Carbon electrode obtained via pyrolysis of plasma-deposited parylene-C for electrochemical immunoassays.

In this study, parylene-C films from plasma deposition as well as thermal deposition were pyrolyzed to prepare a carbon electrode for application in electrochemical immunoassays. Plasma deposition could prepare parylene-C in a faster deposition rate and more precise control over the thickness in comparison with the conventional thermal deposition. To analyze the influence of the deposition method, the crystal and electronic structures of the pyrolyzed parylene-C films obtained via both deposition methods were compared using Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. For application as a carbon electrode in immunoassays, the electrochemical properties of the pyrolyzed carbon films from two both deposition methods were analyzed, including the double layer capacitance (2.10 µF cm-2 for plasma deposition and 2.20 µF cm-2 for thermal deposition), the apparent electron transfer rate (approximately 1.1 × 10-3 cm s-1 for both methods), and the electrochemical window (approximately -1.0 ∼ 2.1 V for both methods). Finally, the applicability of the pyrolyzed carbon electrode from parylene-C was demonstrated for the diagnosis of human hepatitis-C using various amperometric methods, such as cyclic voltammetry, chronoamperometry, square-wave voltammetry and differential pulse voltammetry.

MALDI-TOF Mass Spectrometry Based on Parylene-Matrix Chip for the Analysis of Lysophosphatidylcholine in Sepsis Patient Sera.

In this work, medical diagnosis of sepsis was conducted via quantitative analysis of lysophosphatidylcholine 16:0 (LPC 16:0) by using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry based on a parylene-matrix chip. In the first step, specific mass peaks for the diagnosis of sepsis were searched by comparing MALDI-TOF mass spectra of sepsis patient sera with healthy controls and pneumonia patient sera. Two mass peaks at m/z = 496.3 and 518.3 were chosen as those that are specifically different for sepsis sera to compare with healthy controls and pneumonia patient sera. These mass peaks were identified to be protonated and sodium adducts of LPC 16:0 by using tandem mass spectra (MS2 and MS3) of purely synthesized LPC 16:0 and extracted LPC 16:0 from a healthy control and a sepsis patient. In the next step, a standard curve for LPC 16:0 for the quantitative analysis of LPC 16:0 with MALDI-TOF MS based on the parylene-matrix chip was prepared, and the statistical correlation to the LC-MS analysis results was demonstrated by using the Bland-Altman test and Passing-Bablok regression. Finally, MALDI-TOF MS based on the parylene-matrix chip was used for the quantification of LPC 16:0 with sera from patients with severe sepsis and septic shock (n = 143), pneumonia patients (n = 12), and healthy sera (n = 31). The sensitivity and the selectivity of medical diagnosis of sepsis was estimated to be 97.9% and 95.5% by using MALDI-TOF MS based on the parylene-matrix chip, respectively.

Parylene-Coated Polytetrafluoroethylene-Membrane-Based Portable Urea Sensor for Real-Time Monitoring of Urea in Peritoneal Dialysate.

A portable urea sensor for use in fast flow conditions was fabricated using porous polytetrafluoroethylene (PTFE) membranes coated with amine-functionalized parylene, parylene-A, by vapor deposition. The urea-hydrolyzing enzyme urease was immobilized on the parylene-A-coated PTFE membranes using glutaraldehyde. The urease-immobilized membranes were assembled in a polydimethylsiloxane (PDMS) fluidic chamber, and a screen-printed carbon three-electrode system was used for electrochemical measurements. The success of urease immobilization was confirmed using scanning electron microscopy, and fourier-transform infrared spectroscopy. The optimum concentration of urease for immobilization on the parylene-A-coated PTFE membranes was determined to be 48 mg/mL, and the optimum number of membranes in the PDMS chamber was found to be eight. Using these optimized conditions, we fabricated the urea biosensor and monitored urea samples under various flow rates ranging from 0.5 to 10 mL/min in the flow condition using chronoamperometry. To test the applicability of the sensor for physiological samples, we used it for monitoring urea concentration in the waste peritoneal dialysate of a patient with chronic renal failure, at a flow rate of 0.5 mL/min. This developed urea biosensor is considered applicable for (portable) applications, such as artificial kidney systems and portable dialysis systems.

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.

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.

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.

Functionalized Parylene Films for Enhancement of Antibody Production by Hybridoma Cells.

