Showing papers on "Detection limit published in 2009"

Ultrasensitive Immunosensor for Cancer Biomarker Proteins using Gold Nanoparticle Film Electrodes and Multienzyme-Particle Amplification

Abstract: A densely packed gold nanoparticle platform combined with a multiple-enzyme labeled detection antibody-magnetic bead bioconjugate was used as the basis for an ultrasensitive electrochemical immunosensor to detect cancer biomarkers in serum. Sensitivity was greatly amplified by synthesizing magnetic bioconjugates particles containing 7500 horseradish peroxidase (HRP) labels along with detection antibodies (Ab2) attached to activated carboxyl groups on 1 μm diameter magnetic beads. These sensors had sensitivity of 31.5 μA mL ng−1 and detection limit (DL) of 0.5 pg mL−1 for prostate specific antigen (PSA) in 10 μL of undiluted serum. This represents an ultralow mass DL of 5 fg PSA, 8-fold better than a previously reported carbon nanotube (CNT) forest immunosensor featuring multiple labels on carbon nanotubes, and near or below the normal serum levels of most cancer biomarkers. Measurements of PSA in cell lysates and human serum of cancer patients gave excellent correlations with standard ELISA assays. These ...

Rapid, Direct Analysis of Cholesterol by Charge Labeling in Reactive Desorption Electrospray Ionization

Abstract: Direct and rapid analysis of cholesterol was accomplished in the ambient environment using reactive desorption electrospray ionization (DESI) mass spectrometry. This was achieved by electrospraying reagent solutions in the form of high velocity charged droplets at surfaces such as dried serum samples and animal tissue sections. Betaine aldehyde, incorporated into the spray solvent, reacts selectively and rapidly with the alcohol group of cholesterol by nucleophilic addition, forming a hemiacetal salt. Limits of detection for pure cholesterol and related compounds were ∼1 ng when a solution of cholesterol of 1 μg/mL was spotted onto the surface. Quantitative analysis of free cholesterol in serum using reactive DESI was demonstrated using cholesterol-d7 as internal standard. High throughput analysis of small volumes of serum spotted onto a suitable substrate was achieved at an analysis rate of ∼14 s per sample, with a relative standard deviation (RSD) of ca. 6%. Use of reactive DESI in the imaging mode allo...

Measurement of bisphenol A and bisphenol B levels in human blood sera from healthy and endometriotic women.

Abstract: A sensitive HPLC method with fluorescence detection was developed for the determination of bisphenol A (BPA) and bisphenol B (BPB) in human blood serum. The detection limits of the method were 0.18 and 0.20 ng/mL for BPA and BPB, respectively. A single-step liquid-liquid extraction was used for the pre-treatment of serum samples. The recoveries of BPA and BPB spiked to sera were 85.6 and 87.7%, respectively. The analyses of sera from both healthy and endometriotic women emphasized the absence of bisphenols in all the control cases (11 women), whereas BPA was found in 30 sera (51.7%) and BPB was found in 16 sera (27.6%) in the group of 58 patients with endometriosis; in nine of such sera BPA and BPB were present simultaneously. Only relatively to the sera quantitated, BPA concentrations ranged from 0.79 to 7.12 ng/mL (mean concentration 2.91 +/- 1.74 ng/mL), whereas BPB concentrations ranged from 0.88 to 11.94 ng/mL (mean concentration 5.15 +/- 4.16 ng/mL). Therefore, the presence of at least one of the two bisphenols was verified in a percentage as high as 63.8% in the sera from endometriotic women, suggesting the existence of a relationship between endometriosis and BPA and/or BPB exposure. Indeed, it is well known that bisphenols can work as xenoestrogens, owing to their structural similarity to natural and synthetic estrogens (e.g. estradiol and dietilstilbestrol). However, further studies are necessary to confirm this hypothesis and to assess the actual dose at which exposures to bisphenols are able to increase the sensitivity of the endometriotic cells to estradiol.

Surface-enhanced Raman scattering in nanoliter droplets: towards high-sensitivity detection of mercury (II) ions

Abstract: We report a new method for the trace analysis of mercury (II) ions in water. The approach involves the use of droplet-based microfluidics combined with surface-enhanced Raman scattering (SERS) detection. This novel combination provides both fast and sensitive detection of mercury (II) ions in water. Specifically, mercury (II) ion detection is performed by using the strong affinity between gold nanoparticles and mercury (II) ions. This interaction causes a change in the SERS signal of the reporter molecule rhodamine B that is a function of mercury (II) ion concentration. To allow both reproducible and quantitative analysis, aqueous samples are encapsulated within nanoliter-sized droplets. Manipulation of such droplets through winding microchannels affords rapid and efficient mixing of the contents. Additionally, memory effects, caused by the precipitation of nanoparticle aggregates on channel walls, are removed since the aqueous droplets are completely isolated by a continuous oil phase. Quantitative analysis of mercury (II) ions was performed by calculating spectral peak area of rhodamine B at 1,647 cm−1. Using this approach, the calculated concentration limit of detection was estimated to be between 100 and 500 ppt. Compared with fluorescence-based methods for the trace analysis of mercury (II) ions, the detection sensitivities were enhanced by approximately one order of magnitude. The proposed analytical method offers a rapid and reproducible trace detection capability for mercury (II) ions in water.

