Versatile electrochemiluminescence assays for cancer cells based on dendrimer/CdSe-ZnS-quantum dot nanoclusters

In this work, a novel dendrimer/CdSe-ZnS-quantum dot nanocluster (NC) was fabricated and used as an electrochemiluminescence (ECL) probe for versatile assays of cancer cells for the first time. A large number of CdSe-ZnS-quantum dots (QDs) were labeled on the NCs due to the many functional amine groups within the NCs, which could significantly amplify the QD's ECL signal. Capture DNA was specially designed as a high-affinity aptamer to the target cell; a novel ECL biosensor for cancer cells was directly accomplished by using the biobarcode technique to avoid cross-reaction. Moreover, magnetic beads (MBs) for aptamers immobilization were combined with the dendrimer/QD NCs probe for signal-on ECL assay of cancer cells, which greatly simplified the separation procedures and favored for the sensitivity improvement. In particular, a novel cycle-amplifying technique using a DNA device on MBs was further employed in the ECL assay of cancer cells, which greatly improved the sensitivity. To the best of our knowledge, this is the first study that the novel dendrimer/QD NCs probe combined with a DNA device cycle-amplifying technique was employed in the ECL assays of cells. Excellent discrimination against target and control cells is demonstrated, indicating that the ECL assays have great potential to provide a sensitive, selective, cost-effective, and convenient approach for early and accurate detection of cancer cells.     

Intermolecular and intramolecular quencher based quantum dot nanoprobes for multiplexed detection of endonuclease activity and inhibition

DNA cleavage by endonucleases plays an important role in many biological events such as DNA replication, recombination, and repair and is used as a powerful tool in medicinal chemistry. However, conventional methods for assaying endonuclease activity and inhibition by gel electrophoresis and chromatography techniques are time-consuming, laborious, not sensitive, or costly. Herein, we combine the high specificity of DNA cleavage reactions with the benefits of quantum dots (QDs) and ultrahigh quenching abilities of inter- and intramolecular quenchers to develop highly sensitive and specific nanoprobes for multiplexed detection of endonucleases. The nanoprobe was prepared by conjugating two sets of DNA substrates carrying quenchers onto the surface of aminated QDs through direct assembly and DNA hybridization. With this new design, the background fluorescence was significantly suppressed by introducing inter- and intramolecular quenchers. When these nanoprobes are exposed to the targeted endonucleases, specific DNA cleavages occur and pieces of DNA fragments are released from the QD surface along with the quenchers, resulting in fluorescence recovery. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. The detection was accomplished with a single excitation light. Multiplexed detection was demonstrated by simultaneously assaying EcoRI and BamHI (as model analytes) using two different emissions of QDs. The limits of detection were 4.0 × 10(-4) U/mL for EcoRI and 8.0 × 10(-4) U/mL for BamHI, which were at least 100 times more sensitive than traditional gel electrophoresis and chromatography assays. Moreover, the potential application of the proposed method for screening endonuclease inhibitors has also been demonstrated. The assay protocol presented here proved to be simple, sensitive, effective, and easy to carry out.     

Cucurbiturils: molecular nanocapsules for time-resolved fluorescence-based assays

A new fluorescent host-guest system based on the inclusion of the fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) into the cavity of the molecular container compound cucurbit[7]uril (CB7) has been designed which possesses an exceedingly long-lived emission (690 ns in aerated water). The large binding constant of (4/spl plusmn/1)/spl times/10/sup 5/ M/sup -1/ along with the resistance of the CB7/spl middot/DBO complex toward external fluorescence quenchers allow the use of CB7 as an enhancer in time-resolved fluorescence-based assays, e.g., to screen enzyme activity or inhibition by using DBO-labeled peptides as substrates. The response of CB7/spl middot/DBO to different environmental conditions and possible quenchers are described.     