In this study, the influence of microenvironments on antibody production of hybridoma cells was analyzed using six types of functionalized parylene films, parylene-N and parylene-C (before and after UV radiation), parylene-AM, and parylene-H, and using polystyrene as a negative control. Hybridoma cells were cultured on modified parylene films that produced a monoclonal antibody against the well-known fungal toxin ochratoxin-A. Surface properties were analyzed for each parylene film, such as roughness, chemical functional groups, and hydrophilicity. The proliferation rate of the hybridoma cells was observed for each parylene film by counting the number of adherent cells, and the total amount of produced antibodies from different parylene films was estimated using indirect ELISA. In comparison with the polystyrene, the antibody-production by parylene-H and parylene-AM was estimated to be observed to be as high as 210-244% after the culture of 24 h. These results indicate that the chemical functional groups of the culture plate could influence antibody production. To analyze the influence of the microenvironments of the modified parylene films, we performed cell cycle analysis to estimate the ratio of the G0/G1, S, and G2/M phases of the hybridoma cells on each parylene film. From the normalized proportion of phases of the cell cycle, the difference in antibody production from different surfaces was considered to result from the difference in the proliferation rate of hybridoma cells, which occurred from the different physical and chemical properties of the parylene films. Finally, protein expression was analyzed using an mRNA array to determine the effect of parylene films on protein expression in hybridoma cells. The expression of three antibody production-related genes (CD40, Sox4, and RelB) was analyzed in hybridoma cells cultured on modified parylene films.

Cesium Lead Bromide (CsPbBr3) Perovskite Quantum Dot-Based Photosensor for Chemiluminescence Immunoassays.

Chemiluminescence immunoassays have been widely employed for diagnosing various diseases. However, because of the extremely low intensity chemiluminescence signals, highly sensitive transducers, such as photomultiplier tubes and image sensors with cooling devices, are required to overcome this drawback. In this study, a hypersensitive photosensor was developed based on cesium lead bromide (CsPbBr3) perovskite quantum dots (QDs) with sufficient high sensitivity for chemiluminescence immunoassays. First, CsPbBr3 QDs with a highly uniform size, that is, 5 nm, were synthesized under thermodynamic control to achieve a high size confinement effect. For the fabrication of the photosensor, MoS2 nanoflakes were used as an electron transfer layer and heat-treated at an optimum temperature. Additionally, a parylene-C film was used as a passivation layer to improve the physical stability and sensitivity of the photosensor. In particular, the trap states on the CsPbBr3 QDs were reduced by the passivation layer, and the sensitivity was increased. Finally, a photosensor based on CsPbBr3 QDs was employed in chemiluminescence immunoassays for the detection of human hepatitis B surface antigen, human immunodeficiency virus antibody, and alpha-fetoprotein (AFP, a cancer biomarker). When compared with the conventionally used equipment, the photosensor was determined to be feasible for application in chemiluminescence immunoassays.

Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout.

To date, medical diagnosis of gout and pseudogout has been performed by observing the crystals in the joint fluid of patients under a polarized microscope. Conventional diagnostic methods using a polarized microscope have disadvantages, such as time-consuming analysis, a high false negative rate, and difficulty in distinguishing gout with monosodium urate (MSU) crystals and pseudogout with calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluids. In this study, a chromogenic assay for the diagnosis of gout and pseudogout, without the requirement of a polarized microscope and trained experts, was proposed using Fv antibodies with specific binding activities to MSU and CPPD crystals. The IgG VH chain Fv library with randomized complementarity-determining region 3 (CDR3) region was expressed on the outer membrane of Escherichia coli using autodisplay technology. The target Fv antibodies with binding activity to MSU and CPPD crystals were screened from the autodisplayed Fv library on the E. coli outer membrane, and five clones were selected. On the basis of the binding properties of the screened Fv antibodies, peptides with the selected clone of amino acid sequences of the CDR3 region (15 residues) were chemically synthesized. The binding properties of the synthetic peptides with amino acid sequences of CDR3 regions from the selected clones were analyzed using fluorescence imaging and flow cytometry, and the affinity constants (Kd) of each peptide for binding to MSU and CPPD crystals were calculated by fitting based on the isotherm model. A chromogenic assay configuration for gout and pseudogout was developed using synthetic peptides. In this chromogenic assay, synthetic peptides labeled with biotin and streptavidin-horseradish peroxidase (HRP) complex were used, and crystal detection was possible using a chromogenic reaction between HRP and a chromogenic substrate (TMB). Finally, gout and pseudogout were diagnosed by detecting MSU and CPPD crystals in the synovial fluid in the concentration range of 0-300 µg/mL.

Modified parylene-N films as chemical microenvironments for differentiation and spheroid formation of osteoblast cells.

