A highly sensitive carbapenemase assay using laser desorption/ionization mass spectrometry based on a parylene-matrix chip.

A quantitative carbapenemase assay was developed using laser desorption/ionization mass spectrometry (LDI-MS) based on a parylene-matrix chip. As a first step, the reproducibility (spot-to-spot, shot-to-shot, and day-to-day) of LDI-MS based on a parylene-matrix chip and the quantification ranges for four carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) were determined. A carbapenem-susceptibility test was performed using the four carbapenems and 51 bacterial strains that displayed (1) carbapenem resistance with carbapenemase, (2) carbapenem resistance without carbapenemase, or (3) carbapenem susceptibility. The susceptibility test results showed that LDI-MS based on a parylene-matrix chip was more sensitive and selective for detecting the carbapenemase reaction than conventional MALDI-TOF MS based on a 2,5-dihydroxybenzoic acid matrix.

Newborn screening by matrix-assisted laser desorption/ionization mass spectrometry based on parylene-matrix chip.

Newborn screening for diagnosis of phenylketonuria, homocystinuria, and maple syrup urine disease have been conducted by analyzing the concentration of target amino acids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) based on parylene-matrix chip. Parylene-matrix chip was applied to MALDI-ToF MS analysis reducing the matrix peaks significantly at low mass-to-charge ratio range (m/z < 500). Reproducibility of inter-spot and intra-spot analyses of amino acids was less than 10%. Methanol extraction was adopted for simple and rapid sample preparation of serum before mass spectrometric analysis showing 13.3 to 45% of extraction efficiency. Calibration curves for diagnosis of neonatal metabolic disorders were obtained by analyzing methanol-extracted serum spiked with target amino acids using MALDI-ToF MS. They showed good linearity (R2 > 0.98) and the LODs were ranging from 9.0 to 22.9 µg/mL. Effect of proteins in serum was estimated by comparing MALDI-ToF mass spectra of amino acids-spiked serum before and after the methanol extraction. Interference of other amino acids on analysis of target analyte was determined to be insignificant. From these results, MALDI-ToF MS based on parylene-matrix chip could be applicable to medical diagnosis of neonatal metabolic disorders.

Hypersensitive antibiotic susceptibility test based on a ß-lactamase assay with a parylene-matrix chip.

Many kinds of susceptibility test for ß-lactam antibiotics have been used to determine the antibiotic resistance of bacterial strains. Here, a sensitive antibiotic susceptibility test was presented by using a specialized reaction tool for laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) based on parylene-matrix chip. The ß-lactamase assay was carried out in a specialized reaction tool by (1) concentrating the bacterial strain and (2) incubating the bacteria with penicillin-G. The parylene-matrix chip was produced by deposition of a partially porous parylene-N thin film on a dried organic matrix array, and the products of ß-lactamase reaction in the low range of mass-to-charge ratio (m/z<500) could be effectively analyzed by using a parylene-matrix chip. The sensing parameters were compared with conventional chromogenic antibiotic susceptibility test for ß-lactam antibiotics. Finally, LDI-TOF MS with a specialized reaction tool and parylene-matrix chip could achieve a limit of detection as low as 600 cells/spot for penicillin-G.

Analysis of benzylpenicillin in milk using MALDI-TOF mass spectrometry with top-down synthesized TiO2 nanowires as the solid matrix.

In this work, the wet-corrosion process for the synthesis of titanium oxide (TiO2) nanowires in the anatase phase was optimized as the solid matrix in MALDI-TOF mass spectrometry, and the solid matrix of the TiO2 nanowires was applied to the detection of antibiotics in a daily milk sample. The influence of the alkali concentration and the heat treatment temperature on the crystal structure of the TiO2 nanowires was investigated. The ionization activity of the TiO2 nanowires was estimated for each synthetic condition using amino acids as model analytes with low molecular weights. For the detection of antibiotics in milk, benzylpenicillin was spiked in daily milk samples, and MALDI-TOF mass spectrometry with the TiO2 nanowires was demonstrated to detect the benzylpenicillin at the cut-off concentration of the EU directive.

Highly sensitive bacterial susceptibility test against penicillin using parylene-matrix chip.