Microwave-accelerated metal-enhanced fluorescence: platform technology for ultrafast and ultrabright assays

We describe an exciting assay platform technology that promises to fundamentally address two underlying physical constraints of modern assays and immunoassays, namely, assay sensitivity and rapidity. By combining the use of metal-enhanced fluorescence with low-power microwave heating, we can indeed significantly increase the sensitivity of surface assays as well as >95 % kinetically complete the assay within a few seconds. Subsequently, this new technology promises to fundamentally change the way we currently employ immunoassays in clinical medicine. This new model platform system can be potentially applied to many other important assays, such as to the clinical assessment of myoglobin, where both assay speed and sensitivity is paramount for the assessment and treatment of acute myocardial infarction. To demonstrate the utility of microwave-accelerated metal-enhanced fluorescence (MAMEF), we show that a simple protein-based assay system can be optically amplified approximately 10-fold by using silver nanostructures, while being kinetically complete in less than 20 s. This new platform approach is subsequently over 10-fold more sensitive and approximately 90 times faster than a control assay that operates both at room temperature and without the use of metal-enhanced fluorescence. Finally, we show that low-power heating by microwaves in our model system does not denature proteins, as evidenced by no protein structural changes, probed by fluorescence resonance energy transfer.     

Nanoarrays: a method for performing enzymatic assays

Conventional enzymatic assays for alcohol dehydrogenase, pyruvate kinase, and enolase performed in 96-well microtiter plates were compared with assays monitored in 25-well nanoarrays. All miniaturized reactions could be performed in maximum volumes of 6.3-8 nL and were read out with a conventional fluorescence microscope system equipped with a scientific grade CCD camera. Substrate and cofactor were already present inside the wells after having been presprayed, or they were applied in solution to the wells of the nanoarray shortly before the assays started. For all of the assays, commercially available enzymes and enzymes present in cell-free extracts were used. Assays carried out in premixed nanoarrays gave results comparable to those performed in presprayed nanoarrays. Enzyme activities determined in nanoarrays by using two different methods were in good agreement with assays performed in microtiter plates. Also, good correspondence was found between expected and observed enzyme levels. In short, enzymatic assays performed in premixed and in particular in presprayed nanoarrays are a promising low-volume and low-reagent- and sample-consuming alternative to current methodology and could find applications in many different areas of analytical chemistry.     

Development of a new microtiter plate format for clinically relevant assays

A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The "mix and match" approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care.     

Potentiometric homogeneous enzyme-linked competitive binding assays using adenosine deaminase as the label

Homogeneous enzyme-linked competitive binding assays for biotin are described that are based on the competition between an enzyme-biotin conjugate and free biotin for a fixed number of binding sites of avidin. Unlike conventional homogeneous enzyme immunoassays, in this system the analyte (biotin) is labeled with adenosine deaminase (ADA), an ammonia-producing enzyme. Consequently, potentiometric rather than photometric methods can be used as means of detection. Several ADA-biotin conjugates were prepared and showed as high as 97% inhibition of the enzymatic activity in the presence of avidin. Addition of free biotin reverses this inhibition in an amount proportional to the concentration of analyte. Relatively steep dose-response curves were observed, leading to a precise and accurate assay for biotin. The detection limits of these curves were as low as 1 x 10(-8) M. Varying the concentration of the reagents in the assay allowed the detection limit and working range to be altered to a desired value. The proposed method was applied in the determination of biotin in a horse-feed supplement.     

A theoretical approach to some analytical properties of heterogeneous enzymatic assays

Heterogeneous enzymatic assays (HEA), where an enzyme in solution acts upon an immobilized substrate, are been increasingly used. Given their high throughput and versatility they hold great potential for developing massive enzyme inhibitor screening. However, current HEA lack, in general, rigorous quantitative use. This is in part due to technical problems as a multiplicity of suboptimal substrate populations achieved with traditional immobilization techniques but, more importantly, is due to a poor understanding of the particular kinetic behavior of these systems. This paper addresses the kinetic features of HEA that arise from the very low amount of solid-phase substrate and the resulting inalterability of the free enzyme concentration during the assay, which classify HEA as enzyme quasi-saturable systems (EQSS). We assessed the optimal enzyme concentration working range and time of reaction. We also considered certain attributes of HEA for evaluating isosteric inhibitors. These studies were done on the basis of a simplified model for the kinetics of EQSS and a formal splitting of the functional factor of the analytical sensitivity of an enzymatic assay into [E(o)]/K(m)-dependent and temporal components.     