Ultrasensitive Detection of Bacteria Using Core–Shell Nanoparticles and an NMR‐Filter System

Abstract: Direct detection of pathogens is key in combating human infections, in identifying nosocomial sources, in surveying food chains and in biodefense.[1] Recent advances in nanotechnology have enabled the development of new diagnostic platforms[2] aimed at more sensitive and faster pathogen detection.[3] Many of the reported technologies, albeit elegant, often fail in routine clinical settings[4] because they still require extensive specimen purification, use complex measurement setups, or are not easily scalable for clinical demands. Here we report a new, simple, nanoparticle-based platform that can rapidly detect pathogens in native biological samples. In this approach, bacteria are targeted by highly magnetic nanoparticles (MNP), concentrated into a microfluidic chamber, and detected by nuclear magnetic resonance (NMR). The clinical utility of our diagnostic platform was evaluated by detecting tuberculosis (TB), a leading cause of disease and death worldwide.[5] Using the bacillus Calmette-Guerin (BCG) as a surrogate for Mycobacterium tuberculosis, we demonstrate unprecedented detection speed and sensitivity; as few as 20 colony-forming unit (CFU) in sputum (1 mL) were detected in < 30 min. With the capability for fast, simple and portable operation, the new detection platform could be an ideal point-of-care diagnostic tool, especially in resource-limited settings. The diagnosis starts with specimen collection and incubation with bacteria specific MNP (Supporting Information Figure S1). MNP bind to the bacterial wall, rendering the bacteria superparamagnetic. In a subsequent step the spin-spin relaxation time (T2) of the whole sample is measured by NMR. As the magnetic fields from MNP dephase the precession of nuclear spins in water protons[6, 7], each MNP-tagged bacterium can shorten the T2 of billions of surrounding water molecules. To increase detection sensitivity, we have incorporated signal amplification schemes that made it possible to detect small quantities of bacteria in relatively large sample volumes. At the nanoparticle level, the detection signal has been enhanced by synthesizing Fe-based MNP with the high transverse relaxivity (r2). At the device level, the signal was amplified by concentrating bacteria into a microfluidic chamber where the NMR signal was measured. To provide portable, on-chip bacterial detection, the NMR signal was read out using a miniaturized NMR system we have recently developed.[8]. First, we developed hybrid MNP with a large Fe core and a thin ferrite shell (“cannonballs”; CB), that have very high r2 per particle (Table S1). With the limited number of binding sites per bacterium, the T2 of samples will be shorter and thereby the detection will be more sensitive when the individual MNP have higher r2. Since r2 is ∝ M2•d2, where M and d are the particle magnetization and the diameter respectively[6], we focused on making larger MNP using highly magnetic material (Fe). Figure 1a shows an example of highly mono-disperse CB (d = 16 nm). Initially, we made Fe-only MNP by thermally decomposing Fe(CO)5, followed by controlled air-oxidation to grow the ferrite shell.[9] Compared to chemical oxidation[10], our method produces a thinner shell and thereby leaves a larger Fe core (Figure S2), leading to higher M. The shell showed high crystallinity (Figure 1b) and X-ray diffraction revealed a typical pattern for a spinel structure (Figure 1c), confirming the ferrite nature of the shell.[11] The shell protected the Fe core from oxidation to maintain the magnetic properties of CB (Figure S3). CB showed high magnetization (139 emu g−1 [Fe]) and yet were superparamagnetic at room temperature (Figure 1d). Most importantly, CB assumed high r2 (= 6.1×10−11 m L s−1, 1.5 T; Figure S4), due to their high magnetization and large diameter. Figure 1 Cannonballs (CB) for bacterial targeting. a) The particles have a large metallic core (Fe) passivated with a thin ferrite shell, resulting in high particle relaxivity (> 5×10−11 mL s−1 at 0.5 T). The core diameter and the ... To render CB specific for BCG, we conjugated anti-BCG monoclonal antibodies to their surface (CB-BCG; see Methods in Supporting Information). Bacterial samples were then incubated with CB-BCG for 10 min, followed by a wash step to remove excess particles. Optical microscopy with fluorescent CB-BCG showed excellent targeting (Figure 2a). Binding of CB-BCG was further verified by the element mapping (Figure 2b), which showed high Fe signal on the bacterial membrane. The number of CB-BCG per bacterium, quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Figure S5), was ≈ 105. Magnetic tagging thus made