This work presented a highly sensitive bacterial antibiotic susceptibility test through ß-lactamase assay using Parylene-matrix chip. ß-lactamases (EC 3.5.2.6) are an important family of enzymes that confer resistance to ß-lactam antibiotics by catalyzing the hydrolysis of these antibiotics. Here we present a highly sensitive assay to quantitate ß-lactamase-mediated hydrolysis of penicillin into penicilloic acid. Typically, MALDI-TOF mass spectrometry has been used to quantitate low molecular weight analytes and to discriminate them from noise peaks of matrix fragments that occur at low m/z ratios (m/z<500). The ß-lactamase assay for the Escherichia coli antibiotic susceptibility test was carried out using Parylene-matrix chip and MALDI-TOF mass spectrometry. The Parylene-matrix chip was successfully used to quantitate penicillin (m/z: [PEN+H](+)=335.1 and [PEN+Na](+)=357.8) and penicilloic acid (m/z: [PA+H](+)=353.1) in a ß-lactamase assay with minimal interference of low molecular weight noise peaks. The ß-lactamase assay was carried out with an antibiotic-resistant E. coli strain and an antibiotic-susceptible E. coli strain, revealing that the minimum number of E. coli cells required to screen for antibiotic resistance was 1000 cells for the MALDI-TOF mass spectrometry/Parylene-matrix chip assay.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of small volatile molecules using a parylene-matrix chip.

RATIONALE: In matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), volatile small molecules have been nearly impossible to analyze because (1) such molecules evaporate under drying and vacuum conditions and (2) the organic matrix creates matrix peaks in the low mass-to-charge (m/z) range (m/z <500). In this work, the analysis of volatile small molecules using MALDI-TOFMS was realized using (1) a parylene-matrix chip to eliminate the matrix peaks of the organic matrix and (2) graphene for the effective adsorption of the small volatile molecules. METHODS: The parylene-matrix chip was produced by deposition of a partially porous parylene-N thin film on a dried organic matrix array. The sample solution of volatile small molecules was mixed with the graphene and then placed on the parylene-matrix chip for MALDI-TOFMS. Analogs of chemical agents called dimethyl methyl phosphonate (DMMP) and 2-chloroethylethylsulfide (CEES) were used as model compounds for the small volatile molecules, and the sensing parameters were estimated, such as the limit of detection (LOD) and the detection range. RESULTS: MALDI-TOFMS based on the parylene-matrix chip and graphene as the adsorbent could achieve a LOD of approximately 1 ppb in the detection range of 1 ppm-1 ppb for the highly volatile DMMP and CEES. CONCLUSIONS: The parylene-matrix chip with graphene can be applied for the detection of volatile small molecule analytes in the m/z ratio range of small molecules (m/z <500) using graphene as an effective adsorbent.

Nylon nanoweb with TiO2 nanoparticles as a solid matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: The solid matrices used for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) enable the analysis of small molecules by preventing fragmentations of organic matrix molecules in the low mass-to-charge ratio (m/z) range. In this work, a nylon nanoweb with TiO(2) particles was developed as a solid matrix for MALDI-TOFMS to improve the low intensities of mass peaks, narrow detection ranges and low signal-to-noise levels. METHODS: The nylon nanoweb with TiO(2) particles was prepared by simultaneously electrospinning a nylon nanoweb and electrospraying TiO(2) nanoparticles measuring 25 nm in diameter to form TiO(2) spheres 300 nm in diameter. RESULTS: MS of multiple analytes was demonstrated in the low molecular weight range using eight amino acids. Additionally, leucine-enkephalin (555.6 g/mol) and cyclic citrullinated peptide (1668 g/mol) were used as model analytes to test the feasibility of a nylon nanoweb containing TiO(2) particles as a solid matrix for MALDI-TOFMS. CONCLUSIONS: The nylon nanoweb with TiO(2) particles can be applied for the detection of volatile small molecule analytes in the m/z ratio range of small molecules.

Top-down synthesized TiO2 nanowires as a solid matrix for surface-assisted laser desorption/ionization time-of-flight (SALDI-TOF) mass spectrometry.

Top-down synthesized TiO2 nanowires are presented as an ideal solid matrix to analyze small biomolecules at a m/z of less than 500. The TiO2 nanowires were synthesized as arrays using a modified hydrothermal process directly on the surface of a Ti plate. Finally, the feasibility of the TiO2 nanowires in the anatase phase as a solid matrix. The crystal and electronic structures of the top-down TiO2 nanowires were analyzed at each step of the hydrothermal process, and the optimal TiO2 nanowires were identified by checking their performance toward the ionization of analytes in surface-assisted laser desorption/ionization time-of-flight (SALDI-TOF) mass spectrometry. Finally, the feasibility of the TiO2 nanowires in the anatase phase as a solid matrix for SALDI-TOF mass spectrometry was demonstrated using eight types of amino acids and peptides as model analytes.

