminescence (ECL) to offer an isothermal, highly sensitive and specific assay for the detection of L. monocytogenes. At first, a linear padlock probe was designed to target a specific sequence in the hly gene which is specific to L. monocytogenes and then ligated by Taq DNA ligase. After ligation and digestion, further amplification by HRCA with a biotiny labeled primer and a tris (bipyridine) ruthenium (TBR) labeled primer was performed. The resulting HRCA products were then captured onto streptavidin-coated paramagnetic beads and were analyzed by magnetic beads based ECL platform to confirm the presence of targets. Through this approach, as low as 10 aM synthetic hly gene targets and about 0.0002 ng/μl of genomic DNA from L. monocytogenes can be detected, the ability to detect at such ultratrace levels could be attributed to the powerful amplification of HRCA and the high sensitivity of current magnetic bead based ECL detection platform.     

Detection of Prorocentrum minimum (Pavillard) Schiller with an Electrochemiluminescence–Molecular Probe Assay

The occurrence of harmful algal blooms (HABs) caused by Prorocentrum minimum (Pavillard) Schiller is a crucial subject in the study of HABs. An electrochemiluminescence-molecular probe assay (ECL-MP) was developed to qualitatively and quantitatively detect P. minimum. It was based on the sandwich hybridization integrated with a nuclease protection assay (NPA-SH) and improved by electrochemiluminescence (ECL). An ECL analyzer was established in this study, and it was shown that this analyzer was stable and highly sensitive, with a detection range of 0.4?pmol to 4?nmol Ru(bpy)(3)Cl(2)·6H(2)O under optimal reaction conditions of 1.0?V, 1.0?mA, 1.5?mol·L(-1) TPrA, and pH?7.4. The optimal amount of magnetic beads for separation of labeled NPA probes in a 20-μL hybridization mixture was 4?μg. The ECL counts per second was linear with the number of P. minimum cells in a range of 6.25?×?10(2) to 4?×?10(4), and there was no significant difference between ECL-MP and microscopy, with a 95% confidence level (t test) when individual, mixed cultures and field samples were treated. This study provides a convenient method for fast and accurate detection of P. minimum in the marine environment.     

DNA hybridization biosensors using polylysine modified SPCEs

Two electrochemical DNA hybridization biosensors (genosensors) for the detection of a 30-mer sequence unique to severe acute respiratory syndrome (SARS) virus are described in this work. Both genosensors rely on the hybridization of the oligonucleotide target with its complementary probe, which is immobilized on positively charged polylysine modified screen-printed carbon electrodes (SPCEs), through electrostatic interactions. In one design, a biotinylated target is used and the detection of the hybridization reaction is monitored using alkaline phosphatase labeled streptavidin (S-AP). This enzyme catalyzes the hydrolysis of the substrate 3-indoxyl phosphate (3-IP) to indigo, which is then solubilized to indigo carmine and detected by means of cyclic voltammetry (CV). In the other design, the target is labeled using an Au(I) complex, sodium aurothiomalate, and the duplex formation is detected by measuring, for first time, the current generated by the hydrogen evolution catalyzed by the gold label. Using 30 min of hybridization time, a detection limit of 8 pM is calculated for the enzymatic genosensor. Although this good sensitivity cannot be reached with the metal label (0.5 nM), the use of this label allows a considerable decrease of the analysis time. Both genosensors do not require the modification of the oligonucleotide probe and using stringent experimental conditions (60 min of hybridization time and 50% formamide in the hybridization buffer) can discriminate between a complementary oligonucleotide and an oligonucleotide with a three-base mismatch.     

Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay

We present a novel assay for rapid and highly sensitive detection of specific nucleic acid fragments in human serum. In a magnetic modulation biosensing (MMB) system, magnetic beads and fluorescently labeled probes are attached to the target analyte and form a “sandwich” complex. An alternating external magnetic field gradient condenses the magnetic beads (and hence the target molecules with the fluorescently labeled probes) to the detection volume and sets them in a periodic motion, in and out of a laser beam. A synchronous detection enables the removal of background signal from the oscillating target signal without complicated sample preparation. The high sensitivity of the MMB system, combined with the specificity of a sandwich hybridization assay, enables detection of DNA fragments without enzymatic signal amplification. Here, we demonstrate the sensitivity of the assay by directly detecting the EML4‐ALK oncogenic translocation sequence spiked in human serum. The calculated limit of detection is 1.4 pM, which is approximately 150 times better than a conventional plate reader. In general, the MMB‐assisted SHA can be implemented in many other applications for which enzymatic amplification, such as PCR, is not applicable and where rapid detection of specific nucleic acid targets is required.     