A comparative study of the use of colorimetric assays in the assessment of biocompatibility

A panel of colorimetric assays was assessed for sensitivity, reproducibility, and performance in the investigation of the biocompatibility of a representative range of orthopædic biomaterials, using a commercially available human osteosarcoma-derived cell line. The MTT assay was the most sensitive, with a detection limit of 4×10² cells per well against background, while the NR assay was the least sensitive, with no colour change until the cell density reached 2×10⁴ per well. All of the assays investigated showed a highly significant edge effect when within-plate reproducibility was examined; between-plate reproducibility was good for all assays except the MTT assay. When the assays were tested on cells adherent on biomaterials, there was a wide variation in the results obtained; in particular, the MTS assay showed poor reproducibility in the presence of materials. The MTT and BrdU assays both showed sufficient precision to detect cells on two of the materials studied. The study demonstrates that colorimetric assays are potentially useful in biocompatibility assessment but must be fully validated for the application chosen.     

Nanoencapsulated microcrystalline particles for superamplified biochemical assays

We report on the preparation and utilization of a novel class of particulate labels based on nanoencapsulated organic microcrystals with the potential to create highly amplified biochemical assays. Labels were constructed by encapsulating microcrystalline fluorescein diacetate (FDA; average size of 500 nm) within ultrathin polyelectrolyte layers of poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) via the layer-by-layer technique. Subsequently, the polyelectrolyte coating was used as an "interface" for the attachment of anti-mouse antibodies through adsorption. A high molar ratio of fluorescent molecules present in the microcrystal core to biomolecules on the particle surface was achieved. The applicability of the microcrystal-based label system was demonstrated in a model sandwich immunoassay for mouse immunoglobulin G detection. Following the immunoreaction, the FDA core was dissolved by exposure to organic solvent, leading to the release of the FDA molecules into the surrounding medium. Amplification rates of 70-2000-fold (expressed as an increase in assay sensitivity) of the microcrystal label-based assay compared with the corresponding immunoassay performed with direct fluorescently labeled antibodies are reported. Our approach provides a general and facile means to prepare a novel class of biochemical assay labeling systems. The technology has the potential to compete with enzyme-based labels as it does not require long incubation times, thus speeding up bioaffinity tests.     

Coreactant enhanced anodic electrochemiluminescence of CdTe quantum dots at low potential for sensitive biosensing amplified by enzymatic cycle

This work used sulfite as a coreactant to enhance the anodic electrochemiluminescence (ECL) of mercaptopropionic acid modified CdTe quantum dots (QDs). This strategy proposed the first coreactant anodic ECL of QDs and led to a sensitive ECL emission of QDs in aqueous solution at relatively low potential. In the presence of dissolved oxygen, the stable ECL emission resulted from the excited QDs. Thus, an ECL detection method was proposed at +0.90 V (vs Ag/AgCl) based on the quenching of excited QDs by the analyte. Using tyrosine as a model compound, whose electrooxidized product could quench the excited QDs and thus the ECL emission, an analytical method for detection of tyrosine in a wide concentration range was developed. Furthermore, by combining an enzymatic cycle of trace tyrosinase to produce the oxidized product with an energy-transfer process, an extremely sensitive method for ECL detection of tyrosine with a subpicomolar limit of detection was developed. The sulfite-enhanced anodic ECL emission provided an alternative for traditional ECL light emitters and a new methodology for extremely sensitive ECL detection of mono- and dihydroxybenzenes at relatively low anodic potential. This strategy could be easily realized and opened new avenues for the applications of QDs in ECL biosensing.     

