Sensitive immunoassay for porcine pseudorabies antibody based on fluorescence signal amplification induced by cation exchange in CdSe nanocrystals

We report on a novel immunoassay for porcine pseudorabies virus (PRV) antibody that is based on fluorescence signal amplification induced by silver(I) ion exchange in CdSe nanocrystals. An antigen-antibody-secondary antibody sandwich structure was first formed from PRV, PRV antibody, and CdSe-labeled rabbit anti-pig antibody. Then, the Cd(II) ions in the CdSe labels were released by a cation exchange reaction with Ag(I). Released Cd(II) was finally quantified using the sensitive fluorescent probe Rhodamine 5 N. Due to this signal amplification, the sensitivity and linear range of the immunoassay were largely improved (compared to the traditional ELISA) in having a limit of detection as low as 1.2 ng?mL^?1 of PRV antibody and a linear range from 2.44 to 312 ng?mL^?1. The successful determination of PRV antibody in pig serum samples is proof for the utility of the method.

Multiplexed tyrosine kinase activity detection in cancer cells using a hydrogel immobilized substrate

Kinases play a key role in cellular signaling, and the overactivation or overexpression of these kinases has been linked to a variety of cancers. Tyrosine kinase inhibitors treat the mechanism of these cancers by targeting the specific kinases that are overactive. Some patients, however, do not respond to these inhibitors or develop resistance to these inhibitors during treatment. Additionally, even within cancers of the same tissue type, different kinases may be overactive in different patients. For example, some lung cancers overexpress epidermal growth factor receptor (EGFR) and respond to EGFR inhibitors, whereas other lung cancers do not overexpress EGFR and receive no benefit from this treatment. Even among patients exhibiting EGFR overexpression, some do not respond to EGFR kinase inhibitors because other kinases, such as Met kinase, are also overactivated. Here we describe a quantitative and specific multiplexed microfluidic assay using a hydrogel immobilized substrate for measuring the kinase activity of Met and Abl kinase from cancer cells. We immobilized kinase-specific substrates on macroporous hydrogel micropillars in microchannels. These microchannels were incubated with 6 μl of a kinase reaction solution containing cancer cell lysate, and we measured kinase activity via fluorescence detection of a phosphotyrosine antibody. We showed that the assay can specifically measure the activity of both Met and Abl kinase within one microchannel and has the potential to measure the activity of as many as five kinases within one microchannel. The assay also detected Met kinase inhibition from lysates of cancer cells grown in the Met kinase inhibitor PHA665752.

Colorimetric assay for T4 polynucleotide kinase activity based on the horseradish peroxidase-mimicking DNAzyme combined with λ exonuclease cleavage

T4 polynucleotide kinase (PNK) plays a critical role in various cellular events. Here, we describe a novel colorimetric strategy for estimating the activity of PNK and screening its inhibitors taking advantage of the efficient cleavage of λ exonuclease and the horseradish peroxidase-mimicking DNAzyme (HRPzyme) signal amplification. A label-free hairpin DNA with the sequence of HRPzyme was utilized in the assay. The 5′-hydroxyl terminal of the hairpin DNA was firstly phosphorylated in the presence of PNK and then digested by λ exonuclease. As a result, the blocked ‘HRPzyme’ sequence of the hairpin DNA was released due to the removal of its completely complementary sequence. Using this strategy, the assay for PNK activity was successfully translated into the detection of HRPzyme. Because of the completely blocking and efficiently releasing of HRPzyme, the colorimetric method exhibited an excellent performance in PNK analysis with a low detection limit of 0.06 U mL⁻¹ and a wide detection range from 0.06 to 100 U mL⁻¹. Additionally, the effects of different inhibitors on PNK activity were also evaluated. The proposed strategy holds great potential in the development of high-throughput phosphorylation investigation as well as in the screening of the related drugs.     