In this work, the influence of parylene N film on the spheroid formation of osteoblast-like cells (MG-63) was determined and compared with that of high-hydrophilicity microenvironments, such as hydrophilic culture matrix and ultraviolet-treated parylene N film. To elucidate the change in cell properties due to the microenvironment of parylene N film, global gene expression profiles of MG-63 cells on parylene N film were analyzed. We confirmed the upregulated expression of osteoblast differentiation- and proliferation-related genes, such as Runx2, ALPL, and BGLAP and MKi67 and PCNA, respectively, using the real-time polymerase chain reaction. In addition, the differentiation and proliferation of osteoblast cells cultured on parylene N film were validated using immunostaining. Finally, the formation of spheroids and regulation of differentiation in human mesenchymal stem cells (MSCs) on parylene N film was demonstrated. The results of this study confirm that the microenvironment with the controlled hydrophobic property of parylene N film could effectively trigger the bone differentiation and maintains the proliferation of MSCs, similar to MG-63 cells without any scaffold structures or physical treatments.

Highly sensitive in situ-synthesized cadmium sulfide (CdS) nanowire photosensor for chemiluminescent immunoassays.

Highly sensitive in situ-synthesized cadmium sulfide (CdS) nanowires (NWs) for the detection of chemiluminescence in immunoassays with a photoresist (PR) layer to stabilize the CdS NWs before and after coating with a parylene film were developed. The thickness of the PR layer was controlled by adjusting the viscosity of the PR solution used for spin-coating. PR2005 was the optimal PR for passivation of the NW surface. After the addition of a parylene coating on the CdS NWs, the photocurrent increased by as much as 50% over a broad range of light intensities, and the additional PR layer increased the photoresponse over the whole range of light intensities. When the photoresponses of the CdS NWs with and without the parylene film were compared after the addition of a PR layer, significant differences were observed in the photocurrent behavior after the incident light was turned off. For the CdS NWs with a parylene film and PR layer, the photocurrent reached the baseline within milliseconds of the incident light being turned off. However, the CdS NWs without a parylene film but with a PR layer required >60 s to reach the baseline level. This difference was due to the capacitance arising from the contact between the NWs. The in situ-synthesized CdS NW photosensor passivated by the parylene film and a PR layer was used in a chemiluminescence-based immunoassay. Finally, the detection of human immunodeficiency virus antibodies was demonstrated via a chemiluminescent enzyme-linked immunosorbent assay based on the CdS NW photosensor in comparison with the optical-density measurement for the chromogenic reaction of TMB(3,3',5,5'-Tetramethylbenzidine).

Simultaneous Analysis of Multiple Cancer Biomarkers Using MALDI-TOF Mass Spectrometry Based on a Parylene-Matrix Chip.

Recently, the parylene-matrix chip was developed for quantitative analysis of small molecules less than 1 kDa. In this study, MALDI-TOF MS based on the parylene-matrix chip was performed to clinically diagnose intrahepatic cholangiocarcinoma (IHCC) and colorectal cancer (CRC). The parylene-matrix chip was applied for the detection of small cancer biomarkers, including N-methyl-2-pyridone-5-carboxamide (2PY), glutamine, lysophosphatidylcholine (LPC) 16:0, and LPC 18:0. The feasibility of MALDI-TOF MS based on the parylene-matrix chip was confirmed via analysis of spot-to-spot and shot-to-shot reproducibility. Serum metabolite markers of IHCC, N-methyl-2-pyridone-5-carboxamide (2PY), and glutamine were quantified using MALDI-TOF MS based on the parylene-matrix chip. For clinical diagnosis of CRC, two water-insoluble (barely soluble) biomarkers, lysophosphatidylcholine (LPC) 16:0 and LPC 18:0, were quantified. Finally, glutamine and LPC 16:0 were simultaneously detected at a range of concentrations in sera from colon cancer patients using the parylene-matrix chip. Thus, this method yielded high-throughput detection of cancer biomarkers for the mixture samples of water-soluble analytes (2PY and glutamine) and water-insoluble analytes (LPC 16:0 and LPC 18:0).

Three-Dimensional Paper-Based Microfluidic Analytical Devices Integrated with a Plasma Separation Membrane for the Detection of Biomarkers in Whole Blood.

Paper-based microfluidic analytical devices (µPADs) have recently attracted attention as a point-of-care test kit because of their low cost and nonrequirement for external forces. To directly detect biomarkers in whole blood, however, they need to be assembled with a filter such as a plasma separation membrane (PSM) because the color of the blood cells interferes with the colorimetric assay. However, this assembly process is rather complicated and cumbersome, and the fluid does not uniformly move to the detection zone when the adhesion between the paper and PSM is not perfect. In this study, we report a simple three-dimensional (3D) printing method for fabricating PSM-integrated 3D-µPADs made of plastics without the need for additional assembly. In detail, PSM was coated with parylene C to prevent its dissolution from organic solvent during 3D printing. Then, the coated PSM was superimposed on the paper. Detection zones and a reservoir were printed on the paper and PSM via liquid photopolymerization, using a digital light processing printer. The limit of detection of the PSM-integrated 3D-µPADs for glucose in whole blood was 0.3 mM, and these devices demonstrated clinically relevant performance on diabetes patient blood samples. Our 3D-µPADs can also simultaneously detect multiple metabolic disease markers including glucose, cholesterol, and triglycerides in whole blood. Our results suggest that our printing method is useful for fabricating 3D-µPADs integrated with PSM for the direct detection of biomarkers in whole blood.

