ABSTRACT
Point of care molecular diagnostics benefits from a portable battery-operated device capable of performing a fast turnaround using reliable inexpensive cartridges. We describe a prototype device for performing a molecular diagnostics test for clinical and biodefense samples in 16 minutes using a prototype capable of an 8 minute PCR reaction, followed by hybridization and detection on an electrochemical microarray based on the i-STAT® system. We used human buccal swabs for hemochromatosis testing including in-device DNA extraction. Additional clinical and biodefense samples included influenza A and bacterial select agents Bacillus anthracis, Yersinia pestis and Francisella tularensis.
Subject(s)
Electrochemical Techniques/methods , Molecular Diagnostic Techniques/instrumentation , Point Mutation , Point-of-Care Systems , Bacillus anthracis/genetics , DNA Mutational Analysis/instrumentation , DNA Mutational Analysis/methods , Electrochemical Techniques/instrumentation , Francisella tularensis/genetics , Genotype , Hemochromatosis/diagnosis , Hemochromatosis/genetics , Humans , Influenza A virus/genetics , Lab-On-A-Chip Devices , Molecular Diagnostic Techniques/methods , Polymerase Chain Reaction , Time Factors , Yersinia pestis/geneticsABSTRACT
We present Fourier transform ion cyclotron resonance (FTICR) mass spectra at a magnetic field of 20 T; more than twice the highest field previously used for FTICR. Our instrument is based on a resistive magnet installed at the National High Magnetic Field Laboratory. The magnet has a 50 mm diameter bore and spatial inhomogeneity of approximately 1000 ppm over a 1 cm diameter spherical volume. However, FTICR mass resolving power far in excess of magnet homogeneity is achieved routinely for ions produced by either electron ionization (EI) or matrix-assisted laser desorption/ionization (MALDI). As examples, we show a MALDI mass spectrum of [M + H] quasimolecular ions of the peptide, human luteinizing hormone-releasing hormone (monoisotopic molecular weight, 1181.6 Da) at mass resolving power, m/delta m > 10,000; and an EI mass spectrum of molecular ions of the platinum cluster compound, Pt4(PF3)8 (average molecular weight, 1484 Da at mass resolving power, m/delta m approximately 20,000. Much better FTICR MS performance is predicted for future NHMFL resistive magnets of higher spatial and temporal homogeneity.
Subject(s)
Cyclotrons , Electromagnetic Phenomena , Mass Spectrometry/instrumentation , Fourier Analysis , Gonadotropin-Releasing Hormone/chemistry , Humans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
A single population of multiply charged protein ions formed by electrospray ionization is held in a trapped ion cell and remeasured continuously by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry while undergoing multiple reactions with diethylamine. In a first example, electrosprayed horse myoglobin with an average of 16 attached protons is reacted with base at a pressure of 1.5 x 10(-8) Torr for a period of 60 s. A total of 31 spectra acquired with a duty cycle of 2 s exhibit the charge state-dependent formation of up to three diethylamine adducts and removal of up to nine protons. A realtime measuring experiment is then conducted over a 1 h period to observe charge stripping of electrosprayed myoglobin ions, leaving as few as seven charges. Realtime monitoring is used to evaluate the effect of reagent gas pressure on adduct formation and is used in conjunction with high mass resolution FTICR detection to resolve the isotopic peaks within individual charge states of adduct spectra. The attractive features of real-time reaction monitoring include a dramatic reduction in experiment time and sample consumed while concurrently observing long-lived intermediates and reaction products as they form.
Subject(s)
Mass Spectrometry/methods , Cyclotrons , Cytochrome c Group/chemistry , Diethylamines/chemistry , Fourier Analysis , Gases , Ions , Myoglobin/chemistry , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
A new method for application of quadrupolar excitation to the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is presented. Quadrupolar excitation is conventionally applied to the two pairs of opposed electrodes that normally perform the excitation and detection functions in the FTICR experiment. Symmetry arguments and numerically calculated isopotential contours within the trapped ion cell lead to the conclusion that quadrupolar excitation can be applied to a single pair of opposed side electrodes. Examples of effective quadrupolar axialization via this method include a sevenfold signal-to-noise enhancement derived from 50 remeasurements of a single population of trapped bovine insulin ions and the selective isolation of a single charge state of horse heart myoglobin after an initial measurement that revealed the presence of 14 charge states.
ABSTRACT
A new technique for manipulating the kinetic energy distribution of electrospray ions that arrive at a Fourier transform ion cyclotron resonance trapped-ion cell is presented. Narrow kinetic energy distributions can complicate the selection of appropriate trapping conditions for electrospray ions and introduce charge discrimination in resulting mass spectra. Modulation of the applied skimmer potential controllably broadens the kinetic energy distribution, which improves the reproducibility of acquired spectra and eliminates charge discrimination. Mass spectra of horse heart cytochrome c are presented to demonstrate the utility of the technique. For example, applied static skimmer potentials of 12 and 9 V yield charge state distributions ranging from [M+19H](+19) to [M+12H](+12) and [M+15H](+15) to [M+7H](+7), respectively. A 12 ± 2 V, 100-Hz modulation of the skimmer potential yields an electrospray spectrum with charge states that range from [M+19H](+19) to [M+7H](+7), which is more representative of the source distribution.
