Sub-attomole Levels Research Articles

Urinary adiponectin as a new diagnostic index for chronic kidney disease due to diabetic nephropathy

ObjectiveThe chronic kidney disease (CKD) is widely diagnosed on the basis of albuminuria and the glomerular filtration rate. A more precise diagnosis of CKD, however, requires the assessment of other...

Target-triggered triple isothermal cascade amplification strategy for ultrasensitive microRNA-21 detection at sub-attomole level

MicroRNA-21 (miR-21) is a promising diagnostic biomarker for breast cancer screening and disease progression, thus the method for the sensitive and selective detection of miR-21 is vital to its clinical diagnosis. Herein, we develop a novel method to quantify miR-21 levels as low as attomolar sensitivity by a target-triggered triple isothermal cascade amplification (3TICA) strategy. An ingenious unimolecular DNA template with three functional parts has been designed: 5′-fragment as the miR-21 recognition unit, middle fragment as the miR-21 analogue amplification unit, and 3′-fragment as the 8–17 DNAzyme production unit. Triggered by miR-21 and accompanied by polymerase-nicking enzyme cascade, new miR-21 analogues autonomously generated for the successive re-triggering and cleavage process. Simultaneously, the 8–17 DNAzyme-contained sequence could be exponentially released and activated for the second cyclic cleavage toward a specific ribonucleotide (rA)-contained substrate, inducing a remarkably amplified generation of HRP-mimicking DNAzyme in the presence of hemin. Finally, the amperometric technique was used to record the catalytic reduction current of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. The increase in the steady-state current was proportional with the increase of the miR-21 concentration from 1 aM to 100 pM. An ultra-low detection limit of 0.5 aM with an excellent selectivity for even discriminating differences between 1-base mismatched target and miR-21 was achieved. This simple and cost-effective 3TICA strategy is promising for the detection of any short oligonucleotides, simply by altering the target recognition unit in the template sequence.

Triple cascade reactions: An ultrasensitive and specific single tube strategy enabling isothermal analysis of microRNA at sub-attomole level

Sensitive and specific analysis of microRNAs (miRNAs) in a single tube without the need of thermal cycler instrument would greatly facilitate the investigation of miRNA-associated regulatory circuits and diseases. Homogeneous isothermal amplification assays are attractive in conducting single tube assays that can minimize contamination-prone steps and simplifies assay procedures. However, the relative low amplification efficiency and high detection background remain as bottlenecks restricting their more versatile applications. In this work, we have developed a novel isothermal exponential enzymatic amplification (IEEA) strategy for miRNAs analysis. By rational triple cascade amplification cycles of target recycling, nicking-replication reaction, and DNAzyme catalysis, the strategy exhibited high signal amplification efficiency (104–109 folds of amplification in 1h) with very low detection background and excellent specificity. As a result, the miR-27a target model was rapidly determined with a limit of detection down to 0.79 aM (S/N=3), corresponding to 94 copies of the miRNA molecule in a 200μL sample solution. The levels of miR-27a in atherosclerotic sprague-dawley rats were accurately quantified. The strategy is anticipated to have an important impact on the development of simple and rapid molecular diagnostic applications for any short oligonucleotides.

Rapid amplification/detection of nucleic acid targets utilizing a HDA/thin film biosensor.

Thin film biosensors exploit a flat, optically coated silicon-based surface whereupon formation of nucleic acid hybrids are enzymatically transduced in a molecular thin film that can be detected by the unaided human eye under white light. While the limit of sensitivity for detection of nucleic acid targets is at sub-attomole levels (60 000 copies) many clinical specimens containing bacterial pathogens have much lower levels of analyte present. Herein, we describe a platform, termed HDA/thin film biosensor, which performs helicase-dependant nucleic acid amplification on a thin film biosensor surface to improve the limit of sensitivity to 10 copies of the mecA gene present in methicillin-resistant strains of Staphylococcus. As double-stranded DNA is unwound by helicase it was either bound by solution-phase DNA primers to be copied by DNA polymerase or hybridized to surface immobilized probe on the thin film biosensor surface to be detected. Herein, we show that amplification reactions on the thin film biosensor are equivalent to in standard thin wall tubes, with detection at the limit of sensitivity of the assay occurring after 30 minutes of incubation time. Further we validate the approach by detecting the presence of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) from positive blood culture aliquots with high specificity (signal/noise ratio of 105).