Capture antibody targeted fluorescence in situ hybridization (CAT-FISH): dual labeling allows for increased specificity in complex samples

Pathogen detection using biosensors is commonly limited due to the need for sensitivity and specificity in detecting targets within mixed populations. These issues were addressed through development of a dual labeling method that allows for both liquid-phase fluorescence in situ hybridization (FISH) and capture antibody targeted detection (CAT-FISH). CAT-FISH was developed using Escherichia coli O157:H7 and Staphylococcus aureus as representative bacteria, and processing techniques were evaluated with regard to FISH intensities and antibody recognition. The alternative fixative solution, methacarn, proved to be superior to standard solid-phase paraformaldehyde fixation procedures, allowing both FISH labeling and antibody recognition. CAT-FISH treated cells were successfully labeled with FISH probes, captured by immunomagnetic separation using fluorescent cytometric array beads, and detected using a cytometric array biosensor. CAT-FISH treated cells were detectable with LODs comparable to the standard antibody-based technique, (~ 10³ cells/ml in PBS), and the technique was also successfully applied to two complex matrices. Although immunomagnetic capture and detection using cytometric arrays were demonstrated, CAT-FISH is readily applicable to any antibody-based fluorescence detection platform, and further optimization for sensitivity is possible via inclusion of fluorescently tagged antibodies. Since the confidence level needed for positive identification of a detected target is often paramount, CAT-FISH was developed to allow two separate levels of specificity, namely nucleic acid and protein signatures. With proper selection of FISH probes and capture antibodies, CAT-FISH may be used to provide rapid detection of target pathogens from within complex matrices with high levels of confidence.     

A new infrared fluorescent-labeling agent and labeled antibody for diagnosing microcancers

PURPOSE: We have developed infrared fluorescent labeling agents and infrared-ray fluorescence endoscopes to establish a novel diagnostic technique. Since the fluorescence intensity of the initial labeled antibody (ICG-sulfo-OSu-labeled antibody) was not sufficient for practical use, we synthesized indocyanine green acylthiazolidinethione (ICG-ATT), which was expected to label various target molecules having amino groups efficiently. MATERIALS AND METHODS: To confirm imaging of infrared fluorescence intensity of ICG-ATT- and ICG-sulfo-OSu-labeled anti-MUC1 antibodies, cotton thread was soaked in various concentrations of the antibody solution in 0.1M PBS, and observed under the epi-illumination infrared fluorescence microscope. Localization and the intensity of infrared fluorescence and DAB coloring was compared in paraffin sections of human gastric mucosa. RESULTS: In the study of cotton threads, both labeled antibodies showed relatively clear infrared fluorescence, and significant difference was not observed between the two antibodies. ICG-ATT-labeled anti-MUC1 antibody produced stronger staining than that by ICG-sulfo-OSu-labeled antibody. Localization pattern of infrared fluorescent staining was in good agreement with that by the conventional method with oxidized DAB staining. CONCLUSION: ICG-ATT is useful as a fluorescent-labeling agent for diagnosis of microcancers by infrared fluorescence endoscopes.     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

Synergy of photoacoustic and fluorescence flow cytometry of circulating cells with negative and positive contrasts

In vivo photoacoustic (PA) and fluorescence flow cytometry were previously applied separately using pulsed and continuous wave lasers respectively, and positive contrast detection mode only. This paper introduces a real‐time integration of both techniques with positive and negative contrast modes using only pulsed lasers. Various applications of this new tool are summarized, including detection of liposomes loaded with Alexa‐660 dye, red blood cells labeled with Indocyanine Green, B16F10 melanoma cells co‐expressing melanin and green fluorescent protein (GFP), C8161‐GFP melanoma cells targeted by magnetic nanoparticles, MTLn3 adenocarcinoma cells expressing novel near‐infrared iRFP protein, and quantum dot‐carbon nanotube conjugates. Negative contrast flow cytometry provided label‐free detection of low absorbing or weakly fluorescent cells in blood absorption and autofluorescence background, respectively. The use of pulsed laser for time‐resolved discrimination of objects with long fluorescence lifetime (e.g., quantum dots) from shorter autofluorescence background (e.g., blood plasma) is also highlighted in this paper. The supplementary nature of PA and fluorescence detection increased the versatility of the integrated method for simultaneous detection of probes and cells having various absorbing and fluorescent properties, and provided verification of PA data using a more established fluorescence based technique. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

High sensitive approach for point mutation detection based on electrochemiluminescence

An electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for point mutation detection has been developed. The target is amplified using a tris (bipyridine) ruthenium (TBR)-labeled forward and a biotinylated reverse primer. The amplification products are digested with specific restriction enzyme, then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. The established technique was further applied to detect a specific point mutation in H-ras oncogene in T24 cell line. The results show that the system has a low detection limit of 100 fmol and a linear range of more than 3 orders of magnitude for H-ras amplicon; the two genotypes can be reliably discriminated. In summary, the mutant specific ECL-PCR method can be used to detect a point mutation that creates or destroys a restriction site in any gene. It is useful in single nucleotide polymorphism (SNP) and mutation detection due to its safety, high sensitivity and simplicity.     