Exonuclease I aided enzyme-linked aptamer assay for small-molecule detection

A novel enzyme-linked aptamer assay (ELAA) with the aid of Exonuclease I (Exo I) for colorimetric detection of small molecules was developed. The fluorescein isothiocyanate (FITC)-labeled aptamer was integrated into a double-stranded DNA (dsDNA). In the presence of target, the binding of aptamer with target protected the aptamer from Exo I degradation, which resulted in the FITC tag remaining on the aptamer. Then, the anti-FITC-HRP conjugate was used to produce an optically observable signal. By monitoring the color change, we were able to detect two model molecules, ATP and L-argininamide, with high selectivity and high sensitivity even in the serum matrix. It is expected to be a simple and general ELAA method with wide applicability.

Superior performance of liposomes over enzymatic amplification in a high-throughput assay for myoglobin in human serum

Myoglobin is one of several cardiac markers which become elevated in the blood following an acute myocardial infarction and can aid in the diagnosis of a heart attack. Here, a sandwich immunoassay for myoglobin was developed, including a thorough optimization of fluorescent dye-encapsulating liposomes versus enzymatic amplification (alkaline phosphatase and horseradish peroxidase) at each step. The optimized microtiter plate-based assay was capable of detecting as low as 11.3 pg/mL myoglobin and was successfully applied for the quantification of myoglobin in human serum. In comparison to enzymatic approaches, the liposomes demonstrated lower limits of detection, significantly reduced limits of quantification, improved signal discrimination through substantial signal enhancement, and reduced assay time. Liposomes were stable and functional at ambient temperatures for over 400 days. Finally, ease of use was greater due to lack of reliance on additional reagents, non-time-based signal enhancement, and excellent photostability. Optimal conditions identified for enzymatic approaches can also be used for liposome amplification, which makes substitution of these liposomes into existing assays straightforward. Thus, the extensive studies carried out here suggest that liposomes may be incorporated into formats currently utilizing enzymatic enhanced fluorescence with a potential for increased performance on various levels.

Picoliter droplet microfluidic immunosorbent platform for point-of-care diagnostics of tetanus

We have developed a sensitive, specific, rapid and low cost picoliter microsphere-based platform for bioanalyte detection and quantification. In this method, a biological sample, biosensing microspheres, and fluorescently labeled detection (secondary) antibodies are co-encapsulated to capture the analyte (here: human anti-tetanus immunoglobulin G) on the surface of the microsphere in microfluidic pL-sized droplets. The absorption of the analyte and detecting antibodies on the microsphere concentrate the fluorescent signal in correlation with analyte concentration. Using our platform and commercially available antibodies, we were able to quantify anti-tetanus antibodies in human serum. In comparison to standard bulk immunosorbent assays, the microfluidic droplet platform presented here reduces the reagent volume by four orders of magnitude, while fast reagent mixing reduces the detection time from hours to minutes. We consider this platform to be a major leap forward in the miniaturization of immunosorbent assays and to provide a rapid and low cost tool for global point-of-care.

CdS quantum dots as a fluorescent sensing platform for nucleic acid detection

We demonstrate that CdS quantum dots (QDs) can be applied to fluorescence-enhanced detection of nucleic acids in a two-step protocol. In step one, a fluorescently labeled single-stranded DNA probe is adsorbed on the QDs to quench its luminescence. In step two, the hybridization of the probe with its target ssDNA produces a double-stranded DNA which detaches from the QD. This, in turn, leads to the recovery of the fluorescence of the label. The lower detection limit of the assay is as low as 1?nM. The scheme (that was applied to detect a target DNA related to the HIV) is simple and can differentiate between perfectly complementary targets and mismatches.

Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme whose expression by a broad range of clinical tumors is associated with immunosuppression and poor patient outcome. Here we describe a new fluorescence assay for measuring IDO1 activity suitable for high-throughput screening of compound libraries for novel IDO1 inhibitors. This assay is easy to perform, requiring the addition of only one reagent prior to readout. In place of measuring kynurenine, it uses the in situ formation of an N-formylkynurenine-derived fluorophore (NFKPIP) measured at an excitation wavelength of 400 nm and an emission wavelength of 500 nm. The fluorescence intensity of the NFKPIP formed is directly related to the amount of enzyme activity, and the signal is stable over 8 h. This assay has a lower limit of detection, equating to 153 nM N-formylkynurenine, which is over 30-fold lower than the limits of detection of existing assays for IDO1 activity. When we compared the performance of the new assay with that of the published colorimetric absorbance assay in screening the National Cancer Institute Diversity Set III of 1,597 compounds for IDO1 inhibitors, we obtained an identical list of the 25 most active compounds in the two assays. Although 93 compounds (aldehydes, ketones, and aromatic amines) in the library interfered with the absorbance readout, only 18 compounds (conjugated systems and fused cycles) interfered with the readout of the new fluorescence assay. IC₅₀ values determined using the new assay for three known IDO1 inhibitors—1,4-naphthoquinone, 4-amino-N-(3-chloro-4-fluorophenyl)-N’-hydroxy-1,2,5-oxadiazole-3-carboximidamide and 4-phenyl-1H-imidazole—were consistent with their literature values, further validating the new assay for measuring IDO1 activity.

Sensitive fluorescent detection of Staphylococcus aureus using nanogold linked CdTe nanocrystals as signal amplification labels

A sensitive fluorescent assay was developed for the detection of DNA specifically for Staphylococcus aureus. A sandwich-type detection system was fabricated by first immobilizing biotinylated capture DNA on avidin-modified wells of microplates, then hybridizing the capture DNA with one end of the target DNA, and then recognizing the other end of the target DNA with a signal probe labeled with CdTe nanocrystals and gold nanoparticles (Au-NPs) at the 3′- and 5′-terminus, respectively. Hybridization was monitored by measuring the fluorescent intensity of the assembly. The experimental results demonstrated that the incorporation of Au-NPs in this detection system can significantly enhance the sensitivity and the selectivity because a single Au-NP can be loaded with hundreds of signal DNA probe strands modified with CdTe nanocrystals. Under the optimized conditions, a detection limit of 10 fmol of DNA per L can be achieved and at least 50 colony forming units of Staph. aureus per mL of sample can be detected. The method was assessed by analyzing real samples, and it was validated by comparing it to an official standard method.

Label-free and ultrasensitive electrochemiluminescence detection of microRNA based on long-range self-assembled DNA nanostructures

Electrochemiluminescence (ECL) integrates the advantages of electrochemical detection and chemiluminescent techniques. The method has received particular attention because it is highly sensitive and selective, has a wide linear range but low reagent costs. The use of nanomaterials with their unique physical and chemical properties has led to new kinds of biosensors that exhibit high sensitivity and stability. Compared to other nanomaterials, DNA nanostructures are more biocompatible, more hydrophilic, and thus less prone to nonspecific adsorption onto the electrode surface. We describe here a label-free and ultrasensitive ECL biosensor for detecting a cancer-associated microRNA at a femtomolar level. We have designed two auxiliary probes that cause the formation of a long-range self-assembly in the form of a μm-long 1-dimensional DNA concatamer. These can be used as carriers for signal amplification. The intercalation of the ECL probe Ru(phen)₃ ²⁺ into the grooves of the concatamers leads to a substantial increase in ECL intensity. This amplified sensor shows high selectivity for discriminating complementary target and other mismatched RNAs. The biosensor enables the quantification of the expression of microRNA-21 in MCF-7 cells. It also displays very low limits of detection and provides an alternative approach for the detection of RNA or DNA detection in diagnostics and gene analysis.

Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor

We have developed a sensitive assay for enteropathogenic E. coli (EPEC) by integrating DNA extraction, specific polymerase chain reaction (PCR) and DNA detection using an electrode modified with the bundle-forming pilus (bfpA) structural gene. The PCR amplified products are captured on the electrode and hybridized with biotinylated detection probes to form a sandwich hybrid containing two biotinylated detection probes. The sandwich hybridization structure significantly combined the numerous streptavidin alkaline phosphatase on the electrode by biotin-streptavidin connectors. Electrochemical readout is based on dual signal amplification by both the sandwich hybridization structure and the enzyme. The electrode can satisfactorily discriminate complementary and mismatched oligonucleotides. Under optimal conditions, synthetic target DNA can be detected in the 1 pM to 10 nM concentration range, with a detection limit of 0.3 pM. EPEC can be quantified in the 10 to 10⁷ CFU mL^?1 levels within 3.5 h. The method also is believed to present a powerful platform for the screening of pathogenic microorganisms in clinical diagnostics, food safety and environmental monitoring.

Fluorescent aptasensor for the determination of Salmonella typhimurium based on a graphene oxide platform

We report on an aptamer with high affinity against Salmonella typhimurium (S. typhimurium) and selected from an enriched oligonucleotide pool by a whole-cell SELEX process in a method for the fluorimetric determination of S. typhimurium using a graphene oxide platform. In the absence of target, the fluorescence was fairly weak as result of the FAM-labeled aptamer adjacent to graphene oxide. If, however, the fluorophore is released from the graphene oxide due to the formation of the target/aptamer complexes, fluorescence intensity is substantially increased. Under the optimum conditions, the assay displays a linear response to bacteria in the concentration range from 1?×?10³ to 1?×?10⁸ CFU·mL^?1, with a detection limit of 100 CFU·mL^?1. The method is selective in that fluorescence is not much enhanced in case of other bacteria. This aptasensor displays higher sensitivity and selectivity than others and is believed to possess a large potential with respect to the rapid detection of bacteria.

A Surface Enhanced Raman Scattering (SERS) microdroplet detector for trace levels of crystal violet

We report on a microfluidic platform that integrates a winding microdroplet chip and a surface-enhanced Raman scattering (SERS) detection system for trace determination of crystal violet (CV). Colloidal silver was applied to generate SERS. Compared to the continuous flow microfluidic system, the microdroplet based detection described here effectively eliminates any memory effects. Effects of flow pattern, droplet size, surfactant, and position of detection were optimized. Under optimal conditions, there is a linear correlation between signal and the concentration of CV in the 10 nM to 800 nM range, with a correlation coefficient (R²) of 0.9967. The limit of detection in water is 3.6 nM.

Visual detection and microplate assay for Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification

We have developed a specific method for the visual detection of Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification technology. A biotinylated aptamer specific for S. aureus was immobilized on the surface of the wells of a microplate via biotin-avidin binding. Then, the target bacteria (S. aureus), the biotinylated-aptamer-streptavidin-HRP conjugates, biotinylated tyramine, hydrogen peroxide and streptavidin-HRP were successively placed in the wells of the microplate. After adding TMB reagent and stop solution, the intensity of the yellow reaction product can be visually inspected or measured with a plate reader. Under optimized conditions, there is a linear relationship between absorbance at 450 nm and the concentration of S. aureus in the 10 to 10⁷ cfu mL^?1 concentration range (with an R² of 0.9976). The limit of detection is 8 cfu mL^?1.

Sensitive detection of tuberculosis using nanoparticle-enhanced surface plasmon resonance

We show that the antigen CFP-10 (found in tissue fluids of tuberculosis patients) can be used as a marker protein in a surface-plasmon resonance (SPR) based method for early and simplified diagnosis of tuberculosis. A sandwich SPR immunosensor was constructed by immobilizing the CFP-10 antibody on a self-assembled monolayer on a gold surface, this followed by blocking it with bovine serum albumin. Following exposure of the sensor surface to a sample containing CFP-10, secondary antibody immobilized on nickel oxide nanoparticles are injected which causes a large SPR signal change. The method has a dynamic range from 0.1 to around 150 ng per mL of CFP-10, and a detection limit as low as 0.1 ng per mL. This is assumed to be due to the high amplification power of the NiO nanoparticles.