the bacteria highly efficient T2-shortening agents (Figure 2c). The binding of CB-BCG to BCG could be inhibited by antibody blockade against bacterial epitopes. For control CB or when CB-BCG were used against other bacterial strains, the CB binding was minimal with T2 changes (ΔT2) < 5%. The number of CB-BCG tested against different bacteria species were < 600 per bacterium (Figure S5), confirming the specificity of CB-BCG. Figure 2 Selective BCG targeting with CB-BCG. a) Confocal micrograph of BCG incubated with fluorescently labeled CB-BCG. b) Transmission electron micrograph of BCG targeted with CB-BCG. Element mapping detected high Fe signal on a whole bacterium. c) Specificity ... To further enhance detection sensitivity and to streamline assay procedures, we next developed a chip-based filter system with NMR compatibility (Figure 3). A key component is a microfluidic chamber enclosed by a membrane filter and surrounded by a microcoil. The membrane filter performs two functions. First, it captures bacteria and concentrates them into the microfluidic chamber for NMR detection, providing a way to detect a small number of bacteria from large sample volumes. Second, the filter enables on-chip separation of bacteria from unbound CB, obviating the need for separate off-chip purification steps. Figure 4a illustrates the operating principle of the NMR-filter system. An unpurified sample is introduced into the microfluidic channel. Bacteria are retained by the membrane (pore size ≈ 100 nm), while excess CB permeate. Subsequently, a buffer solution is repeatedly injected to wash off unbound CB and the membrane is backwashed to redisperse the captured bacteria. Figure 4b shows an operation example with a sample containing BCG and CB-BCG. After sample loading, the microfluidic channel was flushed with PBS to remove unbound CB-BCG. Finally, the flow direction was reversed to resuspend the captured BCG for NMR measurements. The number of washing steps to remove unbound CB-BCG was determined by measuring T2 after each wash (Figure 4c). T2 values plateaued in 4–5 washing steps with the whole washing procedure complete in 103 times higher detection sensitivity. Figure 3 NMR-filter system for bacterial concentration and detection. a) The system consists of a microcoil and a membrane filter integrated with a microfluidic channel. The microcoil is used for NMR measurements; the membrane filter concentrates bacteria inside ... Figure 4 Bacterial separation and concentration. a) The unprocessed sample containing bacteria and CB-BCG is introduced into the device. The membrane filter retains bacteria while unbound CB-BCG pass through. To remove free CB-BCG, the microfluidic channel is ... To compare the effect of MNP relaxivity on detection sensitivity, we first used a NMR system without a membrane filter. Two types of nanoparticles, CB and cross-linked iron oxide[12] (CLIO; r2 = 7.0×10−13 mL s−1), were used (Figure 5, black and blue curves). CB-BCG showed much higher mass sensitivity, detecting as few as 6 CFU (in 1 µL sample volume), whereas the detection limit was ~100 CFU with CLIO-BCG. Using the NMR-filter system, we achieved substantially high concentration detection sensitivity (Figure 5, red curve). When BCG samples (100 µL) targeted with CB-BCG were filtered, the concentration detection limit was ~60 CFU mL−1. It is important to note that the concentration limit is theoretically unlimited with the filtering, because bacteria can be concentrated into the NMR detection chamber from large sample volumes. Figure 5 Comparison of detection sensitivity. Measurements were performed on samples with varying BCG counts to determine detection sensitivities. First, a microfluidic chip without a membrane filter was used to determine the intrinsic mass-detection limits. BCG ... To evaluate the clinical utility of the NMR-filter system, we performed comparative detection assays (Table S3). Pulmonary samples were prepared by spiking BCG into human sputa. Following liquefaction, samples were subjected to standard TB diagnostic tests, culture and acid-fast bacilli (AFB) smear microscopy, and CB-based NMR measurements. Without filtration, NMR measurements had a similar sensitivity to AFB smear microscopy, with a detection threshold of ~103 CFU/mL. However, the NMR method was less prone to human error and less labor-intensive. With the NMR-filter system, the detection sensitivity was comparable to that of culture-based detection; when 1-mL of samples were filtered, the detection limit was ~20 CFU. The NMR-based detection was much faster ( 2 weeks) and facility-dependent (e.g. incubators). These results demonstrate that the CB-based NMR-filter system can be readily applied for TB diagnosis in clinical settings.[13]