Magnetoresistive-based biosensors and biochips

Over the past five years, magnetoelectronics has emerged as a promising new platform technology for biosensor and biochip development. The techniques are based on the detection of the magnetic fringe field of a magnetically labeled biomolecule interacting with a complementary biomolecule bound to a magnetic-field sensor. Magnetoresistive-based sensors, conventionally used as read heads in hard disk drives, have been used in combination with biologically functionalized magnetic labels to demonstrate the detection of molecular recognition. Real-world bio-applications are now being investigated, enabling tailored device design, based on sensor and label characteristics. This detection platform provides a robust, inexpensive sensing technique with high sensitivity and considerable scope for quantitative signal data, enabling magnetoresistive biochips to meet specific diagnostic needs that are not met by existing technologies.     

Electrochemiluminescent hybridization chip with electric field aided mismatch discrimination

This paper describes a heterogeneous DNA-hybridization assay based on electrochemiluminescence (ECL) detection on gold electrodes. Short, 15-mer oligonucleotides were conjugated with a synthesized electrochemiluminescent label, bis(2,2'-bipyridine)-5-isothiocyanato-1,10-phenanthroline ruthenium(II) at the amino-modified 5'-end. Gold electrodes were derivatized with 15-mer oligonucleotide probes via 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linking reaction and hybridized with Ru-labeled strands. Two types of self-assembled-monolayers have been utilized for the immobilization reaction, 3-mercaptopropanoic acid (3-MHA) and 16-mercaptohexadecanoic acid (16-MHA). Longer thiols were more stable at high electrode potentials needed for the ECL generation. The system was sensitive down to 1 fmol of labeled complementary strand, detected in 30 microL of buffer. Mismatch discrimination was achieved both passively by washing and actively by application of negative electrode potential on electrodes prior to detection, but active denaturing lead to better results. Two base-pair mismatches were discriminated at room temperature.     

An enzymatic electrochemiluminescence assay for the lethal factor of anthrax

The lethal factor (LF) of anthrax toxin is the toxic component of the exotoxin (lethal toxin) secreted by toxic strains of Bacillus anthracis. The lethal factor is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MAPKK) family of enzymes. We took advantage of this substrate specificity to develop an electrochemiluminescence (ECL) peptide cleavage assay. The ECL assay uses the stable ruthenium (Ru) metal chelate that, in the presence of tripropylamine, generates a light reaction triggered by the application of an electric potential. The Ru label is specifically incorporated into the C-terminal CYS residue of a synthetic peptide (23mer) containing the MAPKK2 cleavage sequence of LF. Streptavidin-coated paramagnetic beads were the solid phase and facilitated separation and characterization of the enzymatic reaction products based upon N-terminal biotinylation of the peptide substrate. Intact peptide bound via the biotin moiety generated high signal due to the Ru label, whereas binding of the cleaved peptide fragment devoid of Ru label reduced the ECL signal. The proposed assay provides a novel opportunity for the screening of potential therapeutics against anthrax.     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

Signal-enhancer molecules encapsulated liposome as a valuable sensing and amplification platform combining the aptasensor for ultrasensitive ECL immunoassay

An innovatory ECL immunoassay strategy was proposed to detect the newly developing heart failure biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP). Firstly, this strategy used small molecules encapsulated liposome as immune label to construct a sandwich immune sensing platform for NT-proBNP. Then the ECL aptasensor was prepared to collect and detect the small molecules released from the liposome. Finally, based on the ECL signal changes caused by the small molecules, the ECL signal indirectly reflected the level of NT-proBNP antigen. In this experiment, the cocaine was chosen as the proper small molecule that can act as signal-enhancer to enhance the ECL of Ru(bpy)(3)(2+). The cocaine-encapsulated liposomes were successfully characterized by TEM. The quantificational calculation proved the ～5.3×10(3) cocaine molecules per liposome enough to perform the assignment of signal amplification. The cocaine-binding ECL aptasensor further promoted the work aimed at amplifying signal. The performance of NT-proBNP assay by the proposed strategy exhibited high sensitivity and high specificities with a linear relationship over 0.01-500 ng mL(-1) range, and a detection limit down to 0.77 pg mL(-1).     

Development of an offline noncompetitive flow immunoassay for the determination of interleukin-8 in cell samples

A noncompetitive flow immunoassay system (FIA) for the analysis of interleukin-8 (IL-8) in cell samples was developed. Affinity interaction assays based on offline incubation of excess labeled antibodies and antigen (IL-8) were carried out. The residual unbound labeled antibody was trapped in an immunoaffinity column with immobilized IL-8 while the immunocomplex, labeled antibody/IL-8, was detected by a fluorescence detector. Two fluorophores, FLUOS and Cy5.5, were conjugated with IL-8 antibody. Optimization and comparison between the two fluorescent labeled antibodies were performed with regard to pH, antibody concentration, flow rate, injection volume, and association time. Additionally, a horseradish peroxidase enzyme label was used for the conjugation to the anti-IL-8. The enzyme substrate reaction was optimized with respect to temperature and length of the substrate reaction coil. The detection limits were found to be 200 amol using the FLUOS-labeled anti-IL-8 and 1 fmol using the Cy5.5 fluorescence label. The developed FIA technique was applied for the analysis of IL-8 in cell samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify IL-8 in the cell samples.