Surface plasmon resonance, fluorescence, and circular dichroism studies for the characterization of the binding of BACE-1 inhibitors

The mechanism of action underlying β-secretase 1 (BACE-1) inhibition was characterized by a surface plasmon resonance (SPR) method using primary amino groups to immobilize OM99-2, a well-known highly potent peptidic BACE-1 inhibitor, on the carboxyl groups of the dextran layer of a sensor chip. The diluted BACE-1 was mixed with buffer or the test compound and the mixture was flushed through the chip. BACE-1 binding to the immobilized peptide inhibitor was quantified. This SPR method was used to identify BACE-1 inhibitor binding sites and the mechanism of action (competitive/noncompetitive) and to validate findings of fluorescence resonance energy transfer (FRET) inhibition studies. To support this, a multimethodological approach (circular dichroism and fluorescence spectroscopy) was applied in parallel to FRET inhibition studies to characterize the binding modes of peptidic and nonpeptidic BACE-1 inhibitors. Circular dichroism spectroscopy served to correlate the conformation of BACE-1 with enzymatic activity and to monitor secondary structure changes upon ligand binding. In a complementary approach, direct fluorescence spectroscopy was used to characterize different BACE-1 inhibitor binding sites. The influence of pH and inhibitors on BACE-1 secondary structure was also elucidated. This multimethodological approach was applied to identify binding modes of bis(7)-tacrine and myricetin in comparison with well-known peptidic inhibitors.

DNA biosensors based on metallo-intercalator probes and electrocatalytic amplification

Strategies for electrochemical sensing of DNA can be classified into label-free and label-based approaches, categories of which include enzyme-, nanomaterial- and redox labels that are attached to DNA either by covalent or non-covalent means. Metallointercalators represent one group of small molecule redox labels that non-covalently enter the groove of a DNA. The metallointercalator plays a dual-role in acting as a structure indicator (for hybridization) and a signal generator. Labeling is not needed, and electrochemical measurements can be carried out in a label-free solution of an electrolyte. However, such metallointercalators lack the option of catalytic signal generation as in the case of enzyme- and nanomaterial-based labels. Therefore, signal amplification becomes crucial. We first survey here recent progress in this area. A signal-amplifying system is presented that relies on the electroatalytic oxidation of a metallointercalator ruthenium(II)bipyridine/phenoxazine complex in the presence of electron donor species such as oxalate, DNA bases, or tripropylamine. Recent work on such DNA sensors is discussed. Results suggest that such metallointercalator-based DNA sensors represent a viable platform for developing high-throughput and automated PCR/lab-on-a-chip devices as well as visualized multifunctional DNA sensors.

Detection of mismatched caspase-3 DNA oligonucleotides with an SPR biosensor following amplification by Taq polymerase

We demonstrate that base mismatches of caspase-3 DNA sequences can be detected by surface plasmon resonance (SPR) following signal amplification by polymerase from Thermus aquaticus (Taq). The concentration of magnesium ions and the respective dNTPs for polymerase binding to the oligonucleotides on the sensing surface were optimized. Taq polymerase binds to double-stranded DNA that is self-assembled on the gold surface of the biosensor to induce an SPR signal. Experiments are presented on the effect of Mg(II) and dNTP concentrations on the activity of the polymerase on the sensing surface. The detection limits are 50 pM, 0.1 nM, 0.7 nM, 7 nM, and 20 nM for correctly matched, single-base mismatched, two-base mismatched, three-base mismatched and four-base mismatched DNA of caspase-3, respectively. This is attributed to the optimized experimental conditions, with samples containing 2 μM of Mg(II) and 0.3 mM of dNTP.

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.