Detection of Trace Heavy Metal Ions Using Carbon Nanotube- Modified Electrodes

Abstract: A sensitive voltammetric method for detection of trace heavy metal ions using chemically modified carbon nanotubes (CNTs) electrode surfaces is described. The CNTs were covalently modified with cysteine prior to casting on electrode surfaces. Cysteine is an amino acid with high affinities towards some heavy metals. In this assay, heavy metals ions accumulated on the cysteine-modified CNT electrode surfaces prior to being subjected to differential pulse anodic stripping voltammetry analysis. The resulting peak currents were linearly related to the concentrations of the metal ions. The method was optimized with respect to accumulation time, reduction time and reduction potential. The detection limits were found to be 1 ppb and 15 ppb for Pb2+ and Cu2+ respectively. The technique was used for the detection of Pb2+ and Cu2+ in spiked lake water. The average recoveries of Pb2+ and Cu2+ were 96.2% and 94.5% with relative standard deviations of 8.43% and 7.53% respectively. The potential for simultaneous detection of heavy metal ions by the modified CNTs was also demonstrated.

Chlorine activation by N 2 O 5 : simultaneous, in situ detection of ClNO 2 and N 2 O 5 by chemical ionization mass spectrometry

Abstract: . We report a new method for the simultaneous in situ detection of nitryl chloride (ClNO2) and dinitrogen pentoxide (N2O5) using chemical ionization mass spectrometry (CIMS). The technique relies on the formation and detection of iodide ion-molecule clusters, I(ClNO2)− and I(N2O5)−. The novel N2O5 detection scheme is direct. It does not suffer from high and variable chemical interferences, which are associated with the typical method of nitrate anion detection. We address the role of water vapor, CDC electric field strength, and instrument zero determinations, which influence the overall sensitivity and detection limit of this method. For both species, the method demonstrates high sensitivity (>1 Hz/pptv), precision (~10% for 100 pptv in 1 s), and accuracy (~20%), the latter ultimately determined by the nitrogen dioxide (NO2) cylinder calibration standard and characterization of inlet effects. For the typically low background signals (

Development of a Heat-Induced Surface-Enhanced Raman Scattering Sensing Method for Rapid Detection of Glutathione in Aqueous Solutions

Abstract: In this paper, a direct and simple detection method based on surface-enhanced Raman scattering (SERS) named “heat-induced SERS sensing method” is proposed for rapid determination of glutathione in aqueous solutions. It was found that highly enhanced SERS spectra of glutathione can be obtained if the silver colloids adsorbed with the analyte were heated up before the SERS measurement. Besides, it was revealed that silver particles with a size of ∼60 nm are suitable for this study and that the SERS intensity is also influenced by the dropped sample volume, drying temperature, buffer concentration, and pH of the solution. It is noted that the thiol group of glutathione has a particularly strong interaction with a silver surface compared with other small biological molecules without a thiol group, validating this method to detect glutathione selectively. Under the optimal conditions, the detection of glutathione can be finished within 5 min, and the detection limit of ca. 50 nM can be reached, which is much b...

Preparation of magnetic molecularly imprinted polymer for rapid determination of bisphenol A in environmental water and milk samples.

Abstract: A magnetic molecularly imprinted polymer (M-MIP) of bisphenol A (BPA) was prepared by miniemulsion polymerization. The morphological and magnetic characteristics of the M-MIP were characterized by Fourier-transform infrared spectroscopy, transmission electron microscopy, and vibrating sample magnetometry. The adsorption capacities of the M-MIP and the nonimprinted polymer were investigated using static adsorption tests, and were found to be 390 and 270 mg g(-1), respectively. Competitive recognition studies of the M-MIP were performed with BPA and the structurally similar compound DES, and the M-MIP displayed high selectivity for BPA. A method based on molecularly imprinted solid-phase extraction assisted by magnetic separation was developed to extract BPA from environmental water and milk samples. Various parameters such as the mass of sorbent, the pH of the sample, the extraction time, and desorption conditions were optimized. Under selected conditions, extraction was completed in 15 min. High-performance liquid chromatography with UV detection was employed to determine BPA after the extraction. For water samples, the developed method exhibited a limit of detection (LOD) of 14 ng L(-1), a relative standard deviation of 2.7% (intraday), and spiked recoveries ranging from 89% to 106%. For milk samples, the LOD was 0.16 microg L(-1), recoveries ranged from 95% to 101%, and BPA was found in four samples at levels of 0.45-0.94 microg L(-1). The proposed method not only provides a rapid and reliable analysis but it also overcomes problems with conventional solid-phase extraction (SPE), such as the packing of the SPE column and the time-consuming nature of the process of loading large-volume samples.