Multifunctional Encoded Self-Assembling Protein Nanofibrils as Platform for High-Throughput and Multiplexed Detection of Biomolecules

A one-dimensional nanofibrillar array formed by the co-assembly of native and biotin-functionalized beta-amyloid (Aβ) peptide was developed for biomolecule sensing. With the presence of biotin moiety, a variety of biomolecular probes can be conjugated onto the nanofibrils, thus converting the protein assembly into a miniature biosensor. In this work, DNA probes were immobilized onto the fibril for the detection of cDNA sequences. The as-developed "DNA-nanoarray" achieved a detection limit at subattomole level (183 fM in 10 μL). This highly sensitive, yet simple, assay requires a trace amount of sample consumption (<10 μL) and is pretreatment-free. In addition, we reported the preparation of alternate-segmented amyloid nanofibrils with multifunctionality. The fibrils hereby serve as an encoded template that can be visualized with various fluorescence labeling dyes for barcode recognition purpose, and, hence, multiplex detection of biomolecules was achieved. Regarding that each protein nanofibril represents a single detection platform, a large number of single fibrils simultaneously are monitored with the dual-color TIRFM in a high-throughput manner.

Perspectives in spicing up proteomics with splicing

In the post-genomics era there has been an acceleration of understanding of cellular and organismal biology and this acceleration has moved the goalposts for proteomics. Higher eukaryotes use alternative promoters, alternative splicing, RNA editing and post-translational modification to produce multiple isoforms of proteins from single genes. Switching amongst these isoforms is a major mechanism for control of cellular function. At present fundamental limitations in sensitivity, in absolute quantitation of proteins and in the characterization of protein structure at functionally important levels strongly limit the applicability of proteomics to higher eukaryotes. Recent developments suggest that quantitative, top-down proteomics analyses of complete proteins at sub-attomole levels are necessary for physiologically relevant studies of higher eukaryotes. New proteomics technologies which will ensure the future of proteomics as an important technology in medicine and cellular biology of higher eukaryotes are becoming available.

Interference-based detection of nucleic acid targets on optically coated silicon.

Sequence-specific detection of polynucleotides typically requires modified reporter probes that are labeled with radioactive, fluorescent, or luminescent moieties. Although these detection methods are capable of high sensitivity, they require instrumentation for signal detection. In certain settings, such as clinical point of care, instrumentation might be impractical or unavailable. Here we describe a detection approach in which formation of a nucleic acid hybrid is enzymatically transduced into a molecular thin film that can be visually detected in white light. The system exploits a flat, optically coated silicon-based surface to which capture oligonucleotides are covalently attached. The optimized system is capable of detection of nucleic acid targets present at sub-attomole levels. To supplement visual detection, signals can be quantitated by a charge-coupled device. The design and composition of the optical surface, optimization of immobilization chemistry for attachment of capture probes, and characterization of the efficiency of the hybridization process are presented. We describe the application of this system to detection of a clinically relevant target, the mecA gene present in methicillin-resistant Staphylococcus aureus.

Subattomole-Sensitivity Microchip Nanoelectrospray Source with Time-of-Flight Mass Spectrometry Detection

A microfabricated microfluidic device coupled with a nanospray tip for electrospray ionization of dilute solutions is described. The device has been interfaced with a time-of-flight mass spectrometer and evaluated for sensitive, high-speed detection of peptides and proteins. The electrospray voltage was applied through the microchip to the nanospray capillary that was attached at the end of a microfabricated channel. Fluid delivery rates were 20-30 nL min(-)(1) through the hybridized structure without any pressure assistance. On-line microchip electrophoresis has been demonstrated and the effect of the capillary-chip junction on band broadening examined. Full mass spectra are acquired within 10-20 ms at 50-100 spectra s(-)(1) storage rates. Detection of subattomole levels of sample from a 100 nM solution is demonstrated for infusion experiments.

Sub-Attomole Molecule Detection in a Single Biological Cell in-vitro by Thermal Lens Microscopy

We have developed a novel thermal lens microscopy coupled with an optical microscope, and presently applied it to the ultratrace molecule detection in a single biological cell (mouse hybridoma) in-vitro. The determination results obtained for individual cells were calibrated by the average values determined by absorption spectrophotometry. The determination limit of the thermal lens microscope, defined as twice the standard deviation of the calibration curve, was 37.8 amol/cell, which was more than one order smaller than that of a fluorescence microscope under our experimental conditions. This superiority should come from escaping light scattering by a cell membrane and by cytoplasm, which is inevitable in fluorescence spectrometry. The absolute determination limit of the thermal lens microscopy was calculated down to sub-attomole level. In addition, the method is more widely applicable, even to non-fluorescent samples without chemical preparation, and therefore, the thermal lens microscope has proved to be very useful in directly quantifying ultratrace chemical species in a single biological cell in-vitro.