Parylene-matrix chip for small molecule analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: In matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), analyte molecules are known to be ionized by mixing with organic matrix molecules. As the organic matrix molecules are ionized, they generate unreproducible mass peaks such that MALDI-TOF MS is nearly impossible in the low mass-to-charge (m/z) range (<1000). In this work, we aimed to develop a parylene-matrix chip for the detection of small molecules in the low m/z range by using MALDI-TOF MS. METHODS: The parylene-matrix chip was fabricated by the deposition of a partially porous parylene-N thin film on a dried organic matrix array. The properties of the parylene thin film were analyzed by atomic force microscopy (AFM) and cyclic voltammetry (CV). Mass spectrometry was performed by using a parylene-matrix chip with eight amino acids as model analytes. RESULTS: The surface roughness and the electric conductivity of the parylene-N film were analyzed by AFM and CV analysis to determine its suitability for a parylene-matrix chip. The ionization of samples on the parylene-matrix chip was optimized by adjusting the laser intensity. The feasibility of applying a parylene-matrix chip for small molecule analysis was tested by using eight kinds of amino acids as model analytes and the simultaneous detection of multiple analytes from the amino acid mixture was also demonstrated. CONCLUSIONS: The parylene-matrix chip can be applied for the detection of multiple analytes in the m/z ratio range of small molecules (<1000) using MALDI-TOF MS.

Covalent protein immobilization with a parylene-H film for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: For the sensitive analysis of receptor-ligand interactions by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), receptor proteins should be immobilized on a target plate with a high surface density. In this work, a parylene-H film with formyl groups was developed for the efficient covalent immobilization of receptor proteins for MALDI-TOF MS. METHODS: The parylene-H film was thermally deposited on a target plate and receptor proteins were covalently immobilized. The surface properties of the parylene-H film were analyzed by atomic force microscopy (AFM) and cyclic voltammetry (CV). The immobilization efficiency of the parylene-H film was analyzed by fluorescence imaging with streptavidin and fluorescence-labeled biotin. MALDI-TOF MS was performed using the parylene-H-coated target plate with streptavidin and different concentrations of biotinylated peptide as the receptor and ligand, respectively. RESULTS: The parylene-H film on a target plate had a flat surface (Rq : ±2.755 nm) without any pinholes and could be regarded to be electrically conductive under an electric potential of 30 kV. The fluorescence image proved that the parylene-H film improved the protein immobilization efficiency as well as ligand detection sensitivity. The mass spectra quantitatively revealed peaks from the ligand molecules without any interference peaks from the immobilized receptor proteins. CONCLUSIONS: A parylene-H film with formyl groups was thermally deposited on a target plate and the receptor protein was covalently immobilized on the target plate. The interactions of ligand molecules with the immobilized receptor proteins were quantitatively analyzed by MALDI-TOF MS.

Autodisplay of streptavidin.

Streptavidin was expressed on the outer membrane of E. coli as a recombinant fusion protein with an autotransporter domain called AIDA-I (adhesin involved in diffuse adherence) using autodisplay technology. The autodisplay of streptavidin was confirmed by SDS-PAGE of the outer membrane proteins, and the number of autodisplayed streptavidin molecules on a single E. coli cell was evaluated with densitometric analysis. The biotin-binding activity of the autodisplayed streptavidin was estimated after treatment with fluorescently labeled biotin by fluorescence microscopy and flow cytometry. The biotin-binding activity of the E. coli with autodisplayed streptavidin was compared with the activity of streptavidin immobilized on magnetic beads. Finally, the outer membrane presenting autodisplayed streptavidin was isolated and layered on a 96-well microplate for an immunoassay.

Diamond-like carbon (DLC) microelectrode for electrochemical ELISA.

A diamond-like carbon (DLC) microelectrode was applied to commercial ELISA kits for medical diagnosis of HIV (human immunodeficiency virus), HBV (human hepatitis B virus), HCV (human hepatitis C virus). In this work, quantification of oxidized 3,3',5,5'-tetramethylbenzidine (TMB) was carried out by using a microelectrode made of boron-doped DLC and cyclic voltammetric analysis method without the conventional quenching step which uses sulfuric acid. The microelectrode provided well-known step-shaped graphs, and limit of detection could be improved by clear determination of electrochemical oxidative and reductive peaks. To demonstrate the applicability of DLC microelectrode to conventional ELISA kits, commercial ELISA kits for detection of HIV antigen, HBV antigen and HCV antigen were also tested. These results proved that the applicability of DLC microelectrode to practical detection is feasible.