Aptamer-based affinity chromatographic assays for thrombin

Affinity chromatographic assays for thrombin were developed using two aptamers as affinity ligands. The efficient capture and step elution of thrombin with NaClO4 enabled the determination of thrombin by using either absorbance or fluorescence detection. Preconcentration of thrombin on the affinity column improved the detection limit of thrombin to 0.1 nM. Using an aptamer for the fibrinogen-binding site of thrombin and a second aptamer for the heparin-binding site, a sandwich chromatographic assay was developed, showing improved selectivity of thrombin detection and eliminating the need for labeling thrombin in the sample. The increased local concentration of aptamers immobilized on monolithic columns favored the formation of aptamer-thrombin complexes, resulting in improved retention and detection of thrombin at trace levels.     

Static and dynamic acute cytotoxicity assays on microfluidic devices

Static and dynamic acute toxicity assays of cells were performed on microfluidic devices where materials were hydraulically transported. Static assays were performed by incubating cells with an agent in a microchip reservoir and optically interrogating the cells after hydrodynamic focusing at a cross intersection. Dynamic assays were performed on a microchip with a 25-cm-long spiral channel where the cells were mixed with an agent and optically monitored 0.1, 12, and 22 cm from the point of mixing. The incubation time was determined by the time needed for cells to transit from the mixing location to the point of detection. Cell viability was determined using the ratio of fluorescence signals from membrane permeant (calcein) and membrane impermeant (propidium iodide) stains. The model system used in this study was the viability of Jurkat cells in the presence of the agent Triton X-100). An average LC50 value of 138 microM for Triton X-100 was obtained for an incubation period of 7-12 min using the static assay. LC50 values obtained with the dynamic assay for 25- and 47-s incubation times were 290 and 250 microM Triton X-100, respectively. Higher LC50 values for the dynamic assay were expected due to the shorter incubation times.     

Sensitive fluorometric nanoparticle assays for cell counting and viability

We have developed easy-to-use homogeneous methods utilizing time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence quenching for quantification of eukaryotic cells. The methods rely on a competitive adsorption of cells and fluorescently labeled protein onto citrate-stabilized colloidal gold nanoparticles or carboxylate-modified polystyrene nanoparticles doped with an Eu(III) chelate. In the gold nanoparticle sensor, the adsorption of the labeled protein to the gold nanoparticles leads to quenching of the fluorochrome. Eukaryotic cells reduce the adsorption of labeled protein to the gold particles increasing the fluorescence signal. In the Eu(III) nanoparticle sensor, the time-resolved fluorescence resonance energy transfer between the nanoparticles and an acceptor-labeled protein is detected; a decrease in the magnitude of the time-resolved energy transfer signal (sensitized time-resolved fluorescence) is proportional to the cell-nanoparticle interaction and subsequent reduced adsorption of the labeled protein. Less than five cells were detected and quantified with the nanoparticle sensors in the homogeneous microtiter assay format with a coefficient of variation of 6% for the gold and 12% for the Eu(III) nanoparticle sensor. The Eu(III) nanoparticle sensor was also combined with a cell impermeable nucleic acid dye assay to measure cell viability in a single tube test with cell counts below 1000 cells/tube. This sensitive and easy-to-use nanoparticle sensor combined with a viability test for a low concentration of cells could potentially replace existing microscopic methods in biochemical laboratories.     

Conjugated polymer-based real-time fluorescence caspase assays

We have developed conjugated polyelectrolyte-based fluorescence turn-on assays for caspase 3 and 8. These assays are composed of a cationic polyphenylene ethynylene polymer PPE4+ and p-nitroaniline modified caspase peptide substrate. The fluorescence of the assay is initially turned-off because of the efficient quenching of the polymer by p-nitroaniline moiety on anionic peptide substrates. A turn-on effect is observed due to the cleavage of the peptide by the enzyme and formation of the neutral p-nitroaniline unit which has no quenching on the polymer. We validated this assay design and obtained kinetic parameters of caspase 3 and caspase 8. These assays demonstrated good sensitivity as in pmol/L (0.1 units/mL) for caspase 3 and nmol/L (0.2 units/mL) for caspase 8. This method also showed high specificity by using caspase 3 assay as a model system and the results demonstrated that other proteases including caspase 8, papain, pepsin, and trypsin did not show observable fluorescence turn-on effect. The dose-response curve of a caspase inhibitor Z-VAD-FMK was evaluated by caspase 3 assay, by which the IC(50) value was determined to be 0.73 μM and was in a good agreement with the literature reported value at 0.62 μM. This design could be applied into the in vitro screening of small molecular inhibitors for drug discovery.     