Sensing antimonite and arsenite at the subattomole level with genetically engineered bioluminescent bacteria.

A highly sensitive and selective optical sensing system for antimonite has been developed using genetically engineered bacteria. The basis of this system is the ability of certain bacteria to survive in environments that are contaminated with antimonite, arsenite, and arsenate. The survival is conferred to the bacteria by the ars operon, which consists of five genes that code for three structural proteins, ArsA, ArsB, and ArsC, and two regulatory proteins, ArsD and ArsR. ArsA, ArsB, and ArsC form a protein pump system that extrudes antimonite, arsenite, and arsenate once these anions reach the cytoplasm of the bacterium. A method was developed for monitoring antimonite and arsenite by using a single plasmid that incorporates the regulatory gene of the extrusion system, arsR, and the genes of bacterial luciferase, luxA and luxB. In the designed plasmid, ArsR regulates the expression of bacterial luciferase in a manner that is dependent on the concentration of antimonite and arsenite in the sample. Thus, the bioluminescence emitted by luciferase can be related to the concentration of antimonite and arsenite in the sample. Concentrations for antimonite and arsenite in the order of 10(-5) M, which corresponds to subattomole levels, can be detected. This bacterial-based sensing system is highly selective for antimonite and arsenite.

Analysis of brevetoxins by micellar electrokinetic capillary chromatography and laser-induced fluorescence detection.

Micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection was used to measure four red tide brevetoxins at sub-attomole levels. The separation of four brevetoxins by MEKC was achieved with a sodium borate/sodium dodecyl sulfate buffer at pH 9.3 Brevetoxins with a terminal alcohol group were derivatized with an acyl azide coumarin to form stable, highly fluorescent products. Brevetoxins with a terminal aldehyde group were reduced to the alcohol with sodium borohydride prior to derivatization with the coumarin. Three derivatized brevetoxins (PbTx-3, PbTx- 5, and PbTx-9) were separated by MEKC and detected using He/Cd laser excitation at 354 nm and fluorescence emission at 410 nm. A fourth brevetoxin (PbTx-2) was converted to PbTx-3 prior to derivatization and was then determined by subtraction. Instrumental detection limits for all four toxins were approximately 0.10 fg or about 10(6)-fold more sensitive than existing liquid chromatographic methods. Brevetoxins were isolated from cell cultures and fish tissue using an alumina column/gel-permeation chromatography procedure. Method detection limits for the brevetoxins in fish tissue were approximately 4 pg/g. These method detection limits are at least 100-fold better than previous chromatographic and/or electrophoretic methods. The MEKC-LIF method reported here allows measurement of brevetoxins at the trace levels considered critical for understanding toxin metabolism and mode of action.

Use of small-diameter capillaries for increasing peptide and protein detection sensitivity in capillary electrophoresis-mass spectrometry.

The use of small ID capillaries is shown to provide a substantial increase in sensitivity for capillary electrophoresis-electrospray ionization/mass spectrometry (CE-ESI/MS). In a comparison using capillaries ranging from either 100 to 10 microns or 50 to 5 microns ID and chemically modified with aminopropylsilane, a 25- to 50-fold increase in sensitivity was observed for both peptide and protein mixtures. This enhanced solute sensitivity allowed the detection of approximately 150 attomoles of melittin (2845 Da) with selected ion monitoring and 600 attomoles of carbonic anhydrase (29,157 Da) while scanning for CE-MS with a quadrupole mass spectrometer. For the protein mixture, mass spectra of sufficient quality for precise molecular weight determination (< or = 0.05%) were obtained for 600 attomole injections using a 5 microns ID capillary. The increase in sensitivity with small capillary diameters can be primarily attributed to a reduced mass flow rate of buffer and other background constituents into the electrospray source, which allows for greater sample ionization efficiency. A model that qualitatively accounts for the results is presented, but quantitative agreement is precluded due to difficulties in accounting for contributions due to a liquid sheath flow used with the electrospray source. The model accounts for the observation that the ESI/MS appears to function as a concentration-sensitive detector under many conditions using large-diameter capillaries. A transition occurs, however, to a regime where the ESI/MS functions as a mass flow-sensitive detector for small-diameter capillaries, where the ESI current is limited by the rate of delivery to the ESI source of charge carrying species in solution. These results suggest peptide and protein analysis at low attomole and subattomole levels should be obtainable with alternative types of mass spectrometers.