UV-irradiated parylene surfaces for proliferation and differentiation of PC-12 cells.

PC-12 cells originate from neuroblastic cells, which have an ability to differentiate into neuronlike cells. In this work, the purpose was to estimate the influence of microenvironments on cell attachment and neuritogenesis capacity of PC-12 cells on parylene-N and parylene-C films with and without ultraviolet (UV) light treatment. The estimate of total cell number after incubation for 72h, the ratio of adherent to suspended cells, counting of neurite outgrowths on parylene-N or parylene-C films after UV exposure suggested that these films were suitable for proliferation as well as differentiation of PC-12 cells. The differences in surface properties of parylene-N and parylene-C films with and without UV exposure were analyzed by contact angle measurement, Fourier-transform infrared spectrometry, and X-ray photoelectron spectroscopy. According to these analyses, introduction of oxygen-related chemical functional groups was presumed to result in increased hydrophilicity and efficiency of protein immobilization on parylene-N and parylene-C films after UV treatment. According to fluorescent staining, western blotting, and cell cycle analysis, UV-treated parylene-C and parylene-N films appear to effectively facilitate simultaneous proliferation and differentiation of PC-12 cells with neurite outgrowth.

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.

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.

In situ-synthesized cadmium sulfide nanowire photosensor with a parylene passivation layer for chemiluminescent immunoassays.

The direct in situ synthesis of cadmium sulfide (CdS) nanowires (NWs) was presented by direct synthesis of CdS NWs on the gold surface of an interdigitated electrode (IDE). In this work, we investigated the effect of a strong oxidant on the surfaces of the CdS NWs using X-ray photoelectron spectroscopy, transmission electron microscopy, and time-of-flight secondary ion mass spectrometry. We also fabricated a parylene-C film as a surface passivation layer for in situ-synthesized CdS NW photosensors and investigated the influence of the parylene-C passivation layer on the photoresponse during the coating of parylene-C under vacuum using a quartz crystal microbalance and a photoanalyzer. Finally, we used the in situ-synthesized CdS NW photosensor with the parylene-C passivation layer to detect the chemiluminescence of horseradish peroxidase and luminol and applied it to medical detection of carcinoembryonic antigen.

Band-type microelectrodes for amperometric immunoassays.

A band-type microelectrode was made using a parylene-N film as a passivation layer. A circular-type, mm-scale electrode with the same diameter as the band-type microelectrode was also made with an electrode area that was 5000 times larger than the band-type microelectrode. By comparing the amperometric signals of 3,5,3',5'-tetramethylbenzidine (TMB) samples at different optical density (OD) values, the band-type microelectrode was determined to be 9 times more sensitive than the circular-type electrode. The properties of the circular-type and the band-type electrodes (e.g., the shape of their cyclic voltammograms, the type of diffusion layer used, and the diffusion layer thickness per unit electrode area) were characterized according to their electrode area using the COMSOL Multiphysics software. From these simulations, the band-type electrode was estimated to have the conventional microelectrode properties, even when the electrode area was 100 times larger than a conventional circular-type electrode. These results show that both the geometry and the area of an electrode can influence the properties of the electrode. Finally, amperometric analysis based on a band-type electrode was applied to commercial ELISA kits to analyze human hepatitis B surface antigen (hHBsAg) and human immunodeficiency virus (HIV) antibodies.

Chemiluminescence lateral flow immunoassay based on Pt nanoparticle with peroxidase activity.

A lateral flow immunoassay (LF-immunoassay) with an enhanced sensitivity and thermostability was developed by using Pt nanoparticles with a peroxidase activity. The Pt nanoparticles were synthesized by citrate reduction method, and the peroxidase activity of Pt nanoparticles was optimized by adjusting reaction conditions. The peroxidase activity was estimated by using Michaelis-Menten kinetics model with TMB as a chromogenic substrate. The kinetics parameters of KM and Vmax were calculated and compared with horseradish peroxidase (HRP). The thermal stability of the Pt nanoparticles was compared with horseradish peroxidase (HRP) according to the storage temperature and long-term storage period. The feasibility of lateral flow immunoassay with a chemiluminescent signal band was demonstrated by the detection of human chorionic gonadotropin (hCG) as a model analyte, and the sensitivity was determined to be improved by as much as 1000-fold compared to the conventional rapid test based on colored gold-colloids.

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.