Reverse-phase versus sandwich antibody microarray, technical comparison from a clinical perspective

Protein microarrays are powerful tools to quantify and characterize proteins in multiplex assays. They have great potential within clinical diagnostics and prognostics, as they minimize consumption of both analyte and biological sample. Assays that do not require labeling of the biological specimen, henceforth called label-free, are vital for ease of clinical sample processing. Here, we evaluate two label-free techniques, reverse-phase and sandwich antibody assays, using microarrays on high-performance porous silicon surfaces and fluorescence detection. In view of increasing interest in reverse microarrays, this paper focuses on analytical sensitivity of the reverse assays compared to the more complex but highly sensitive sandwich assay. Sensitivity, linear range, and reproducibility of the two assays were compared using prostate-specific antigen (PSA) in buffer. The sandwich assay displayed 5 orders of magnitude lower detection limit (0.7 ng/mL) compared to the reverse assay (70 microg/mL). PSA at 50 nM (1.5 microg/mL) in cell lysates was detected by the sandwich assay but not by the reverse assay, demonstrating again a far lower detection limit for sandwich microarrays. In independent assay runs of PSA spiked in female serum, the sandwich assay had good linearity (R2 > 0.99) and reproducibility (coefficient of variation < or =15%), and the detection limit could be improved to 0.14 ng/mL. Without further signal amplification, the sandwich assay would be our choice for PSA analysis of clinical samples using a microarray technology platform.     

Hairpin-DNA probe for enzyme-amplified electrochemical detection of Legionella pneumophila

An electrochemical genosensor for the detection of nucleic acid sequences specific of Legionella pneumophila is reported. An immobilized thiolated hairpin probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the signaling probe, and streptavidin-alkaline phosphatase as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 2 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The sensor allows discrimination between L. pneumophila and L. longbeachae with high sensitivity under identical assay conditions (no changes in stringency). A limit of detection of 340 pM L. pneumophila DNA, and a linear relationship between the analytical signal and the logarithm of the target concentration to 2 muM were obtained. Experimental results show the superior sensitivity and selectivity of the hairpin-based assay when compared with analogous sandwich-type assays using linear capture probes.     

Development of binding assays in microfabricated picoliter vials: an assay for biotin

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.     

Direct, ultrasensitive, and selective optical detection of protein toxins using multivalent interactions.

Three highly sensitive, selective, and reagent-free optical signal transduction methods for detection of polyvalent proteins have been developed by directly coupling distance-dependent fluorescence self-quenching and/or resonant-energy transfer to the protein-receptor binding events. The ganglioside GM1, as the recognition unit for cholera toxin (CT), was covalently labeled with fluorophores and then incorporated into a biomimetic membrane surface. The presence of CT with five binding sites for GM1 causes dramatic change for the fluorescence of the labeled GM1. (1) In the scheme using fluorescence self-quenching as a signal-transduction mechanism, the fluorescence intensity drops significantly as a result of aggregation of the fluorophore-labeled GM1 on a biomimetic surface. (2) By labeling GM1 with a fluorescence energy transfer pair, aggregation of the labeled GM1 results in a decrease in donor fluorescence and an increase in acceptor fluorescence, providing a unique signature for selective protein-receptor binding. (3) In the third scheme, using the biomimetic surface as part of signal transduction and combining both fluorescence self-quenching and energy-transfer mechanisms to enhance the signal transduction, a signal amplification was achieved. The detection systems can reliably detect less than 0.05 nM CT with fast response (less than 5 min). This approach can easily be adapted to any biosensor scheme that relies on multiple receptors or co-receptors. The methods can also be applied to investigate the kinetics and thermodynamics of the multivalent interactions.     

Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.